Sommaire du brevet 2807993 - Base de données sur les brevets canadiens (2024)

BISMUTH-THIOLS AS ANTISEPTICS FOR AGRICULTURAL, INDUSTRIAL
AND OTHER USES
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BACKGROUND
Technical field
The presently disclosed invention embodiments relate to
compositions and methods for the treatment of microbial infections. In
particular, the present embodiments relate to improved treatments for
managing bacterial infections in agricultural, industrial, manufacturing,
clinical,
personal healthcare, and other contexts, including treatment of bacterial
biofilms and other conditions.
Description of the Related Art
The complex series of coordinated cellular and molecular
interactions that contribute to responding to and resisting microbial
infections
and/or to healing or maintenance of plant and animal (including human) bodily
tissues generally, may be adversely impacted by a variety of external factors,

such as opportunistic and nosocomial infections (e.g., clinical regimens that
can
increase the risk of infection), local or systemic administration of
antibiotics
(which may influence cell growth, migration or other functions and can also
select for antibiotic-resistant microbes), and/or other factors.
Unfortunately, systemically or locally introduced antibiotics are
often not effective for the treatment of many chronic infections, and are
generally not used unless an acute bacterial infection is present. Current
approaches include administration or application of antibiotics, but such
remedies may promote the advent of antibiotic-resistant bacterial strains
and/or
may be ineffective against bacterial biofilms. It therefore may become
especially important to use antiseptics when drug resistant bacteria (e.g.,
methicillin resistant Staphylococcus aureus, or MRSA) are detected. There are
many antiseptics widely in use, but bacterial populations or subpopulations
that
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are established may not respond to these agents, or to any other currently
available treatments. Additionally, a number of antiseptics may be toxic to
host
cells at the concentrations that may be needed to be effective against an
established bacterial infection, and hence such antiseptics are unsuitable.
This
problem may be particularly acute in the case of efforts to clear infections
from
natural surfaces, including surface features on commercially and/or
agriculturally important plants such as many crop plants, and also including
internal epithelial surfaces, such as respiratory (e.g., airway,
nasopharyngeal
and laryngeal paths, tracheal, pulmonary, bronchi, bronchioles, alveoli, etc.)
or
gastrointestinal (e.g., buccal, esophageal, gastric, intestinal, rectal, anal,
etc.)
tracts, or other epithelial surfaces.
Particularly problematic are infections composed of bacterial
biofilms, a relatively recently recognized organization of bacteria by which
free,
single-celled ("planktonic") bacteria assemble by intercellular adhesion into
organized, multi-cellular communities (biofilms) having markedly different
patterns of behavior, gene expression, and susceptibility to environmental
agents including antibiotics. Biofilnns may deploy biological defense
mechanisms not found in planktonic bacteria, which mechanisms can protect
the biofilm community against antibiotics and host immune responses.
Established biofilms can arrest the tissue-healing process.
Common microbiologic contaminants that underlie persistent and
potentially deleterious infections include S. aureus, including MRSA
(Methicillin
Resistant Staphylococcus aureus), Enterococci, E. coli, P. aeruginosa,
Streptococci, and Acinetobacter baumannii. Some of these organisms exhibit
an ability to survive on non-nutritive clinical surfaces for months. S.
aureus, has
been shown to be viable for four weeks on dry glass, and for between three and

six months on dried blood and cotton fibers (Domenico et al., 1999 Infect.
lmmun. 67:664-669). Both E. coli and P. aeruginosa have been shown to
survive even longer than S. aureus on dried blood and cotton fibers (ibid).
Microbial biofilms are associated with substantially increased
resistance to both disinfectants and antibiotics. Biofilm morphology results
when bacteria and/or fungi attach to surfaces. This attachment triggers an
altered transcription of genes, resulting in the secretion of a remarkably
resilient
and difficult to penetrate polysaccharide matrix, protecting the microbes.
Biofilms are very resistant to the mammalian immune system, in addition to
their very substantial resistance to antibiotics. Biofilnns are very difficult
to
eradicate once they become established, so preventing biofilm formation is a
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very important clinical priority. Recent research has shown that open wounds
can quickly become contaminated by biofilms. These microbial biofilms are
thought to delay wound healing, and are very likely related to the
establishment
of serious wound infections.
Maintenance of intact, functioning skin and other epithelial tissues
(e.g., generally avascular epithelial surfaces that form barriers between an
organism and its external environment, such as those found in skin and also
found in the linings of respiratory and gastrointestinal tracts, glandular
tissues,
etc.) is significant to the health and survival of humans and other animals.
Bismuth Thiol- (BT) based Antiseptics
A number of natural products (e.g., antibiotics) and synthetic
chemicals having antimicrobial, and in particular antibacterial, properties
are
known in the art and have been at least partially characterized by chemical
structures and by antimicrobial effects, such as ability to kill microbes
("cidal"
.. effects such as bacteriocidal properties), ability to halt or impair
microbial
growth ("static" effects such as bacteriostatic properties), or ability to
interfere
with microbial functions such as colonizing or infecting a site, bacterial
secretion
of exopolysaccharides and/or conversion from planktonic to biofilm populations

or expansion of biofilm formation. Antibiotics, disinfectants, antiseptics and
the
.. like (including bismuth-thiol or BT compounds) are discussed, for example,
in
U.S. 6,582,719, including factors that influence the selection and use of such

compositions, including, e.g., bacteriocidal or bacteriostatic potencies,
effective
concentrations, and risks of toxicity to host tissues.
Bismuth, a group V metal, is an element that (like silver)
possesses antimicrobial properties. Bismuth by itself may not be
therapeutically useful and may exhibit certain inappropriate properties, and
so
may instead be typically administered by means of delivery with a connplexing
agent, carrier, and/or other vehicle, the most common example of which is
Pepto Bismo10, in which bismuth is combined (chelated) with subsalicylate.
Previous research has determined that the combination of certain thiol- (-SH,
sulfhydryl) containing compounds such as ethane dithiol with bismuth, to
provide an exemplary bismuth thiol (BT) compound, improves the antimicrobial
potency of bismuth, compared to other bismuth preparations currently
available.
There are many thiol compounds that may be used to produce BTs (disclosed,
for example, in Domenico et al., 2001 Antimicrob. Agent. Chemotherap.
45(5):1417-1421, Domenico et al., 1997 Antimicrob. Agent. Chemother.
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41(8):1697-1703, and in U.S. RE37,793, U.S. 6,248,371, U.S. 6,086,921, and
U.S. 6,380,248; see also, e.g., U.S. 6,582,719) and several of these
preparations are able to inhibit biofilm formation.
BT compounds have proven activity against MRS/A (methicillin
resistant S. aureus), MRSE (methicillin resistant S. epidermidis),
Mycobacterium tuberculosis, Mycobacterium avium, drug-resistant P.
aeruginosa, enterotoxigenic E. coli, enterohemorrhagic E. coli, Klebsiella
pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila,

Enterococcus faecalis, Enterobacter cloacae, Salmonella typhimurium, Proteus
vulgaris, Yersinia enterocolitica, Vibrio cholerae, and Shigella Flexneri
(Domenico et al., 1997 Antimicrob. Agents Chemother. 41:1697-1703). There
is also evidence of activity against cytomegalovirus, herpes simplex virus
type 1
(HSV-1) and HSV-2, and yeasts and fungi, such as Candida alb/cans. BT roles
have also been demonstrated in reducing bacterial pathogenicity, inhibiting or
killing a broad spectrum of antibiotic-resistant microbes (gram-positive and
gram-negative), preventing biofilm formation, preventing septic shock,
treating
sepsis, and increasing bacterial susceptibility to antibiotics to which they
previously exhibited resistance (see, e.g., Domenico et al., 2001 Agents
Chemother. 45:1417-1421; Domenico et al., 2000 Infect. Med. 17:123-127;
Domenico et al., 2003 Res. Adv. In Antimicrob. Agents & Chemother. 3:79-85;
Domenico et al., 1997 Antimicrob. Agents Chemother. 41(8):1697-1703;
Domenico et al., 1999 Infect. lmmun. 67:664-669: Huang et al. 1999 J
Antimicrob. Chemother. 44:601-605; Veloira et al., 2003 J Antimicrob.
Chemother. 52:915-919; Wu et al., 2002 Am J Respir Cell Mol Biol. 26:731-
738).
Despite the availability of BT compounds for well over a decade,
effective selection of appropriate BT compounds for particular infectious
disease indications has remained an elusive goal, where behavior of a
particular BT against a particular microorganism cannot be predicted, where
synergistic activity of a particular BT and a particular antibiotic against a
particular microorganism cannot be predicted, where BT effects in vitro may
not
always predict BT effects in vivo, and where BT effects against planktonic
(single-cell) microbial populations may not be predictive of BT effects
against
microbial communities, such as bacteria organized into a biofilm.
Additionally,
limitations in solubility, tissue permeability, bioavailability,
biodistribution and the
like may in the cases of some BT compounds hinder the ability to deliver
clinical
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benefit safely and effectively. The presently disclosed invention embodiments
address these needs and offer other related advantages.
Protection of Plants and Agricultural Products: Description of the Related Art

In the agricultural and botanical arts there is a recognized need
for formulations to reduce biofilms and disease in plants, and for methods of
using such formulations on, e.g., seeds, plants, fruits and flowers, soil, and
on
cut flowers, trees, fruits, leaves, stems and other plant parts.
In agriculture, every year billions of dollars of crops are lost due to
the formation of biofilms. The problem of anthracnose and biofilm-related
diseases in plants is well known despite numerous unsatisfactory approaches
that have attempted to address it. Plant diseases also affect industries
involved
in transporting and preserving fruit, vegetables, cut flowers and trees, and
other
plant products, as the normal protective mechanisms employed by intact living
plants are no longer operative in the harvested product.
It is therefore desirable for agricultural purposes to reduce the
amount of microbial growth on the surfaces of leaves, stems, fruits and
flowers
in situ, in transit or at commercial venues while maintaining compliance with
environmental regulations. At the same time, it is desirable to allow for the
flow
of water within cut flowers, plants and trees to maintain plant tissue
turgidity,
integrity and quality in order to enhance the desirable characteristics of
these
products.
Organisms that cause infectious disease in plants include fungi,
bacteria, viruses, protozoa, nematodes and parasitic plants. Insects and other

pests also affect plant health by consumption of plant tissues, and by
exposure
of plant tissues to microbes.
Biofilms occur when bacteria bind to a surface, typically in an
aqueous milieu such as under aquatic conditions or in water droplets or other
conditions of high humidity, and after binding the biofilm formers begin to
excrete a sticky substance which can then bind to a variety of materials
including metals, plastics, medical implants and tissues. These biofilms can
cause many problems, including degradation of materials and clogging of pipes,

in industrial and agricultural environments, and infection of surrounding
tissue
when occurring in a medical environment. The medical field is particularly
susceptible to problems caused by biofilm formation; implanted medical
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devices, catheters (urinary, venous, dialysis, cardiac) and slow-healing
wounds
are easily infiltrated by the bacteria present in biofilms. In agriculture,
biofilms
can cause mastitis, Pierce's disease, ring rot in potatoes, various crop
blights
and anthracnoses in many types of plants. Biofilnns also reduce the quality
and
product life of cut flowers and trees.
Many plant diseases are caused by biofilm-producing bacteria
indigenous to soil. Most microorganisms in the natural environment exist in
multicellular aggregates generally described as biofilms. Cells adhere to
surfaces and to each other through a complex matrix comprising a variety of
extracellular polymeric substances (EPS) including exopolysaccharides,
proteins and DNA. Plant-associated bacteria interact with host tissue surfaces

during pathogenesis and symbiosis, and in commensal relationships.
Observations of bacteria associated with plants increasingly reveal biofilm-
type
structures that vary from small clusters of cells to extensive biofilms. The
surface properties of the plant tissue, nutrient and water availability, and
the
proclivities of the colonizing bacteria strongly influence the resulting
biofilm
structure (Ramey et al., 2004 Carr Opinion Microbiol. 7:602-9).
The terrestrial environment harbors abundant and diverse
microbial populations that can compete for and modify resource pools. In this
complex and competitive environment, plants offer protective oases of nutrient-

rich tissues. Plants are colonized by bacteria on their leaves, roots, seeds
and
internal vasculature. Each tissue type has unique chemical and physical
properties that represent challenges and opportunities for microbial
colonists.
Biofilms may form upon association or at later stages, with significant
potential
to direct or modulate the plant¨microbe interaction. Additional temporal and
spatial complexity arises as many microbes actively modify the colonized plant

environment.
Surface-associated bacteria have a significant impact on
agriculture. In developed countries, the losses caused by plant diseases reach
up to 25% of crop yields, a percentage that is much higher in developing
countries. Epiphytic populations constitute a reservoir and future source of
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infection, and can be found on host and non-host plants. Xylophylus
ampelinus, a bacterial pathogen of grapevines, forms thick biofilms in the
vasculature of these plants (Grail & Manceau 2003). Xylella fastidiosa is the
causal agent of Pierce's disease in grapevines. X. fastidiosa is able to form
biofilms within xylem vessels of many economically important crops. The
mechanisms of pathogenicity are largely due to occlusion of xylem vessels by
aggregation of X. fastidiosa and biofilm formation. Vessel blockage is
believed
to be a major contributor to disease development, with xylem sap providing a
natural medium that facilitates the virulence of Pierce's disease of grapevine
and citrus variegated chlorosis (Zaini et al., 2009 FEMS Microbiol LETT.
295:129-34).
One of the most relevant plant pathogens, Pseudomonas
syringae, causes brown spot disease on bean. It colonizes the leaf surface
sparsely in solitary small groups (fewer than ten cells), while larger
populations
(more than 1000 cells) primarily develop near trichomes or veins with higher
nutrient availability. Large aggregates survive desiccation stress better than

solitary cells. P. syringae survives as an epiphyte (i.e., colonizer of the
aerial
parts of plants) when not causing infections on host plant tissues (Monier et
al.
PNAS 2003;100:15977-82).
Pseudomonas putida can respond rapidly to the presence of root
exudates in soils, converging at root colonization sites and establishing
stable
biofilms (Espinosa-Urgel et al. Microbiol 2002;148:341-3).
Xanthom*onas campestris pv. campestris (Xcc) causes black rot
on cruciferous plants, accessing the vasculature through wound sites in roots.
Virulence involves degradative exoenzynnes and the exopolysaccharide
xanthan gum, which is necessary for virulence (Dow et al. PNAS
2003;100:10995-1000).
Xanthom*onas smithii subsp. citri is responsible for the disease,
citrus canker. This disease has been found in most continents of the world
except Europe. The pathogen has been eradicated in many countries.
Xanthom*onas smithii forms canker lesions on fruit, leaves and twigs of citrus
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plants. Wind-driven rain can spread the bacteria up to 15 km from the source
to infect citrus trees via stomata or wounds (Sosnowski, et at. Plant Pathol
2009;58:621-35).
Pantoea stewartii subsp. stewartii causes Stewart's wilt disease in
maize and is transmitted by the corn flea beetle. The bacteria reside
primarily
in the host xylem and produce large amounts of exopolysaccharide (von
Bodman et al. PNAS 1998;95:7687-92).
Ralstonia solanacearum is a soil-borne pathogen that causes
lethal wilt on many plants. Virulence depends on EPS and cell-wall-degrading
enzymes controlled by a complex regulatory network (Kong et al. Mol Microbiol
2002; 46:427-37).
Clavibacter michiganensis subsp. sepedonicus is a Gram-positive
phytopathogen that causes bacterial ring rot in potato. Marques and colleagues

showed large bacterial, matrix-encased aggregates attached to the xylem
vessels (Marques et al. Phytopathol 2003;93:S57).
Biofilm-producing Erwinia chrysanthemi causes soft-rot disease
through rapid maceration of plant tissue. The production of pectic enzymes
may be quorum-sensing (QS)-regulated, and therefore the inability to form
bacterial aggregates may preclude pectinolytic enzyme secretion. Erwinia
amylovora, a related plant pathogen, infects approximately 75 different
species
of plants, all in the family Rosaceae. Hosts for this bacterium include apple,

pear, blackberry, cotoneaster, crabapple, firethorn (Pyracantha), hawthorn,
Japanese or flowering quince, mountain-ash, pear, quince, raspberry,
serviceberry, and spiraea. The cultivated apple, pear, and quince are the most
seriously affected species. A single fire blight epidemic in Michigan in 2000
resulted in the death of over 220,000 trees with a total loss of $42 million.
Annual losses to fire blight and cost of control in the U.S. are estimated at
over
$100 million (Norelli et al. Plant Dis 2003;87:26-32).
E. amylovora produces two exopolysaccharides, amylovoran and
levan, which cause the characteristic fire blight wilting symptom in host
plants
(Koczan et al. Phytopathol 2009;99:1237-44). In addition, other genes, and
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their encoded proteins, have been characterized as virulence factors of E.
amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic
activity and iron harvesting (Oh & Beer. FEMS Microbiology Lett 2005;253:185-
192).
No matter which part of the plant is attacked by a microbial plant
pathogen such as a biofilm-former, the effect is usually to weaken or kill the

plant. By infecting the leaves, the pathogen compromises the plant's ability
to
produce its food (e.g., via photosynthesis). Some plant pathogens block the
fluid transport vessels in the stems that supply the leaves, and when such
.. pathogens attack the roots, the uptake of water and nutrients is reduced or
stopped completely. Blockage of plant vasculature often involves biofilm-
producing bacteria that clog the flow of water and nutrients, both in growing
plants in soil and in cut plants in vase water.
When a plant is attacked by one of these microorganisms, the
.. resulting damage provides an opportunity for additional microbial invasion
of
plant tissue and it is the combined onslaught that ultimately damages and
destroys the plant. Plants that are under environmental stresses, such as
drought or poor nutrition, are particularly e susceptible to microbial attack.
Sometimes the microbial 'infection' is symbiotic, where both
.. organisms derive a benefit. A good example of this is the well known
nitrogen
fixing bacteria (Rhizobium) which reside in nodules on the roots of leguminous

(pea family) plants-- the plant provides food and protection, while the
bacteria
take nitrogen from the air and convert it to a form usable by the host. As
another example, the Mycorrhizae are a whole Order of fungi that have a
.. symbiotic relationship with plant roots. In view of such mutually
beneficial
symbioses, preservation or protection of plants against harmful microbial
pathogens may desirably employ antimicrobial agents that do not disrupt these
symbiotic relationships, wherever possible.
Saprophytic fungi are essential in breaking down dead organic
.. matter to produce the humus which is needed for good soil structure. They
do
not have any chlorophyll and so cannot use light to capture energy (e.g., via
photosynthesis); instead they derive their energy by breaking down plant and
animal material - alive or dead. They can also live in a symbiotic
relationship
with certain plant species, e.g., the micorrhizae in the fine roots of
conifers,
which cannot survive without them to take up vital nutrients. The widespread
use of chemical agents to control harmful plant pathogens can damage the
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balance of these beneficial fungi, and runs counter to the principals of
organic
management.
There are, however, other less welcome fungi, which attack living
plants and weaken or kill them. Another category of microbial plant pathogens,
viruses, may be resident within the cells of plant tissues and thus often
cannot
be treated with topically applied chemicals, such that affected plants must be

destroyed. There are currently no antibiotics specifically developed for the
treatment of plants (although some antibiotics developed for other purposes
have found uses on plants), leaving a number of economically significant plant
species vulnerable to pathogenic bacterial attacks. For instance, fireblight
infestations of numerous plant species of the family Rosaceae have proven
untreatable. Many harmful fungi, by contrast, can be killed with topically
applied chemicals without damaging the plant host, because thefungal growth
habitat is different, i.e., a number of undesirable pathogenic fungi tend to
grow
on plant surfaces and not within plant tissues, using root-like structures to
extract nourishment.
Because killing many plant pathogens is often difficult or
impossible, a number of strategies for protecting plants against deleterious
microbial pathogens adopt the philosophy that "prevention is better than
cure".
By observing good hygiene when propogating and growing plants, many
microbial plant diseases can be prevented by blocking the opportunity for a
microbial infection to be established. Often, significantly lower quantities
of
pesticides or microbicides can be effective when such agents are used
prophylactically, rather than in response to an established infection.
Plants are also more susceptible to disease if they are not
growing under optimal or near-optimal conditions, for example, due to poor
soil
quality (e.g., dearth of nutrients) by itself or in combination with drought
or
excessive rainfall or flooding. Extremely wet conditions can, for instance,
promote pathogenic fungal and/or bacterial growth. Quorum sensing in P.
syringae, for example, is dictated by water availability on the leaf surface
(DuIla
& Lindow. PNAS 2008;105:3-082-7). Of course not all plant diseases can be
prevented by good agricultural hygiene, insofar as some plant diseases are
transmitted by insects and others are wind-borne. Aphids and other sap-
sucking insects, for example, are the main vectors of viruses. Spores of
fungal
diseases are carried in the air, and in rain drops and splashes.

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Bio films on seeds and sprouts
Bacterial adherence to seeds is a process that strongly influences
rhizosphere colonization. Seed suppliers often deliberately coat seed stocks
with microbial biofilms to inoculate the developing rhizosphere. Conversely,
biofilms on seeds and sprouts used for human consumption are often common
sources of gastrointestinal infection. P. putida adheres effectively to seeds
and
will subsequently colonize the rhizosphere. Endophytic populations of
nonpathogenic actinobacteria found in wheat tissues were derived from interior

colonization by the actinobacteria of surface-sterilized seeds. Endophytic
seed
populations of beneficial nitrogen-fixing bacteria can help ensure future
rhizosphere colonization. Other studies of seed colonization have reported rod

shaped and coccal bacteria embedded within EPS in scanning
electronmicrographs of alfalfa seeds and sprouts. Biofilms are notoriously
resistant to washing and other common antibacterial treatments on seeds and
sprouts. Fett et al. found that both Escherichia coli 0157:H7 and Salmonella
populations on alfalfa sprouts required treatments much harsher than simple
water washing to reduce the numbers of adherent microbes, and full removal
was never achieved. The surviving bacteria likely resided within biofilms
(Ramey et al. Curr Opinion Microbiol 2004;7:602-9).
Cut flowers and trees
Vascular pathogens inhabit the xylem or phloem of plant hosts
and generally depend on insect vectors or wounding for dissemination. Cutting
flowers or trees is a similar type of wounding that is especially prone to
vascular
infection. Biofilm bacteria enter and clog the vasculature at the cut surface,
and
interfere with the flow of water, minerals and nutrients. Cut flower
preservatives
diluted in vase water often contain salicylate or aspirin to reduce biofilm
formation (Domenico et al., J Antimicrob Chemo 1991;28:801-10; Salo et al.,
Infection 1995;23:371-7), and provide a low pH to prevent bacterial growth and
disrupt biofilms.
Antimicrobial Agents in Agriculture. Eradication of plant pathogen
incursions is very important for the protection of plant industries, managed
gardens and natural environments worldwide. The consequence of a pathogen
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becoming endemic can be serious, in some cases having an impact on the
national economy. The current strategy for eradication of a pathogen relies on

techniques for the treatment, removal and disposal of affected host plants.
There are many examples where these techniques have been successful but
many where they have not. Success relies on a sound understanding of the
biology and epidemiology of the pathogen and its interaction with the host. In

examining examples of dealing with plant pathogens and diseased host
material around the world, particularly Australasia, various techniques
including
burning, burying, pruning, composting, soil- and biofumigation, solarization,
steam sterilization and biological vector control have been used (Sosnowski,
et
al. Plant Pathol 2009; 58:621-35).
Antibiotics have also been used since the 1950s, to control certain
bacterial diseases of high-value fruit, vegetable, and ornamental plants.
Today,
the antibiotics most commonly used on plants are oxytetracycline and
streptomycin. In the USA, antibiotics applied to plants account for less than
0.5% of total antibiotic use. Resistance of plant pathogens to oxytetracycline
is
rare, but the emergence of streptomycin-resistant strains of Erwinia
amylovora,
Pseudomonas spp., and Xanthom*onas campestris has impeded the control of
several important diseases. Thus, the role of antibiotic use on plants in the
antibiotic-resistance crisis in human medicine is the subject of debate
(McManus et al. Annu Rev Phytopathol 2002;40:443-65).
The emergence of streptomycin-resistant (SmR) plant pathogens
has complicated the control of bacterial diseases of plants. For example, in
the
United States, streptomycin is permitted on tomato and pepper for control of
X.
campestris pv. vesicatoria, but it is rarely used for this purpose because
resistant strains are now widespread. Resistance in E. amylovora, the fire
blight pathogen, has had widespread economic and political implications. Other

phytopathogenic bacteria in which SmR has been reported include
Pectobacterium carotovora, Pseudomonas chichorii, Pseudomonas
lachrymans, Pseudomonas syringae pv. papulans, Pseudomonas syringae pv.
syringae, and Xanthom*onas dieffenbachiae (McManus et al. Annu Rev
Phytopathol 2002;40:443-65). The emergence SmR E. amylovora has
intensified fire blight epidemics in the western USA and Michigan.
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Streptomycin and oxytetracycline have been assigned the lowest
toxicity category by the U.S. Environmental Protection Agency (EPA), and
carcinogenic or mutagenic activities have not been observed for either
antibiotic.
Alternatives to antibiotics are available and, at least to some
extent, practical. Indeed, bacterial disease management in most cropping
systems is based on the integration of genetic resistance of the host,
sanitation
(avoidance or removal of inoculum), and cultural practices that create an
environment unfavorable for disease development. Biocontrol of plants using
various species of bacteria and fungi is of growing interest. Rhizobacteria
are
considered as efficient microbial competitors in the root zone.
Representatives
of many different bacterial genera have been introduced into soils, onto
seeds,
roots, tubers or other planting materials to improve crop growth. These
bacterial genera include Acinetobacter, Agrobacterium, Arthrobacter,
Azospirillum, Bacillus, Bra dyrhizobium, Frankia, Pseudomonas, Rhizobium,
Serratia, Thiobacillus, and many others. Certain species of Bacillus, for
example, can induce systemic resistance in many plants (Choudhary &Johri.
Microbiol Res 2009;164:493-513).
Application of copper compounds is effective in reducing
populations of some bacterial plant pathogens, although several species have
become resistant to copper (Cooksey Annu Rev Phytopathol 1990;28:201-14),
and most tree-fruit crops are sensitive to copper injury.
A number of synthetic and natural remedies exist for various plant
diseases. Natural remedies include apple cider vinegar for leafspot, mildew
and scab; baking soda spray for anthracnose, early tomato blight, leaf blight,
powdery mildew and as a general fungicide; neem oil; sulfur; garlic; hydrogen
peroxide; compost teas, etc. Numerous synthetic chemicals are used to
prevent or treat plant disease, and come in water-soluble or water-insoluble
formulations. Microbicides include phenoxarsine or a phenarsazine, maleimide,
isoindole dicarboximide, halogenated aryl alkanol, 4-thioxopyrimidine
derivatives (US Patent 6384040), heterocyclic organosilyl compounds and
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isothiazolinone. Many microbicides are combined with pyrithione derivatives to

make synergistic compounds (e.g., EP1468607). Certain
isothiazolecarboxamides can be employed for the control of plant pests (e.g.,
US 6552056; WO 2001/064644)
Recognizing the toxicity problem of microbicides in powder or
crystalline form, U.S. Pat. No. Re. 29,409 teaches dissolving microbicides in
liquid solvents, which may be added to the formulation mixture from which the
end-use resin compositions are fabricated. Although liquid dispersions may be
safely used at the site of preparing end-use resin compositions, careless use
or
disposal of the liquids may still pose environmental and health hazards.
Alternatively, microbicides can also be administered in water-soluble
thermoplastic resins. Microbicides can be added to rigid thermoplastic resin
compositions and impart biocidal activity thereto so as to inhibit microbial
growth on the surfaces thereof (US 5,229,124). This is a solid, melt-blended
solution consisting essentially of a microbicide dissolved in a carrier resin
that is
a copolymer of vinyl alcohol and (alkyleneoxy) acrylate. Although a
microbicide
may be a highly toxic chemical, its low concentration in the end-use product
and its retention by the resin composition ensures that the microbicide in the

end-use product poses no hazard to humans or animals.
Isothiazolinones are often used as microbicides in agriculture, for
example, N-alkylbenzenesulfonylcarbamoy1-5-chloroisothiazole derivatives
(e.g., US 5,045,555). This microbicide is widely useful in, for example, the
paper industry, textile industry, for producing coatings and adhesives, in
painting, metal processing, in the resin industry, wood industry, construction
industry, agriculture, forestry, fisheries, food industry and petroleum
industry as
well as in medicine. It exhibits an intense microbicidal effect, and can be
added, in an appropriate amount, to processing water, circulating water, a raw

material or a product. Further, it may be employed for disinfecting or
sterilizing
facilities, plants, livestock barns or instruments as well as seeds, seedlings
and
raw materials. Other derivatives of isothiazolone are also known (U.S Pat. No.
3,523,121 and J. Heterocyclic Chem., 8, 587 (1971)). However, every known
14

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derivative compound is highly toxic to animals and fishes, which significantly

restricts their application.
Sodium bicarbonate commonly has also been found to possess
fungicidal properties when applied to plants, but typically requires frequent
.. reapplication in order to realize efficacy.
The role of iron in plant host-parasite relationships has been
elucidated in diseases as different as the soft rot and fire blight incited by

Erwinia chrysanthemi and E amylovora, respectively (Expert. Annu Rev
Phytopathol 1999;37:307-34). Because of its unique position in biological
systems, iron controls the activities of plant pathogens. The production of
siderophores by pathogens not only represents a powerful strategy to acquire
iron from host tissues but may also act as a protective agent against iron
toxicity. The need of the host to bind and possibly sequester the metal during

pathogenesis is another central issue. Antimicrobials that interfere with
bacterial iron uptake and cell respiration may play an important role in plant
disinfection.
Many natural products (e.g., antibiotics) and synthetic chemicals
with antimicrobial, antiseptic and in particular antibacterial, properties are

known and have been at least partially characterized chemically and
biologically. Exemplary characteristics include the ability to kill microbes
(bactericidal effects), ability to halt or impair microbial growth
(bacteriostatic
effects), or ability to interfere with microbial functions such as colonizing
or
infecting a site, bacterial secretion of metabolites (some of which are
malodorous), and/or conversion from planktonic to biofilm populations or
expansion of biofilrn formation (anti-biofilm effects). Antibiotics,
disinfectants,
antiseptics and the like (including bismuth-thiol or BT compounds) are
discussed in U.S. 6,582,719, including factors that influence the selection
and
use of such compositions, including, e.g., bactericidal, bacteriostatic, or
anti-
biofilm potencies, effective concentrations, and risks of toxicity to host
tissues.
Bacterial microcolonies protected within the biofilm are typically
resistant to antiseptics or disinfectants. Antibiotic doses that kill free-
floating
bacteria, for example, need to be increased as much as 1,500 times to kill
biofilm bacteria. At this high concentration, some antimicrobials can be
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Oxidizing brominated and chlorinated compounds, for example, are highly toxic
and corrosive.
Suppression of the blossom-blight phase is a key to the
management of fire blight. For blossom infection to occur, Erwinia amylovora
to
needs proliferate on stigmatic surfaces in an epiphytic phase. Rain is
necessary for infection because it dilutes sugars on the hypanthium to osmotic

potentials not inhibitory to E. amylovora. Rain is also important as an agent
for
redistribution of the bacterium from the stigmas to the hypanthium. These
observations suggest that the optimal timing for use of antibiotic sprays is
during this epiphytic phase, and after excessive rain (Johnson & Stockwell.
Annu Rev Phytopathol 1998;36:227-48).
Other bacterial epiphytes also colonize stigmas where they can
interact with and suppress epiphytic growth of the pathogen. A commercially
available bacterial antagonist of E. amylovora (BlightBan, Pseudomonas
fluorescens A506) can be included in antibiotic spray programs. Integration of

bacterial antagonists with chemical methods suppresses populations of the
pathogen and concomitantly, fills the ecological niche provided by the stigma
with a nonpathogenic, competing microorganism (Johnson & Stockwell. Annu
Rev Phytopathol 1998;36:227-48).
Pyrithione is the conjugate base derived from 2-mercaptopyridine-
N-oxide (CAS# 1121-31-9), a derivative of pyridine-N-oxide. Its antifungal
effect resides in its ability to disrupt membrane transport by blocking the
proton
pump that energizes the transport mechanism. Experiments have suggested
that fungi are capable of inactivating pyrithione in low concentrations
(Chandler
& Segel. Antimicrob. Agents Chemother 1978;14:60-8). Zinc pyrithione is a
coordination complex of zinc. This colorless solid is used as an antifungal
and
antibacterial agent. Due to its low solubility in water (8 ppm at neutral pH),
zinc
pyrithione is suitable for use in outdoor paints, cements and other products
that
provide protection against mildew and algae. It is an effective algaecide. It
is
chemically incompatible, however, with paints that rely on metal carboxylate
curing agents. When used in latex paints comprising water that contains high
amount of iron, a sequestering agent that will preferentially bind the iron
ions is
needed.
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Particularly problematic in agriculture are infections composed of
bacterial biofilms, a relatively recently recognized organization of bacteria
by
which free, single-celled ("planktonic") bacteria assemble by intercellular
adhesion into organized, multi-cellular communities (biofilms) having markedly
different patterns of behavior, gene expression, and susceptibility to
environmental agents including antibiotics. Biofilms may deploy biological
defense mechanisms not found in planktonic bacteria, which mechanisms can
protect the biofilm community against antibiotics and host immune responses.
Established biofilms can arrest growth, development or wound-healing
processes in plants.
Microbial biofilms are associated with substantially increased
resistance to both disinfectants and antibiotics. Biofilm morphology results
when bacteria and/or fungi attach to surfaces. This attachment triggers an
altered transcription of genes, resulting in the secretion of a remarkably
resilient
and difficult to penetrate polysaccharide matrix, protecting the microbes.
Biofilms are very resistant to the plant immune defense mechanisms, in
addition to their very substantial resistance to antibiotics. Biofilms are
very
difficult to eradicate once they become established, so preventing biofilm
formation is a very important agricultural priority. Recent research has shown
that open wounds can quickly become contaminated by biofilms. These
microbial biofilms are thought to impair growth, development and/or wound
healing, and are very likely related to the establishment of serious and often

intractable infections.
Clearly there is a need for improved compositions and methods
for treating and preventing microbial infections in and on plants, including
microbial infections that occur as biofilms. Certain embodiments described
herein address this need and provide other related advantages.
BRIEF SUMMARY
As disclosed herein, and without wishing to be bound by theory,
according to certain embodiments described for the first time herein, bismuth-
thiol (BT) compounds may be used as antiseptic agents for use in a wide
variety of agricultural, industrial, manufacturing and other contexts, as well
as in
the treatment or prevention of infectious diseases and related conditions and
in
personal healthcare, while also decreasing the costs incurred for the
treatment
of such infections, including savings that are realized by prevention or
prophylaxis mediated at least in part by BTs.
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Also, in certain embodiments described herein there are
contemplated formulations for treating plants or plant tissues (e.g., a root,
bulb,
stem, leaf, branch, vine, runner, bud, flower or a part thereof, greentip,
fruit,
seed, seed pod, or the like) and animal tissues and/or natural and artificial
surfaces that contain bacterial biofilms or bacteria related to biofilm
formation
(e.g., bacteria that are capable of forming or otherwise promoting biofilms),
which formulations comprise one or more BT compound and one or more
antibiotic compound, as described herein, where according to non-limiting
theory, appropriately selected combinations of BT compound(s) and
antibiotic(s) based on the present disclosure provide heretofore unpredicted
synergy in the antibacterial (including anti-biofilm) effects of such
formulations,
and/or unpredicted enhancing effects, for prevention, prophylaxis and/or
therapeutically effective treatment against microbial infections including
infections that contain bacterial biofilms.
Also provided herein for use in these and related embodiments
are bismuth-thiol compositions that advantageously comprise substantially
nnonodisperse microparticulate suspensions, and methods for their synthesis
and use.
According to certain embodiments of the invention described
herein there is thus provided a method for protecting a plant against a
bacterial,
fungal or viral pathogen, comprising contacting the plant or a part thereof
(e.g.,
all or part of a root, bulb, stem, leaf, branch, vine, runner, bud, flower or
a part
thereof, greentip, fruit, seed, seed pod, or the like) with an effective
amount of a
bismuth-thiol (BT) composition under conditions and for a time sufficient for
one
or more of: (i) prevention of infection of the plant by the bacterial, fungal
or viral
pathogen, (ii) inhibition of cell viability or cell growth of substantially
all
planktonic cells of the bacterial, fungal or viral pathogen, (iii) inhibition
of biofilm
formation by the bacterial, fungal or viral pathogen, and (iv) inhibition of
biofilm
viability or biofilm growth of substantially all biofilm-form cells of the
bacterial,
fungal or viral pathogen, wherein the BT composition comprises a substantially

monodisperse suspension of nnicroparticles that comprise a BT compound, said
microparticles having a volumetric mean diameter of from about 0.4 Jim to
about 10 p.m. In a further embodiment the bacterial pathogen comprises
Erwinia amylovora cells. In another embodiment the bacterial pathogen is
selected from Erwinia amylovora, Xanthom*onas campestris pv die ffenbachiae,
Pseudomonas syringae, Xylella fastidiosa; Xylophylus ampelinus; Monilinia
fructicola, Pantoea stewartii subsp. Stewartii, Ralstonia solanacearum, and
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Clavibacter michiganensis subsp. sepedonicus. In certain embodiments the
bacterial pathogen exhibits antibiotic resistance. In certain embodiments the
bacterial pathogen exhibits streptomycin resistance. In certain embodiments
the plant is a food crop plant, which in certain further embodiments is a
fruit
tree. In certain still further embodiments the fruit tree is selected from an
apple
tree, a pear tree, a peach tree, a nectarine tree, a plum tree, an apricot
tree. In
certain other embodiments the food crop plant is a banana tree of genus Musa.
In certain other embodiments the food crop plant is a plant selected from a
tuberous plant, a leguminous plant, and a cereal grain plant. In certain
further
embodimenst the tuberous plant is selected from Solanum tuberosum (potato),
and Ipomoea batatas (sweet potato). In certain embodiments of the above
described method, the step of contacting is performed one or a plurality of
times. In certain further embodiments at least one step of contacting
comprises
one of spraying, dipping, coating and painting the plant. In certain other
further
embodiments at least one step of contacting is performed at a flower blossom,
green-tip or growth site of the plant. In certain embodiments at least one
step
of contacting is performed within 24, 48 or 72 hours of first flower blooming
on
the plant.
In certain embodiments of the above described method, the BT
composition comprises one or more BT compounds selected from BisBAL,
BisEDT, Bis-dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr,
Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-

Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-
EDT/2-hydroxy-1-propanethiol. In certain embodiments the bacterial pathogen
exhibits antibiotic resistance.
In certain further embodiments of the above described methods,
the method comprises contacting the plant with a synergizing or enhancing
antibiotic, simultaneously or sequentially and in any order with respect to
the
step of contacting the plant with the BT composition. In certain embodiments
the synergizing or enhancing antibiotic comprises an antibiotic that is
selected
from an aminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporin
antibiotic, a fluoroquinolone antibiotic, a penicillinase-resistant penicillin

antibiotic, and an aminopenicillin antibiotic. In certain embodiments the
synergizing or enhancing antibiotic is an aminoglycoside antibiotic that is
selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin,
netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and
apramycin.
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According to certain other embodiments there is provided a
method for overcoming antibiotic resistance in a plant in or on which an
antibiotic-resistant bacterial plant pathogen is present, comprising (a)
contacting the plant with an effective amount of a BT composition under
conditions and for a time sufficient for one or more of: (i) prevention of
infection
of the plant by the antibiotic-resistant bacterial pathogen, (ii) inhibition
of cell
viability or cell growth of substantially all planktonic cells of the
antibiotic-
resistant bacterial pathogen, (iii) inhibition of biofilm formation by the
antibiotic-
resistant bacterial pathogen, and (iv) inhibition of biofilm viability or
biofilm
.. growth of substantially all biofilm-form cells of the antibiotic-resistant
bacterial
pathogen, wherein the BT composition comprises a substantially monodisperse
suspension of microparticles that comprise a BT compound, said microparticles
having a volumetric mean diameter of from about 0.5 ii.rn to about 10 pm; and
(b) contacting the plant with a synergizing or enhancing antibiotic,
simultaneously or sequentially and in any order with respect to the step of
contacting the plant with the BT composition.
In certain embodiments of the above described methods, the
bismuth-thiol composition comprises a plurality of microparticles that
comprise
a bismuth-thiol (BT) compound, substantially all of said microparticles having
a
volumetric mean diameter of from about 0.4 p.m to about 5 pm and being
formed by a process that comprises: (a) admixing, under conditions and for a
time sufficient to obtain a solution that is substantially free of a solid
precipitate,
(i) an acidic aqueous solution that comprises a bismuth salt comprising
bismuth
at a concentration of at least 50 mM and that lacks a hydrophilic, polar or
organic solubilizer, with (ii) ethanol in an amount sufficient to obtain an
admixture that comprises about 25% ethanol by volume; and (b) adding to the
admixture of (a) an ethanolic solution comprising a thiol-containing compound
to obtain a reaction solution, wherein the thiol-containing compound is
present
in the reaction solution at a molar ratio of from about 1:3 to about 3:1
relative to
the bismuth, under conditions and for a time sufficient for formation of a
precipitate which comprises the microparticles comprising the BT compound.
In certain embodiments the bismuth salt is Bi(NO3)3. In certain
embodiments the acidic aqueous solution comprises at least 5%, 10%, 15%,
20%, 22% or 22.5% bismuth by weight. In certain embodiments the acidic
aqueous solution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,
4.5% or 5% nitric acid by weight. In certain embodiments the thiol-containing
compound comprises one or more agents selected from 1,2-ethane dithiol, 2,3-

CA 02807993 2013-02-08
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dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-
butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-
propanol, dithioerythritol, alpha-lipoic acid, dithiothreitol, methanethiol
(CH3SH
[m-mercaptan]), ethanethiol (C2H5SH [e- mercaptan]), 1-propanethiol (C3H7SH
[n-P mercaptan]), 2-propanethiol (CH3CH(SH)CH3 [2C3 mercaptan]),
butanethiol (C4H9SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH3)3SH [t-
butyl nnercaptan]), pentanethiol (C5H11SH [pentyl mercaptan]), coenzyme A,
lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol,
dithioerythritol, 2-mercaptoindole, transglutaminase, (11-
mercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene
glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functional ized gold
nanoparticles, 1,1',4',1"-terpheny1-4-thiol, 1,11-undecanedithiol, 1,16-
hexadecanedithiol, 1,2-ethanedithiol technical grade, 1,3-propanedithiol, 1,4-
benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-
pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,
adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-
heptanethiol,
1-heptanethiol purum, 1-hexadecanethiol, 1-hexanethiol, 1-nnercapto-
(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether functional
ized
gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-octadecanethiol,
1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-pentanethiol, 1-
propanethiol,
1-tetradecanethiol, 1-tetradecanethiol pururn, 1-undecanethiol, 11-(1H-pyrrol-
1-
yOundecane-1-thiol, 11-amino-l-undecanethiol hydrochloride, 11-bromo-1-
undecanethiol, 11-mercapto-1-undecanol, 11-mercapto-1-undecanol, 11-
mercaptoundecanoic acid, 11-mercaptoundecanoic acid, 11-mercaptoundecyl
trifluoroacetate, 11-mercaptoundecylphosphoric acid, 12-mercaptododecanoic
acid, 12-mercaptododecanoic acid, 15-mercaptopentadecanoic acid, 16-
mercaptohexadecanoic acid, 16-mercaptohexadecanoic acid, 1 H,1H,2H,2H-
perfluorodecanethiol, 2,2'-(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-
butanethiol, 2-ethylhexanethiol, 2-methyl-1 -propanethiol, 2-methyl-2-
propanethiol, 2-phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol
purum, 3-(dimethoxymethylsily1)-1-propanethiol, 3-chloro-1-propanethiol, 3-
mercapto-1-propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide,
3-mercaptopropionic acid, 3-nnercaptopropyl-functionalized silica gel, 3-
methyl-
1-butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-(6-

mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-
butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol, 6-mercaptohexanoic
acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-mercapto-1-nonanol,
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biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate, copper(I) 1-butanethiolate,

cyclohexanethiol, cyclopentanethiol, decanethiol functionalized silver
nanoparticles, dodecanethiol functionalized gold nanoparticles, dodecanethiol
functionalized silver nanoparticles, hexa(ethylene glycol)mono-11-
(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-
mercaptopropionate, nanoTether BPA-HH, NanoThinks TM 18, NanoThinkerm 8,
NanoThinks TM ACID11, NanoThinks TM ACID16, NanoThinks TM ALC011,
NanoThinksTm THI08, octanethiol functionalized gold nanoparticles, PEG dithiol

average Mn 8,000, PEG dithiol average mol wt 1,500, PEG dithiol average mol
wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4-cyanobutyl)thioacetate,
thiophenol, triethylene glycol mono-11-mercaptoundecyl ether,
trimethylol propane tris(3-mercaptopropionate), [11-
(methylcarbonylthio)undecyl]tetra(ethylene glycol), m-carborane-9-thiol, p-
terpheny1-4,4"-dithiol, tert-dodecylmercaptan, and tert-nonyl mercaptan.
In certain embodiments the bacterial pathogen comprises at least
one of: (i) one or more gram-negative bacteria; (ii) one or more gram-positive

bacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one or more
antibiotic-resistant bacteria; (v) a bacterial pathogen that is selected from
Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus),
Staphylococcus epidermidis, MRSE (methicillin-resistant S. epidermidis),
Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,
drug-resistant P. aeruginosa, Escherichia co/i, enterotoxigenic E. coli,
enterohemorrhagic E. coil, Klebsiella pneumoniae, Clostridium difficile,
Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, methicill
in-
susceptible Enterococcus faecalis, Enterobacter cloacae, Salmonella
typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholera,
Shigella
flexneri, vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia
complex, Francisella tularensis, Bacillus anthracis, Yersinia pestis,
Pseudomonas aeruginosa, Streptococcus pneumonia, penicillin-resistant
Streptococcus pneumonia, Escherichia co/i, Burkholderia cepacia, Bukholderia
multivorans, Mycobacterium smegm*tis and Acinetobacter baumannii.
In certain embodiments the method comprises contacting the
plant with at least one of (i) a synergizing antibiotic and (ii) a cooperative

antimicrobial efficacy enhancing antibiotic, simultaneously or sequentially
and in
any order with respect to the step of contacting the surface with the BT
composition. In certain further embodiments the synergizing antibiotic or the
cooperative antimicrobial efficacy enhancing antibiotic comprises an
antibiotic
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that is selected from an aminoglycoside antibiotic, a carbapenem antibiotic, a

cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide
antibiotic, a
lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and
an
aminopenicillin antibiotic. In certain further embodiments the synergizing
antibiotic or the cooperative antimicrobial efficacy enhancing antibiotic is
an
aminoglycoside antibiotic that is selected from amikacin, arbekacin,
gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,
streptomycin, tobramycin and apramycin.
In certain other embodiments there is provided a method for
overcoming antibiotic resistance in or on a plant where an antibiotic-
resistant
bacterial pathogen is present, comprising: contacting the plant simultaneously

or sequentially and in any order with an effective amount of (1) at least one
bismuth-thiol (BT) composition and (2) at least one antibiotic that is capable
of
enhancing or acting synergistically with the at least one BT composition,
under
conditions and for a time sufficient for one or more of: (i) prevention of
infection
of the plant by the bacterial pathogen, (ii) inhibition of cell viability or
cell growth
of substantially all planktonic cells of the bacterial pathogen, (iii)
inhibition of
biofilm formation by the bacterial pathogen, and (iv) inhibition of biofilm
viability or biofilm growth of substantially all biofilm-form cells of the
bacterial
pathogen, wherein the BT composition comprises a plurality of microparticles
that comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a volumetric mean diameter of from about 0.4 Jim to
about 5 turn; and thereby overcoming antibiotic resistance on the epithelial
tissue surface. In certain further embodiments the bacterial pathogen exhibits
resistance to an antibiotic that is selected from nnethicillin, vancomycin,
naficilin,
gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin
and gatifloxacin. In certain other embodiments the BT composition comprises
one or more BT compounds selected from BisBAL, Bis EDT, Bis-dinnercaprol,
Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT,
Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-
Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-
propanethiol. In certain embodiments the synergizing or enhancing antibiotic
comprises an antibiotic that is selected from clindamicin, gatifloxacin, an
aminoglycoside antibiotic, a carbapenem antibiotic, a cephalosporin
antibiotic, a
fluoroquinolone antibiotic, a penicillinase-resistant penicillin antibiotic,
and an
aminopenicillin antibiotic. In certain further embodiments the synergizing or
enhancing antibiotic is an aminoglycoside antibiotic that is selected from
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amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
According to certain other embodiments there is provided a
bismuth-thiol composition, comprising a plurality of microparticles that
comprise
a bismuth-thiol (BT) compound, substantially all of said microparticles having
a
volumetric mean diameter of from about 0.4 lam to about 5 lam, wherein the BT
compound comprises bismuth or a bismuth salt and a thiol-containing
compound. In another embodiment there is provided a bismuth-thiol
composition, comprising a plurality of microparticles that comprise a bismuth-
thiol (BT) compound, substantially all of said microparticles having a
volumetric
mean diameter of from about 0.41.1m to about 5 gn and being formed by a
process that comprises (a) admixing, under conditions and for a time
sufficient
to obtain a solution that is substantially free of a solid precipitate, (i) an
acidic
aqueous solution that comprises a bismuth salt comprising bismuth at a
concentration of at least 50 mM and that lacks a hydrophilic, polar or organic
solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture
that
comprises at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume;
and (b) adding to the admixture of (a) an ethanolic solution comprising a
thiol-
containing compound to obtain a reaction solution, wherein the thiol-
containing
compound is present in the reaction solution at a molar ratio of from about
1:3
to about 3:1 relative to the bismuth, under conditions and for a time
sufficient for
formation of a precipitate which comprises the microparticles comprising the
BT
compound. In certain embodiments the bismuth salt is Bi(NO3)3. In certain
embodiments the acidic aqueous solution comprises at least 5%, 10%, 15%,
20%, 22% or 22.5% bismuth by weight. In certain embodiments the acidic
aqueous solution comprises at least 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%,
4.5% or 5% nitric acid by weight. In certain embodiments the thiol-containing
compound comprises one or more agents selected from 1,2-ethane dithiol, 2,3-
dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-
butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-
propanol, dithioerythritol, alpha-lipoic acid and dithiothreitol.
In another embodiment there is provided a method for preparing a
bismuth-thiol composition that comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles
having a volumetric mean diameter of from about 0.4 ium to about 5 um, said
method comprising the steps of (a) admixing, under conditions and for a time
sufficient to obtain a solution that is substantially free of a solid
precipitate, (i)
24

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an acidic aqueous solution that comprises a bismuth salt comprising bismuth at

a concentration of at least 50 mM and that lacks a hydrophilic, polar or
organic
solubilizer, with (ii) ethanol in an amount sufficient to obtain an admixture
that
comprises at least about 5%, 10%, 15%, 20%, 25% or 30% ethanol by volume;
and (b) adding to the admixture of (a) an ethanolic solution comprising a
thiol-
containing compound to obtain a reaction solution, wherein the thiol-
containing
compound is present in the reaction solution at a molar ratio of from about
1:3
to about 3:1 relative to the bismuth, under conditions and for a time
sufficient for
formation of a precipitate which comprises the microparticles comprising the
BT
compound. In certain embodiments the method further comprises recovering
the precipitate to remove impurities. In certain embodiments the bismuth salt
is
Bi(NO3)3. In certain embodiments the acidic aqueous solution comprises at
least 5%, 10%, 15%, 20%, 22% or 22.5% bismuth by weight. In certain
embodiments the acidic aqueous solution comprises at least 0.5%, 1%, 1.5%,
2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5% nitric acid by weight. In certain
embodiments the thiol-containing compound comprises one or more agents
selected from the group consisting of 1,2-ethane dithiol, 2,3-
dimercaptopropanol, pyrithione, dithioerythritol, 3,4-dimercaptotoluene, 2,3-
butanedithiol, 1,3-propanedithiol, 2-hydroxypropane thiol, 1-mercapto-2-
propanol, dithioerythritol, dithiothreitol, alpha-lipoic acid, methanethiol
(CH3SH
[m-mercaptan]), ethanethiol (C2H5SH [e- mercaptan]), 1-propanethiol (C3H7SH
[n-P mercaptan]), 2-propanethiol (CH3CH(SH)CH3 [2C3 mercaptan]),
butanethiol (C4H9SH ([n-butyl mercaptan]), tert-butyl mercaptan (C(CH3)3SH [t-
butyl mercaptan]), pentanethiols (C5H11SH [pentyl mercaptan]), coenzyme A,
lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol, dithiothreitol,
dithioerythritol, 2-mercaptoindole, transglutaminase, (11-
mercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene
glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functional ized gold
nanoparticles, 1,1`,4`,1"-terphenyl-4-thiol, 1,11-undecanedithiol, 1,16-
hexadecanedithiol, 1,2-ethanedithiol technical grade, 1,3-propanedithiol, 1,4-
benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-
pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,
adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-
heptanethiol, 1-heptanethiol purum, 1-hexadecanethiol, 1-hexanethiol, 1-
mercapto-(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether
functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-
octadecanethiol, 1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-

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pentanethiol, 1-propanethiol, 1-tetradecanethi01, 1-tetradecanethi01 purum, 1-
undecanethiol, 11-(1H-pyrrol-1-yOundecane-1-thiol, 11-amino-1-undecanethiol
hydrochloride, 11-bromo-1-undecanethiol, 11-mercapto-1-undecanol, 11-
mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanoic
acid, 11-nnercaptoundecyl trifluoroacetate, 11-mercaptoundecylphosphoric
acid, 12-mercaptododecanoic acid, 12-mercaptododecanoic acid, 15-
nnercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-
mercaptohexadecanoic acid, 1H,1H,2H,2H-perfluorodecanethiol, 2,2'-
(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-
ethylhexanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-
phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purum, 3-
(dinnethoxymethylsily1)-1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-
propanol, 3-mercapto-2-butanol, 3-mercapto-N-nonylpropionamide, 3-
nnercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3-methyl-
1-butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-
(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-
butanol, 6-(ferrocenyl)hexanethiol, 6-nnercapto-1-hexanol, 6-
mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-
mercapto-1-nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate,
copper(I) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol
functionalized silver nanoparticles, dodecanethiol functionalized gold
nanoparticles, dodecanethiol functionalized silver nanoparticles,
hexa(ethylene
glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-
mercaptopropionate, nanoTether BPA-HH, NanoThinksTm 18, NanolhinksTM 8,
NanoThinks TM ACID11, NanoThinks TM ACID16, NanoThinks TM ALC011,
NanoThinksTm THI08, octanethiol functionalized gold nanoparticles, PEG
dithiol average Mn 8,000, PEG dithiol average mol wt 1,500, PEG dithiol
average mol wt 3,400, S-(11-bronnoundecyl)thioacetate, S-(4-
cyanobutypthioacetate, thiophenol, triethylene glycol mono-11-
mercaptoundecyl ether, trimethylolpropane tris(3-mercaptopropionate), [11-
(methylcarbonylthio)undecyl]tetra(ethylene glycol), m-carborane-9-thiol, p-
terpheny1-4,4"-dithiol, tert-dodecylmercaptan, tert-nonyl nnercaptan.
In another embodiment there is provided a method for protecting
a natural or artificial surface, including a biological tissue surface such as
a
plant surface (e.g., all or part of a surface of a root, bulb, stem, leaf,
branch,
vine, runner, bud, flower or a part thereof, greentip, fruit, seed, seed pod,
or the
like) or an epithelial tissue surface, against one or more of a bacterial
pathogen,
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a fungal pathogen and a viral pathogen, comprising contacting the epithelial
tissue surface with an effective amount of a BT composition under conditions
and for a time sufficient for one or more of (i) prevention of infection of
the
surface by the bacterial, fungal or viral pathogen, (ii) inhibition of cell
viability or
cell growth of substantially all planktonic cells of the bacterial, fungal or
viral
pathogen, (iii) inhibition of biofilm formation by the bacterial, fungal or
viral
pathogen, and (iv) inhibition of biofilm viability or biofilm growth of
substantially
all biofilm-form cells of the bacterial, fungal or viral pathogen, wherein the
BT
composition comprises a plurality of microparticles that comprise a bismuth-
thiol (BT) compound, substantially all of said microparticles having a
volumetric
mean diameter of from about 0.4 pm to about 5 pm. In certain embodiments
the bacterial pathogen comprises at least one of (i) one or more gram-negative

bacteria; (ii) one or more gram-positive bacteria; (iii) one or more
antibiotic-
sensitive bacteria; (iv) one or more antibiotic-resistant bacteria; (v) a
bacterial
pathogen that is selected from Staphylococcus aureus (S. aureus), MRSA
(methicillin-resistant S. aureus), Staphylococcus epidermidis , MRSE
(methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,
Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P. aeruginosa,
Escherichia coli, enterotoxigenic E. coli, enterohemorrhagic E. coli,
Klebsiella
pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophila,
Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis,
Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia
enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
Enterococcus (VRE), Burkholderia cepacia complex, Fran cisella tularensis,
Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin-
resistant enterococci, Streptococcus pneumonia, penicillin-resistant
Streptococcus pneumonia, Escherichia co/i, Burkholderia cepacia, Bukholderia
multi vorans, Mycobacterium smegm*tis and Acinetobacter baumannii. In
certain embodiments the bacterial pathogen exhibits antibiotic resistance. In
certain embodiments the bacterial pathogen exhibits resistance to an
antibiotic
that is selected from methicillin, vancomycin, naficilin, gentamicin,
ampicillin,
chloramphenicol, doxycycline and tobramycin.
In certain embodiments the natural or artificial surface comprises
an oral/buccal cavity surface, prosthetic device, ceramic, plastic, polymer,
rubber, metal article of manufacture, painted surface, marine structure
including
ship hull, rudder, propeller, anchor, hold, ballast tank, dock, dry dock,
pier,
piling, bulkhead, or other natural or artificial surface.
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In certain embodiments the surface comprises an epithelial tissue
surface that comprises a tissue that is selected from epidermis, dermis,
respiratory tract, gastrointestinal tract and glandular linings.
In certain embodiments the step of contacting is performed one or
.. a plurality of times. In certain embodiments at least one step of
contacting
comprises one of spraying, irrigating, dipping and painting the natural or
artifical
surface. In certain embodiments at least one step of contacting comprises one
of inhaling, ingesting and orally irrigating. In certain embodiments least one

step of contacting comprises administering by a route that is selected from
topically, intraperitoneally, orally, parenterally, intravenously,
intraarterially,
transdermally, sublingually, subcutaneously, intramuscularly, transbuccally,
intranasally, via inhalation, intraoccularly, intraauricularly,
intraventricularly,
subcutaneously, intraadiposally, intraarticularly and intrathecally. In
certain
embodiments the BT composition comprises one or more BT compounds
selected from the group consisting of BisBAL, BisEDT, Bis-dimercaprol, Bis-
DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-
PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-
Pyr/Ery, bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-
propanethiol.
In certain embodiments the bacterial pathogen exhibits antibiotic
resistance. In certain other embodiments the above described method further
comprises contacting the natural or artificial surface with a synergizing
antibiotic
and/or with an enhancing antibiotic, simultaneously or sequentially and in any

order with respect to the step of contacting the surface with the BT
composition.
In certain embodiments the synergizing and/or enhancing antibiotic comprises
an antibiotic that is selected from an aminoglycoside antibiotic, a carbapenem

antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a
glycopeptide
antibiotic, a lincosannide antibiotic, a penicillinase-resistant penicillin
antibiotic,
and an aminopenicillin antibiotic. In certain embodiments the synergizing
and/or enhancing antibiotic is an aminoglycoside antibiotic that is selected
from
amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
In another embodiment of the invention described herein there is
provided a method for overcoming antibiotic resistance (e.g., for a bacterial
.. pathogen that is resistant to at least one anti-bacterial effect of at
least one
antibiotic known to have an anti-bacterial effect against bacteria of the same
bacterial species, rendering such a pathogen susceptible to an antibiotic) on
a
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natural or artificial surface where an antibiotic-resistant bacterial pathogen
is
present, comprising contacting the surface simultaneously or sequentially and
in any order with an effective amount of (1) at least one bismuth-thiol (BT)
composition and (2) at least one antibiotic that is enhanced by, and/or that
is
capable of acting synergistically with the at least one BT composition, under
conditions and for a time sufficient for one or more of: (i) prevention of
infection
of the surface by the bacterial pathogen, (ii) inhibition of cell viability or
cell
growth of substantially all planktonic cells of the bacterial pathogen, (iii)
inhibition of biofilm formation by the bacterial pathogen, and (iv) inhibition
of
biofilm viability or biofilm growth of substantially all biofilm-form cells of
the
bacterial pathogen, wherein the BT composition comprises a plurality of
microparticles that comprise a bismuth-thiol (BT) compound, substantially all
of
said microparticles having a volumetric mean diameter of from about 0.4 p.m to

about 5 i_tm; and thereby overcoming antibiotic resistance on the epithelial
tissue surface. In certain embodiments the bacterial pathogen comprises at
least one of: (i) one or more gram-negative bacteria; (ii) one or more gram-
positive bacteria; (iii) one or more antibiotic-sensitive bacteria; (iv) one
or more
antibiotic-resistant bacteria; (v) a bacterial pathogen that is selected from
Staphylococcus aureus (S. aureus), MRSA (methicillin-resistant S. aureus),
Staphylococcus epidermidis , MRSE (methicillin-resistant S. epidermidis),
Mycobacterium tuberculosis, Mycobacterium avium, Pseudomonas aeruginosa,
drug-resistant P. aeruginosa, Escherichia co/i, enterotoxigenic E. coli,
enterohemorrhagic E. coil, Klebsiella pneumoniae, Clostridium difficile,
Heliobacter pylori, Legionella pneumophila, Enterococcus faecalis, methicill
in-
susceptible Enterococcus faecalis, Enterobacter cloacae, Salmonella
typhimurium, Proteus vulgaris, Yersinia enterocolitica, Vibrio cholera,
Shigella
flexneri, vancomycin-resistant Enterococcus (VRE), Burkholderia cepacia
complex, Francisella tularensis, Bacillus anthracis, Yersinia pestis,
Pseudomonas aeruginosa, vancomycin-resistant enterococci, Streptococcus
pneumonia, penicillin-resistant Streptococcus pneumonia, Escherichia coil,
Burkholderia cepacia, Bukholderia multivorans, Mycobacterium smegm*tis and
Acinetobacter baumannii.
In certain embodiments the bacterial pathogen exhibits resistance
to an antibiotic that is selected from methicillin, vancomycin, naficilin,
gentarnicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin

and gatifloxacin.
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In certain embodiments the natural or artificial surface comprises
an oral/buccal cavity surface, prosthetic device, ceramic, plastic, polymer,
rubber, metal article of manufacture, painted surface, marine structure
including
ship hull, rudder, propeller, anchor, hold, ballast tank, dock, dry dock,
pier,
piling, bulkhead, or other natural or artificial surface.
In certain embodiments the surface comprises a tissue that is
selected from the group consisting of epidermis, dermis, respiratory tract,
gastrointestinal tract and glandular linings. In certain embodiments the step
of
contacting is performed one or a plurality of times. In certain embodiments at
least one step of contacting comprises one of spraying, irrigating, dipping
and
painting the surface. In certain other embodiments at least one step of
contacting comprises one of inhaling, ingesting and orally irrigating. In
certain
embodiments at least one step of contacting comprises administering by a
route that is selected from topically, intraperitoneally, orally,
parenterally,
intravenously, intraarterially, transdermally, sublingually, subcutaneously,
intramuscularly, transbuccally, intranasally, via inhalation, intraoccularly,
intraauricularly, intraventricularly, subcutaneously, intraadiposally,
intraarticularly and intrathecally. In certain embodiments the BT composition
comprises one or more BT compounds selected from BisBAL, BisEDT, Bis-
dimercaprol, Bis-DTT, Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-
Tol, Bis-BDT, Bis-PDT, Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT,
Bis-Pyr/Tol, Bis-Pyr/Ery, bismuth-1 -mercapto-2-propanol, and Bis-EDT/2-
hydroxy-1-propanethiol. In certain embodiments the synergizing and/or
enhancing antibiotic comprises an antibiotic that is selected from
clindamicin,
gatifloxacin, an aminoglycoside antibiotic, a carbapenem antibiotic, a
cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide
antibiotic, a
lincosamide antibiotic, a penicillinase-resistant penicillin antibiotic, and
an
anninopenicillin antibiotic. In certain embodiments the synergizing and/or
enhancing antibiotic is an aminoglycoside antibiotic that is selected from
amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin and apramycin.
Turning to another embodiment there is provided an antiseptic
composition, comprising (a) at least one BT compound; (b) at least one
antibiotic compound that is enhanced by and/or is capable of acting
synergistically with the BT compound; and (c) a pharmaceutically acceptable
excipient or carrier, including a carrier for topical use. In certain
embodiments
the BT compound is selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,

CA 02807993 2013-02-08
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Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT,
Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,
bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. In
certain embodiments the BT composition comprises a plurality of microparticles
that comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles having a volumetric mean diameter of from about 0.4 1.trn to
about 5 p.m. In certain embodiments the BT compound is selected from
BisEDT and BisBAL. In certain embodiments the antibiotic compound
comprises an antibiotic that is selected from methicillin, vancomycin,
naficilin,
gentamicin, ampicillin, chloramphenicol, doxycycline, tobramycin, clindamicin,

gatifloxacin and an aminoglycoside antibiotic. In certain embodiments the
aminoglycoside antibiotic is selected from amikacin, arbekacin, gentamicin,
kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin,
streptomycin, tobramycin and apramycin. In certain embodiments the
aminoglycoside antibiotic is amikacin.
In certain other embodiments there is provided a method for
treating a natural or artificial surface that supports or contains bacterial
biofilm,
comprising (a) identifying a bacterial infection on or in the surface as
comprising
one of (i) gram positive bacteria, (ii) gram negative bacteria, and (iii) both
(i) and
(ii); (b) administering a formulation that comprises one or more bismuth thiol
(BT) compositions to the surface, wherein (i) if the bacterial infection
comprises
gram positive bacteria, then the formulation comprises therapeutically
effective
amounts of at least one BT compound and at least one antibiotic that is
rifamycin, (ii) if the bacterial infection comprises gram negative bacteria,
then
the formulation comprises therapeutically effective amounts of at least one BT
compound and amikacin, (iii) if the bacterial infection comprises both gram
positive and gram negative bacteria, then the formulation comprises
therapeutically effective amounts of one or a plurality of BT compounds,
rifamycin and amikacin, and thereby treating the surface.
In certain embodiments the biofilm comprises one or a plurality of
antibiotic-resistant bacteria. In certain embodiments treating the surface
comprises at least one of: (i) eradicating the bacterial biofilm, (ii)
reducing the
bacterial biofilm, and (iii) impairing growth of the bacterial biofilm. In
certain
embodiments the BT composition comprises a plurality of microparticles that
comprise a bismuth-thiol (BT) compound, substantially all of said
microparticles
having a volumetric mean diameter of from about 0.4 pm to about 5 p.m.
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CA 2807993
These and other aspects of the herein described invention embodiments will be
evident upon reference to the following detailed description and attached
drawings. Aspects and
embodiments of the invention can be modified, if necessary, to employ concepts
of the various
patents, applications and publications to provide yet further embodiments.
Various embodiments of the claimed invention relates to a use of a bismuth-
thiol (BT) composition for the treatment of an open wound, a chronic wound, or
an acute
wound that has or that is at risk for having a bacterial biofilm infection,
wherein the BT
composition comprises a monodisperse suspension of solid microparticles of one
or more
BT compounds, wherein at least 80% of said microparticles have a volumetric
mean
diameter of from 0.4 pm to 10 pm.
Various embodiments of the claimed invention relates to a use of a bismuth-
thiol (BT) composition in the manufacture of a medicament for the treatment of
an open
wound, a chronic wound, or an acute wound that has or that is at risk for
having a bacterial
biofilm infection, wherein the BT composition comprises a monodisperse
suspension of solid
microparticles of one or more BT compounds, wherein at least 80% of said
microparticles
have a volumetric mean diameter of from 0.4 pm to 10pm.
Various embodiments of the claimed invention relates to a bismuth-thiol (BT)
composition for the treatment of an open wound, a chronic wound, or an acute
wound that
has or that is at risk for having a bacterial biofilm infection, wherein the
BT composition
comprises a monodisperse suspension of solid microparticles of one or more BT
compounds, wherein at least 80% of said microparticles have a volumetric mean
diameter
of from 0.4 pm to 10 pm.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Figure 1 shows surviving numbers (log CFU; colony forming units) from
Pseudomonas aeruginosa colony biofilms grown for 24 hours on 10% tryptic soy
agar (TSA) at
37 C, followed with indicated treatment for 18 hours. Indicated antibiotic
treatments are TOB,
tobramycin 10X MIC; AMK, amikacin 100X MIC; IPM, imipenem 10X MIC; CEF,
cefepime 10X
MIC; CIP, ciprofloxacin 100X MIC; Cpd 2B, compound 2B (Bis-BAL, 1:1.5). (MIC;
minimum
inhibitory concentration, e.g., lowest concentration that prevents 20
bacterial growth).
Figure 2 shows surviving numbers (log CFU) from Staphylococcus aureus colony
biofilms grown for 24 hours on 10% tryptic soy agar, followed by the indicated
treatment.
Indicated antibiotic treatments are Rffampicin, RIF 100X MIC; daptomycin, DAP
320X MIC;
32
Date Recue/Date Received 2021-08-30

CA 2807993
minocycline, MIN 100X MIC; ampicillin, AMC 10X MIC; vancomycin, VAN 10X MIC;
Cpd 2B,
compound 2B (Bis-BAL, 1:1.5), Cpd 8-2, compound 8-2 (Bis-Pyr/BDT (1:1/0.5).
Figure 3 shows scratch closure over time of keratinocytes exposed to biofilms.

(*) Significantly different from control (P<0.001).
Figure 4A and 4B show the subinhibitory BisEDT reversing antibiotic-resistance

to several antibiotics. Effects of antibiotics with and without BisEDT (0.05
pg/ml) on a lawn of
MRSA (Methicillin-resistant S. aureus) is shown. Panel A shows standard
antibiotic-soaked
discs alone, and Panel B shows discs combined with a BisEDT (BE). [GM=
gentamicin, CZ=
cefazolin, FEP= cefepime, IPM= imipenim,SAM= ampicillin/ sulbactam, LVX=
levofloxacin.
32a
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Figure 5 shows the effect of BisEDT and antibiotics on biofilm
formation. S. epidermidis grown in TSB + 2% glucose in polystyrene plates for
48h at 37 C. Gatifloxacin (GF), clindamycin (CM), minocycline (MC),
gentamicin (GM), vancomycin (VM), cefazolin (CZ), nafcillin (NC), and
rifampicin (RP). Results were expressed as the mean change in the BPC (in
serial 2-fold dilution steps) at 0.25 p.M BisEDT (n=3).
Figure 6 shows the effect of BisEDT and antibiotics on growth of
S. epidermidis grown in TSB plus 2% glucose for 48h at 37 C. Results are
expressed as the mean change in MIC (dilution steps) with increasing BisEDT
(n=3). See legend in Figure 5 for antibiotic definitions.
Figure 7 is a bar graph showing the mean S. aureus bacteria
levels detected on the bone and hardware samples from open fractures in an in
vivo rat model following treatment with three BT formulations, Bis-EDT , MB-11

and MB-8-2 with or without Cefazolin antibiotic treatment. Standard errors of
the mean are shown as error bars. Animals euthanized early are not excluded
from the analysis, however samples from one animal in group 2 have been
excluded due to gross contamination.
DETAILED DESCRIPTION
Particular embodiments of the invention disclosed herein are
based on the surprising discovery that certain bismuth-thiol (BT) compounds as
provided herein (preferably including BT microparticles having a volumetric
mean diameter of from about 0.4 pm to about 5 p.m), but not certain other BT
compounds (even if provided as microparticles), exhibited potent antiseptic,
antibacterial and/or anti-biofilm activity against particular bacteria,
including
bacteria associated with a number of clinically significant infections
including
infections that can comprise bacterial biofilms.
Unexpectedly, not all BT compounds were uniformly effective
against such bacteria in a predictable fashion, but instead exhibited
different
potencies depending on the target bacterial species. In particular and as
described herein, certain BT compounds (preferably including BT microparticles

having a volumetric mean diameter of from about 0.4 p.m to about 5 iim) were
found to exhibit higher potency against gram-negative bacteria, while certain
other BT compounds (preferably including BT microparticles having a
volumetric mean diameter of from about 0.4 pm to about 5 pm) were found to
exhibit greater potency against gram-positive bacteria, in a manner that,
according to non-limiting theory, may for the first time afford clinically
relevant
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strategies for the management of bacterial infections, including bacterial
biofilm
infections.
Additionally, and as described in greater detail below, certain
embodiments of the invention described herein relate to surprising advantages
that are provided by novel bismuth-thiol (BT) compositions that, as disclosed
herein, can be made in preparations that comprise a plurality of BT
microparticles that are substantially monodisperse with respect to particle
size
(e.g., having volumetric mean diameter from about 0.4 pm to about 5 pm). In
certain of these and related embodiments, the microparticulate BT is not
provided as a component of a lipid vesicle or liposome such as a multilamellar

phosphocholine-cholesterol liposome or other nnultilamellar or unilamellar
liposomal vesicle.
As also disclosed herein, with respect to certain embodiments, it
has been discovered that antibacterial and anti-biofilm efficacies of certain
antibiotics, which antibiotics have previously been found to lack potent
therapeutic effect against such bacterial infections, may be significantly
enhanced (e.g., increased in a statistically significant manner) by treating
the
infection (e.g., by direct application on or in an infected site such as a
natural or
artificial surface) with one or more of these antibiotics in concert,
simultaneously or sequentially and in either order, with a selected BT
compound. In a manner that could not be predicted prior to the present
disclosure, certain BT compounds can be combined with certain antibiotics to
provide a synergizing or enhancing combination as provided herein with respect

to antibacterial and/or anti-biofilm activity against certain bacterial
species or
bacterial strains. The unpredicted nature of such combinations, as described
in
greater detail below, is evidenced by the observations that while certain
BT/antibiotic combinations acted synergistically or exhibited enhancement
against certain bacteria, certain other BT/antibiotic combinations failed to
exhibit such synergistic or enhanced antibacterial and/or anti-biofilm
activity.
According to these and related embodiments, the antibiotic and
the BT compound may be administered simultaneously or sequentially and in
either order, and it is noteworthy that the specific synergizing or enhancing
combinations of one or more antibiotic and one or more BT compound as
disclosed herein for treatment of a particular infection (e.g., a biofilm
formed by
gram-negative or gram-positive bacteria) did not exhibit predictable (e.g.,
merely additive) activities but instead acted in an unexpectedly synergistic
or
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enhancing (e.g., supra-additive) fashion, as a function of the selected
antibiotic,
the selected BT compound and the specifically identified target bacteria.
For example, by way of illustration and not limitation, disclosed
herein in the context of a wide variety of actually or potentially microbially
infected natural and/or artificial surfaces, and further in the context of
improved
substantially monodisperse microparticulate BT formulations, either or both of
a
particular antibiotic compound and a particular BT compound may exert limited
antibacterial effects when used alone against a particular bacterial strain or

species, but the combination of both the antibiotic compound and the BT
compound exerts a potent antibacterial effect against the same bacterial
strain
or species, which effect is greater in magnitude (with statistical
significance)
than the simple sum of the effects of each compound when used alone, and is
therefore believed according to non-limiting theory to reflect antibiotic-BT
synergy (e.g., FICI < 0.5) or an enhancing effect (e.g., 0.5 < FICI < 1.0) of
the
BT on the antibiotic potency and/or of the antibiotic on the BT potency.
Accordingly, not every BT compound may synergize with, or be enhancing for,
every antibiotic, and not every antibiotic may synergize with, or be enhancing

for, every BT compound, such that antibiotic-BT synergy and BT-antibiotic
enhancement generally are not predictable. Instead, and according to certain
embodiments as disclosed herein, specific combinations of synergizing or
enhancing antibiotic and BT compounds surprisingly confer potent antibacterial

effects against particular bacteria, including in particular environments such
as
natural and/or artificial surfaces as described herein, and further including
in
certain situations antibacterial effects against biofilms formed by the
particular
bacteria.
That is, certain BT-synergizing antibiotics are described herein,
which includes an antibiotic that is capable of acting synergistically (FICI <
0.5)
with at least one BT composition that comprises at least one BT compound as
provided herein, where such synergy manifests as a detectable effect that is
greater (i.e., in a statistically significant manner relative to an
appropriate
control condition) in magnitude than the effect that can be detected when the
antibiotic is present but the BT compound is absent, and/or when the BT
compound is present but the antibiotic is absent. Similarly, certain BT-
antibiotic
combinations exhibit enhancement (0.5 < FICI < 1.0), where such enhancement
manifests as a detectable effect that is greater in a statistically
significant
manner relative to an appropriate control condition) in magnitude than the
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that can be detected when the antibiotic is present but the BT compound is
absent, and/or when the BT compound is present but the antibiotic is absent.
Examples of such a detectable effect may in certain embodiments
include (i) prevention of infection by a bacterial pathogen, (ii) inhibition
of cell
viability or cell growth of substantially all planktonic cells of a bacterial
pathogen, (iii) inhibition of biofilm formation by a bacterial pathogen, and
(iv)
inhibition of biofilm viability or biofilm growth of substantially all biofilm-
form
cells of a bacterial pathogen, but the invention is not intended to be so
limited,
such that in other contemplated embodiments antibiotic-BT synergy may
manifest as one or more detectable effects that may include alteration (e.g.,
a
statistically significant increase or decrease) of one or more other
clinically
significant parameters, for example, the degree of resistance or sensitivity
of a
bacterial pathogen to one or more antibiotics or other drugs or chemical
agents,
the degree of resistance or sensitivity of a bacterial pathogen to one or more
chemical, physical or mechanical conditions (e.g., pH, ionic strength,
temperature, pressure), and/or the degree of resistance or sensitivity of a
bacterial pathogen to one or more biological agents (e.g., a virus, another
bacterium, a biologically active polynucleotide, an immunocyte or an
innnnunocyte product such as an antibody, cytokine, chemokine, enzyme
including degradative enzymes, membrane-disrupting protein, a free radical
such as a reactive oxygen species, or the like).
Persons familiar with the art will appreciate these and a variety of
other criteria by which the effects of particular agents on the structure,
function
and/or activity of a bacterial population may be determined (e.g., Coico et
al.
(Eds.), Current Protocols in Microbiology, 2008, John Wiley & Sons, Hoboken,
NJ; Schwalbe et al., Antimicrobial Susceptibility Testing Protocols, 2007, CRC

Press, Boca Raton, FL), for purposes of ascertaining antibiotic-BT synergy or
enhancement which, as provided herein, is present when the effects of the
synergizing or enhancing antibiotic-BT combination exceed the mere sum of the
effects observed when one component of the combination is not present.
For example, in certain embodiments synergy may be determined
by determining an antibacterial effect such as those described herein using
various concentrations of candidate agents (e.g., a BT and an antibiotic
individually and in combination) to calculate a fractional inhibitory
concentration
index (FICI) and a fractional bactericidal concentration index (FBCI),
according
to Eliopoulos et al. (Eliopoulos and Moellering, (1996) Antimicrobial
combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 330-

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96, Williams and Wilkins, Baltimore, MD, USA). Synergy may be defined as an
FICI or FBCI index of 1:1.5, and antagonism at >4. (e.g., Odds, FC (2003)
Synergy, antagonism, and what the chequerboard puts between them. Journal
of Antimicrobial Chemotherapy 52:1). Synergy may also be defined
conventionally as .4-fold decrease in antibiotic concentration, or
alternatively,
using fractional inhibitory concentration (FIC) as described, e.g., by
Hollander et
al. (1998 Antimicrob. Agents Chemother. 42:744). In certain embodiments,
synergy may be defined as an effect that results from a combination of two
drugs (e.g., an antibiotic and a BT composition) wherein the effect of the
combination is greater (e.g., in a statistically significant manner) than it
would
be if the concentration of the second drug is replaced by the first drug.
Accordingly as described herein and in certain preferred
embodiments, a combination of BT and antibiotic will be understood to
synergize when a FICI value that is less than or equal to 0.5 is observed.
(Odds, 2003). As also described herein, in certain other preferred
embodiments and according to non-limiting theory, it is disclosed that certain

BT-antibiotic combinations may exhibit a FICI value between 0.5 and 1.0 that
signifies a high potential for such synergy, and which may be observed using
non-optimal concentrations of at least one BT and at least one antibiotic that
exhibit unilateral or mutually enhanced cooperative antimicrobial efficacy.
Such
an effect may also be referred to herein as "enhanced" antibiotic activity or
"enhanced" BT activity.
Enhanced antibiotic and/or BT activity may be detected according
to certain embodiments when the presence both (i) of at least one BT at a
concentration that is less (in a statistically significant manner) than the
characteristic minimum inhibitory concentration (MIC) for that BT for a given
target microbe (e.g., a given bacterial species or strain), and (ii) of at
least one
antibiotic at a concentration that is less (in a statistically significant
manner)
than the characteristic IC50 (concentration that inhibits the growth of 50% of
a
microbial population; e.g., Soothill et al., 1992 J Antimicrob Chemother
29(2):137) and/or that is less than the biofilm-prevention concentration (BPC)
of
that antibiotic for the given target microbe, results in enhanced (in a
statistically
significant manner) antimicrobial efficacy of the BT-antibiotic combination
relative to the antimicrobial effect that would be observed if either
antimicrobial
agent (e.g., the BT or the antibiotic) were used at the same concentration in
the
absence of the other antimicrobial agent (e.g., the antibiotic or the BT). In
preferred embodiments, "enhanced" antibiotic and/or BT activity is present
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when a FICI value that is less than or equal to 1.0, and greater than 0.5, is
determined.
As will be appreciated by the skilled person based on the present
disclosure, in certain embodiments synergistic or enhanced antibiotic and/or
BT
activity may be determined according to methods known in the art, such as
using Loewe additivity-based models (e.g., FIC index, Greco model), or Bliss
independence based models (e.g., non-parametric and semi-parametric
models) or other methods described herein and known in the art (e.g.,
Meletiadis et al., 2005 Medical Mycology 43:133-152). Illustrative methods for
determining synergy or enhanced antibiotic and/or BT activity are thus
described, for instance, in Meletiadis etal., 2005 Medical Mycology 43:133-152

and references cited therein (see also, Meletiadis et al., 2002 Rev Med
Microbiol 13:101-117; White et al., 1996 Antimicrob Agents Chemother
40:1914-1918; Mouton et al., 1999 Antimicrob Agents Chemother 43:2473-
2478).
Certain other embodiments contemplate specific combinations of
one or more antibiotic and one or more BT compound as disclosed herein that
may exhibit synergizing or enhancing effects in vivo for treatment of a
particular
infection (e.g., a biofilm formed by gram-negative or gram-positive bacteria),
even where the BT compound(s) and antibiotic(s) did not exhibit predictable
(e.g., merely additive) activities in vivo but instead acted in an
unexpectedly
synergistic or enhancing (e.g., supra-additive; or conferring an effect when
two
or more such agents are present in combination that is greater (e.g., in a
statistically significant manner) than the effect that is obtained if the
concentration of the second agent is replaced by the first agent) fashion, as
a
function of the selected antibiotic, the selected BT compound and one or more
of the specifically identified target bacterial species of which the infection
is
comprised. It will therefore be appreciated, according to these and related
embodiments, that in certain in vivo situations FICI or FBCI values (which are
determined in vitro) may not be readily available, but that instead BT-
antibiotic
synergizing or enhancing effects may be determined in a manner afforded by
the quantifiable metrics of the infection.
For example, in one embodiment, such as in the in vivo open
fracture Rattus norvegicus femur critical defect model as described in Example
11, a statistically significant reduction in bacterial counts observed post-
treatment for the BT-antibiotic combination as compared to the antibiotic
treatment or BT compound alone, is an indication of synergizing or enhancing
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effects. Statistical significance can be determined using methods well-known
to
the skilled person. In certain other embodiments, a reduction observed in this

or other in vivo models by at least 5%, 10%, 20%, 30%, 40%, or 50% of
bacterial counts observed in the injury post-treatment for the BT-antibiotic
combination as compared to the antibiotic treatment or BT compound alone is
considered an indication of synergizing or enhancing effects.
Other exemplary indicia of in vivo infections may be determined
according to established methodologies that have been developed for
quantification of the severity of the infection, such as a variety of wound
scoring
systems known to the skilled person (see e.g., scoring systems reviewed in
European Wound Management Association (EWMA), Position Document:
Identifying criteria for wound infection. London: MEP Ltd, 2005). Illustrative

wound scoring systems that may be used in assessing synergistic or
enhancement activity of BT-antibiotic combinations as described herein include
ASEPSIS (Wilson AP, J Hosp Infect 1995; 29(2): 81-86; Wilson et al., Lancet
1986; 1: 311-13), the Southampton Wound Assessment Scale (Bailey IS,
Karran SE, Toyn K, et al. DMJ 1992; 304: 469-71). See also, Horan IC,
Gaynes P, Marione WJ, etal. ,1992 Infect Control Hosp Epidemiol 1992; 13:
606-08. Additionally, recognized clinical indicia of wound healing known to
the
skilled clinician may also be measured in the presence or absence of BT
compounds and/or antibiotics, such as wound size, depth, granulation tissue
condition, infection, etc. Accordingly, and based on the present disclosure,
the
skilled person will readily appreciate a variety of methods for determining
whether a BT composition ¨antibiotic combination alters (e.g., increases or
decreases in a statistically significant manner relative to appropriate
controls) in
vivo wound healing.
In view of these and related embodiments, there are provided
herein a wide variety of methods for treating nnicrobially infected natural
and
artificial surfaces such as surfaces that support or contain bacterial
biofilms,
with an effective amount (e.g., in certain embodiments a therapeutically
effective amount) of a composition or formulation that comprises one or more
BT compounds and, optionally, one or more antibiotic compounds, such as one
or more synergizing antibiotics, or one or more enhancing antibiotics, as
provided herein. It will be appreciated that based on the present disclosure,
certain antibiotics are now contemplated for use in the treatment of given
types
of infections, where such antibiotics had previously been viewed by persons
familiar with the art as ineffective against infections of the same type.
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Certain embodiments thus contemplate compositions that
comprise one or more BT compounds for use as antiseptics. An antiseptic is a
substance that kills or prevents the growth of microorganisms, and may be
typically applied to living tissue, distinguishing the class from
disinfectants,
which are usually applied to inanimate objects (Goodman and Gilman's "The
Pharmacological Basis of Therapeutics", Seventh Edition, Gilman et al.,
editors, 1985, Macmillan Publishing Co., (hereafter, Goodman and Gilman") pp.
959-960). Common examples of antiseptics are ethyl alcohol and tincture of
iodine. Germicides include antiseptics that kill microbes such as microbial
pathogens.
Certain embodiments described herein may contemplate
compositions that comprise one or more BT compounds and one or more
antibiotic compound (e.g., a synergizing antibiotic and/or an enhancing
antibiotic as provided herein). Antibiotics are known in the art and typically
comprise a drug made from a compound produced by one species of
microorganism to kill another species of microorganism, or a synthetic product

having an identical or similar chemical structure and mechanism of action,
e.g.,
a drug that destroys microorganisms within or on the body of a living
organism,
including such drug when applied topically. Among embodiments disclosed
herein are those in which an antibiotic may belong to one of the following
classes: anninoglycosides, carbapenenns, cephalosporins, fluoroquinolones,
glycopeptide antibiotics, lincosannides (e.g., clindamycin), penicillinase-
resistant
penicillins, and aminopenicillins. Antibiotics thus may include, but need not
be
limited to, oxacillin, piperacillin, cefuroxime, cefotaxime, cefepime,
imipenem,
aztreonam, streptomycin, tobramycin, tetracycline, minocycline, ciprofloxacin,

levalloxacin, erythromycin, linezolid, phosphomycin, capreomycin, isoniazid,
ansamycin, carbacephem, monobactam, nitrofuran, penicillin, quinolone,
sulfonamide, Clofazimine, Dapsone, Capreonnycin, Cycloserine, Ethannbutol,
Ethionamide, lsoniazid, Pyrazinamide, Rifampicin, Rifampin, Rifabutin,
Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin,
Fusidic acid, Linezolid, Metronidazole, Mupirocin, Platensimycin,
Quinupristin,
Dalfopristin, Rifaximin, Thiamphenicol, Tinidazole, aminoglycoside, beta-
lactam, penicillin, cephalosporin, carbapenem, fluroquinolone, ketolide,
lincosamide, macrolide, oxazolidinone, stretogramin, sulphonamide,
tetracycline, glycylcycline, methicillin, vancomycin, naficilin, gentamicin,
ampicillin , chloramphenicol, doxycycline, tobramycin, amikacin, arbekacin,
gentamicin, kanamycin, neomycin, netilmicin, paromomycin,

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rhodostreptomycin, streptomycin, tobramycin, apramycin, clindamicin,
gatifloxacin, aminopenicillin, and others known to the art. Compendia of these

and other clinically useful antibiotics are available and known to those
familiar
with the art (e.g., Washington University School of Medicine, The Washington
Manual of Medical Therapeutics (32nd Ed.), 2007 Lippincott, Williams and
Wilkins, Philadelphia, PA; Hauser, AL, Antibiotic Basics for Clinicians, 2007
Lippincott, Williams and Wilkins, Philadelphia, PA).
An exemplary class of antibiotics for use with one or more BT
compounds in certain herein disclosed embodiments is the aminoglycoside
class of antibiotics, which are reviewed in Edson RS, Terrell CL. The
aminoglycosides. Mayo Clin Proc. 1999 May; 74(5):519-28. This class of
antibiotics inhibits bacterial growth by impairing bacterial protein
synthesis,
through binding and inactivation of bacterial ribosomal subunits. In addition
to
such bacteriostatic properties, anninoglycosides also exhibit bacteriocidal
effects through disruption of cell walls in gram-negative bacteria.
Aminoglycoside antibiotics include gentamicin, amikacin,
streptomycin, and others, and are generally regarded as useful in the
treatment
of gram-negative bacteria, mycobacteria and other microbial pathogens,
although cases of resistant strains have been reported. The anninoglycosides
are not absorbed through the digestive tract and so are not generally regarded
as being amenable to oral formulations. Amikacin, for example, although often
effective against gentamicin-resistant bacterial strains, is typically
administered
intravenously or intramuscularly, which can cause pain in the patient.
Additionally, toxicities associated with aminoglycoside antibiotics such as
amikacin can lead to kidney damage and/or irreversible hearing loss.
Despite these properties, certain embodiments disclosed herein
contemplate oral administration of a synergizing BT/antibiotic combination
(e.g.,
where the antibiotic need not be limited to an aminoglycoside) for instance,
for
treatment of an epithelial tissue surface at one or more locations along the
oral
cavity, gastrointestinal tract/ alimentary canal. Also contemplated in certain

other embodiments may be use of compositions and methods described herein
as disinfectants, which refers to preparations that kill, or block the growth
of,
microbes on an external surface of an inanimate object.
As also described elsewhere herein, a BT compound may be a
composition that comprises bismuth or a bismuth salt and a thiol- (e.g., -SH,
or
sulfhydryl) containing compound, including those that are described (including

their methods of preparation) in Domenico et al., 1997 Antimicrob. Agent.
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Chemother. 41(8):1697-1703, Domenico et al., 2001 Antimicob.Agent.
Chemother. 45(5):1417-1421, and in U.S. RE37,793, U.S. 6,248,371, U.S.
6,086,921, and U.S. 6,380,248; see also, e.g., U.S. 6,582,719. Certain
embodiments are not so limited, however, and may contemplate other BT
compounds that comprise bismuth or a bismuth salt and a thiol-containing
compound. The thiol-containing compound may contain one, two, three, four,
five, six or more thiol (e.g., -SH) groups. In preferred embodiments the BT
compound comprises bismuth in association with the thiol-containing compound
via ionic bonding and/or as a coordination complex, while in some other
embodiments bismuth may be associated with the thiol-containing compound
via covalent bonding such as may be found in an organometallic compound.
Certain contemplated embodiments, however, expressly exclude a BT
compound that is an organometallic compound such as a compound in which
bismuth is found in covalent linkage to an organic moiety.
Exemplary BT compounds are shown in Table 1:
TABLE 1
Exemplary BT Compounds*
1) CPD 1B-1 Bis-EDT (1:1) BiC2H4S2
2) CPD 1B-2 Bis-EDT (1:1.5) BiC3H6S3
3) CPD 1B-3 Bis-EDT (1:1.5) BiC3H6S3
4) CPD 1C Bis-EDT (1:1.5) BiC3H6S3
5) CPD 2A Bis-Bal (1:1) BiC3H6S20
6) CPD 2B Bis-Bal (1:1.5) BiC4.6H901.5S3
7) CPD 3A Bis-Pyr (1:1.5) BiC7.5H6N1.501.5S1.5
8) CPD 3B Bis-Pyr (1:3) BiC15H12N303S3
9) CPD 4 Bis-Ery (1:1.5) BiC6H1203S3
10) CPD 5 Bis-Tol (1:1.5) BiC105H9S3
11) CPD 6 Bis-BDT (1:1.5) BiC6H12S3
12) CPD 7 Bis-PDT (1:1.5) B1645H9S3
13) CPD 8-1 Bis-Pyr/BDT (1:1/1)
14) CPD 8-2 Bis-Pyr/BDT (1:1/0.5)
15) CPD 9 Bis-2hydroxy, propane thiol (1:3)
16) CPD 10 Bis-Pyr/Bal (1:1/0.5)
17) CPD 11 Bis-Pyr/EDT (1:1/0.5)
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18) CPD 12 Bis-Pyr/Tol (1:1/0.5)
19) CPD 13 Bis-Pyr/PDT (1:1/0.5)
20) CPD 14 Bis-Pyr/Ery (1:1/0.5)
21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)
*Shown are atomic ratios relative to a single bismuth atom, for comparison,
based
on the stoichiometric ratios of the reactants used and the known propensity of

bismuth to form trivalent complexes with sulfur containing compounds. Atomic
ratios as shown may not be accurate molecular formulae for all species in a
given
preparation. The numbers in parenthesis are the ratios of bismuth to one (or
more)
thiol agents. (e.g. Bi:thio11/thi012) "CPD", compound.
BT compounds for use in certain of the presently disclosed
embodiments may be prepared according to established procedures (e.g., U.S.
RE37,793, U.S. 6,248,371, U.S. 6,086,921, and U.S. 6,380,248; Domenico et
al., 1997 Antimicrob. Agent. Chemother. 41(8):1697-1703, Domenico et al.,
2001 Antimicob.Agent. Chemother. 45(5):1417-1421) and in certain other
embodiments BT compounds may also be prepared according to
methodologies described herein. Certain preferred embodiments thus
contemplate the herein described synthetic methods for preparing BT
compounds, and in particular for obtaining BT compounds in substantially
monodisperse microparticulate form, in which an acidic aqueous bismuth
solution that contains dissolved bismuth at a concentration of at least 50 mM,
at
least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300
mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at
least 700 mM, at least 800 mM, at least 900 mM or at least 1 M and that lacks
a
hydrophilic, polar or organic solubilizer is admixed with ethanol to obtain a
first
ethanolic solution, which is reacted with a second ethanolic solution
comprising
a thiol-containing compound to obtain a reaction solution, wherein the thiol-
containing compound is present in the reaction solution at a molar ratio of
from
about 1:3 to about 3:1 relative to the bismuth, under conditions and for a
time
sufficient for formation of a precipitate which comprises the microparticles
comprising the BT compound (such as the conditions of concentration, solvent
strength, temperature, pH, mixing and/or pressure, and the like, as described
herein and as will be appreciated by the skilled person based on the present
disclosure).
Accordingly, exemplary BTs include compound 1B-1, Bis-EDT
(bismuth-1,2-ethane dithiol, reactants at 1:1); compound 1B-2, Bis-EDT
(1:1.5);
compound 1B-3, Bis-EDT (1:1.5); compound 1C, Bis-EDT (soluble Bi
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preparation, 1:1.5); compound 2A, Bis-Bal (bismuth-British anti-Lewisite
(bismuth-dimercaprol, bismuth-2,3-dimercaptopropanol), 1:1); compound 2B,
Bis-Bal (1:1.5); compound 3A Bis-Pyr (bismuth-pyrithione, 1:1.5); compound 3B
Bis-Pyr (1:3); compound 4, Bis-Ery (bismuth-dithioerythritol, 1:1.5); compound
5, Bis-Tol (bismuth-3,4-dimercaptotoluene, 1:1.5); compound 6, Bis-BDT
(bismuth-2,3-butanedithiol, 1:1.5); compound 7, Bis-PDT (bismuth-1,3-
propanedithiol, 1:1.5); compound 8-1 Bis-Pyr/BDT (1:1/1); compound 8-2, Bis-
Pyr/BDT (1:1/0.5); compound 9, Bis-2-hydroxy, propane thiol (bismuth-1-
mercapto-2-propanol, 1:3); compound 10, Bis-Pyr/Bal (1:1/0.5); compound 11,
Bis-Pyr/EDT (1:1/0.5); compound 12 Bis-Pyr/Tol (1:1/0.5); compound 13, Bis-
Pyr/PDT (1:1/0.5); compound 14 Bis-Pyr/Ery (1:1/0.5); compound 15, Bis-
EDT/2-hydroxy, propane thiol (1:1/1) (see, e.g., Table 1).
Without wishing to be bound by theory, it is believed that the
presently disclosed methods of preparing a BT compound, which in certain
preferred embodiments may comprise preparing or obtaining an acidic aqueous
liquid solution that comprises bismuth such as an aqueous nitric acid solution

comprising bismuth nitrate, may desirably yield compositions comprising BT
compounds where such compositions have one or more desirable properties,
including ease of large-scale production, improved product purity, uniformity
or
consistency (including uniformity in particle size), or other properties
useful in
the preparation and/or administration of the present topical formulations.
In particular embodiments it has been discovered that BT
compositions, prepared according to the methods described herein for the first
time, exhibit an advantageous degree of hom*ogeneity with respect to their
occurrence as a substantially monodisperse suspension of microparticles each
having a volumetric mean diameter (VMD) according to certain presently
preferred embodiments of from about 0.4 pm to about 5 pm. Measures of
particle size can be referred to as volumetric mean diameter (VMD), mass
median diameter (MMD), or mass median aerodynamic diameter (MMAD).
These measurements may be made, for example, by impaction (MMD and
MMAD) or by laser (VMD) characterization. For liquid particles, VMD, MMD
and MMAD may be the same if environmental conditions are maintained, e.g.,
standard humidity. However, if humidity is not maintained, MMD and MMAD
determinations will be smaller than VMD due to dehydration during impactor
measurements. For the purposes of this description, VMD, MMD and MMAD
measurements are considered to be under standard conditions such that
descriptions of VMD, MMD and MMAD will be comparable. Similarly, dry
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powder particle size determinations in [AVID, and MMAD are also considered
comparable,
As described herein, preferred embodiments relate to a
substantially monodisperse suspension of ST-containing microparticles.
Generation of a defined ST particle size with limited geometric standard
deviation (GSD) may, for instance, optimize BT deposition, accessibility to
desired target sites in or on a natural or artificial surface, andior
tolerability by a
subject to whom the ST microparticles are administered. Narrow GSD limits
the number of particles outside the desired VMD or MMAD size range,
In one embodiment, a liquid or aerosol suspension of
microparticles containing one or more ST compounds disclosed herein is
provided having a VMD from about 0.5 microns to about 5 microns. In another
embodiment, a liquid or aerosol suspension having a VIVID or MMAD from
about 0.7 microns to about 4,0 microns is provided. In another embodiment, a
liquid or aerosol suspension having aVMD or MMAD from about 1.0 micron to
about 3,0 microns is provided, hi certain other preferred embodiments there is

provided a liquid suspension comprising one or a plurality of DT compound
particles of from about 0.1 to about 5,0 microns VMD, or of from about 0.1,
about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8 or
about 0.9 microns to about 1.0, about 1.5, about 2.0, about 2.5, about 3.0,
about 3,5, about 4.0, about 4,5, about 5.0, about 5,5, about 6.0, about 6,5,
about 7.0, about 7,5 or about 8.0 microns, the particle comprising a BT
compound prepared as described herein.
Accordingly and in certain preferred embodiments, a ST
preparation described for the first time herein which is "substantially"
monodisperse, for example, a ST composition that comprises a ST compound
in microparticuiate form wherein "substantially" all of the microparticles
have a
volumetric mean diameter (VMD) within a specified range (e.g., from about 0.4
p.m to about 5 pm), includes those compositions in which at least 80%, 85%,
90%, 91%, 92%, 93%, or 94%, more preferably at least 95%, 96%, 97%, 98%,
99% or more of the particles have a VMD that is within the recited size range.

These and related properties of BT compositions prepared
according to the herein described synthetic methods offer unprecedented
advantages over previously described BTs, including lower cost and ease of
production, and uniformity within the composition that may permit its
characterization in a manner that facilitates regulatory compliance according
to
one or more of pharmaceutical, formulary and cosmeceutical standards.

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Additionally or alternatively, the herein described substantially
monodisperse BT microparticles may advantageously be produced without the
need for micronization, i.e., without the expensive and labor-intensive
milling or
supercritical fluid processing or other equipment and procedures that are
.. typically used to generate microparticles (e.g., Martin et al. 2008 Adv.
Drug
Deliv. Rev. 60(3):339; Moribe et al., 2008 Adv. Drug Delhi. Rev. 60(3):328;
Cape et al., 2008 Pharm. Res. 25(9):1967; Rasenack et al. 2004 Pharm. Dev.
Technol. 9(1):1-13). Hence, the present embodiments offer beneficial effects
of
substantially uniform microparticulate preparations, including without
limitation
enhanced and substantially uniform solubilization properties, suitability for
desired administration forms such as oral, inhaled or dermatological/ skin
wound topical forms, increased bioavailability and other beneficial
properties.
The BT compound microparticulate suspension can be
administered as aqueous formulations, as suspensions or solutions in aqueous
as well as organic solvents including halogenated hydrocarbon propellants, as
dry powders, or in other forms as elaborated below, including preparations
that
contain wetting agents, surfactants, mineral oil or other ingredients or
additives
as may be known to those familiar with formulary, for example, to maintain
individual microparticles in suspension. Aqueous formulations may be
aerosolized by liquid nebulizers employing, for instance, either hydraulic or
ultrasonic atomization. Propellant-based systems may use suitable pressurized
dispensers. Dry powders may use dry powder dispersion devices, which are
capable of dispersing the BT-containing microparticles effectively. A desired
particle size and distribution may be obtained by choosing an appropriate
device.
As also noted above, also provided herein according to certain
embodiments is a method for preparing a bismuth-thiol (BT) composition that
comprises a plurality of microparticles that comprise a BT compound,
substantially all of such microparticles having a volumetric mean diameter
(VMD) of from about 0.1 to about 8 microns, and in certain preferred
embodiments from about 0.4 microns to about 5 microns.
In general terms, the method comprises the steps of (a) admixing,
under conditions and for a time sufficient to obtain a solution that is
substantially free of a solid precipitate, (i) an acidic aqueous solution that
comprises a bismuth salt comprising bismuth at a concentration of at least 50
mM and that lacks a hydrophilic, polar or organic solubilizer, with (ii)
ethanol in
an amount sufficient to obtain an admixture that comprises at least about 5%,
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10%, 15%, 20%, 25% or 30%, and preferably about 25% ethanol by volume;
and (b) adding to the admixture of (a) an ethanolic solution comprising a
thiol-
containing compound to obtain a reaction solution, wherein the thiol-
containing
compound is present in the reaction solution at a molar ratio of from about
1:3
to about 3:1 relative to the bismuth, under conditions and for a time
sufficient for
formation of a precipitate which comprises the BT compound.
In certain preferred embodiments the bismuth salt may be
Bi(NO3)3, but it will be appreciated according to the present disclosure that
bismuth may also be provided in other forms. In certain embodiments the
bismuth concentration in the acidic aqueous solution may be at least 100 mM,
at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least
350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM,
at least 800 mM, at least 900 mM or at least 1 M. In certain embodiments the
acidic aqueous solution comprises at least 5%, 10%, 15%, 20%, 22% or 22.5%
bismuth by weight. The acidic aqueous solution may in certain preferred
embodiments comprise at least 5% or more nitric acid by weight, and in certain

other embodiments the acidic aqueous solution may comprise at least 0.5%, at
least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least
3.5%, at
least 4%, at least 4.5% or at least 5% nitric acid by weight.
The thiol-containing compound may be any thiol-containing
compound as described herein, and in certain embodiments may comprise one
or more of 1,2-ethane dithiol, 2,3-dimercaptopropanol, pyrithione,
dithioerythritol, 3,4-dimercaptotoluene, 2,3-butanedithiol, 1,3-
propanedithiol, 2-
hydroxypropane thiol, 1-mercapto-2-propanol, dithioerythritol and
dithiothreitol.
Other exemplary thiol-containing compounds include alpha-lipoic acid,
methanethiol (CH3SH [m-mercaptan]), ethanethiol (C2H5SH [e- mercaptan]), 1-
propanethiol (C3H7SH [n-P mercaptan]), 2-Propanethiol (CH3CH(SH)CH3 [2C3
nnercaptan]), butanethiol (C4H9SH ([n-butyl mercaptan]), tert-butyl nnercaptan

(C(CH3)3SH [t-butyl mercaptan]), pentanethiols (C5H11SH [pentyl mercaptan]),
coenzyme A, lipoamide, glutathione, cysteine, cystine, 2-mercaptoethanol,
dithiothreitol, dithioerythritol, 2-mercaptoindole, transglutaminase and any
of the
following thiol compounds available from Sigma-Aldrich (St. Louis, MO): (11-
nnercaptoundecyl)hexa(ethylene glycol), (11-mercaptoundecyl)tetra(ethylene
glycol), (11-mercaptoundecyl)tetra(ethylene glycol) functional ized gold
nanoparticles, 1,1 `,4`,1"-terpheny1-4-thiol, 1 ,1 1 -undecaned ithiol, 1 ,1 6-

hexadecanedithiol, 1,2-ethanedithiol technical grade, 1,3-propanedithiol, 1,4-
benzenedimethanethiol, 1,4-butanedithiol, 1,4-butanedithiol diacetate, 1,5-
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pentanedithiol, 1,6-hexanedithiol, 1,8-octanedithiol, 1,9-nonanedithiol,
adamantanethiol, 1-butanethiol, 1-decanethiol, 1-dodecanethiol, 1-
heptanethiol, 1-heptanethiol purum, 1-hexadecanethiol, 1-hexanethiol, 1-
mercapto-(triethylene glycol), 1-mercapto-(triethylene glycol) methyl ether
functionalized gold nanoparticles, 1-mercapto-2-propanol, 1-nonanethiol, 1-
octadecanethiol, 1-octanethiol, 1-octanethiol, 1-pentadecanethiol, 1-
pentanethiol, 1-propanethiol, 1-tetradecanethiol, 1-tetradecanethiol purum, 1-
undecanethiol, 11-(1H-pyrrol-1-yOundecane-1-thiol, 11-amino-l-undecanethiol
hydrochloride, 11-bromo-1-undecanethiol, 11-mercapto-1-undecanol, 11-
mercapto-1-undecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecanoic
acid, 11-mercaptoundecyl trifluoroacetate, 11-mercaptoundecylphosphoric
acid, 12-nnercaptododecanoic acid, 12-mercaptododecanoic acid, 15-
mercaptopentadecanoic acid, 16-mercaptohexadecanoic acid, 16-
nnercaptohexadecanoic acid, 1H,1H,2H,2H-perfluorodecanethiol, 2,2'-
(ethylenedioxy)diethanethiol, 2,3-butanedithiol, 2-butanethiol, 2-
ethylhexanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-
phenylethanethiol, 3,3,4,4,5,5,6,6,6-nonafluoro-1-hexanethiol purunn, 3-
(dimethoxymethylsily1)-1-propanethiol, 3-chloro-1-propanethiol, 3-mercapto-1-
propanol, 3-mercapto-2-butanol, 3-nnercapto-N-nonylpropionamide, 3-
mercaptopropionic acid, 3-mercaptopropyl-functionalized silica gel, 3-methyl-
1-butanethiol, 4,4'-bis(mercaptomethyl)biphenyl, 4,4'-dimercaptostilbene, 4-
(6-mercaptohexyloxy)benzyl alcohol, 4-cyano-1-butanethiol, 4-mercapto-1-
butanol, 6-(ferrocenyl)hexanethiol, 6-mercapto-1-hexanol, 6-
mercaptohexanoic acid, 8-mercapto-1-octanol, 8-mercaptooctanoic acid, 9-
mercapto-1-nonanol, biphenyl-4,4'-dithiol, butyl 3-mercaptopropionate,
copper(1) 1-butanethiolate, cyclohexanethiol, cyclopentanethiol, decanethiol
functionalized silver nanoparticles, dodecanethiol functionalized gold
nanoparticles, dodecanethiol functionalized silver nanoparticles,
hexa(ethylene
glycol)mono-11-(acetylthio)undecyl ether, mercaptosuccinic acid, methyl 3-
mercaptopropionate, nanoTether BPA-HH, NanoThinksTm 18, NanoThinksTm 8,
NanoThinksTm AC1D11, NanoThinks TM AC1D16, NanoThinks TM ALC011,
NanoThinksTm TH108, octanethiol functionalized gold nanoparticles, PEG
dithiol average Mn 8,000, PEG dithiol average nnol wt 1,500, PEG dithiol
average mol wt 3,400, S-(11-bromoundecyl)thioacetate, S-(4-
cyanobutyl)thioacetate, thiophenol, triethylene glycol mono-11-
mercaptoundecyl ether, trimethylolpropane tris(3-mercaptopropionate), [11-
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(methylcarbonylthio)undecylltetra(ethylene glycol), m-carborane-9-thiol, p-
terpheny1-4,4"-dithiol, tert-dodecylmercaptan, and tert-nonyl mercaptan.
Exemplary reaction conditions, including temperature, pH,
reaction time, the use of stirring or agitation to dissolve solutes and
procedures
for collecting and washing precipitates, are described herein and employ
techniques generally known in the art.
Unlike previously described methodologies for producing BT
compounds, according to the present methods for preparing BT, BT products
are provided as microparticulate suspensions having substantially all
microparticles with VMD from about 0.4 to about 5 microns in certain preferred
embodiments, and generally from about 0.1 microns to about 8 microns
according to certain other embodiments. Further unlike previous approaches,
according to the instant embodiments bismuth is provided in an acidic aqueous
solution that comprises a bismuth salt at a concentration of from at least
about
50 mM to about 1 M, and nitric acid in an amount from at least about 0.5% to
about 5% (w/w), and preferably less than 5% (weight/weight), and that lacks a
hydrophilic, polar or organic solubilizer.
In this regard the present methods offer surprising and
unexpected advantages in view of generally accepted art teachings that
bismuth is not water soluble at 50 pM (e.g., U.S. RE37793), that bismuth is
unstable in water (e.g., Kuvshinova et al., 2009 Russ. J lnorg. Chem
54(11):1816), and that bismuth is unstable even in nitric acid solutions
unless a
hydrophilic, polar or organic solubilizer is present. For example, in all of
the
definitive descriptions of BT preparation methodologies (e.g., Domenico et
al.,
1997 Antimicrob. Agents. Chemother. 41:1697; U.S. 6,380,248; U.S. RE37793;
U.S. 6,248,371), the hydrophilic solubilizing agent propylene glycol is
required
to dissolve bismuth nitrate, and the bismuth concentration of solutions
prepared
for reaction with thiols is well below 15 nnM, thereby limiting the available
production modalities for BT compounds.
By contrast, according to the present disclosure there is no
requirement for a hydrophilic, polar or organic solubilizer in order dissolve
bismuth, yet higher concentrations are surprisingly achieved. Hydrophilic,
polar
or organic solubilizers include propylene glycol (PG) and ethylene glycol (EG)

and may also include any of a large number of known solubility enhancers,
including polar solvents such as dioxane and dimethylsulfoxide (DMSO),
polyols (including, e.g., PG and EG and also including polyethylene glycol
(PEG), polypropyleneglycol (PPG), pentaerythritol and others), polyhydric
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alchohols such as glycerol and mannitol, and other agents. Other water-
miscible organic of high polarity include dimethylsulfoxide (DrviSO),
dimethylformamide (DMF) and NMP (N-methyl-2-pyrrolidone).
Thus, it will be appreciated by those familiar with the art that
solvents, including those commonly used as hydrophilic, polar or organic
solubilizers as provided herein, may be selected, for instance, based on the
solvent polarity/ polarizability (SPP) scale value using the system of Catalan
et
al. (e.g., 1995 Liebigs Ann. 241; see also Catalan, 2001 In: Handbook of
Solvents, Wypych (Ed.), Andrew Publ., NY, and references cited therein),
according to which, for example, water has a SPP value of 0.962, toluene a
SPP value of 0.655, and 2-propanol a SPP value of 0.848. Methods for
determining the SPP value of a solvent based on ultraviolet measurements of
the 2-N,N-dimethy1-7-nitrofluorene/ 2-fluoro-7-nitrofluorene probe/ hom*omorph
pair have been described (Catalan et a/., 1995).
Solvents with desired SPP values (whether as pure single-
component solvents or as solvent mixtures of two, three, four or more
solvents;
for solvent miscibility see, e.g., Godfrey 1972 Chem. Technol. 2:359) based on

the solubility properties of a particular BT composition can be readily
identified
by those having familiarity with the art in view of the instant disclosure,
although
as noted above, according to certain preferred embodiments regarding the
herein described synthetic method steps, no hydrophilic, polar or organic
solubilizer is required in order dissolve bismuth.
Solubility parameters may also include the interaction parameter
C. Hildebrand solubility parameter d, or partial (Hansen) solubility
parameters:
bp, oh and 6d, describing the solvent's polarity, hydrogen bonding potential
and
dispersion force interaction potential, respectively. In certain embodiments,
the
highest value for a solubility parameter that describes a solvent or co-
solvent
system in which the bismuth salt comprising bismuth will dissolve may provide
a limitation for the aqueous solution that comprises the bismuth salt, for
instance, according to the presently described method for preparing a
microparticulate BT composition. For example, higher Oh values will have a
greater hydrogen bonding ability and would therefore have a greater affinity
for
solvent molecules such as water. A higher value of maximum observed Oh for
a solvent may therefore be preferred for situations where a more hydrophilic
environment is desired.

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By way of non-limiting example, BisEDT having the structure
shown below in formula I may be prepared according to the following reaction
scheme:
/
SH Et0H \s ,S¨Bi
Bi(NO3)3 HS
\s/
(I)
Briefly, and as a non-limiting illustrative example, to an excess
(11.4 L) of 5% aqueous HNO3 at room temperature may be slowly added 0.331
L (about 0.575 moles) of an aqueous acidic bismuth solution such as a Bi(NO3)3
solution (e.g., 43% Bi(NO3)3 (w/w), 5% nitric acid (w/w), 52% water (w/w),
available from Shepherd Chemical Co., Cincinnati, OH) with stirring, followed
by slow addition of absolute ethanol (4 L). An ethanolic solution (1.56 L) of
a
thiol compound such as 1,2-ethanedithiol [-0.55 M] may be separately
prepared by adding, to 1.5 L of absolute ethanol, 72.19 mL (0.863 moles) of
1,2-ethanedithiol using a 60 mL syringe, and then stirring for five minutes.
1,2-
ethanedithiol (CAS 540-63-6) and other thiol compounds are available from,
e.g., Sigma-Aldrich, St. Louis, MO. The ethanolic solution of the thiol
compound may then be slowly added to the aqueous Bi(NO3)3/ HNO3 solution
with stirring overnight to form a reaction solution. The thiol-containing
compound may be present in the reaction solution, according to certain
preferred embodiments, at a molar ratio of from about 1:3 to about 3:1
relative
to the bismuth. The formed product is allowed to settle as a precipitate
comprising microparticles as described herein, which is then collected by
filtration and washed sequentially with ethanol, water and acetone to obtain
BisEDT as a yellow amorphous powdered solid. The crude product may be
redissolved in absolute ethanol with stirring, then filtered and washed
sequentially with ethanol several times followed by acetone several times. The

washed powder may be triturated in 1M NaOH (500mL), filtered and washed
sequentially with water, ethanol and acetone to afford purified
microparticulate
BisEDT.
According to non-limiting theory, bismuth inhibits the ability of
bacteria to produce extracellular polymeric substances (EPS) such as bacterial
exopolysaccharides, and this inhibition leads to impaired biofilm formation.
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Bacteria are believed to employ the glue-like EPS for biofilm cohesion.
Depending on the nature of an infection, biofilm formation and elaboration of
EPS may contribute to bacterial pathogenicity such as interference with wound
healing. However, bismuth alone is not therapeutically useful as an
intervention
agent, and is instead typically administered as part of a complex such as a
BT.
Bismuth-thiols (BTs) are thus a family of compositions that includes compounds

that result from the chelation of bismuth with a thiol compound, and that
exhibit
dramatic improvement in the antimicrobial therapeutic efficacy of bismuth. BTs

exhibit remarkable anti-infective, anti-biofilm, and immunomodulatory effects.
Bismuth thiols are effective against a broad-spectrum of microorganisms, and
are typically not affected by antibiotic-resistance. BTs prevent biofilm
formation
at remarkably low (sub-inhibitory) concentrations, prevent many pathogenic
characteristics of common wound pathogens at those same sub-inhibitory
levels, can prevent septic shock in animal models, and may be synergistic with
many antibiotics.
As described herein, such synergy in the antibacterial effects of
one or more specified BT when combined with one or more specified antibiotic
compound is not readily predictable based on profiles of separate antibiotic
and
BT effects against a particular bacterial type, but surprisingly may result
from
selection of particular BT-antibiotic combinations in view of the specific
bacterial
population, including identification of whether gram-negative or gram-positive

(or both) bacteria are present. For instance, as disclosed herein, antibiotics

that synergize with certain BTs may include one or more of amikacin,
ampicillin,
aztreonam, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin
(or
other lincosamide antibiotics), daptomycin (Cubicin ), doxycycline,
gatifloxacin,
gentamicin, imipenim, levofloxacin, linezolid (Zyvox0), minocycline, nafcilin,

paromomycin, rifampin, sulphamethoxazole, tetracycline, tobramycin and
vancomycin. In vitro studies showed, for example, that MRSA, which was
poorly or not at all susceptible to gentamicin, cefazolin, cefepime,
suphamethoxazole, imipenim or levofloxacin individually, exhibited marked
sensitivity to any one of these antibiotics if exposed to the antibiotic in
the
presence of the BT compound BisEDT. Certain embodiments contemplated
herein thus expressly contemplate compositions and/or methods in which may
be included the combination of a BT compound and one or more antibiotics
selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or another lincosamide antibiotic), daptomycin
(Cubicin0),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
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linezolid (Zyvox0), minocycline, nafcilin, paromomycin, rifannpin,
sulphamethoxazole, tobramycin and vancomycin, whilst certain other
embodiments contemplated herein contemplate compositions and/or methods
in which may be included the combination of a BT compound and one or more
antibiotics from which expressly excluded may be one or more antibiotic
selected from amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or other lincosamides), daptonnycin (Cubicin0),
doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin, linezolid
(Zyvox0),
minocycline, nafcilin, paromomycin, rifampin, sulphamethoxazole, tobramycin
and vancomycin. It is noted in this context that gentamicin and tobramycin
belong to the aminoglycoside class of antibiotics. Also expressly excluded
from
certain contemplated embodiments are certain compositions and methods
described in Domenico et al., 2001 Agents Chemother. 45:1417-1421;
Domenico et al., 2000 Infect. Med. 17:123-127; Domenico et al., 2003 Res.
Adv. In Antimicrob. Agents & Chemother. 3:79-85; Domenico et al., 1997
Antimicrob. Agents Chemother. 41(8):1697-1703; Domenico et al., 1999 Infect.
lmmun. 67:664-669: Huang et al. 1999 J Antimicrob. Chemother. 44:601-605;
Veloira et al., 2003 J Antimicrob. Chemother. 52:915-919; Wu et al., 2002 Am J

Respir Cell Mol Biol. 26:731-738; Halwani et al., 2008 Int. J Pharmaceut.
358:278; Halwani et al., 2009 Int. J. Pharmaceut. 373:141-146; where it will
be
noted that none of these publications teach or suggest the mondisperse
microparticulate BT compositions that are disclosed herein.
Accordingly and as described herein, in certain preferred
embodiments there are provided compositions and methods for treating a plant,
animal or human subject, or an article of manufacture, with a composition that
comprises the herein described microparticulate BT and that optionally and in
certain other embodiments also comprises a synergizing and/or an enhancing
antibiotic. Persons familiar with the relevant art will, based on the present
disclosure, recognize appropriate agricultural, clinical, commercial,
industrial,
manufacturing, domestic and other contexts and situations in which such
treatment may be desired, criteria for which are established in the medical
arts,
including inter alia, e.g., surgical, military surgical, dermatological,
trauma
medicine, gerontological, cardiovascular, metabolic diseases (e.g., diabetes,
obesity, etc.), infection and inflammation (including in the epithelial
linings of the
respiratory tract or the gastrointestinal tract, or other epithelial tissue
surfaces
such as in glandular tissues), and other relevant medical specialties and
subspecialities.
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Preferred compositions for treating a microbial infection on or in a
natural or artificial surface for use according to the embodiments described
herein, may include in certain embodiments compositions that comprise
bismuth-thiol (BT) compounds as described herein, and which may in certain
distinct but related embodiments also include other compounds that are known
in the art such as one or more antibiotic compounds as described herein. BT
compounds and methods for making them are disclosed herein and are also
disclosed, for example, in Domenico et al. (1997 Antimicrob. Agent. Chemother.

41(8):1697-1703; 2001 Antimicrob. Agent. Chemother. 45(5)1417-1421) and in
U.S. RE37,793, U.S. 6,248,371, U.S. 6,086,921, and U.S. 6,380,248. As also
noted above, certain preferred BT compounds are those that contain bismuth or
a bismuth salt ionically bonded to, or in a coordination complex with, a thiol-

containing compound, such as a composition that comprises bismuth chelated
to the thiol-containing compound, and certain other preferred BT compounds
are those that contain bismuth or a bismuth salt in covalent bond linkage to
the
thiol-containing compound. Also preferred are substantially monodisperse
nnicroparticulate BT compositions as described herein. Neither from previous
efforts to treat bacterial infections, nor from previous characterization in
other
contexts of any compounds described herein for the first time as having use in
compositions and methods for promoting the herein described treatment of
natural and/or artificial surfaces, could it be predicted that the present
methods
of using such compounds would have the herein described beneficial effects.
According to preferred embodiments there are thus provided
methods for treating a natural or artificial surface, comprising administering
to
the surface at least one microparticulate BT compound as described herein. In
certain embodiments the method further comprises administering,
simultaneously or sequentially and in either order, at least one antibiotic
compound, which in certain preferred embodiments may be a synergizing
antibiotic as described herein, and which in certain other preferred
embodiments may be an enhancing antibiotic as described herein. The
antibiotic compound may be an aminoglycoside antibiotic, a carbapenem
antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a
glycopeptides antibiotic, a lincosannide antibiotic, a penicillinase-resistant

penicillin antibiotic, or an aminopenicillin antibiotic. Clinically useful
antibiotics
are discussed elsewhere herein and are also described in, e.g., Washington
University School of Medicine, The Washington Manual of Medical
Therapeutics (32nd Ed.), 2007 Lippincott, Williams and Wilkins, Philadelphia,
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PA; and in Hauser, AL, Antibiotic Basics for Clinicians, 2007 Lippincott,
Williams and Wilkins, Philadelphia, PA.
As described herein, certain embodiments derive from the
unpredictable discovery that for a bacterial infection that comprises gram
positive bacteria, a preferred therapeutically effective formulation may
comprise
a BT compound (e.g., BisEDT, bismuth:1,2-ethanedithiol; BisPyr,
bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-ethanedithiol/pyrithione) and
rifamycin, or a BT compound and daptomycin (Cubicin0, Cubist
Pharmaceuticals, Lexington, MA), or a BT compound and linezolid (Zyvox0,
Pfizer, Inc., NY, NY), or a BT compound (e.g., BisEDT, bismuth:1,2-
ethanedithiol; BisPyr, bismuth:pyrithione; BisEDT/Pyr, bismuth:1,2-
ethanedithiol/pyrithione) and one or more of ampicillin, cefazolin, cefepime,
chloramphenicol, clindamycin (or another lincosamide antibiotic), daptomycin
(Cubicin0), doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
linezolid (Zyvox0), nafcilin, paromomycin, rifampin, sulphamethoxazole,
tobramycin and vancomycin.
As also described herein, certain embodiments derive from the
unpredictable discovery that for a bacterial infection that comprises gram
negative bacteria, a preferred therapeutically effective formulation may
comprise a BT compound and amikacin. Certain related embodiments
contemplate treatment of an infection comprising gram negative bacteria with a

BT compound and another antibiotic, such as another aminoglycoside
antibiotic, which in certain embodiments is not gentamicin or tobramycin.
Accordingly and in view of these embodiments, other related embodiments
contemplate identifying one or more bacterial populations or subpopulations in

or on a natural or artificial surface by the well known criterion of being
gram
positive or gram negative, according to methodologies that are familiar to
those
skilled in the medical microbiology art, as a step for selecting appropriate
antibiotic compound(s) to include in a formulation to be administered
according
to the present methods.
The presently described compositions and methods may find use
in the treatment of microbes (e.g., bacteria, viruses, yeast, molds and other
fungi, microbial parasites, etc.) in a wide variety of contexts, typically by
application or administration of the herein described compounds (e.g., one or
.. more rnicroparticulate BTs alone or in combination with one or more
synergizing and/or enhancing antibiotics as disclosed herein) to a microbial
site
such as a microbial presence on or in a natural or artificial surface. Such

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natural surfaces include but are not limited to surfaces found on plants
(e.g., all
or a portion of a surface of a root, bulb, stern, leaf, branch, vine, runner,
bud,
flower or a part thereof, greentip, fruit, seed, seed pod, or the like),
mammalian
tissues (e.g., epithelia including skin, scalp, gastrointestinal tract lining,
buccal
cavity, etc.; endothelia, cell and tissue membranes such as peritoneal
membrane, pericardial membrane, pleural membrane, periosteal membrane,
meningeal membranes, sarcolennal membranes, and the like; cornea, sclera,
mucous membranes, etc.; and other mammalian tissues such as muscle, heart,
lung, kidney, liver, spleen, gall bladder, pancreas, bladder, nerve, teeth,
bone,
joint, tendon, ligament, etc.) and can also include any site on an article of
manufacture where a microbial presence may be found (e.g., commercial,
residential, industrial, educational, health care and other institutional
building
walls, windows, floors, crawlspaces, attics, basem*nts, fences, roofs,
ceilings,
light and plumbing fixtures, vents, ducts, conduits, doorknobs, switches,
sanitation systems, drains, cisterns, water lines; medical and dental devices,

implants, tools, instruments, equipment and the like; metal, glass, plastic,
wood,
rubber and paper goods; transportation equipment including shipping
containers, automobiles, railroad equipment, boats, ships (e.g., exterior
hull,
rudder, anchor and/or propeller surfaces, interior holds and ballast tank and
other interior surfaces), barges and other maritime equipment including docks,

bulkheads, piers and the like; etc.).
The rnicroparticulate antimicrobial agents described herein may
be used to suppress microbial growth, reduce microbial infestation, treat
products including natural and/or artificial surfaces to improve product
resistance to microbial infestation, reduce biofilm, prevent conversion of
bacteria to biofilm, prevent or inhibit microbial infection, prevent spoilage,
and
any other use described herein. These agents are also useful for a number of
antiviral purposes, including prevention or inhibition of viral infection by
herpes
family viruses such as cytomegalovirus, herpes simplex virus Type 1, and
herpes simplex virus Type 2, and/or infection by other viruses. In this
regard,
the agents are useful for the prevention or inhibition of viral infection by a

variety of viruses, such as, single stranded RNA viruses, single stranded DNA
viruses, Rous sarcoma virus (RSV), hepatitis A virus, hepatitis B virus (HBV),

Hepatitis C (HCV), Influenza viruses, west nile virus (WNV), Epstein-Barr
virus
(EBV), eastern equine encephalitis virus (FEEV), severe acute respiratory
virus
(SARS), human immunodeficiency virus (HIV), human papilloma virus (HPV),
and human T cell lymphoma virus (HTLV),and also including viruses that are
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known as plant pathogens (e.g., potato leaf roll virus; potato virus A, M, S,
X, or
Y; tomato spotted wilt virus; grapevine leaf roll-associated virus 3; plum pox

virus; lettuce mosaic virus; pepino mosaic virus; pepper mild mottle virus;
tomato mosaic virus; tobacco mosaic virus; Calibrachoa mottle virus; Impatiens
necrotic spot virus; etc.).
Other internal and external pharmaceutical uses of the herein
described antimicrobial agents include, but are not limited to, treatment or
prevention of bacterial infection, of tuberculosis, of fungal infections such
as
yeast and mold infections (for example, Candida (e.g., Candida albicans,
Can dida glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis) or
Cryptococcus or other fungi), of Helicobacter pylori infection, and of peptic
ulcer
disease. In one embodiment, the agent is used at a dosage not generally lethal

to bacteria but which is nonetheless sufficient to reduce protective
polysaccharide coatings that would otherwise resist natural immune response.
This technique is thus believed to aid immune system-mediated eradication of
bacterial infection without harming human symbiotic microorganisms (e.g.,
normal intestinal flora and the like) to the extent that may be the case with
antibiotics. By way of illustration and not limitation, certain contemplated
embodiments are now described.
Microoarticulate Bismuth-Thiols for Coating and Treating Water
Lines. In one embodiment, methods are provided herein for preventing and/or
controlling (i.e., slowing, retarding, inhibiting) biofilm development,
disrupting a
biofilm, or reducing the amount of biofilm on the interior or exterior surface
of a
water line (such as a water line used by dentists, dental hygienists, and
other
oral care specialists and caregivers), or other water delivery vehicle
including a
tube, pipe, faucet, water fountain, showerhead, or any other instrument or
apparatus (e.g., dental instruments including a high speed dental drill, air-
water
syringe, and cleaning apparatus or instrument (e.g., Cavitron0)) that contacts

or delivers water that is consumed by or applied to a human or non-human
animal. These methods may also be useful for preventing, reducing, inhibiting,

eliminating, or abrogating growth and division of bacteria, fungi, and/or
protozoa
in a water line or water delivery vehicle. These methods comprise applying,
flushing, attaching, or adhering of microparticulate BT compound to a surface
of
a water line or water delivery vehicle.
Biofilms are microscopic communities that consist primarily of
naturally occurring bacteria and fungi. The microorganisms form thin layers on
surfaces, including dental water delivery systems and other water delivery
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vehicles, such as showerheads, faucets and tubes. Water used as a coolant
and irrigant during dental procedures can be heavily contaminated with
microorganisms (see, e.g., Environmental Protection Agency web site at
epa.gov/safewater/mcl/html). Pathogenic microorganisms or opportunitistic
pathogens that have been found in water from dental water lines and
instruments include Actinomyces, Bacteroides, Bacillus, Cryptosporidium, E.
coil, Flavobacterium, Klebsiella, Legionella, Moraxella, Mycobacterium,
Peptostreptococcus, Pseudomonas, Staphylococcus, Streptococcus, and
Veillonella. In addition, as a result of biofilm formation, Legionella spp.
and
protozoa can proliferate in the water line or water delivery vehicle. Bacteria
from the biofilm and other microorganisms present in a water line or water
delivery vehicle are continuously released as water flows through the line or
vehicle. Patients and clinical staff are exposed to the microorganisms present
in
tiny droplets or fine mist sprayed out of the line or delivery vehicle.
For use and consumption of water in dental applications, the
Center for Disease Control has recommended that the number of bacteria in
water used as a coolant/irrigant for nonsurgical dental procedures should have

an aerobic heterotrophic plate count (H PC) of 500 CFU/ml. The American
Dental Association (ADA) has proposed a more stringent standard,
recommending that water used in dental treatment contain a bacterial level of
CFU/ml. Measures taken to maintain low level of bacterial count in dental
water systems include use of antimicrobial agents (see, e.g., McDowell et al.,
J.
Am. Dent. Assoc. 135:799-805 (2004)); hydrogen peroxide-based disinfectants
(see, e.g., Linger et al., J. Am. Dent. Assoc. 132:1287-91 (2001)); routine
flushing of water lines before and after use; maintenance of water lines and
delivery systems; use of filtering systems; use of chemicals as disinfectants
(e.g., diluted bleach 1:10, glutaraldehyde, food grade ethyl, alcohol,
chlorhexidine-based products); thermal eradication; copper-silver ionization;
chlorine dioxide; ultraviolet light; ozone; disinfectant combinations (e.g.,
Adece
ICX (Adex, Newburg, OR): sodium percarbonate, silver nitrate, and cationic
surfactants and silver ion catalyst.
An alternative antimicrobial that may be used for preventing
and/or controlling (i.e., slowing, retarding, inhibiting) biofilm development,

disrupting a biofilm, or reducing the amount of biofilm on the interior or
exterior
surface of a water line or water delivery vehicle include the microparticulate
BT
compounds (or compositions comprising at least one microparticulate BT
compound) described herein. Microparticulate BT compounds may be
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introduced into water lines, water conduit systems, and water delivery
vehicles
manually or automatically as gels, sprays, pastes, liquids, or powders or
other
forms known to a person skilled in the art. In particular embodiments, a
microparticulate BT compound, either in powder or liquid form is mixed with at
least one or more additional ingredients, which may include at least one
additional biologically active ingredient and/or a biologically inactive
excipient,
to formulate the product, which is delivered or injected periodically into the

water line, water delivery vehicle, or water conduit system. Compositions may
be prepared by a person skilled in the art using any number of methods known
in the art. By way of example, a microparticulate BT compound in an anti-
microbial effective amount may be combined with DMSO may be used. With
routine use, a level of microparticulate BT compound that is sufficient to
prevent
biofilm formation is desired. However, in other embodiments, the level of
microparticulate BT compound may be higher for reducing, removing,
disrupting, or eliminating existing biofilms present in a water line, water
delivery
vehicle, or water conduit system.
A microparticulate BT compound may also be formulated to
release slowly from the composition comprising the microparticulate BT
compound applied to the water line, water delivery vehicle, or water conduit
system. A microparticulate BT compound can also be incorporated into a
coating, which can be applied to, adfixed to, adhered to, or in some manner
placed into contact with the interior surface of a waterline, vehicle, or
system.
The composition comprising a microparticulate BT compound may be a gel
(e.g., a hydrogel, thiomer, aerogel, or organogel) or liquid. An organogel may
comprise an organic solvent, lipoic acid, vegetable oil, or mineral oil. A
slow-
release composition may deliver an antimicrobially effective amount of
microparticulate BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for
2, 3,
4, 5, 6, 7 weeks, or 1, 2, 3, 4, 5, or 6 months.
The microparticulate BT compound (or a composition comprising
the microparticulate BT compound) may be combined with at least one other
antimicrobial agent (i.e., a second, third, fourth, etc. antimicrobial agent)
that
when administered in combination have enhanced or synergistic antimicrobial
effects as described herein. By way of example, an enhanced antimicrobial
effect may be observed when microparticulate BT compound is administered
together with an antimicrobial agent that chelates iron. A microparticulate BT
compound may be combined with at least one of an oxidizing agent,
microbicide, or disinfectant. Microparticulate BT compounds that are prepared
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with hydrophobic thiols (e.g., thiochlorophenol) may be used and which may
exhibit greater capability than less hydrophobic BT compounds to adhere to
surfaces of water lines and water delivery vehicles and systems. BT
compounds that have a net negative charge, such as those having a 1:2 molar
ratio (bismuth to thiol) may also have favorable adhesive properties.
A microparticulate BT compound (and compositions comprising
microparticulate BT compound) may be combined with baking soda or another
alkaline compound or substance. Because of the chemical and physical
properties of baking soda, it has wide range of applications, including
cleaning,
deodorizing, and buffering. Baking soda neutralizes odors chemically, rather
than masking or absorbing them. Baking soda can be combined with
microparticulate BT compound either as a mixture of powders, or dissolved or
suspended in a powder, spray, gel, paste, or liquid described herein. In other

embodiments, microparticulate BT compound can be combined with other alkali
metal bicarbonate or carbonate substances (e.g., potassium bicarbonate or
calcium carbonate) that help maintain a desired alkaline pH and that also
possess cleansing and deodorizing properties.
By way of an additional example, a microparticulate BT compound
(or a composition comprising microparticulate BT compound) may be combined
with one or more of the following. Antimicrobial agents: for example,
chlorhexidine; sanguinarine extract; metronidazole; quaternary ammonium
compounds (such as cetylpyridinium chloride); bis-guanides (e.g.,
chlorhexidine
digluconate, hexetidine, octenicline, alexidine); halogenated bisphenolic
compounds (e.g., 2,2' methylenebis-(4-chloro-6-bromophenol) or other phenolic
antibacterial compounds; alkylhydroxybenzoate; cationic antimicrobial
peptides;
anninoglycosides; quinolones; lincosamides; penicillins; cephalosporins,
macrolides; tetracyclines; other antibiotics known in the art; Coleus
forskohlii
essential oil; silver or colloidal silver antimicrobials; tin- or copper-based

antimicrobials; Manuka oil; oregano; thyme; rosemary; or other herbal
extracts;
and grapefruit seed extract. Anti-caries agents: for example, sodium- and
stannous fluoride, aminefluorides, sodium monofluorophosphate, sodium
trimetaphosphate, zinc citrate or other zinc agents, and casein. Plaque
buffers:
for example, urea, calcium lactate, calcium glycerophosphate, and strontium
polyacrylates. Vitamins: for example, Vitamins A, C and E. Plant extracts.
Anti-calculus agents: for example, alkali-metal pyrophosphates, hypophosphite-
containing polymers, organic phosphonates and phosphocitrates etc.
Bionnolecules: for example, bacteriocins. Preservatives. Opacifying agents.

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pH-adjusting agents. Sweetening agents. Surfactants: for example, anionic,
nonionic, cationic and zwitterionic or amphoteric surfactants, saponins from
plant materials (see, e.g., U.S. Patent No. 6,485,711). Particulate abrasive
materials: for example, silicas, alunninas, calcium carbonates, dicalcium
phosphates, calcium pyrophosphates, hydroxyapatites, trinnetaphosphates,
insoluble hexametaphosphates, agglomerated particulate abrasive materials,
chalk, fine ground natural chalk and the like. Humectants: for example,
glycerol, sorbitol, propyleneglycol, xylitol, lactitol etc. Binders and
thickeners:
for example, sodium carboxy methyl cellulose, hydroxyethyl cellulose
(Natrosol0), xanthan gum, gum arabic, synthetic polymers (e.g., polyacrylates
and carboxyvinyl polymers such as Carbopol0). Polymeric compounds that
enhance the delivery of active ingredients such as antimicrobial agents.
Buffers
and salts to buffer the pH and ionic strength of the oral care composition.
Bleaching agents: for example, peroxy compounds (e.g., potassium
peroxydiphosphate). Effervescing systems: for example, sodium
bicarbonate/citric acid systems.
In another embodiment, a microparticulate BT compound
described herein (or composition comprising the microparticulate BT
compound) may be combined with at least one or more anti-biofilm agents for
controlling biofilm development, disrupting a biofilm, or reducing the amount
of
biofilm. As understood in the art, interspecies quorum sensing is related to
biofilm formation. Certain agents that increase LuxS-dependent pathway or
interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408)
contribute to controlling development and/or proliferation of a biofilm.
Exemplary agents include, by way of example, N-(3-oxododecanoyI)-L-
hom*oserine lactone (OdDHL) blocking compounds and N-butyryl-L-hom*oserine
lactone (BHL) analogs, either in combination or separately (see, e.g., U.S.
Patent No. 6,455,031). An oral hygiene composition comprising a
microparticulate BT compound and at least one anti-biofilm agent can be
delivered locally for disruption and inhibition of bacterial biofilm and for
treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
The effectiveness of a microparticulate BT compound as an anti-
biofilm agent may be enhanced by heating the water line, water delivery
vehicle, or water conduit system to which the microparticulate BT compound is
applied by heating the line, vehicle, or system. In certain embodiments, the
line, vehicle, or system is heated to between about 37 C to about 60 C or to

about 37 C to about 100 C. In other embodiments, the line, vehicle, or
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system is heated to between about 450 C to about 50 C; to between about 50
C to about 55 C; between about 55 C to about 60 C; to between about 60 C
to about 70 C; to between about 70 C to about 80 C; to between about 80 C
to about 90 C; or to between about 90 C to about 100 C. In particular
embodiments, the line, vehicle, or system is heated to about 37 C. In another

particular embodiment, the line, vehicle, or system is heated to about 55 C.
As
would be understood by a person skilled in the art, the length of time that
the
line, vehicle, or system, is heating may vary depending on the temperature
applied. For example, the length of time required to achieve the same
antimicrobial effect will be longer when the line, vehicle, or system is
heated to
a lower temperature than needed when heated to the higher temperatures.
Determining the appropriate length of time for exposure of the line, vehicle,
or
system at each temperature may readily be determined by a person skilled in
the art.
A microparticulate BT compound (or compositions comprising a
microparticulate BT compound) can be employed in conjunction with other
modalities to reduce or prevent development of biofilnn. By way of example,
microparticulate BT compounds may be combined with oxidative chemicals,
descaling compounds, biofilnn disruptors, or flushing systems, which are
described herein and used in the art.
Compositions Comprising Microparticulate Bismuth-Thiols and
Uses for Dental Restoration. In another embodiment provided herein are
compositions comprising a microparticulate BT compound and dental amalgam
and microparticulate BT compound and dental composites for use in prevention
and/or treatment of dental caries. Currently, the only treatment for carious
lesions is tooth restoration by placement of an inert material that acts as a
block
to further decay. Dental amalgam and dental composites are most commonly
used for restoration of teeth affected by dental caries.
Recurrent marginal decay is an important contributor to
restoration failure, particularly when dental composites are used for
restoration.
The presence of bacteria located at the interface between a composite material

and dental tissues may an important factor in restoration failure (see, e.g.,
Hansel et al, J. Dent. Res. 77:60-67 (1998)). In a study in Portugal (Casa Pia

Study, 1986-1989), 1,748 posterior restorations were placed and 177(10.1%)
of them failed during the course of the study. Recurrent marginal decay was
the main reason for failure in both amalgam and composite restorations,
accounting for 66% (32/48) and 88% (113/129) failure, respectively (see
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Bernardo et al. JADA 2007;138:775-83). Polymerization shrinkage, which is
the shrinkage that occurs during the composite curing process, has been
implicated as the primary reason for postoperative marginal leakage (see,
e.g.,
Estefan et al., Gen. Dent. 2003;51:506-509).
Incorporation of antimicrobial compounds and agents into
restoration materials, such as dentin bonding systems (DBS), have been
attempted but with limited success. Development of composites and amalgam
and other restoration materials that have antimicrobial properties may
contribute to prevention of secondary dental caries (see, e.g., Imazato, Dent.
Materials 19:449 (2003)). The present embodiments contemplate replacement
of antimicrobials formulated with restoration compositions described herein,
which are described in the art, with the presently described microparticulate
BT
compounds to provide the advantages disclosed herein, including the range of
antimicrobial activities, solubility and bioavailability, anti-biofilm
effects, non-
toxicity, enhancement of antibiotic efficacies, and other properties as
described
herein.
In certain embodiments, a composition is provided comprising a
microparticulate BT compound and a dental composite. Dental composites
typically contain a polymerizable resin base containing ceramic filler. A
.. microparticulate BT compound may be combined with any one of the dental
composites known in the art using methods practiced in the art (see, e.g.,
O'Brien, Dental Materials and Their Selection (Chicago: Quintessence
Publishing Co.) (2002); Powers et al., Dental Materials: Properties and
Manipulation (New York: Mosby) (2007); Roeters et al., J. Dent. 32:371-77
(1998)).
In other embodiments, a composition is provided comprising a
microparticulate BT compound and amalgam. An amalgam is an alloy of
mercury with one or more other metals. Most dental amalgams are called silver
amalgams because silver is the principal constituent that reacts with mercury.
The kinetics of reactions between mercury and silver are not appropriate for
clinical use, so that the silver is provided as an alloy with other elements.
This
alloy is often referred to as a dental amalgam alloy or, collectively, the
alloys
are known as 'alloys for dental amalgam' (see, e.g., International Standars
Organization Standard ISO 1559, Dental Materials ¨ Alloys for Dental Amalgam
.. (1995)). Several types of dental amalgam alloy are known, and all include
tin
and most have some copper and, to a lesser extent, zinc. Some of the dental
amalgam alloys themselves contain a little mercury to facilitate the
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amalgamation reaction. A conventional dental amalgam alloy will contain
between 67% and 74% silver, with 25-28% tin, and up to 6% copper, 2% zinc
and 3% mercury. The so-called dispersion type amalgam alloys have about
70% silver, 16% tin and 13% copper. A different group of amalgam alloys may
contain up to 30% copper, which are known as high-copper content amalgam
alloys. The amalgam alloys are mixed with mercury before clinical placement
at a 1 to 1 weight ratio. The mercury content of a finished dental amalgam
restoration is therefore approximately 50% by weight. In the conventional
dental amalgam alloys, the ratio of silver to tin results in a crystal
structure that
is essentially the intermetallic compound Ag3Sn, referred to as the gamma (y)
phase. The exact percentage of this phase controls the kinetics of the
amalgamation reaction and many properties of the resulting amalgam structure.
With the higher copper dispersion alloys, the microstructure is usually a
mixture
of the gamma phase with the eutectic silver-copper phase. Different
manufacturers present the amalgam alloy in different formats, although they
are
usually made available as fine particles, either spherical or irregular in
shape,
with particle sizes around 25-35 microns. (See Scientific Committee on
Emerging and Newly Identified Health Risks (SCENIHR), European
Commission: Directorate-General, Health & Consumer Protection, May 6, 2008
at Internet site:
ec.europa.eu/health/ph_risk/comnnittees/04_scenihr/docs/scenihr_o_016.pdf.)
A microparticulate BT compound may also be used for preventing
or treating caries and/or inflammation (i.e., reducing the likelihood of
occurrence
or recurrence of caries and/or inflammation, respectively) by administering
the
microparticulate BT compound to the surface of the teeth, amalgam, or
composite. A composition comprising a microparticulate BT compound may be
a mucoadhesive composition that is applied to the surface of a tooth and/or
gum or oral mucous membrane may be in any form that adheres to some
extent to a surface or that delivers a pharmaceutically effective amount of
the
active ingredient(s) to the desired surface. A microparticulate BT compound
can also be formulated to release slowly from the composition applied to the
tooth. For example, the composition may be a gel (e.g., a hydrogel, thiomer,
aerogel, or organogel) or liquid. An organogel may comprise an organic
solvent, lipoic acid, vegetable oil, or mineral oil. Such gel or liquid
coating
formulations may be applied interior or exterior to an amalgam or composite or

other restorative composition. A slow-release composition may deliver a
pharmaceutically effective amount of microparticulate BT compound for 1, 2, 3,
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4, 5, 6, or 7 (a week) days or for 2, 3, 4, 5, 6, 7 weeks, or 1, 2, 3, 4, 5,
or 6
months. Such compositions can be prepared by a person skilled in the art
using any number of methods known in the art.
Compositions comprising a microparticulate BT compound that
are useful for dental restoration may comprise glass iononner cements; giomers
(formed by reacting fluoride containing glass and a liquid polyacid);
compomers
(a polymerizable dinnethacrylate resin and ion-leachable glass filler
particles).
Compomers may further comprise fluoride.
Compositions comprising a microparticulate BT compound that
are applied to the surface of the teeth, amalgam, or composite may further
comprise one or more other surface active agents that enhance the
antimicrobial effect. Exemplary antimicrobial agents for use in the
compositions
comprising a microparticulate BT compound include, for example,
chlorhexidine, sanguinarine extract, metronidazole, quaternary ammonium
compounds, such as cetylpyridinium chloride; bis-guanides, such as
chlorhexidine digluconate, hexetidine, octenidine, alexidine; and halogenated
bisphenolic compounds, such as 2,2' nnethylenebis-(4-chloro-6-bronnophenol),
or other phenolic antibacterial compounds, alkylhydroxybenzoate, cationic
antimicrobial peptides, aminoglycosides, quinolones, lincosamides,
penicillins,
cephalosporins, macrolides, tetracyclines, and other antibiotics, taurolidine
or
taurultann, A-dec ICX, Coleus forskohlii essential oil, silver or colloidal
silver
antimicrobials, tin- or copper-based antimicrobials, chlorine or bromine
oxidants, Manuka oil, oregano, thyme, rosemary or other herbal extracts, and
grapefruit seed extract; anti-inflammatory or antioxidant agents such as
ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric, olive
leaf
extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-linolenic acid

(GLA), green tea, ginger, grape seed, etc.
The compositions may also further comprise one or more
pharmaceutically acceptable carriers, such as, starch, sucrose, water or
water/alcohol systems, DMSO, etc. The compositions may also include a
surfactant, such as an anionic, nonionic, cationic and zwitterionic or
amphoteric
surfactants, or may include sapon ins from plant materials (see, e.g., U.S.
Patent No. 6,485,711). Buffers and salts to buffer the pH and ionic strength
of
composition for oral use my also be included. Other optional ingredients that
may be included are bleaching agents such as peroxy compounds; potassium
peroxydiphosphate; effervescing systems such as sodium bicarbonate/citric
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Compositions Comprising Microparticulate Bismuth-Thiols and
Uses for Oral Hygiene and for Treating Inflammation and Infection of the
Mouth.
In another embodiment, compositions comprising microparticulate BT
compounds are formulated for oral use and may be used in methods for
preventing or reducing microbial growth in the mouth and for preventing and/or

treating microbial infections and inflammation of the oral cavity. These
compositions are therefore useful for preventing or treating (i.e., reducing
or
inhibiting development of, reducing the likelihood of occurrence or recurrence

of) dental plague, halitosis, periodontal disease, gingivitis, and other
infections
of the mouth. The oral compositions comprising microparticulate BT compound
may also be useful for preventing and/or controlling (i.e., slowing,
retarding,
inhibiting) biofilm development, disrupting a biofilm, or reducing the amount
of
biofilm present on an oral surface, particularly a tooth or gums.
Trapped food particles, poor oral hygiene and poor oral health,
and improper cleaning of dentures can promote microbial growth between
teeth, around the gums, and on the tongue. Continued microbial growth and
the presence of dental caries may result in halitosis, dental plaque (i.e., a
biofilm formed by colonization of microorganisms), gingivitis, and
inflammation.
In the absence of proper oral care (e.g., tooth brushing, flossing), more
serious
infections, such as periodontal disease and infections of the jaw, may ensue.
Good oral hygiene is important not only for oral health, but for
prevention of several chronic conditions. Controlling bacterial growth in the
mouth may help lower risk of heart disease, preserve memory, and reduce the
risk of infection and inflammation in other areas of the body. People with
diabetes are at greater risk for developing severe gum problems, and reducing
the risk of gingivitis by maintaining good oral health may help control blood
sugar. Pregnant women may be more likely to experience gingivitis, and some
research suggests a relationship between gum disease in pregnant women and
delivery of preterm, low-birth-weight infants.
Bacteria are the primary etiologic agents in periodontal disease.
More than 500 bacterial strains may be found in dental plague (Kroes et al.,
Proc. Natl. Acad. Sci. USA 96:14547-52 (1999)). Bacteria have evolved to
survive in the environment of the tooth surface, gingival epithelium, and oral

cavity as biofilms, which contributes to the difficulty in treating
periodontitis.
Bactericidal agents as well as antibiotics that are currently used to treat
such
infections often do not kill all of offending organisms. Use of an agent that
is
ineffective against certain bacteria species may result in proliferation of
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resistant bacterial species. Moreover, these agents may cause unpleasant side
effects, such allergic reactions, inflammation, and tooth discoloration.
Dental bacterial plaque is a biofilm that adheres tenaciously to
tooth surfaces, restorations, and prosthetic appliances. The primary means to
control biofilms in the mouth is through mechanical cleaning (i.e.,
tootbrushing,
flossing, etc.). Within the first two days after which no such cleaning has
been
undertaken, the tooth's surface is colonized predominantly by gram-positive
facultative cocci, which are primarily streptococci species. The bacteria
excrete
an extracellular slime layer that helps anchor the bacteria to the surface and
provides protection for the attached bacteria. Microcolony formation begins
once the surface of the tooth has been covered with attached bacteria. The
biofilm grows primarily through cell division of adherent bacteria, rather
than
through the attachment of new bacteria. Doubling times of bacteria forming
plaque are rapid in early development and slower in more mature biofilms.
Coaggregation occurs when bacterial colonizers subsequently
adhere to bacteria already attached to the pellicle. The result of
coaggregation
is the formation of a complex array of different bacteria linked to one
another.
After a few days of undisturbed plaque formation, the gingival margin becomes
inflamed and swollen. Inflammation may result in creation of a deepened
gingival sulcus. The biofilm extends into this subgingival region and
flourishes
in this protected environment, resulting in the formation of a mature
subgingival
plaque biofilm. Gingival inflammation does not appear until the biofilm
changes
from one composed largely of gram-positive bacteria to one containing gram-
negative anaerobes. A subgingival bacterial microcolony, composed
predominantly of gram-negative anaerobic bacteria, becomes established in the
gingival sulcus between 3 and 12 weeks after the beginning of supragingival
plaque formation. Most bacterial species currently suspected of being
periodontal pathogens are anaerobic, gram-negative bacteria.
Bacterial microcolonies protected within the biofilm are typically
resistant to antibiotics (administered systemically), antiseptics or
disinfectants
(administered locally), and immune defenses. Antibiotic doses that kill free-
floating bacteria, for example, need to be increased as much as 1,500 times to

kill biofilm bacteria. At this high concentration, these antimicrobials tend
to be
toxic to the patient as well (see, e.g., Coghlan 1996, New Scientist 2045:32-
6;
Elder et al., 1995, Eye 9:102-9).
Diligent and frequent physical removal of bacterial plaque biofilms
is the most effective means of eliminating and controlling plaque. However,
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subgingival plaque within pockets cannot be reached by brushes, floss, or oral

rinses. Therefore, frequent periodontal debridement of subgingival root
surfaces by a dental hygienist or dentist is an essential component in
prevention and treatment of periodontitis.
In certain embodiments, a microparticulate BT compound may be
incorporated into oral hygiene compositions and onto (such as a coating) or
into
devices, such as but not limited to, toothpaste, mouthwash (Le., mouth rinse),

oral gels, dentifrice powders, oral sprays (including a spray dispersed by an
oral
inhaler), edible film, chewing gum, oral slurry, denture liquid cleaners,
denture
storage liquids, and dental floss, which may be routinely used by any subject.
A microparticulate BT compound may be incorporated into oral hygiene
compositions and onto devices that are used primarily by dental care
professions, including for example, fluoride liquid treatments, cleaning
compositions, buffing compositions, oral rinses, dental floss, and cleaning
tools.
.. The present embodiments contemplate replacement of antimicrobials
formulated with oral hygiene compositions and/or coated onto devices, which
are described in the art, with the presently described microparticulate BT
compounds to provide the advantages disclosed herein, including the range of
antimicrobial activities, solubility and bioavailability, anti-biofilm
effects, non-
toxicity, enhancement of antibiotic efficacies, and other properties as
described
herein.
A microparticulate BT compound may also be used for preventing
or treating caries and/or inflammation (i.e., reducing the likelihood of
occurrence
or recurrence of caries and/or inflammation, respectively) by administering
the
.. microparticulate BT compound to the surface of the teeth. A composition
comprising a microparticulate BT compound may be a nnucoadhesive
composition that is applied to the surface of a tooth and/or gum or oral
mucous
membrane may be in any form that adheres to some extent to a surface or that
delivers a pharmaceutically effective amount of the active ingredient(s) to
the
desired surface. A microparticulate BT compound can also be formulated to
release slowly from the composition applied to the tooth. For example, the
composition may be a gel (e.g., a hydrogel, thiomer, aerogel, or organogel) or

liquid. An organogel may comprise an organic solvent, lipoic acid, vegetable
oil, or mineral oil. Such gel or liquid coating formulations may be applied
interior or exterior to an amalgam or composite or other restorative
composition.
A slow-release composition may deliver a pharmaceutically effective amount of
microparticulate BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for
2, 3,
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4, 5, 6, 7 weeks, or 1, 2, 3, 4, 5, or 6 months. Such compositions can be
prepared by a person skilled in the art using any number of methods known in
the art.
In certain other embodiments, and as described herein,
antimicrobial compositions are provided for oral use that comprise
microparticulate BT compound and one or more additional antimicrobial
compounds or agents. Particularly useful are the compositions comprising s
and a second antimicrobial agent that when administered in combination have
enhanced or synergistic antimicrobial effects, as described herein. By way of
example, an enhanced antimicrobial effect may be observed when a
microparticulate BT compound is administered together with an antimicrobial
agent that chelates iron. In other particular embodiments, a microparticulate
BT
compound is formulated with an anti-inflammatory agent, compound, small
molecule, or macromolecule (such as a peptide or polypeptide).
Any of the microparticulate BT compounds described herein may
be formulated for oral use. In certain embodiments, microparticulate BT
compounds that are prepared with hydrophobic thiols (e.g., thiochlorophenol)
may be used and which may exhibit greater capability than less hydrophobic BT
compounds to adhere to teeth and tissues of the mouth. BT compounds that
have a net negative charge, such as those having a 1:2 molar ratio (bismuth to

thiol) may also have favorable adhesive properties.
The oral hygiene compositions comprising a microparticulate BT
compound may further comprise one or more active ingredients and/or one or
more orally suitable excipients or carriers. In one embodiment, the oral
hygiene
compositions may further comprise baking soda or another alkaline compound
or substance. Because of the chemical and physical properties of baking soda,
it has wide range of applications, including cleaning, deodorizing, and
buffering.
Baking soda neutralizes odors chemically, rather than masking or absorbing
them. Baking soda can be combined with a microparticulate BT compound
either as a mixture of powders, or dissolved or suspended in any one of the
dentifrice powders, gels, pastes, and liquids described herein. In other
embodiments, a microparticulate BT compound can be combined with other
alkali metal bicarbonate or carbonate substances (e.g., potassium bicarbonate
or calcium carbonate) that help maintain a desired alkaline pH and that also
possess cleansing and deodorizing properties.
Oral hygiene compositions comprising a microparticulate BT
compound may further comprise one or more of the following ingredients.
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Antimicrobial agents: for example, chlorhexidine; sanguinarine extract;
metronidazole; quaternary ammonium compounds (such as cetylpyridinium
chloride); bis-guanides (e.g., chlorhexidine digluconate, hexetidine,
octenidine,
alexidine); halogenated bisphenolic compounds (e.g., 2,2' methylenebis-(4-
chloro-6-bromophenol) or other phenolic antibacterial compounds;
alkylhydroxybenzoate; cationic antimicrobial peptides; aminoglycosides;
quinolones; lincosannides; penicillins; cephalosporins, macrolides;
tetracyclines;
other antibiotics known in the art; Coleus forskohlii essential oil; silver or

colloidal silver antimicrobials; tin- or copper-based antimicrobials; Manuka
oil;
oregano; thyme; rosemary; or other herbal extracts; and grapefruit seed
extract.
Anti-inflammatory or antioxidant agents: for example, ibuprofen, flurbiprofen,

aspirin, indomethacin, aloe vera, turmeric, olive leaf extract, cloves,
panthenol,
retinol, omega-3 fatty acids, gamma-linolenic acid (GLA), green tea, ginger,
grape seed, etc. Anti-caries agents: for example, sodium- and stannous
fluoride, aminefluorides, sodium monofluorophosphate, sodium
trimetaphosphate, zinc citrate or other zinc agents, and casein. Plaque
buffers:
for example, urea, calcium lactate, calcium glycerophosphate, and strontium
polyacrylates. Vitamins: for example, Vitamins A, C and E. Plant extracts.
Desensitizing agents: for example, potassium citrate, potassium chloride,
potassium tartrate, potassium bicarbonate, potassium oxalate, potassium
nitrate, and strontium salts. Anti-calculus agents: for example, alkali-metal
pyrophosphates, hypophosphite-containing polymers, organic phosphonates
and phosphocitrates etc. Biomolecules: for example, bacteriocins,
bacteriophages, antibodies, enzymes, etc. Flavors: for example, peppermint
and spearmint oils, fennel, cinnamon, etc. Proteinaceous materials: for
example, collagen. Preservatives. Opacifying agents. Coloring agents. pH-
adjusting agents. Sweetening agents. Pharmaceutically acceptable carriers:
for example, starch, sucrose, water or water/alcohol systems etc. Surfactants:

for example, anionic, nonionic, cationic and zwitterionic or amphoteric
surfactants, sapon ins from plant materials (see, e.g., U.S. Patent No.
6,485,711). Particulate abrasive materials: for example, silicas, aluminas,
calcium carbonates, dicalcium phosphates, calcium pyrophosphates,
hydroxyapatites, trimetaphosphates, insoluble hexannetaphosphates,
agglomerated particulate abrasive materials, chalk, fine ground natural chalk
and the like. Humectants: for example, glycerol, sorbitol, propyleneglycol,
xylitol, lactitol etc. Binders and thickeners: for example, sodium carboxy
methyl
cellulose, hydroxyethyl cellulose (Natrosol0), xanthan gum, gum arabic,

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synthetic polymers (e.g., polyacrylates and carboxyvinyl polymers such as
Carbopol0). Polymeric compounds that enhance the delivery of active
ingredients such as antimicrobial agents. Buffers and salts to buffer the pH
and
ionic strength of the oral care composition. Bleaching agents: for example,
peroxy compounds (e.g., potassium peroxydiphosphate). Effervescing systems:
for example, sodium bicarbonate/citric acid systems. Color change systems. In
particular embodiments, an abrasive is silica or fine ground natural chalk.
The oral hygiene compositions comprising a microparticulate BT
compound that are formulated for use as a toothpaste may further comprise a
humectant (for example, glycerol or sorbitol), a surface-active agent, binding
agent, and/or a flavoring agent. The toothpastes may also include a
sweetening agent, whitening agent, preservative, and antimicrobial agent. The
pH of a toothpaste and other compositions for oral use is typically between pH

5.5 and 8.5. In certain embodiments, oral hygiene compositions, including
toothpaste, have a pH between 7 and 7.5, between 7.5 and 8, between 8 and
8.5, or between 8.5 and 9, which may enhance the antimicrobial activity of the

microparticulate BT compound. The toothpaste compositions described herein
may include one or more of chalk, dicalcium phosphate dihydrate, sorbitol,
water, hydrated aluminum oxide, precipitated silica, sodium lauryl sulfate,
sodium carboxymethyl cellulose, flavoring, sorbitan monooleate, sodium
saccharin, tetrasodiunn pyrophosphate, methyl paraben, propyl paraben. One
or more coloring agents, for example, FD&C Blue, can be employed if desired.
Other suitable ingredients that may be including in a toothpaste formulation
are
described in the art, for example, in U.S. Pat. No. 5,560,517.
In one particular embodiment, the oral hygiene composition is a
nnouthspray and comprises a microparticulate BT compound, an alkaline buffer
(e.g., potassium bicarbonate), an alcohol, a sweetener component, and a flavor

systenn. The flavor system may also have or more of the following: a
flavorant,
a humectant, a surfactant, a sweetener, and a colorant agent (see, e.g., U.S.
Patent No. 6,579,513). Surfactants described herein and known in the art for
use in oral hygiene compositions may be anionic, nonionic, or amphoteric.
In another embodiment, the microparticulate BT-containing oral
hygiene composition may be combined with additional active ingredients such
as taurolidine and taurultam, which have been described in the art as useful
for
including in toothpastes, tooth gels, and mouthwashes for treating treat
serious
infections (see, e.g., United Kingdom Patent Application No., GB 1557163, U.S.
Patent No. 6,488,912). As described herein, microparticulate BT can also be
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combined with one or more additional antimicrobial agents that when combined
with microparticulate BT, the combination has additive or synergistic effects.
In yet another particular embodiment, an oral hygiene composition
described herein may further comprise at least one or more anti-biofilm agents
for controlling biofilm development, disrupting a biofilm, or reducing the
amount
of biofilm. As understood in the art, interspecies quorum sensing is related
to
biofilm formation. Certain agents that increase LuxS-dependent pathway or
interspecies quorum sensing signal (see, e.g., U.S. Patent No. 7,427,408)
contribute to controlling development and/or proliferation of a biofilm.
Exemplary agents include, by way of example, N-(3-oxododecanoyI)-L-
hom*oserine lactone (OdDHL) blocking compounds and N-butyryl-L-hom*oserine
lactone (BHL) analogs, either in combination or separately (see, e.g., U.S.
Patent No. 6455031). An oral hygiene composition comprising a
microparticulate BT compound and at least one anti-biofilm agent can be
delivered locally for disruption and inhibition of bacterial biofilm and for
treatment of periodontal disease (see, e.g., U.S. Patent No.6,726,898).
An oral hygiene composition described herein may contain a
sufficient amount of a microparticulate BT compound that effects substantial
antimicrobial action during the time required for a normal tooth brushing,
mouth
rinsing, or flossing. As described herein a microparticulate BT compound may
be retained on oral surfaces (such as tooth, amalgam, composite, mucous
membrane, gums). A microparticulate BT compound retained on the teeth and
gums after completion of brushing, rinsing, flossing, for example, may
continue
to provide extended anti-biofilm and anti-inflammatory action.
In other embodiments, microparticulate BT compounds are slowly
released from muco-adhesive polymers or other agents that contribute to
retention of microparticulate BT compound on mucosal, tooth, and restoration
surfaces. Microparticulate BTconnpounds may be added to stable, viscous,
mucoadhesive aqueous compositions, which may also be used for the
prevention and treatment of ulcerative, inflammatory, and/or erosive disorders
of mucous membranes and/or the delivery of pharmaceutically active
compounds to mucosal surfaces for topical treatment or transfer to the
systemic
circulation (see, e.g., U.S. Patent No. 7,547,433).
In another embodiment, oral hygiene compositions comprising a
microparticulate BT compound further comprise olive oil, which may enhance
plaque removal. The use of olive oil in a product intended for oral hygiene,
such as a toothpaste, a mouthwash, a spray, oral inhaler, or chewing gum, may
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contribute to elimination or reduction (a decrease) of bacterial plaque and/or
to
elimination or reduction (decrease of) in the numbers of bacteria present in
the
buccal cavity, thereby achieving a reduction in the occurrence of dental
diseases (e.g., tooth decay, periodontal disease) and halitosis (see, e.g.,
U.S.
__ Patent No. 7,074,391).
In other embodiments, an oral hygiene composition comprising a
microparticulate BT compound may further comprise a mucosal disinfectant
preparation for topical application in the mouth. An oral hygiene composition
may further comprise an aqueous slurry useful for cleaning the tongue and
throat (see, e.g., U.S. Patent No. 6,861,049). In still another embodiment, an

oral hygiene composition comprising a microparticulate BT compound may
further comprise at least one mint that is used for preventing (i.e., reducing
the
likelihood of occurrence) formation of a cavity (dental caries) or reducing
the
number of cavities. One such mint, called CaviStatO (Ortek Therapeutics, Inc.,
Roslyn Heights, NY), contains arginine and calcium, which helps neutralize
acid
pH and promotes adherence of calcium to enamel surfaces. The inclusion of
mint in an oral hygiene composition comprising a microparticulate BT
compound may thus increase pH and enhance adherence of a microparticulate
BT compound to oral surfaces.
Adhesive Compositions Comprising Microparticulate Bismuth-
Thiols Formulated for Dental and Orthopedic Use. In another embodiment,
compositions comprising a microparticulate BT compound are formulated for
use in methods for preventing or reducing microbial growth on a bone or joint
prosthesis or of the tissue and skeletal structure adjacent to the bone or
joint
__ prosthesis. In a particular embodiment, methods are provided for using
compositions comprising a microparticulate BT compound for preventing and/or
treating microbial infections and inflammation resulting from an orthopedic
procedure (e.g., orthopedic surgery, orthopedic therapy, arthroplasty
(including
two-step arthoplasty), orthodontic therapy). In certain embodiments, the
compositions comprise a microparticulate BT compound and bone cement, and
in other certain embodiments, comprise a microparticulate BT compound and
dental cement. These compositions are therefore useful for preventing and/or
treating (i.e., reducing or inhibiting development of, reducing the likelihood
of
occurrence or recurrence of) microbial infections of the skeleton and
supporting
structure (Le., bones, joints, muscles, ligaments, tendons) such as
osteomyelitis. The compositions described herein comprising a
microparticulate BT compound and a bone cement or dental cement may also
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be useful for preventing and/or controlling (La, slowing, retarding,
inhibiting)
biofilm development, disrupting a biofilm, or reducing the amount of biofilm
present in a joint or on a surface, such as the surface of a joint, bone,
ligament,
tendon, or tooth or a replacement joint, bone (partial or total), ligament,
tendon,
or tooth.
A cement as described herein and known in the art is a binder
substance that binds materials together and that is capable of hardening. Such

a substance is capable of binding tissues together or capable of binding a
prosthetic or artificial device (e.g., prosthetic joint, bone, or tooth) to
the
adjacent tissue. Bone cements include, for example, polymethyl methacrylate
(PMMA), magnesium phosphate, and calcium phosphate. Forms of calcium
phosphate are used as "replacement bone" for treating fissures and breaks in
bone that may not heal sufficiently quickly and/or properly without an
implanted
material. The compositions that comprise a bone cement (e.g., calcium
phosphate) and a microparticulate BT compound may also be used for treating
cancellous bone defects by providing mechanical integrity to the cancellous
bone. Cements may be resorbed or may remain at the implantation site.
In particular embodiments, the compositions described herein that
are useful as bone cements comprise a BT compound or microparticulate BT
compound and a preparation of calcium phosphate or magnesium phosphate
suitable for use as a bone cement. A preparation of calcium phosphate or
magnesium sulfate may also be called herein a calcium phosphate-containing
bone cement or calcium phosphate bone cement or magnesium phosphate-
containing bone cement or magnesium phosphate bone cement, respectively.
Calcium phosphate may be included in the compositions in any one of several
forms known and used in the art and include, by way of non-limiting example,
hydroxyapatite (Caio(PO4)6(OH)2); brush ite (CaHPO4*2H20); monetite
(CaHPO4); calcium deficient hydroxyapatite (CDHA, Ca9(PO4)5HP040H);
calcium sulfate/phosphate (CSPC) (see, e.g., Hu et al., J. Mater. Sci. Mater.
Med. 2009 October 13, e-publication ahead of print) cements. A magnesium
phosphate used in the art is also called struvite (MgNH4PO4* 6H20) cement
(see, e.g., Grosshardt et al., Tissue Eng. Part A, 2010 Jul 30, e-pub ahead of

print; see also, e.g., Bohner et al., J. Pharm. Sci. 86:565-72; (1997); Fulmer
et
al., 3:299-305 (1992); Lobenhoffer et al., J. Orthopaedic Trauma 16:143-49
(2002); Lee et al., J. Camiofac. Surg. 21:1084-88 (2010)). In a particular
embodiment, the compositions described herein comprising a microparticulate
BT compound and a calcium phosphate-containing bone cement comprise
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calcium sulfate/phosphate (CSPC) as the form of calcium phosphate (see, e.g.,
Hu et al., J. Mater. Sci. Mater. Med. 2009 October 13, e-publication ahead of
print). In certain other embodiments, the compositions comprising a
microparticulate BT compound and calcium phosphate or magnesium
phosphate cement may further comprise chitosan (biopolynner from crustacean
cells); at least one or more antibiotics or antimicrobial agents; and/or at
least
one or more anti-inflammatory agents.
Bone cements have been used in the art for release of drugs and
agents. In certain particular embodiments, a calcium phosphate cement may
be in the form, at least in part, as a hydroxyapatite microsphere that
encapsulates an agent (such as an antimicrobial agent) for therapeutic use
(see, e.g., U.S. Patent No. 6,730,324). Such cements that include
microspheres are useful for slow release of the agent included within the
nnicrosphere. Contemplated herein are compositions comprising calcium
phosphate microspheres that comprise a microparticulate BT compound.
Also provided herein are compositions comprising a
microparticulate BT compound and a PMMA bone cement. The PMMA bone
cement may be formulated with a microparticulate BT compound according to
methods described in the art for formulating PMMA with other agents having
antimicrobial activity (see, for example, European Patent Application No.
EP1649874).
Also provided herein are compositions comprising a
microparticulate BT compound and a dental cement (i.e., dental adhesive),
which compositions may be used for inhibiting, preventing, or treating a
.. microbial infection of the tooth or gums. Dental cements may comprise any
one
of the following compounds or compositions: zinc phosphate, glass ionomers,
alpha-tricalcium phosphate (a-TCP), alkyl methacrylate (see, e.g., U.S. Patent

No. 6,071,528); bismuth oxide (see, e.g., Bueno et al., Oral Surg. Oral Med.
Oral Pathol. Oral Radio!. Endod 107:e65-69 (2009)); and mineral trioxide
aggregate (MTA) (see, e.g., Hwang et al., Oral Surg. Oral Med. Oral Pathol.
Oral Radio!. Endod. 107:e96-102 (2009)).
The present embodiments contemplate replacement of
antimicrobials formulated with dental cement or bone cement, which are
described in the art, with the presently described microparticulate BT
compounds to provide the advantages disclosed herein, including the range of
antimicrobial activities, solubility and bioavailability, anti-biofilm
effects, non-
toxicity, enhancement of antibiotic efficacies, and other properties as
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herein. Bone and dental cements may be formulated with a microparticulate BT
compound and one or more additional antibiotics according to methods
described in the art (see, e.g., U.S. Patent Application Publication No.
2006/0205838; Alt et al., Antimicrob. Agents Chemother. 48:4-84-88 (2004);
Bohner et al., supra; Bueno et al., supra; Chuard et al., Antimicrob. Agents
Chemother. 37:625-32 (1993); J. Orthopaed. Res. 27:1008-15 (2009); De LaIla,
J. Chemother. 13:48-53 (2001); Domenico et al., Peptides 25:2047-53 (2004);
Widmer et al., Antimicrob. Agents Chemother. 35:741-46 (1991)).
The amount of a BT compound used in a microparticulate BT-
containing composition comprising bone cement or dental cement may range
from between about 10-500 pg BT per gram of the respective cement. The
microparticulate BT compounds, alone or in combination with at least one
additional antibiotic, provide advantages as described herein over presently
used antibiotics in bone and dental cements. The compositions described
herein that comprise a microparticulate BT compound and a bone cement (e.g.,
calcium phosphate) or dental cement may further comprise one or more
additional antimicrobial compounds or agents. Particularly useful are the
compositions comprising a microparticulate BT compound and a second
antimicrobial agent that when administered in combination have enhanced or
synergistic antimicrobial effects, as described herein. By way of an
additional
example, an enhanced antimicrobial effect may be observed when a
microparticulate BT compound is administered together with an antimicrobial
agent that chelates iron. In other particular embodiments, a microparticulate
BT
compound is formulated with an anti-inflammatory agent, compound, small
molecule, or macromolecule (such as a peptide or polypeptide).
Compositions comprising a microparticulate BT compound and a
bone cement as described herein may also be used for coating hardware (for
example, screws, plates, staples, pins, and wires and the like) that is used
to
attach, stabilize, or fixate a fracture, fusion, osteotomy, or replacement
joint.
Compositions comprising a microparticulate BT compound and a dental cement
as described herein may be used for coating tooth pulp, a tooth cap, a liner,
a
tooth, or a dental filling or restoration composition within a tooth, or the
like.
These compositions may be formulated into a coating that can be applied to,
adfixed to, adhered to, or in some manner placed into contact with the surface
of bone and/or joint related hardware. In particular embodiments, the coating
comprises a microparticulate BT compound and a calcium phosphate or
magnesium phosphate bone cement. The microparticulate BT compound and
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calcium phosphate or magnesium phosphate are formulated together for
application to bone hardware according to methods practiced in the art. For
example, a composition comprising a microparticulate BT compound and a
bone cement (e.g., calcium phosphate or magnesium phosphate bone cement)
may be in the form of a liquid, gel, paste, or spray (e.g., a thermal spray,
which
includes a plasma spray) for application to the hardware. The composition
comprising a microparticulate BT compound and a bone cement may be a gel
(e.g., a hydrogel, thiomer, aerogel, or organogel) or liquid. An organogel may

comprise an organic solvent, lipoic acid, vegetable oil, or mineral oil. A
slow-
release composition may deliver an antimicrobially effective amount of
microparticulate BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for
2, 3,
4, 5, 6, 7 weeks, or 1, 2, 3, 4, 5, or 6 months. The rate of release may be
controlled, at least in part, according to the porosity of the cement (see,
e.g.,
Bohner et al., supra).
Compositions comprising a microparticulate BT compound and a
bone cement or dental cement may be combined with at least one other
antimicrobial agent (i.e., a second, third, fourth, etc. antimicrobial agent)
that
when administered in combination have enhanced or synergistic antimicrobial
effects (i.e., greater than an additive effect). By way of example, an
enhanced
antimicrobial effect may be observed when a microparticulate BT compound is
administered together with an antimicrobial agent that chelates iron. In
particular embodiments, compositions comprising a microparticulate BT
compound and a bone cement or dental cement may be combined with at least
one other antimicrobial agent and/or anti-inflammatory agent selected from the
following: Antimicrobial agents: for example, chlorhexidine; sanguinarine
extract; metronidazole; quaternary ammonium compounds (such as
cetylpyridinium chloride); bis-guanides (e.g., chlorhexidine digluconate,
hexetidine, octenidine, alexidine); halogenated bisphenolic compounds (e.g.,
2,2' methylenebis-(4-chloro-6-bromophenol) or other phenolic antibacterial
compounds; alkylhydroxybenzoate; cationic antimicrobial peptides;
aminoglycosides; quinolones; lincosamides; penicillins; cephalosporins,
macrolides; tetracyclines; other antibiotics known in the art; Coleus
forskohlii
essential oil; silver or colloidal silver antimicrobials; tin- or copper-based

antimicrobials; Manuka oil; oregano; thyme; rosemary; or other herbal
extracts;
and grapefruit seed extract. Anti-inflammatory or antioxidant agents: for
example, ibuprofen, flurbiprofen, aspirin, indomethacin, aloe vera, turmeric,
olive leaf extract, cloves, panthenol, retinol, omega-3 fatty acids, gamma-
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linolenic acid (GLA), green tea, ginger, grape seed, etc. In particular
embodiments, the compositions comprising microparticulate BT compound and
a bone cement or dental cement may further comprise an antibiotic selected
from clindamycin, vancomycin, daptomycin, cefazolin, gentannicin, tobramycin,
metronidazole, cefaclor, ciprofloxacin, or other antimicrobial such as a
quaternary ammonium compound (e.g., benzalkonium chloride, cetyl pyridinium
chloride), an anti-microbial zeolite, alkali metal hydroxide, or an alkaline
earth
metal oxide. The compositions may optionally comprise one or more
pharmaceutically suitable carriers (i.e., excipients), surfactants, buffers,
diluents, and salts, and bleaching agents, which are described herein.
Antimicrobial agents may be formulated with dental cements and bone cements
as described herein and in the art (see, e.g., Akashi et al., Biomaterials
22:2713-17(2001); U.S. Patent No. 6,071,528; Alt et al., supra).
Animal models of foreign body infection may be used to
characterize the antimicrobial activity of the compositions comprising a
microparticulate BT compound and a dental cement or bone cement (see, e.g.,
Chuard et al. Antimicrob. Agents Chemother. 1993;37:625-32). In vivo efficacy
of antibiotics in these models correlates with the ability of antimicrobials
to kill
stationary-phase microorganisms and those that are adherent to foreign
material (see, e.g., Widmer et al. J. Infect. Dis. 1990;162:96-102; Widmer et
al.
Antimicrob Agents Chemother 1991;35:741-6; see also, e.g., Karchmer. Clin.
Infect. Dis. 1998;27:714-6).
By way of non-limiting example and for illustration purposes only,
a bone cement may comprise a microparticulate BT compound in 75% (2/2)
methyl methacrylate styrene copolymer, 15% polymethylmethacrylate (to assist
handling of the composition), and 10% barium sulfate (for radio-opaqueness),
and from about 10 to about 500 pg of a microparticulate BT compound per
gram of cement powder (i.e., 0.001 - 0.05% w/w). In other particular
embodiments, at least one additional antimicrobial agent may be added.
Compositions Comprising Microparticulate Bismuth-Thiols
Formulated with Paints and Paint Coatings. Certain other embodiments
contemplate incorporation of the microparticulate BT compounds described
herein into paints or onto paints as paint coatings for reducing biofouling
and
preventing and/or controlling (i.e., slowing, retarding, inhibiting) biofilm
development, disrupting a biofilm, or reducing the amount of biofilm present
on
a painted surface. The compositions described herein comprising a
microparticulate BT compound may be formulated with a paint or paint coating
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that is applied to any one of numerous articles of manufacture, including but
not
limited to, medical devices, orthopedic devices, dental devices, industrial
devices, electronic devices, walls, floors, ceilings, roofs, pilings, docks,
piers,
pipes, pipelines and piping structures (e.g., intake screens, cooling towers),
heat exchangers, dams, and textiles, and other surfaces, such as those present
in and on vehicles of all types, including automobiles, trains, planes, and
water
vessels such as ships, boats, submarines, and other water vessels.
In a particular embodiment, the compositions and methods
described herein are useful for preventing and/or reducing biofouling or
biofilms
that form on articles of manufacture that are exposed to water. The formation
of biofilm on surfaces in the marine environment is believed to be an
important
factor contributing to the colonization and recruitment of some sessile
invertebrate communities on marine structures (see, e.g., Siboni et al., FEMS
Microbiol Lett 2007;274:24-9). Subsequent interactions of macrobiota with
these microbial films lead within days or weeks to the attachment and growth
of
invertebrates and algae, which account for most of the hydrodynamic drag
associated with biofouling (see, e.g., Schultz, Biofouling 2007;23:331-41).
Old
biofilms on surfaces supported barnacle larval attachment, irrespective of the

type of substrata (see, e.g., Hunga et al., J Exptl Marine Biol Ecol
2008;361:36-
41). Blofilms also significantly increased adhesion strength in the ascidian
Phallusia nigra, the polychaete tubewornn Hydroides elegans, and the barnacle
Balanus amphitrite at one or more developmental stages (see, e.g., Zardus et
al., Biol Bull 2008;214:91-8). Biofilms can also enhance attachment of Zebra
mussels (Dressena polymorpha) to some artificial surfaces (see, e.g., Kavouras
& Maki. lnverteb Biol 2005;122:138-51), which has resulted in millions if not
billions of dollars in lost revenues and costs to the seafood, power
generation,
and manufacturing industries and to water and wastewater treatment facilities
and has caused significant damage to ecosystems into which the mussel is
introduced.
In marine, brackish, and freshwater environments, organisms
collect, settle, attach, and grow on submerged structures and vessels. Such
organisms include algae, fungi and other microorganisms, and aquatic animals,
such as tunicates, hydroids, bivalves, bryozoans, polychaete worms, sponges,
and barnacles. The presence of these organisms, known as the "fouling" of a
structure, can be detrimental, for example, by adding to the weight of the
structure and/or hampering its hydrodynamics thereby reducing its operating
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efficiency, increasing susceptibility to corrosion, and degrading or
fracturing the
structure.
Certain paints and coatings used to date to prevent or reduce
biofouling and biofilm production include toxic components that while
inhibiting
biofouling and biofilm formation may be toxic to desired and beneficial flora
and
fauna. Exemplary biocides and chemical toxins include copper and copper
containing compounds (e.g., cuprous oxide), mercury, arsenic, tributyltin
oxide
(TBT), organotins (i.e., tin with one or more carbon groups attached), hexio
two-
part bisphenol-A-(epichlrohydrin expoxy compounds, difunctional glycidyl ether
epoxy resin, glycidyl ether expoxy, and barium metaborate epoxy.
The presently described microparticulate BT compounds provide
a non-toxic alternative and provide the advantages disclosed herein, including

the range of antimicrobial activities, solubility and bioavailability, anti-
biofilm
effects, enhancement of antibiotic efficacies, and other properties as
described
herein. The microparticulate BT compounds be substituted for other
antimicrobial agents in paints and paint coatings and may be incorporated into

these paints and paint coatings by integration of the microparticulate BT
compounds and methods described herein, with processes that are known for
producing paints and paint coating that include biocidal agents (see, e.g.,
U.S.
Patent Nos. 4,596,724; 4,410,642; 4,788,302; 5,470,586; 6,162,487; 5,384,176;
U.S. Patent Application Publication Nos. 2007/125703 and 2009/0197003;
Gerhart et al., J. Chem. Ecol. 14:1905-17 (1988); Sears et al., J. Chem. Ecol.

16:791-99 (1990); Ganguli et al., Smart Mater. Struct. 18:104027 (2009); Cao
et
al., ACS Applied Materials Interfaces 1:494 (2009); Kumar et al., Nature
Materials 7:236-41 (2008)). Paints into which microparticulate BT compounds
may be incorporated include epoxy, silicone, or acrylic based paints. In more
particular embodiments, microparticulate BT compounds may be incorporated
into paints formulated for marine use and exposure to seawater and which
include, for example, alkyd resin based, Bitumen based, Gilsonite based
paints,
chlorinated rubber based, and epoxy resin based paints.
Antimicrobial agents may be released in a controlled manner by
incorporating the agents into paint coatings. Methods of enhancing drug
release
rate from a composite material are known in the art. Composite material can
include a natural or synthetic, bioabsorbable polymer matrix and a drug
particle
phase dispersed therein (see, e.g., U.S. Patent Nos. 7,419,681 and 5,028,664;
see also, e.g., U.S. Patent Application No. 2009/0043388). By way of example,
a drug eluting pain coating composition may comprise at least one

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microparticulate BT compound dispersed in a modified, biologically active
binders.
A microparticulate BT compound may also be formulated to
release slowly from the composition comprising the microparticulate BT
compound applied to a painted surface. A microparticulate BT compound can
also be incorporated into a coating (e.g., an epoxy coating), which can be
applied to, adfixed to, adhered to, or in some manner placed into contact with
a
surface of a painted structure or article of manufacture. A microparticulate
BT
compound may be slowly released from such compositions. A slow-release
composition comprising a microparticulate BT compound may be a gel (e.g., a
hydrogel, thiomer, aerogel, or organogel) or liquid. An organogel may comprise
an organic solvent, lipoic acid, vegetable oil, or mineral oil. A slow-release

composition may deliver an antimicrobially effective amount of
microparticulate
BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for 2, 3, 4, 5, 6, 7
weeks,
or 1, 2, 3, 4, 5, or 6 months.
Other coatings used in the art and with which the microparticulate
BT compounds described herein may be formulated include polysaccharides
including a polysaccharide matrix reversibly cross-linked with polyvalent
metal
cations (see, e.g., U.S. Patent Application Publication No. 2009/0202610);
titania nanotubes; nanostructu red surfaces; biocompatible dextran-coated
nanoceria with pH-dependent antioxidant properties; polysulfone block
polymers; and other biodegradable coatings (see also, e.g., U.S. Patent No.
6,162,487). Other coatings contemplated herein are formulating
microparticulate BT compounds with anti-corrosion and antifouling antiseptic
coatings used in industry, and include by way of non-limiting example,
Carnauba wax fluoropolymer, Xylan , PTFE, and moly materials.
The microparticulate BT compound concentration (by weight)
within the paint or paint coating may, for example, vary from as low as about
0.001% to about 0.1%, depending on the intended use and desired properties
of the paint or paint coating. The microparticulate BT compound (or a
composition comprising the microparticulate BT compound) incorporated into a
paint or paint coating may be combined with at least one other antimicrobial
agent (i.e., a second, third, fourth, etc. antimicrobial agent) that when
administered in combination have enhanced or synergistic antimicrobial effects
as described herein. By way of non-limiting example, an antimicrobial agent
that may be included in a composition comprising a microparticulate BT
compound includes chlorhexidine; sanguinarine extract; metronidazole;
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quaternary ammonium compounds (such as cetylpyridinium chloride); bis-
guanides (e.g., chlorhexidine digluconate, hexetidine, octenidine, alexidine);

halogenated bisphenolic compounds (e.g., 2,2' methylenebis-(4-chloro-6-
bromophenol) or other phenolic antibacterial compounds;
alkylhydroxybenzoate; cationic antimicrobial peptides; aminoglycosides;
quinolones; lincosamides; penicillins; cephalosporins, madrolides;
tetracyclines;
other antibiotics known in the art; Coleus forskohlii essential oil; silver or

colloidal silver antimicrobials; tin- or copper-based antimicrobials; Manuka
oil;
oregano; thyme; rosemary; or other herbal extracts; and grapefruit seed
extract.
The compositions may also further optionally comprise a surfactant, diluent or

carrier, buffer, and/or bleaching agent, which are described above and herein.
Compositions Comprising Microparticulate Bismuth-Thiols
Formulated with Concrete and Cement Compounds. Certain other
embodiments contemplate incorporation of the microparticulate BT compounds
described herein in industrial cements and in or on concrete, mortar, and
grout,
including coating of concrete, mortar, and grout for preventing and/or
controlling
(i.e., slowing, retarding, inhibiting) biofilnn development, disrupting a
biofilnn, or
reducing the amount of biofilm present on a concrete surface. Microorganisms
that grow on and within concrete structures reduce the useful life of the
product
and can pose health hazards to animals and humans who are exposed to
microorganisms present on a concrete surface (see, e.g., ldachaba et al.,
Waste Manag. Res. 19:284-91(2001); Idachaba et al., J. Hazard. Mater.
90:279-95 (2002); Tazaki, Canadian Mineralogist 30:431-34 (1992)).
As used herein and in the art, cement refers to the dry powder
substance (typically limestone that may also contain additional substances)
that
is used to bind the aggregate materials of concrete. Exemplary cements that
are described in the art are called Ordinary Portland Cement, Portland blast
furnace cement, masonry cements, slag-lime cements, and calcium alum mate
cements. Upon the addition of water and/or additives the cement mixture is
referred to as concrete, especially if aggregates have been added. Concrete is

a composite material consisting of aggregate (e.g., gravel and sand), cement,
and water. Cements used in construction are characterized as hydraulic or
non-hydraulic. Hydraulic cements are typically used for finishing brick
buildings
in wet climates; for masonry construction of harbor works and the like that
are
in contact with seawater; and development of strong concretes.
The compositions described herein that comprise microparticulate
BT compounds may be used to coat or may be mixed with cement that is used
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for concrete structures including, for example, bridges, buildings, pipes,
elevated highways, tunnels, parking garages, offshore oil platforms, piers,
dam
walls, water systems and pipelines, floors, counter tops, sidewalks,
driveways,
loading docks, skate park structures, and radioactive waste holding
structures.
The microparticulate BT compounds described herein may be incorporated into
cements as described in the art (see, e.g., U.S. Patent No. 7,507,281). The
alkalinity of the cement or concrete may also enhance the anti-microbial
effect
of the microparticulate BT compounds.
Cement can also be degraded by acidifying bacteria, such as
Thiobacillus thiooxidans. As non-limiting examples by way of illustration and
not limitation, a bismuth thiol compound, BisEDT (but not a presently
described
microparticulate BT compound), was shown to retard the growth of T.
thiooxidans in concrete used for waste and nuclear disposal systems. The
effective antibacterial range of BisEDT in concrete was shown to be 10-
500pg/g, or 0.001-0.05%. Higher BisEDT levels interfered with concrete
strength. Other compounds, such as BisPYR, may be useful for inhibiting
fouling and biofilnn development by molds and algae. The present
embodiments contemplate replacement of bismuth thiol compounds and other
antimicrobials with the presently described microparticulate BT compounds to
provide the advantages disclosed herein, including the range of antimicrobial
activities, solubility and bioavailability, anti-biofilm effects, non-
toxicity,
enhancement of antibiotic efficacies, and other properties as described
herein.
Microparticulate BT compounds may be introduced onto a
concrete surface manually or automatically as a gel, spray, paste, liquid, or
powder or other forms known to a person skilled in the art. In particular
embodiments, a microparticulate BT compound, either in powder or liquid form
is mixed with at least one or more additional ingredients, which may include
at
least one additional biologically active ingredient and/or a biologically
inactive
excipient, to formulate the product, which is delivered or injected
periodically
into or onto the concrete structure (i.e., onto a surface of the concrete
structure
that is exposed, particularly a surface exposed to water). Compositions may be

prepared by a person skilled in the art using any number of methods known in
the art. By way of example, a microparticulate BT compound in an anti-
microbial effective amount combined with DMSO may be used (e.g., 1 mg/ml
microparticulate BT compound in DMSO). With routine use, a level of
microparticulate BT compound that is sufficient to prevent biofilm formation
is
desired. However, in other embodiments, the level of microparticulate BT
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compound may be higher for reducing, removing, disrupting, or eliminating
existing biofilms present on a concrete surface.
A microparticulate BT compound may also be formulated to
release slowly from the composition comprising the microparticulate BT
compound applied to a surface of a concrete structure. A microparticulate BT
compound can also be incorporated into a coating (e.g., an epoxy coating),
which can be applied to, adfixed to, adhered to, or in some manner placed into

contact with a surface of a concrete structure. A microparticulate BT compound

may be slowly released from such compositions. A slow-release composition
comprising a microparticulate BT compound may be a gel (e.g., a hydrogel,
thiomer, aerogel, or organogel) or liquid. An organogel may comprise an
organic solvent, lipoic acid, vegetable oil, or mineral oil. A slow-release
composition may deliver an antimicrobially effective amount of
microparticulate
BT compound for 1, 2, 3, 4, 5, 6, or 7 (a week) days or for 2, 3, 4, 5, 6, 7
weeks,
or 1, 2, 3, 4, 5, or 6 months.
The rnicroparticulate BT compound (or a composition comprising
the microparticulate BT compound) may be combined with at least one other
antimicrobial agent (i.e., a second, third, fourth, etc. antimicrobial agent)
that
when administered in combination have enhanced or synergistic antimicrobial
effects as described herein. By way of example, an enhanced or synergistic
antimicrobial effect may be observed when a microparticulate BT compound is
administered together with an antimicrobial agent that chelates iron. A
microparticulate BT compound described herein may be combined with at least
one other antimicrobial agent, including a fungicide or an algicide. By way of
non-limiting example, an antimicrobial agent that may be included in a
composition comprising a microparticulate BT compound includes
chlorhexidine; sanguinarine extract; metronidazole; quaternary ammonium
compounds (such as cetylpyridiniunn chloride); bis-guanides (e.g.,
chlorhexidine
digluconate, hexetidine, octenidine, alexidine); halogenated bisphenolic
compounds (e.g., 2,2' methylenebis-(4-chloro-6-bromophenol) or other phenolic
antibacterial compounds; alkylhydroxybenzoate; cationic antimicrobial
peptides;
aminoglycosides; quinolones; lincosamides; penicillins; cephalosporins,
nnacrolides; tetracyclines; other antibiotics known in the art; Coleus
forskohlii
essential oil; silver or colloidal silver antimicrobials; tin- or copper-based
antimicrobials; Manuka oil; oregano; thyme; rosemary; or other herbal
extracts;
and grapefruit seed extract. The compositions may also further optionally
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comprise a surfactant, diluent or carrier, buffer, and/or bleaching agent,
which
are described above and herein.
Microparticulate BT compounds that are prepared with
hydrophobic thiols (e.g., thiochlorophenol) may be used and may exhibit
greater
capability than less hydrophobic BT compounds to adhere to concrete surfaces,
particularly those exposed to water. BT compounds that have a net negative
charge, such as those having a 1:2 molar ratio (bismuth to thiol) may also
have
favorable adhesive properties.
Microparticulate BTs in Rubber, Silicone and Plastic Products.
Certain embodiments contemplate incorporation of the herein described
microparticulate BT compounds in or on artificial surfaces that comprise
fabricated natural and synthetic rubber and/or rubber coatings, including
silicone and silicone coatings, to reduce biofilms and biofouling of such
rubber
surfaces, for example, in medical devices (e.g., catheters, stents, Foley
catheters and other urological catheters, gastrostomy tubes, feeding tubes,
etc.), orthopedic devices, dental devices, industrial devices, electronic
devices,
surfaces, such as those present in and on vehicles of all types, including
automobiles, tires, door and window profiles, hoses, belts, matting, flooring
and
dampeners (anti-vibration mounts), trains, planes, ships, boats, submarines,
pilings, pipes, pipelines, tubing and textiles, plumbing/water fixtures,
houseware
products, flooring materials, footwear products, athletic apparatus, mobile
phones, computer equipment and compounds that use organic fillers, outdoor
products including decking, awnings, tarps, roofing membranes, and swimming
pool liners, and also including disinfection products and systems for food and
beverage preservation, pharmaceuticals manufacturing, and chemical and
water disinfection.
The presently described microparticulate BT compounds may be
incorporated into these and other natural and artificial rubber products by
integration of the BT compositions and methods described herein, with
fabrication processes that are known for these categories of articles of
manufacture. As non-limiting examples by way of illustration and not
limitation,
BTs (but not the presently described microparticulate BTs) have been
incorporated into hydrogel-coated polyurethane rods and Dacron grafts
(Domenico et al. Antimicrob Agents Chemother 2001;45:1417-1421; Domenico
et al., Peptides 2004;25:2047-53). WO/2002/077095 and Japanese Patent
Application 1997-342076 describe pre-vulcanized and/or vulcanized raw rubber
formulations containing silver-based compounds to provide antimicrobial

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characteristics; U.S. Pat. Nos. 6,448,306, 6,555,599, 6,638,993, 6,848,871,
6,852,782, 6,943,205, and 7,060,739 teach the use of silver-based
antimicrobial agents in a rubber matrix. Drug eluting silicone compositions
may
comprise an antimicrobial agent dispersed in modified, biologically active
binders that can be applied to medical devices or other surfaces without using
inert polymer carriers (US Application Pub. No. 2009/0043388).
Silicone oils generally have molecular weights in the range of
2,000 to 30,000 with viscosities ranging from 20 to 1,000 centistokes.
Silicone
rubbers generally have molecular weights in the range of 40,000 to 100,000
with viscosities ranging from 10 to 1,000 stokes. Silicone is used in a
variety of
materials that are typically subject to microbial fouling. These include
sealants,
caulk, grease, oil, spray, rubber, hose and implants. Silicone-based
antifouling
and other antimicrobial coatings have been described but suffer from
shortcomings associated with poor efficacy, poor durability, poor
biocompatibility, loss of antimicrobial activity, short useful lifetime, high
cost of
materials and other issues (e.g., Schultz J Fluids Eng 2004;126:1039-47; US
Patent 4,025,693; Yan & Li. Ophthalmologica 2008;222:245-8; US Patent
6,221,498; US Patent 7,381,751; European Patent Application EP0506113;
Sawada et al. JPRAS 1990;43:78-82; Tiller et al. Surface Coatings
International
Part B: Coatings Transactions 2005;88:1-82; Juhni & Newby Proceedings
Annual Meeting Adhesion Society 2005;28:179-181; Ozdannar et al. Retina
1999;19:122-6; Piccirillo et al. J Mater Chem 2009;19:6167; US Pub.
2009/0215924; Bayston et al. Biomaterials 2009;30:3167-73; Gottenbos et al.
Biomaterials 2002;23:1417-23; Millsap et al. Antonie Van Leeuwenhoek
2001;79:337-43). While these publications describe methods for the
incorporation of antimicrobial materials into rubber articles of manufacture,
none of the products or processes that they describe offer the advantages
provided by the herein described microparticulate BTs.
The present embodiments thus contemplate substitution of the
herein described microparticulate BTs in these and similar rubber (including
silicone) products and processes, as well as in plastics and polymer
fabrication
methodologies such as those to which reference is made below. In each of
these and other known fabrication contexts, the presently described
microparticulate BTs may be incorporated based on the disclosure herein, in
place of other antimicrobial agents, to afford the herein disclosed advantages
as provided by these microparticulate BTs, including the range of
antimicrobial
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activities, solubility and bioavailability, anti-blofilm effects, non-
toxicity,
enhancement of antibiotic efficacies, and other properties as described
herein.
BT compounds can also be formulated, for instance, at low
concentrations that do not interfere with the rubber fabrication process, into
products for reducing biofilms and preventing fouling in or on silicone
products.
The microparticulate BT concentration (by weight) within the silicone may, for

example, vary from as low as about 0.0001% to about 0.1%, depending on the
intended uses and properties of the silicone rubber product. The herein
described microparticulate BTs similarly may be incorporated as coatings on
silicone, or in silicone gels or oils, to prevent or treat biofilms on
silicone
surfaces for extended time periods. Silicone rubber injection port valves are
described in WO/2008/064173 that exude silicone oil periodically, such that
the
presence in such exudates of effective antimicrobial levels of the herein
described microparticulate BT confers anti-biofilm and/or anti-fouling
capabilities on manufactured articles containing such valves or similarly
configured silicone rubber devices. The erodible oil spreads across any
surface
in the vicinity of the valve, providing a renewable source of protection for
extended time periods. This configuration may, for instance, be built into the

under-surfaces of ship hulls, or into other surfaces exposed to water or
humidity.
For enhanced retention of BT on rubber surfaces, the herein
described microparticulate BTs may be selected to possess greater
hydrophobicity by virtue of the particular thiol moiety, for example by using
a
hydrophobic thiol (e.g., thiochlorophenol), which may have enhanced adhesive
properties, and/or by including BTs that are made to have a net negative
charge (e.g., 1:2 molar ratio of bismuth to thiol) which may also possess
enhanced adhesive properties. Silicone materials can, for example, be
assembled in the presence of appropriate concentrations of the herein
described microparticulate BTs at temperatures of 100 C or below.
Bioerodible materials can also be produced to allow gradual release of such
BTs at levels that thwart biofilm formation, for example, around 1-2 ppm. In
other embodiments, rubber and/or plastic components are contemplated that
are fabricated from materials which slowly elute microparticulate BT
compounds and which can be replaced regularly, to prevent biofouling in
various industrial systems or medical devices.
In certain other embodiments, and in a manner analogous to that
described above for compositions and methods that relate to BT incorporation
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into rubber (including silicone) items, the presently described
microparticulate
BT compounds may also be incorporated into these and other plastic and
polymeric products by integration of the BT compositions and methods
described herein, with fabrication processes that are known for these
categories of articles of manufacture.
Non-limiting examples of uses for such microparticulate BT-
containing plastic products include plastics and plastic coatings in medical
devices, orthopedic devices, dental devices, industrial devices, electronic
devices, walls, floors, ceilings, roofs, and other surfaces, such as those
present
in and on vehicles of all types, including automobiles, trains, planes, ships,
boats, submarines, pilings, pipes, pipelines, and textiles, sprinkler heads,
hair
care products, plumbing/water fixtures, houseware products, footwear products,

athletic apparatus, mobile phones, compounds that use organic fillers, outdoor

products that include decking, awnings, tarps, roofing membranes, and
.. swimming pool liners, and other products that include those used in food
and
beverage preservation, and in pharmaceutical, chemical and water disinfection.

Modern plastic materials have been in use since the 1930s.
Plastics are typically made of polymers and, usually, additives. Typical
polymers include: synthetic resins, styrenes, polyolefins, polyamides,
fluoropolymers, vinyls, acrylics, polyurethanes, cellulosics, imides, acetals,

polycarbonates, and polysulfphones. In order to improve physical
characteristics of polymers, additives such as plasticizers are often used,
which
serve as a source of nutrients for microorganisms. Examples of such modern
plasticizers include phthalates, adipates, and other esters. These and other
plasticizers may be particularly susceptible to bacteria and fungi, especially
in
high moisture areas, leading to microbial surface growth and development of
spores, which may result in one or more of infections in humans and animals,
allergic reactions, unpleasant odors, staining, embrittlennent of the plastic,

premature product failure and other undesirable consequences.
Modifying plastic products during or after the fabrication process
by the Introduction of antifouling and other antimicrobial coatings has been
described, but typically suffers from shortcomings associated with poor
efficacy,
poor durability, poor biocompatibility, loss of antimicrobial activity, short
useful
lifetime, high cost of materials and other issues (e.g., U.S. Pat. Nos.
3,624,062;
4,086,297; 4,663,077; 3,755,224; 3,890,270; 6,495,613; 4,348,308; 5,654,330;
5,281,677; 6,120,790; 5,906,825; 7,419,681, 5,028,664; 6,162,487; Markarian,
Plastics, Additives and Compounding 2009, 11:18-22; EP 927 222 Bl; JP 08-
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157641; CN 1528470 A; Masatoshi et al. 2006;51:18-23; U.S. Pub. Nos.
2008/0071229, 2009/0202610 and 2009/0043388); none of the existing
approaches offers the advantages provided by the herein described
microparticulate BTs. Nevertheless, generally known to the artisan will be
.. incorporation of an antimicrobial agent into or onto a plastic product
according
to a strategy such as (a) adsorption of the agent on the polymer surface
(passively or via surfactants); (b) introduction into a polymer of an
antimicrobial
coating which is applied on the surface of a molding device; (c) incorporation

into the bulk phase of the polymeric substrate material; (d) covalent bonding
of
.. the agent to the polymer surface; and/or (e) mixing an antimicrobial agent
with
a polymer-forming (e.g., polyurethane) component prior to the polymerization
reaction, to give the finished polymer.
For example, the herein described microparticulate BTs can be
introduced into these and similar systems manually or automatically, as gels,
sprays, liquids or powders. In one embodiment, for instance, the
microparticulate BT in powder or liquid form is mixed with the ingredients for

plastic fabrication, including active components (e.g., polymeric precursors,
catalysts, reaction initiators, crosslinkers, etc.) and excipients (e.g.,
carrier
solvents, mold-releasing agents, dyes or colorants, plasticizers, etc.),
involved
in the production mixture, which is injected periodically into the fabrication
system. For example, a 1 nng/m1 solution or suspension of microparticulate BT
in DMSO may be injected periodically into the polymer-forming reaction liquor,

or sprayed into the working parts of a molding unit, to achieve desired anti-
biofilm concentrations in the finished product.
Accordingly, these and certain of the related herein disclosed
embodiments contemplate inclusion in such products and processes of the
presently disclosed microparticulate BT compositions, which may include one
or more microparticulate BT, and which may also optionally further include an
antibiotic such as a synergizing or an enhancing antibiotic as described
herein.
Non-limiting examples of bacteria against which the herein
described compositions and methods may find beneficial use, according to
certain embodiments as described herein, include Staphylococcus aureus (S.
aureus), MRSA (methicillin-resistant S. aureus), Staphylococcus epidermidis ,
MRSE (methicillin-resistant S. epidermidis), Mycobacterium tuberculosis,
Mycobacterium avium, Pseudomonas aeruginosa, drug-resistant P. aeruginosa,
Escherichia coli, enterotoxigenic E. coli, enterohemorrhagic E. coli,
Klebsiella
pneumoniae, Clostridium difficile, Heliobacter pylori, Legionella pneumophilaõ
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Enterococcus faecalis, methicillin-susceptible Enterococcus faecalis,
Enterobacter cloacae, Salmonella typhimurium, Proteus vulgaris, Yersinia
enterocolitica, Vibrio cholera, Shigella flexneri, vancomycin-resistant
Enterococcus (VRE), Burkholderia cepacia complex, Francisella tularensis,
Bacillus anthracis, Yersinia pestis, Pseudomonas aeruginosa, vancomycin¨
sensitive and vancomycin-resistant enterococci (e.g., E. faecalis, E.
faecium),
methicillin-sensitive and methicillin-resistant staphylococci (e.g., S. aureus
, S.
epidermidis) and Acinetobacter baumannii, Staphylococcus haemolyticus,
Staphylococcus hominis, Enterococcus faecium, Streptococcus pyo genes,
Streptococcus agalactiae, Bacillus anthracis, Klebsiella pneumonia, Proteus
mirabilis, Proteus vulgaris, Yersinia enterocolytica, Stenotrophom*onas
maltophilia, Streptococcus pneumonia, penicillin-resistant Streptococcus
pneumonia, Burkholderia cepacia, Bukholderia multivorans, Mycobacterium
smegm*tis and E. cloacae.
The practice of certain embodiments of the present invention will
employ, unless indicated specifically to the contrary, conventional methods of

microbiology, molecular biology, biochemistry, cell biology, virology and
immunology techniques that are within the skill of the art, and reference to
several of which is made below for the purpose of illustration. Such
techniques
are explained fully in the literature. See, e.g., Sambrook, et al. Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); Man iatis et al. Molecular
Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I

& II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic

Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and
Translation (B. Harries & S. Higgins, eds., 1984); Animal Cell Culture (R.
Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
Unless the context requires otherwise, throughout the present
specification and claims, the word "comprise" and variations thereof, such as,

"comprises" and "comprising" are to be construed in an open, inclusive sense,
that is as "including, but not limited to".
Reference throughout this specification to "one embodiment" or
"an embodiment" or "an aspect" means that a particular feature, structure or
characteristic described in connection with the embodiment is included in at
least one embodiment of the present invention. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to the same

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embodiment. Furthermore, the particular features, structures, or
characteristics
may be combined in any suitable manner in one or more embodiments.
As noted above, certain invention embodiments described herein
relate to agricultural, industrial, manufacturing and other formulations of
the
described BT compounds (e.g., BisEDT and/or BisBAL), which formulations
may in certain further embodiments comprise one or more antibiotic
compounds as described herein, for instance, amikacin, ampicillin, cefazolin,
cefepime, chloramphenicol, ciprofloxacin, clindamycin (or another lincosannide

antibiotic), daptomycin (CubicinO),_doxycycline, gatifloxacin, gentamicin,
imipenim, levofloxacin, linezolid (Zyvox0), rninocycline, nafcilin,
paromomycin,
rifampin, sulphamethoxazole, tobramycin and vancomycin; or a carbapenem
antibiotic, a cephalosporin antibiotic, a fluoroquinolone antibiotic, a
glycopeptide
antibiotic, a lincosamide antibiotic, a penicillinase-resistant penicillin
antibiotic,
and/or an aminopenicillin antibiotic, and/or an aminoglycoside antibiotic such
as
amikacin, arbekacin, gentamicin, kanamycin, neomycin, netilmicin,
paromomycin, rhodostreptomycin, streptomycin, tobramycin or apramycin,
and/or a lipopeptide antibiotic such as daptomycin (Cubicin0), or an
oxazolidinone antibiotic such as linezolid (Zyvox0). These and related
formulations may comprise the BT compound(s) (and optionally one or more
antibiotics) in a suitable carrier, excipient or diluent and in an effective
amount,
as disclosed herein, when administered to a plant or animal or applied to a
natural or artificial surface, such as a plant, animal or article of
manufacture in
or on which is present a bacterial infection which may be biofilm-related
(e.g., in
which bacteria capable of promoting biofilm formation may be present but a
biofilm is not yet detectable) or that contains a bacterial infection such as
a
biofilm or other bacterial presence.
Administration or incorporation of the BT compounds described
herein, or their salts, in pure form or in an appropriate agricultural,
manufacturing or other industrial composition, can be carried out via any of
the
accepted modes of administration or incorporation of agents for serving
similar
utilities. Application, incorporation or administration of a composition
includes,
in preferred embodiments, directly contacting the composition with the subject

plant or animal or article of manufacture undergoing treatment, which may be
at
one or more localized or widely distributed surface sites and which may
generally refer to contacting the topical formulation with an acute or chronic
infection site (e.g., a wound site on a plant surface) that is surrounded by
intact
tissue but need not be so limited; for instance, certain embodiments
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contemplate as a topical application the administration of a topical
formulation
described herein to injured, abraded or damaged natural or artificial
surfaces.
The formulations (e.g., agricultural compositions) may be
prepared by combining the described BT compound (e.g., comprising a
compound described in U.S. RE37,793, U.S. 6,248,371, U.S. 6,086,921, and/or
U.S. 6,380,248 and/or prepared according to the present disclosure such as the

herein described nnicroparticulate BT suspensions), and in certain related
embodiments as described herein by combining one or more desired antibiotics
(e.g., an anninoglycoside antibiotic such as amikacin) separately or together
with the BT compound, with an appropriate vehicle, dispersant, carrier,
diluent
or excipient for use in preparation of the formulation as may vary depending
upon the intended use, and may be formulated into preparations in solid,
semi-solid, gel, cream, colloid, suspension or liquid or other topically
applied
forms, such as powders, granules, ointments, solutions, washes, gels, pastes,
plasters, paints, bioadhesives, microsphere suspensions, and aerosol sprays.
Compositions of these and related embodiments are formulated
so as to allow the active ingredients contained therein, and in particularly
preferred embodiments the herein described BT compound(s) alone or in
combination with one or more desired antibiotics (e.g., a carbapenem
antibiotic,
a cephalosporin antibiotic, a fluoroquinolone antibiotic, a glycopeptide
antibiotic,
a lincosannide antibiotic, a penicillinase-resistant penicillin antibiotic,
and an
aminopenicillin antibiotic, or an aminoglycoside antibiotic such as amikacin,
or
rifamycin) which may be applied simultaneously or sequentially and in either
order, to be bioavailable upon administration of the formulation containing
the
BT compound(s) and/or antibiotic composition(s) to a desired site and
optionally
to surrounding natural or artificial surfaces of a plant or animal (including
human) subject or of an article of manufacture. Certain embodiments disclosed
herein contemplate administration to and/or incorporation into such a subject
or
article of a BT compound and of an antibiotic, including administration that
may
be simultaneous or sequential and in either order, but the invention is not
intended to be so limited and in other embodiments expressly contemplates a
distinct route of administration for the BT compound relative to the route of
administration of the antibiotic. Thus, the antibiotic may be administered by
any
route of administration as described herein, while the BT compound may be
administered by a route that is independent of the route used for the
antibiotic.
The formulations described herein deliver an effective amount of
the antiseptic agent(s) (and optionally the antibiotic(s)) to the desired
site, such
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as an infection site or a site where it is desired to prevent infection or
biofilnn
formation.
As noted above, the present formulations may take any of a wide
variety of forms, and include, for example, liquids, suspensions, plasters,
creams, lotions, solutions, sprays, gels, ointments, pastes or the like,
and/or
may be prepared so as to contain liposomes, micelles, and/or microspheres.
See, e.g., U.S. Patent No. 7,205,003. For instance, creams, as is well known
in
the arts of pharmaceutical and cosmeceutical formulation, are viscous liquids
or
semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are
water-
washable, and contain an oil phase, an emulsifier, and an aqueous phase. The
oil phase, also called the "internal" phase, is generally comprised of
petrolatum
and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase
usually, although not necessarily, exceeds the oil phase in volume, and
generally contains a humectant. The emulsifier in a cream formulation is
generally a nonionic, anionic, cationic or amphoteric surfactant.
Solutions are hom*ogeneous mixtures prepared by dissolving one
or more chemical substances (solutes) in a liquid such that the molecules of
the
dissolved substance are dispersed among those of the solvent. The solution
may contain other chemicals to buffer, stabilize or preserve the solute.
Common examples of solvents used in preparing solutions are ethanol, water,
propylene glycol or any other vehicle.
Gels are semisolid, suspension-type systems. Single-phase gels
contain organic macromolecules distributed substantially uniformly throughout
the carrier liquid, which is typically aqueous, but also, preferably, contain
an
alcohol, and, optionally, an oil. Preferred "organic macromolecules," i.e.,
gelling
agents, may be chemically crosslinked polymers such as crosslinked acrylic
acid polymers, for instance, the "carbomer" family of polymers, e.g.,
carboxypolyalkylenes, that may be obtained commercially under the Carbopol
trademark. Also preferred in certain embodiments may be hydrophilic polymers
such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers
and polyvinylalcohol; cellulosic polymers such as hydroxypropyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and
xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel,
dispersing agents such as alcohol or glycerin can be added, or the gelling
agent
can be dispersed by trituration, mechanical mixing or stirring, or
combinations
thereof.
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Ointments, as also well known in the art, are semisolid
preparations that are typically based on petrolatum or other petroleum
derivatives. The specific ointment base to be used, as will be appreciated by
those skilled in the art, is one that will provide for a number of desirable
characteristics, e.g., emolliency or the like. As with other carriers or
vehicles,
an ointment base should be inert, stable, nonirritating, and nonsensitizing.
As
explained in Remington: The Science and Practice of Pharmacy, 19th Ed.
(Easton, Pa.: Mack Publishing Co., 1995), at pages 1399-1404, ointment bases
may be grouped in four classes: oleaginous bases; emulsifiable bases;
emulsion bases; and water-soluble bases. Oleaginous ointment bases include,
for example, vegetable oils, fats obtained from animals, and semisolid
hydrocarbons obtained from petroleum. Emulsifiable ointment bases, also
known as absorbent ointment bases, contain little or no water and include, for

example, hydroxystearin sulfate, anhydrous lanolin, and hydrophilic
petrolatum.
Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-
water
(0/W) emulsions, and include, for example, cetyl alcohol, glyceryl
nnonostearate, lanolin, and stearic acid. Preferred water-soluble ointment
bases are prepared from polyethylene glycols of varying molecular weight (see,

e.g., Remington, Id.).
Pastes are semisolid dosage forms in which the active agent is
suspended in a suitable base. Depending on the nature of the base, pastes are
divided between fatty pastes or those made from single-phase aqueous gels.
The base in a fatty paste is generally petrolatum or hydrophilic petrolatum or

the like. The pastes made from single-phase aqueous gels generally
incorporate carboxymethylcellulose or the like as a base.
Formulations may also be prepared with liposomes, micelles, and
microspheres. Liposomes are microscopic vesicles having one (unilamellar) or
a plurality (multilannellar) of lipid walls comprising a lipid bilayer, and,
in the
present context, may encapsulate and/or have adsorbed to their lipid
membranous surfaces one or more components of the formulations herein
described, such as the antiseptic, or certain carriers or excipients.
Liposomal
preparations herein include cationic (positively charged), anionic (negatively

charged), and neutral preparations. Cationic liposomes are readily available.
For example, N[1-2,3-dioleyloxy)propyI]-N,N,N-triethylammonium (DOTMA)
liposomes are available under the tradename Lipofectin0 (GIBCO BRL, Grand
Island, N.Y.). Similarly, anionic and neutral liposomes are readily available
as
well, e.g., from Avanti Polar Lipids (Birmingham, AL), or can be easily
prepared
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using readily available materials. Such materials include phosphatidyl
choline,
cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphoshatidyl ethanolamine
(DOPE), among others. These materials can also be mixed with DOTMA in
appropriate ratios. Methods for making liposomes using these materials are
well known in the art.
Micelles are known in the art as comprised of surfactant
molecules arranged so that their polar headgroups form an outer spherical
shell, while the hydrophobic, hydrocarbon chains are oriented towards the
center of the sphere, forming a core. Micelles form in an aqueous solution
containing surfactant at a high enough concentration so that micelles
naturally
result. Surfactants useful for forming micelles include, but are not limited
to,
potassium laurate, sodium octane sulfonate, sodium decane sulfonate, sodium
dodecane sulfonate, sodium lauryl sulfate, docusate sodium,
decyltrimethylammonium bromide, dodecyltrimethylammonium bromide,
tetradecyltrimethylammonium bromide, tetradecyltrimethyl-ammonium chloride,
dodecylannnnoniunn chloride, polyoxy1-8 dodecyl ether, polyoxyl-12 dodecyl
ether, nonoxynol 10, and nonoxynol 30.
Microspheres, similarly, may be incorporated into the presently
described topical formulations. Like liposomes and micelles, microspheres
essentially encapsulate one or more components of the present formulations.
They are generally, but not necessarily, formed from lipids, preferably
charged
lipids such as phospholipids. Preparation of lipidic microspheres is well
known
in the art.
Various additives, as known to those skilled in the art, may also
be included in the formulations. For example, solvents, including relatively
small amounts of alcohol, may be used to solubilize certain formulation
components. Examples of suitable enhancers include, but are not limited to,
ethers such as diethylene glycol monoethyl ether (available commercially as
Transcuto10) and diethylene glycol monomethyl ether; surfactants such as
sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide,
benzalkonium chloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80),
and lecithin (U.S. Pat. No. 4,783,450); alcohols such as ethanol, propanol,
octanol, benzyl alcohol, and the like; polyethylene glycol and esters thereof
such as polyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat. No.
4,568,343); amides and other nitrogenous compounds such as urea,
dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1 -methyl-

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2-pyrrolidone, ethanolamine, diethanolamine, and triethanolannine; terpenes;
alkanones; and organic acids, particularly citric acid and succinic acid.
Azone0
and sulfoxides such as DMSO and C10MS0 may also be used, but are less
preferred.
Certain permeation enhancers may include those lipophilic co-
enhancers typically referred to as "plasticizing" enhancers, i.e., enhancers
that
have a molecular weight in the range of about 150 to 1000 daltons, an aqueous
solubility of less than about 1 wt %, preferably less than about 0.5 wt (Yo,
and
most preferably less than about 0.2 wt %. The Hildebrand solubility parameter
of plasticizing enhancers is in the range of about 2.5 to about 10, preferably
in
the range of about 5 to about 10. Preferred lipophilic enhancers are fatty
esters, fatty alcohols, and fatty ethers. Examples of specific and most
preferred
fatty acid esters include methyl lau rate, ethyl oleate, propylene glycol
nnonolaurate, propylene glycerol dilaurate, glycerol nnonolaurate, glycerol
monooleate, isopropyl n-decanoate, and octyldodecyl myristate. Fatty alcohols
include, for example, stearyl alcohol and oleyl alcohol, while fatty ethers
include
compounds wherein a diol or trio!, preferably a C2-C4 alkane diol or trio!,
are
substituted with one or two fatty ether substituents. Additional permeation
enhancers will be known to those of ordinary skill in the art of topical drug
delivery, and/or are described in the relevant literature. See, e.g.,
Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, Boca
Raton, FL, 1995).
Various other additives may be included in the topical
formulations according to certain embodiments of the present invention, in
addition to those identified above. These include, but are not limited to,
antioxidants, astringents, perfumes, preservatives, emollients, pigments,
dyes,
humectants, propellants, and sunscreen agents, as well as other classes of
materials whose presence may be cosmetically, medicinally or otherwise
desirable. Typical examples of optional additives for inclusion in the
formulations of certain embodiments of the invention are as follows:
preservatives such as sorbate; solvents such as isopropanol and propylene
glycol; astringents such as menthol and ethanol; emollients such as
polyalkylene methyl glucosides; humectants such as glycerine; emulsifiers such

as glycerol stearate, PEG-100 stearate, polyglycery1-3 hydroxylauryl ether,
and
polysorbate 60; sorbitol and other polyhydroxyalcohols such as polyethylene
glycol; sunscreen agents such as octyl methoxyl cinnamate (available
commercially as Parsol MCX) and butyl methoxy benzoyl methane (available
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under the tradename Parsol 1789); antioxidants such as ascorbic acid (vitamin
C), a-tocopherol (Vitamin E), f3-tocopherol , y-tocopherol, 6-tocopherol, e-
tocopherol , 1-tocopherol, 2-tocophero1,11-tocopherol , and retinol (vitamin
A);
essential oils, ceramides, essential fatty acids, mineral oils, wetting agents
and
.. other surfactants such as the PLURONIC series of hydrophilic polymers
available from BASF (Mt. Olive, NJ), vegetable oils (e.g., soy bean oil, palm
oil,
liquid fraction of shea butter, sunflower oil), animal oils (e.g.,
perhydrosqualene), mineral oils, synthetic oils, silicone oils or waxes (e.g.,

cyclomethicone and dimethicone), fluorinated oils (generally
perfluoropolyethers), fatty alcohols (e.g., cetyl alcohol), and waxes (e.g.,
beeswax, carnauba wax, and paraffin wax); skin-feel modifiers; and thickeners
and structurants such as swelling clays and cross-linked carboxypolyalkylenes
that may be obtained commercially under the Carbopol trademark.
Other additives include agents such as, by way of example,
pyrrolidine carboxylic acid and amino acids; organic antimicrobial agents such
as 2,4,4'-trichloro-2-hydroxy diphenyl ether (triclosan) and benzoic acid;
anti-
inflammatory agents such as acetylsalicylic acid and glycyrrhetinic acid; anti-

seborrhoeic agents such as retinoic acid; vasodilators such as nicotinic acid;

inhibitors of melanogenesis such as kojic acid; and mixtures thereof. Other
.. advantageously included active agents may be present, for example, a-
hydroxyacids, a-ketoacids, polymeric hydroxyacids, moisturizers, collagen,
marine extracts, and antioxidants such as ascorbic acid (vitamin C), a-
tocopherol (Vitamin E) or other tocopherols such as those described above,
and retinol (vitamin A), and/or suitable salts, esters, amides, or other
derivatives
thereof. Additional agents include those that are capable of improving oxygen
supply in living tissue, as described, for example, in WO 94/00098 and WO
94/00109. Sunscreens may also be included.
The formulations of certain embodiments of the invention may
also include conventional additives such as opacifiers, fragrance, colorant,
gelling agents, thickening agents, stabilizers, surfactants, and the like.
Other
agents may also be added, such as antimicrobial agents, to prevent spoilage
upon storage, i.e., to inhibit growth of microbes such as yeasts and molds.
Suitable antimicrobial agents are typically selected from methyl and propyl
esters of p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium
benzoate, sorbic acid, imidurea, and combinations thereof.
The topical formulations may also contain, in addition to the BT
compound, (e.g., as substantially hom*ogeneous microparticles as provided
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herein, and optionally in combination with one or more synergizing antibiotics

as described herein), an effective amount of one or more additional active
agents suitable for a particular mode of administration or incorporation.
A pharmacologically acceptable carrier may also be incorporated
in the topical formulation of certain present embodiments and may be any
carrier conventionally used in the art. Examples include water, lower
alcohols,
higher alcohols, honey, polyhydric alcohols, nnonosaccharides, disaccharides,
polysaccharides, sugar alcohols such as, for example, glycols (2-carbon),
glycerols (3-carbon), erythritols and threitols (4-carbon), arabitols,
xylitols and
ribitols (5-carbon), mannitols, sorbitols, dulcitols and iditols (6-carbon),
isomaltols, maltitols, lactitols and polyglycitols, hydrocarbon oils, fats and
oils,
waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants,
silicone
surfactants, and water-based mixtures and emulsion-based mixtures of such
carriers.
Topical formulation embodiments of the present invention may be
applied regularly to whatever natural (e.g., plant or animal, including human)
or
artificial (e.g., article of manufacture) surface requires treatment with the
frequency and in the amount necessary to achieve the desired results. The
frequency of treatment depends on the nature of the application, the strength
of
the active ingredients (e.g., BT compound and optionally one or more
additional
active ingredients, such as an antibiotic, e.g., amikacin or other antibiotic)
in the
particular embodiment, the effectiveness of the vehicle used to deliver the
active ingredients, and the ease with which the formula is removed by
environmental factors (e.g., physical contact with other materials or objects,
precipitation, wind, temperature).
Typical concentrations of active substances such as the BT
compound in the compositions described herein can range, for example, from
about 0.001-30% by weight based on the total weight of the composition, to
about 0.01-5.0%, and more preferably to about 0.1-2.0%. As one
representative example, compositions of these embodiments of the present
invention may be applied to a natural or artificial surface at a rate equal to
from
about 1.0 mg/cm2 to about 20.0 mg/cnn2. Representative examples of topical
formulations include, but are not limited to, aerosols, alcohols, anhydrous
bases, aqeuous solutions, creams, emulsions (including either water-in-oil or
oil-in-water emulsions), fats, foams, gels, hydro-alcoholic solutions,
liposomes,
lotions, microemulsions, ointments, oils, organic solvents, polyols, polymers,

powders, salts, silicone derivatives, and waxes. The formulations may include,
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for example, chelating agents, conditioning agents, emollients, excipients,
humectants, protective agents, thickening agents, or UV absorbing agents.
One skilled in the art will appreciate that formulations other than those
listed
may be used in embodiments of the present invention.
Chelating agents may be optionally included in certain
formulations, and may be selected from any natural or synthetic chemical agent

which has the ability to bind divalent cationic metals such as Ca2+, Mn2+, or
Mg2+. Examples of chelating agents include, but are not limited to EDTA,
disodium EDTA, EGTA, citric acid, and dicarboxylic acids.
Conditioning agents may also be optionally included in certain
formulations. Examples of conditioning agents include, but are not limited to,

acetyl cysteine, N-acetyl dihydrosphingosine, acrylates/behenyl
acrylate/dimethicone acrylate copolymer, adenosine, adenosine cyclic
phosphate, adensosine phosphate, adenosine triphosphate, alanine, albumen,
algae extract, allantoin and deriviatives, aloe barbadensis extracts, aluminum

PCA, amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk
powder, butylene glycol, caffeine, calcium gluconate, capsaicin,
carbocysteine,
carnosine, beta-carotene, casein, catalase, cephalins, ceramides, chamomilla
recutita (matricaria) flower extract, cholecalciferol, cholesteryl esters,
coco-
betaine, coenzyme A, corn starch modified, crystallins, cycloethoxymethicone,
cysteine DNA, cytochronne C, darutoside, dextran sulfate, dinnethicone
copolyols, dimethylsilanol hyaluronate, DNA, elastin, elastin amino acids,
epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA,
fibronectin,
folic acid, gelatin, gliadin, beta-glucan, glucose, glycine, glycogen,
glycolipids,
glycoproteins, glycosaminoglycans, glycosphingolipids, horseradish peroxidase,

hydrogenated proteins, hydrolyzed proteins, jojoba oil, keratin, keratin amino

acids, and kinetin, lactoferrin, lanosterol, lauryl PCA, lecithin, linoleic
acid,
linolenic acid, lipase, lysine, lysozynne, malt extract, nnaltodextrin,
melanin,
methionine, mineral salts, niacin, niacinamide, oat amino acids, oryzanol,
palmitoyl hydrolyzed proteins, pancreatin, papain, PEG, pepsin, phospholipids,

phytosterols, placental enzymes, placental lipids, pyridoxal 5-phosphate,
quercetin, resorcinol acetate, riboflavin, RNA, saccharomyces lysate extract,
silk amino acids, sphingolipids, stearamidopropyl betaine, stearyl palmitate,
tocopherol, tocopheryl acetate, tocopheryl linoleate, ubiquinone, vitis
vinifera
(grape) seed oil, wheat amino acids, xanthan gum, and zinc gluconate.
Conditioning agents other than those listed above may be combined with a
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disclosed composition or preparation provided thereby, as can be readily
appreciated by one skilled in the art.
In certain embodiments the herein described formulations may
also optionally include one or more emollients, examples of which include, but
are not limited to, acetylated lanolin, acetylated lanolin alcohol,
acrylates/C10_30
alkyl acrylate crosspolymer, acrylates copolymer, alanine, algae extract, aloe

barbadensis extract or gel, althea officinalis extract, aluminum starch
octenylsuccinate, aluminum stearate, apricot (prunus armeniaca) kernel oil,
arginine, arginine aspartate, arnica montana extract, ascorbic acid, ascorbyl
palmitate, aspartic acid, avocado (persea gratissima) oil, barium sulfate,
barrier
sphingolipids, butyl alcohol, beeswax, behenyl alcohol, beta-sitosterol, BHT,
birch (betula alba) bark extract, borage (borago officinalis) extract, 2-bromo-
2-
nitropropane-1,3-diol, butcherbroom (ruscus aculeatus) extract, butylene
glycol,
calendula officinalis extract, calendula officinalis oil, candelilla
(euphorbia
cerifera) wax, canola oil, caprylic/capric triglyceride, cardamon (elettaria
cardarnomum) oil, carnauba (copernicia cerifera) wax, carrageenan (chondrus
crispus), carrot (daucus carota sativa) oil, castor (ricinus connmunis) oil,
ceram ides, ceresin, ceteareth-5, ceteareth-12, ceteareth-20, cetearyl
octanoate, ceteth-20, ceteth-24, cetyl acetate, cetyl octanoate, cetyl palm
itate,
chamomile (anthem is nobilis) oil, cholesterol, cholesterol esters,
cholesteryl
hydroxystearate, citric acid, clary (salvia sclarea) oil, cocoa (theobroma
cacao)
butter, coco-caprylate/caprate, coconut (cocos nucifera) oil, collagen,
collagen
amino acids, corn (zea mays) oil, fatty acids, decyl oleate, dextrin,
diazolidinyl
urea, dimethicone copolyol, dimethiconol, dioctyl adipate, dioctyl succinate,
dipentaerythrityl hexacaprylate/hexacaprate, DM DM hydantoin, DNA, erythritol,

ethoxydiglycol, ethyl linoleate, eucalyptus globulus oil, evening primrose
(oenothera biennis) oil, fatty acids, tructose, gelatin, geranium maculatum
oil,
glucosannine, glucose glutamate, glutannic acid, glycereth-26, glycerin,
glycerol,
glyceryl distearate, glyceryl hydroxystearate, glyceryl laurate, glyceryl
linoleate,
glyceryl myristate, glyceryl oleate, glyceryl stearate, glyceryl stearate SE,
glycine, glycol stearate, glycol stearate SE, glycosaminoglycans, grape (vitis

vinifera) seed oil, hazel (corylus americana) nut oil, hazel (corylus
avellana) nut
oil, hexylene glycol, honey, hyaluronic acid, hybrid safflower (carthamus
tinctorius) oil, hydrogenated castor oil, hydrogenated coco-glycerides,
hydrogenated coconut oil, hydrogenated lanolin, hydrogenated lecithin,
hydrogenated palm glyceride, hydrogenated palm kernel oil, hydrogenated
soybean oil, hydrogenated tallow glyceride, hydrogenated vegetable oil,
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hydrolyzed collagen, hydrolyzed elastin, hydrolyzed glycosaminoglycans,
hydrolyzed keratin, hydrolyzed soy protein, hydroxylated lanolin,
hydroxyproline, imidazolidinyl urea, iodopropynyl butylcarbamate, isocetyl
stearate, isocetyl stearoyl stearate, isodecyl oleate, isopropyl isostearate,
isopropyl lanolate, isopropyl myristate, isopropyl palmitate, isopropyl
stearate,
isostearamide DEA, isostearic acid, isostearyl lactate, isostearyl
neopentanoate, jasmine (jasnninum officinale) oil, jojoba (buxus chinensis)
oil,
kelp, kukui (aleurites moluccana) nut oil, lactamide MEA, laneth-16, laneth-10

acetate, lanolin, lanolin acid, lanolin alcohol, lanolin oil, lanolin wax,
lavender
(lavandula angustifolia) oil, lecithin, lemon (citrus medica limonum) oil,
linoleic
acid, linolenic acid, macadamia ternifolia nut oil, magnesium stearate,
magnesium sulfate, maltitol, matricaria (chamomilla recutita) oil, methyl
glucose
sesquistearate, methylsilanol PCA, microcrystalline wax, mineral oil, mink
oil,
mortierella oil, myristyl lactate, myristyl myristate, myristyl propionate,
neopentyl
glycol dicaprylate/dicaprate, octyldodecanol, octyldodecyl myristate,
octyldodecyl stearoyl stearate, octyl hydroxystearate, octyl palmitate, octyl
salicylate, octyl stearate, oleic acid, olive (olea europaea) oil, orange
(citrus
aurantium dulcis) oil, palm (elaeis guineensis) oil, palmitic acid,
pantethine,
panthenol, panthenyl ethyl ether, paraffin, PCA, peach (prunus persica) kernel
oil, peanut (arachis hypogaea) oil, PEG-8 012 18 ester, PEG-15 cocamine,
PEG-150 distearate, PEG-60 glyceryl isostearate, PEG-5 glyceryl stearate,
PEG-30 glyceryl stearate, PEG-7 hydrogenated castor oil, PEG-40
hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-20 methyl
glucose sesquistearate, PEG-40 sorbitan peroleate, PEG-5 soy sterol, PEG-10
soy sterol, PEG-2 stearate, PEG-8 stearate, PEG-20 stearate, PEG-32
stearate, PEG-40 stearate, PEG-50 stearate, PEG-100 stearate, PEG-150
stearate, pentadecalactone, peppermint (mentha piperita) oil, petrolatum,
phospholipids, polyannino sugar condensate, polyglycery1-3 diisostearate,
polyquaternium-24, polysorbate 20, polysorbate 40, polysorbate 60,
polysorbate 80, polysorbate 85, potassium myristate, potassium palmitate,
potassium sorbate, potassium stearate, propylene glycol, propylene glycol
dicaprylate/dicaprate, propylene glycol dioctanoate, propylene glycol
dipelargonate, propylene glycol laurate, propylene glycol stearate, propylene
glycol stearate SE, PVP, pyridoxine dipalmitate, quaternium-15, quaternium-18
hectorite, quaternium-22, retinol, retinyl palmitate, rice (oryza sativa) bran
oil,
RNA, rosemary (rosmarinus officinalis) oil, rose oil, safflower (carthamus
tinctorius) oil, sage (salvia officinalis) oil, salicylic acid, sandalwood
(santalum
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album) oil, serine, serum protein, sesame (sesamum indicum) oil, shea butter
(butyrospermum parkii), silk powder, sodium chondroitin sulfate, sodium DNA,
sodium hyaluronate, sodium lactate, sodium palmitate, sodium PCA, sodium
polyglutamate, sodium stearate, soluble collagen, sorbic acid, sorbitan
laurate,
sorbitan oleate, sorbitan palnnitate, sorbitan sesquioleate, sorbitan
stearate,
sorbitol, soybean (glycine soja) oil, sphingolipids, squalane, squalene,
stearannide MEA-stearate, stearic acid, stearoxy dinnethicone,
stearoxytrimethylsilane, stearyl alcohol, stearyl glycyrrhetinate, stearyl
heptanoate, stearyl stearate, sunflower (helianthus annuus) seed oil, sweet
almond (prunus amygdalus dulcis) oil, synthetic beeswax, tocopherol,
tocopheryl acetate, tocopheryl linoleate, tribehen in, tridecyl neopentanoate,

tridecyl stearate, triethanolamine, tristearin, urea, vegetable oil, water,
waxes,
wheat (triticum vulgare) germ oil, and ylang ylang (cananga odorata) oil.
Surfactants may also desirably be included in certain formulations
contemplated herein, and can be selected from any natural or synthetic
surfactants suitable for use in cosmetic compositions, such as cationic,
anionic,
zwitterionic, or non-ionic surfactants, or mixtures thereof. (See Rosen, M.,
"Surfactants and Interfacial Phenomena," Second Edition, John Wiley & Sons,
New York, 1988, Chapter 1, pages 431). Examples of cationic surfactants may
include, but are not limited to, DMDAO or other amine oxides, long-chain
primary amines, diamines and polyamines and their salts, quaternary
ammonium salts, polyoxyethylenated long-chain amines, and quaternized
polyoxyethylenated long-chain amines. Examples of anionic surfactants may
include, but are not limited to, SDS; salts of carboxylic acids (e.g., soaps);
salts
of sulfonic acids, salts of sulfuric acid, phosphoric and polyphosphoric acid
esters; alkylphosphates; monoalkyl phosphate (MAP); and salts of
perfluorocarboxylic acids. Examples of zwitterionic surfactants may include,
but
are not limited to, cocoannidopropyl hydroxysultaine (CAPHS) and others which
are pH-sensitive and require special care in designing the appropriate pH of
the
formula (i.e., alkylaminopropionic acids, imidazoline carboxylates, and
betaines)
or those which are not pH-sensitive (e.g., sulfobetaines, sultaines). Examples

of non-ionic surfactants may include, but are not limited to, alkylphenol
ethoxylates, alcohol ethoxylates, polyoxyethylenated polyoxypropylene glycols,

polyoxyethylenated mercaptans, long-chain carboxylic acid esters,
alkonolamides, tertiary acetylenic glycols, polyoxyethylenated silicones, N-
alkylpyrrolidones, and alkylpolyglycosidases. Wetting agents, mineral oil or
other surfactants such as non-ionic detergents or agents such as one or more
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members of the PLURONICS series (BASF, Mt. Olive, NJ) may also be
included, for example and according to non-limiting theory, to discourage
aggregation of BT microparticles within the microparticulate suspension. Any
combination of surfactants is acceptable. Certain embodiments may include at
least one anionic and one cationic surfactant, or at least one cationic and
one
zwitterionic surfactant which are compatible, i.e., do not form complexes
which
precipitate appreciably when mixed.
Examples of thickening agents that may also be present in certain
topical formulations include, but are not limited to, acrylamides copolymer,
agarose, amylopectin, bentonite, calcium alginate, calcium carboxymethyl
cellulose, carbomer, carboxymethyl chitin, cellulose gum, dextrin, gelatin,
hydrogenated tallow, hydroxytheylcellulose, hydroxypropylcellulose,
hydroxpropyl starch, magnesium alginate, methylcellulose, microcrystalline
cellulose, pectin, various PEG's, polyacrylic acid, polymethacrylic acid,
polyvinyl
alcohol, various PPG's, sodium acrylates copolymer, sodium carrageenan,
xanthan gum, and yeast beta-glucan. Thickening agents other than those listed
above may also be used in embodiments of this invention.
According to certain embodiments contemplated herein, a BT
formulation may comprise one or more sunscreening or UV absorbing agents.
Where ultraviolet light- (UVA and UVB) absorbing properties are desired, such
agents may include, for example, benzophenone, benzophenone-1,
benzophenone-2, benzophenone-3, benzophenone-4, benzophenone-5,
benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9,
benzophenone-10, benzophenone-11, benzophenone-12, benzyl salicylate,
butyl PABA, cinnamate esters, cinoxate, DEA-nnethoxycinnamate, diisopropyl
methyl cinnamate, ethyl dihydroxypropyl PABA, ethyl diisopropylcinnamate,
ethyl methoxycinnamate, ethyl PABA, ethyl urocanate, glyceryl octanoate
dimethoxycinnamate, glyceryl PABA, glycol salicylate, honnosalate, isoannyl p-
methoxycinnamate, oxides of titanium, zinc, zirconium, silicon, manganese, and
cerium, PABA, PABA esters, Parsol 1789, and isopropylbenzyl salicylate, and
mixtures thereof. One skilled in the art will appreciate that sunscreening and

UV absorbing or protective agents other than those listed may be used in
certain embodiments of the present invention.
The BT formulations disclosed herein are typically effective at pH
.. values between about 2.5 and about 10Ø Preferably, the pH of the
composition is at or about the following pH ranges: about pH 5.5 to about pH
8.5, about pH 5 to about pH 10, about pH 5 to about pH 9, about pH 5 to about
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pH 8, about pH 3 to about pH 10, about pH 3 to about pH 9, about pH 3 to
about pH 8, and about pH 3 to about pH 8.5. Most preferably, the pH is about
pH 7 to about pH 8. One of ordinary skill in the art may add appropriate pH
adjusting ingredients to the compositions of the present invention to adjust
the
pH to an acceptable range. "About" a specified pH is understood by those
familiar with the art to include formulations in which at any given time the
actual
measured pH may be less or more than the specified value by no more than
0.7, 0.6, 0.5, 0.4., 0.3, 0.2 or 0.1 pH units, where it is recognized that
formulation composition and storage conditions may result in drifting of pH
from
an original value.
A cream, lotion, gel, ointment, paste or the like may be spread on
the affected surface and gently rubbed in. A solution may be applied in the
same way, but more typically will be applied with a dropper, swab, or the
like,
and carefully applied to the affected areas. The application regimen will
depend on a number of factors that may readily be determined, such as the
severity of the infection and its responsiveness to initial treatment. One of
ordinary skill may readily determine the optimum amount of the formulation to
be administered, administration methodologies and repetition rates. In
general,
it is contemplated that the formulations of these and related embodiments of
the
invention will be applied in the range of once or twice or more weekly up to
once, twice, thrice, four times or more daily.
As also discussed above, the BT formulations useful herein thus
also may contain an acceptable carrier, including any suitable diluent or
excipient, which includes any agent that does not itself harm the subject
(e.g.,
plant or animal including a human) or article of manufacture receiving the
composition, and which may be administered without undue toxicity.
Acceptable carriers may include, but are not limited to, liquids, such as
water,
saline, glycerol and ethanol, and the like, and may also include viscosity
enhancers (e.g., balsam fir resin) or film-formers such as colloidion or
nitrocellulose solutions. A thorough discussion of pharmaceutically acceptable
carriers, diluents, and other excipients is presented in REMINGTON'S
PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).
The BT formulation may include an agent that binds to the BT
compound and thereby assists in its delivery to or retention at a desired site
on
a subject or article of manufacture. Suitable agents that may act in this
capacity include clathrating agents such as cyclodextrins; other agents may
include a protein or a liposome.
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The BT formulations,are administered, applied or incorporated in
an effective amount, which will vary depending upon a variety of factors
including the nature of the delivery site (where relevant), the activity of
the
specific BT compound employed (including the inclusion or absence from the
formulation of an antibiotic, such as an anninoglycoside antibiotic, e.g.,
amikacin); the metabolic stability and length of action of the compound; the
condition of the (plant or animal, including a human) subject or article of
manufacture; the mode and time of administration; the rate of loss of the BT
compound in the ordinay course of activities undertaken by the subject or
article; and other factors. Generally, a therapeutically effective daily dose
is (for
a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg
(i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg
mammal)
from about 0.01 mg/kg (i.e., 7 mg) to about 50 mg/kg (i.e., 3.5 g); more
preferably a therapeutically effective dose is (for a 70 kg mammal) from about
1
mg/kg (L e . , 70 mg) to about 25 mg/kg (i.e., 1.75 g). Effective doses for
plants
may be expected to be lower by about 10, 20, 50 or 75 percent or more.
The ranges of effective doses provided herein are not intended to
be limiting and represent preferred dose ranges. However, the most preferred
dosage will be tailored to the individual subject, as is understood and
determinable by one skilled in the relevant arts. (see, e.g., Berkow et al.,
eds.,
The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Goodman
et al., eds., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, 10th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001);
Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and
Therapeutics, 3rd edition, ADIS Press, Ltd., Williams and Wilkins, Baltimore,
MD. (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985);
Osolci al., eds., Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology,
Appleton and Lange, Norwalk, CT (1992)).
The total dose required for each treatment can be administered by
multiple doses or in a single dose over the course of the day, if desired.
Certain
preferred embodiments contemplate a single application of the BT formulation
per day, per week, per 10 days, per 14 days or per longer time periods.
Generally, and in distinct embodiments, treatment may be initiated with
smaller
dosages, which are less than the optimum dose of the compound. Thereafter,
the dosage is increased by small increments until the optimum effect under the

circ*mstances is reached.
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Bismuth-Thiols for Protection of Plants and Agricultural Products
Certain herein disclosed embodiments relate to compositions and
methods for protecting plants and flowers from microbial infections and
infestations including biofilms, to reduce blight and increase product life.
According to certain herein described embodiments, including
those summarized above, there is provided a method for protecting a plant
against a bacterial, fungal or viral pathogen, comprising contacting the plant

with an effective amount of a BT composition under conditions and for a time
sufficient for one or more of (i) prevention of infection of the plant by the
bacterial, fungal or viral pathogen, (ii) inhibition of cell viability or cell
growth of
substantially all planktonic cells of the bacterial, fungal or viral pathogen,
(iii)
inhibition of biofilm formation by the bacterial, fungal or viral pathogen,
and (iv)
inhibition of biofilm viability or biofilm growth of substantially all biofilm-
form
cells of the bacterial, fungal or viral pathogen, wherein the BT composition
comprises a substantially monodisperse suspension of nnicroparticles that
comprise a BT compound, said microparticles having a volumetric mean
diameter of from about 0.5 lam to about 10 .m.
In certain embodiments the bacterial pathogen comprises Erwinia
amylovora cells and in certain embodiments the bacterial pathogen is selected
from Erwinia amylovora, Xanthom*onas cam pestris pv die ffenbachiae,
Pseudomonas syringae, Xylella fastidiosa; Xylophylus ampelinus; Monilinia
fructicola, Pantoea stewartii subsp. Stewartii, Ralstonia solanacearum, and
Clavibacter michiganensis subsp. sepedonicus. In certain embodiments the
bacterial pathogen exhibits antibiotic resistance and in certain other
embodiments the bacterial pathogen exhibits streptomycin resistance. In
certain embodiments the plant is a food crop plant, which in certain further
embodiments is a fruit tree that in certain still further embodiments is
selected
from an apple tree, a pear tree, a peach tree, a nectarine tree, a plum tree,
and
an apricot tree. In certain embodiments the food crop plant is a banana tree
of
genus Musa. In certain other embodiments the food crop plant is a plant
selected from a tuberous plant, a leguminous plant, and a cereal grain plant.
In
certain further embodiments the tuberous plant is selected from Solanum
tuberosum (potato), and 1pomoea batatas (sweet potato).
In certain embodiments the step of contacting is performed one or
a plurality of times. In certain embodiments at least one step of contacting
comprises one of spraying, dipping, coating and painting the plant. In certain

embodiments at least one step of contacting is performed at a flower blossom,
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green-tip or growth site of the plant, or on, at or in other plant parts such
as a
root, bulb, stem, leaf, branch, vine, runner, bud, flower or a part thereof,
greentip, fruit, seed, seed pod, or the like. In certain embodiments at least
one
step of contacting is performed within 24, 48 or 72 hours of first flower
blooming
on the plant. In certain embodiments the BT composition comprises one or
more BT compounds selected from BisBAL, BisEDT, Bis-dimercaprol, Bis-DTT,
Bis-2-mercaptoethanol, Bis-DTE, Bis-Pyr, Bis-Ery, Bis-Tol, Bis-BDT, Bis-PDT,
Bis-Pyr/Bal, Bis-Pyr/BDT, Bis-Pyr/EDT, Bis-Pyr/PDT, Bis-Pyr/Tol, Bis-Pyr/Ery,
bismuth-1-mercapto-2-propanol, and Bis-EDT/2-hydroxy-1-propanethiol. In
certain embodiments the bacterial pathogen exhibits antibiotic resistance.
In certain embodiments of the above described methods, the
method further comprises contacting the plant with a synergizing or enhancing
antibiotic, simultaneously or sequentially and in any order with respect to
the
step of contacting the plant with the BT composition. In certain further
embodiments the synergizing or enhancing antibiotic comprises an antibiotic
that is selected from an aminoglycoside antibiotic, a carbapenem antibiotic, a

cephalosporin antibiotic, a fluoroquinolone antibiotic, a penicillinase-
resistant
penicillin antibiotic, and an aminopenicillin antibiotic. In certain
embodiments
the synergizing or enhancing antibiotic is an aminoglycoside antibiotic that
is
selected from amikacin, arbekacin, gentamicin, kanamycin, neomycin,
netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin and
apramycin.
In another embodiment there is provided a method for overcoming
antibiotic resistance in a plant in or on which an antibiotic-resistant
bacterial
plant pathogen is present, comprising (a) contacting the plant with an
effective
amount of a BT composition under conditions and for a time sufficient for one
or
more of (i) prevention of infection of the plant by the antibiotic-resistant
bacterial
pathogen, (ii) inhibition of cell viability or cell growth of substantially
all
planktonic cells of the antibiotic-resistant bacterial pathogen, (iii)
inhibition of
biofilm formation by the antibiotic-resistant bacterial pathogen, and (iv)
inhibition
of biofilm viability or biofilm growth of substantially all biofilm-form cells
of the
antibiotic-resistant bacterial pathogen, wherein the BT composition comprises
a
substantially nnonodisperse suspension of microparticles that comprise a BT
compound, said microparticles having a volumetric mean diameter of from
about 0.5 tam to about 10 turn; and (b) contacting the plant with a
synergizing or
enhancing antibiotic, simultaneously or sequentially and in any order with
respect to the step of contacting the plant with the BT composition.
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Bismuth Thiol- (BT) based Antiseptics
As also noted above, a number of natural products (e.g.,
antibiotics) and synthetic chemicals having antimicrobial (e.g.,
antibacterial,
antiviral, antifungal), and in particular antibacterial, properties are known
in the
art and have been at least partially characterized by chemical structures and
by
antimicrobial effects, such as ability to kill microbes ("cidal" effects such
as
bacteriocidal properties), ability to halt or impair microbial growth
("static"
effects such as bacteriostatic properties), or ability to interfere with
microbial
functions such as colonizing or infecting a site, bacterial secretion of
exopolysaccharides and/or conversion from planktonic to biofilm populations or

expansion of biofilm formation. Antibiotics, disinfectants, antiseptics and
the
like (including bismuth-thiol or BT compounds) are discussed herein above and,
for example, in U.S. 6,582,719, including factors that influence the selection

and use of such compositions, e.g., bacteriocidal or bacteriostatic potencies,
effective concentrations, and risks of toxicity to host tissues.
Bismuth thiols (BTs), and related thiol compounds having a
different group V metal (e.g., arsenic, antimony) substituting for the
bismuth,
are discussed above. Also discussed herein are compositions and methods
directed to advantageous microparticulate BT compositions microparticles
having a volumetric mean diameter of from about 0.511M to about 10 p.m.
Certain exemplary embodiments thus pertain to the use of herein described
antimicrobial, including antibiofilm, agents to treat or prevent infections
and
biofilms in plants, said agents typically present in compositions that contain
one
or more microparticulate bismuth thiols at a concentration that is between
0.0001% and 0.001% by weight, preferably in alkaline form. The compositions
may comprise BTs and one or more carriers or excipients, and/or may further
comprise other ingredients such as other compatible germicides, which in
certain preferred embodiments comprise synergizing or enhancing antibiotics
as described herein.
Target crops to be protected within certain contemplated but non-
limiting embodiments include, for example, the following species of plants:
cereals (e.g., wheat, barley, rye, oats, rice, sorghum and related crops),
beets
(e.g., sugar beet and fodder beet), ponnes, drupes and softfruit (e.g.,
apples,
pears, plums, peaches, almonds, cherries, strawberries, raspberries and
blackberries), leguminous plants, (e.g., beans, lentils, peas, soybeans), oil
plants (e.g., rapeseed, mustard, poppy, olives, sunflowers, coconut, castor
oil
plants, cocoa beans, ground nuts), cucumber plants (e.g., cucumber, marrows,
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melons), fiber plants (e.g., cotton, flax, hemp, jute), citrus fruit (e.g.,
oranges,
lemons, grapefruit, mandarins), vegetables (e.g., spinach, lettuce, asparagus,

cabbages, carrots, onions, tomatoes, potatoes, paprika), lauraceae (e.g.,
avocados, cinnamon, camphor), and other plants such as maize, tobacco, nuts,
coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, as
well
as ornamentals (composites) including flowering plants and harvested cut
flowers therefrom. Certain embodiments thus contemplate extending the
product lifetime (e.g., prolonging the period of time during which the item is

commercially, nutritionally and/or aesthetically useful, in a statistically
significant
manner relative to a control group that is not contacted with the presently
described microparticulate BT) of a harvested target crop item such as a cut
flower or a target-crop derived foodstuff (e.g., fruit, vegetable, grain,
seed, etc.)
by contacting the crop item with a composition that comprises one or more of
the microparticulate BT compounds as provided herein.
Effective concentrations of microparticulate BTs as described
herein, for use in these and related embodiments, will depend on many factors,

including the choice of BT, pH, temperature, molar ratio of BT components, and

the offending microorganisms. Effectiveness also depends on whether
prevention of an infection or treatment of an existing infection (e.g., a
biofilm) is
the goal of a particular application. A preventive dose will suffice in most
instances. The effective sustained concentration of BTs is likely to be around

the MIC of the most resistant organism. This concentration is likely to be in
the
range of 1-2 pg/ml, but may go up to 8 pg/ml or beyond, depending on the
specific microparticulate BT compound(s). In one exemplary embodiment,
microparticulate BisPyrithione (BisPyr) is provided at a 5:1 molar ratio
(bismuth
to pyrithione) for application to plants. In another embodiment, a dual
bismuth
thiol in microparticulate form, BisPyr/Ery (Bis-pyrithione/ dithioerythritol)
may be
provided as a broad-spectrum antimicrobial. In yet another embodiment,
microparticulate BTs may be combined with specific antibiotics as provided
herein, preferably a synergizing or an enhancing antibiotic, to provide
targeted
and potent protection against microbial infections for plants and cut
flowers/trees. Based on observed synergy between BisEDT and gentamicin,
this BT-antibiotic combination is preferred in certain embodiments for
agricultural applications.
In other embodiments, the addition to a microparticulate BT
formulation of baking soda (sodium bicarbonate) or other alkaline substance(s)

(e.g., potassium bicarbonate, calcium carbonate) may add to or enhance the
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antimicrobial effects of the BT. Other ingredients in the microparticulate BT
formulations for agricultural uses may include surface-active agents and other

antimicrobial agents, e.g., chlorhexidine, sanguinarine extract,
metronidazole,
quaternary ammonium compounds, such as cetylpyridinium chloride; bis-
guanides, such as chlorhexidine digluconate, hexetidine, octenidine,
alexidine;
and halogenated bisphenolic compounds, such as 2,2' methylenebis-(4-chloro-
6-bromophenol), or other phenolic antibacterial compounds,
alkylhydroxybenzoate, cationic antimicrobial peptides, aminoglycosides,
quinolones, lincosamides, penicillins, cephalosporins, macrolides,
tetracyclines,
and other antibiotics, taurolidine or taurultam, A-dec ICX, Coleus forskohlii
essential oil, silver or colloidal silver antimicrobials, tin- or copper-based

antimicrobials, chlorine or bromine oxidants, Manuka oil, oregano, thyme,
rosemary or other herbal extracts, grapefruit seed extract; anti-inflammatory
or
antioxidant agents such as ibuprofen, flurbiprofen, aspirin, indomethacin,
aloe
vera, turmeric, olive leaf extract, cloves, panthenol, retinol, omega-3 fatty
acids,
gamma-linolenic acid (GLA), green tea, ginger, grape seed, etc.;
pharmaceutically acceptable carriers, e.g., starch, sucrose, water or
water/alcohol systems, DMSO, etc.; surfactants, such as anionic, nonionic,
cationic and zwitterionic or amphoteric surfactants, or sapon ins from plant
materials (e.g., U.S. Patent 6,485,711); buffers and salts; and other optional

ingredients that may be included, e.g., bleaching agents such as peroxy
compounds, potassium peroxydiphosphate, effervescing systems such as
sodium bicarbonate/citric acid systems, and the like.
Microparticulate BT compositions for agricultural use and use on
plants can, in certain embodiments, also be combined with these and optionally

other agents that produce additive, enhancing or synergistic effects as
described herein, or in liposomal or nanoparticle form to enhance activity and

delivery. Certain embodiments expressly exclude microparticulate BT
formulations that comprise liposomes such as phospholipid (e.g.,
phosphocholine) and/or cholesterol-containing liposomes, while certain other
embodiments are not so limited and may include these and other liposomes.
Specific formulations of microparticulate BTs can also be made that contain
carriers, excipients or other additives that promote adherence of the
formulation
to surfaces (e.g., glucose, starch, citric acid, carrier oils, emulsions,
dispersants, surfactants, and the like, etc.).
In other contemplated embodiments, microparticulate BT
formulations for use as anti-biofilm agents on plants or agricultural crops
can be
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combined with other agents for controlling biofilm development. It is known,
for
example, that interspecies quorum sensing is related to biofilm formation.
Certain agents that increase LuxS-dependent pathway or interspecies quorum
sensing signal (e.g., U.S. Pat. Nos. 7,427,408 and 6,455,031) help control
biofilms, such as N-(3-oxododecanoyI)-L-honnoserine lactone (OdDHL) blocking
compounds and/or N-butyryl-L-hom*oserine Iactone (BHL) analogs. These anti-
biofilm agents combined with the herein described microparticulate BTs may be
delivered in foliar sprays for inhibition of bacterial biofilm development or
for
treatment of pre-formed biofilms. In another embodiment, these anti-biofilm
agents are contained within a biodegradable microparticle for controlled
release, and/or in liposomal form with other antimicrobial agents.
The presently described microparticulate BTs thus may, according
to certain embodiments, be used with other existing technologies to improve
anti-biofilm effects. The present microparticulate BTs may synergize or
enhance the activity against certain plant pathogens of the antibiotics
streptomycin and/or gentamicin. Streptomycin does not kill bacteria but
instead
inhibits their multiplication and thus reduces the rate at which flower
stigmata
are colonized, thereby diminishing the subsequent multiplication of the
bacteria
within the nectarthodes. (See, e.g., Domenico et al. J Antimicrob Chemo
1991;28:801-10; Domenico et al. Research Advances in Antimicrob Agents
Chemother 2003;3:79-85). Further benefits may accrue through the use of an
activator-type spray adjuvant (e.g., RegulaidTM) that improves the coverage
and
penetration of streptomycin enough to allow reduced amounts of this antibiotic

to be used safely.
The present microparticulate BTs may be combined with any of
the active ingredients currently in use for combating agricultural and plant
microbial pathogens, including those having antibiofilm activity, such as
oxidizing agents, chelating agents (e.g., iron chelators), germicides and
disinfectants. Preferred combinations may be additive, or may be enhancing or
synergistic according to the present disclosure, with regard to their anti-
biofilm
effects. Certain embodiments contemplate microparticulate BT compositions
that are formulated to be hydrophobic in order to enhance retention of the BT
on surfaces, for example by using hydrophobic thiols (e.g., thiochlorophenol)
that confer enhanced adhesive properties. BTs with a net negative charge
(e.g., 1:2 molar ratio of bismuth to thiol) may also possess enhanced adhesive

properties.
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The BT compound microparticulate suspension can be
administered as aqueous formulations, as suspensions or solutions in organic
solvents including halogenated hydrocarbon propellants, dispersion oils, or as

dry powders. Aqueous formulations may be aerosolized by liquid nebulizers
employing either hydraulic or ultrasonic atomization. Propellant-based systems

may use suitable pressurized dispensers. Dry powders may use dry powder
dispersion devices, which are capable of dispersing the BT-containing
microparticles effectively. A desired particle size and distribution may be
obtained by choosing an appropriate device.
Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will be
understood to imply the inclusion of a stated step or element or group of
steps
or elements but not the exclusion of any other step or element or group of
steps
or elements. By "consisting of" is meant including, and limited to, whatever
follows the phrase "consisting of." Thus, the phrase "consisting of' indicates

that the listed elements are required or mandatory, and that no other elements

may be present. By "consisting essentially of" is meant including any elements

listed after the phrase, and limited to other elements that do not interfere
with or
contribute to the activity or action specified in the disclosure for the
listed
elements. Thus, the phrase "consisting essentially of" indicates that the
listed
elements are required or mandatory, but that no other elements are required
and may or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
In this specification and the appended claims, the singular forms
"a," "an" and "the" include plural references unless the content clearly
dictates
otherwise. As used herein, in particular embodiments, the terms "about" or
"approximately" when preceding a numerical value indicates the value plus or
minus a range of 5%, 6%, 7%, 8% or 9%. In other embodiments, the terms
"about" or "approximately" when preceding a numerical value indicates the
value plus or minus a range of 10%, 11%, 12%, 13% or 14%. In yet other
embodiments, the terms "about" or "approximately" when preceding a numerical
value indicates the value plus or minus a range of 15%, 16%, 17%, 18%, 19%
or 20`)/0.
References: Bad ireddy et al., Biotechnol Bioengineering
2008;99:634-43; Bad ireddy et al., Biomacromolecules, 2008;9:3079-89;
Bayston et al., Biomaterials 2009;30:3167-73. Codony et al., J Applied
112

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Microbiol 2003;95:288-93. Domenico et al., J Antimicrob Chemo 1991;28:801-
810. Domenico et al., Antimicrob Agents Chemother 1997;41:1697-703.
Domenico et al., 1999 Infect Immun 67:664-669. Domenico et al., Antimicrob
Agents Chemother 2001;45:1417-21. Domenico et al., Research Advances in
Antimicrob Agents Chemother 2003;3:79-85. Domenico et al., Peptides
2004.;25:2047-53. Domenico et al., 2005 Antibiotics for Clinicians 9:291-297.
Dufrene, J Bacteriol 2004;186:3283-5. Eboigbodin et al., Biomacromolecules
2008;9:686-95. Feazel LM, Baumgartner LK, Peterson KL, et al. Opportunistic
pathogens enriched in showerhead biofilms. PNAS 2009 (epub ahead of print).
Geesey GG, Lewandowski Z, Flemming H-C (eds). Biofouling and biocorrosion
in industrial water systems. CRC Press, Boca Raton, FL, 1994. Huang et al., J
Antimicrob Chemother 1999;44:601-5; Juhni et al., Proceedings Annual
Meeting Adhesion Society 2005;28:179-181. Omoike et al.,
Biomacromolecules 2004;5:1219-30. Ouazzani K, Bentama J. Bio-fouling in
membrane processes: micro-organism/surface interactions, hydrodynamic
detachment method. Congres 2008;220:290-4. Ozdamar et al., Retina
1999;19:122-6. Piccirillo et al., J Mater Chem 2009;19:6167. Reunala et al.,
Curr Opin Allergy Clin lmmunol 2004;4:397-401. Ronno et al., Environ Progress
1999;18:107-12. Saha DC, Shahin S, Rackow EC, Astiz ME, Domenico P.
2000. Cytokine modulation by bismuth-ethanedithiol in experimental sepsis.
10th Intl. Conf. Inflamm. Res. Assoc., Hot Springs, VA. Sawada et al., JPRAS
1990;43:78-82. Schultz, J Fluids Eng 2004;126:1039-47. Tiller JC, Hartmann
L, Scherble J. Reloadable antimicrobial coatings based on amphiphilic silicone

networks. Surface Coatings International Part B: Coatings Transactions
2005;88:1-82. Tsuneda et al., FEMS Microbiol Lett 2003;223:287-92. Vu et al.,
Molecules 2009;14:2535-54. Yan et al., Ophthalmologica 2008;222:245-8.
Yeo et al., Water Sci Technol 2007;55:35-42.
Additional References (including re: Plant Protection and
Related): Chandler et al., Antimicrob. Agents Chemother 1978;14:60-8.
Choudhary et al., Microbiol Res 2009;164:493-513. Cooksey, Annu Rev
Phytopathol 1990;28:201-14. Dill K, McGown EL. The biochemistry of arsenic,
bismuth and antimony. In S. Patai (ed.), The chemistry of organic arsenic,
antimony and bismuth compounds. John Wiley & Sons, New York, 1994, pp.
695-713. Domenico et al., 1996 J Antimicrob Chemother 38:1031-1040.
Domenico et al., 2000 Infect Med 17:123-127. Dow et al., Proc Natl Aced Sci
USA 2003;100:10995-1000. Dulla et al., PNAS 2008;105:3-082-7. Espinosa-
Urgel et al., Microbiol 2002;148:341-3. Expert, Annu Rev Phytopathol
113

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1999;37:307-34. Ganguli et al., Smart Mater. Struct. 2009;18:104027. Huang
et al., J Antimicrob Chemother 1999;44:601-5. Hung et al., J Exptl Marine Blot

Ecol 2008;361:36-41. Johnson et al., Annu Rev Phytopathol 1998;36:227-48.
Kang et al., Ma/ Microbiol 2002; 46:427-37. Kavouras et al., Inverteb Biol
2005;122:138-51. Koczan et al., Phytopathol 2009;99:1237-44. Kumar et al.,
Nature Materials 2008;7:236-41. Marques et al., Phytopathol 2003;93:S57.
McManus et al., Annu Rev Phytopathol 2002;40:443-65. Monier et al., Proc
Natl Acad Sci USA 2003;100:15977-82. Norelli JL., Holleran HT, Johnson WC
et al. Resistance of Geneva and other apple root- stocks to Erwinia amylovora.
Plant Dis 87:26-32. Oh et al., FEMS Microbiology Lett 2005;253:185-192.
Omoike et al., Biomacromolecules 2004;5:1219-30. Ramey et al., Curr Opinion
Microbiol 2004;7:602-9. Salo et al., Infection 1995;23:371-7. Schultz et al.,
Biofouling 2007;23:331-41. Siboni et al., FEMS Microbiol Lett 2007;274:24-9.
Sosnowski et al., Plant Pathol 2009;58:621-35. Tsuneda et al., FEMS
Microbiol Lett 2003;223:287-92. von Bodman et al., Proc Natl Acad Sci USA
1998,95:7687-7692. Vu et al., Molecules 2009;14:2535-54. Zaini et al., FEMS
Microbiol LETT 2009;295:129-34.
The following Examples are presented by way of illustration and
not limitation.
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EXAMPLES
EXAMPLE 1
PREPARATION OF BT COMPOUNDS
The following BT compounds were prepared either according to
the methods of Domenico et al. (U.S. RE37,793, U.S. 6,248,371, U.S.
6,086,921, U.S. 6,380,248) or as microparticles according to the synthetic
protocol described below for BisEDT. Shown are atomic ratios relative to a
single bismuth atom, for comparison, based on the stoichiometric ratios of the

reactants used and the known propensity of bismuth to form trivalent
complexes with sulfur containing compounds. The numbers in parenthesis are
the ratios of bismuth to one (or more) thiol agents (e.g. Bi:thio11/thi012;
see also
Table 1).
1) CPD 1B-1 Bis-EDT (1:1) BiC2H4S2
2) CPD 1B-2 Bis-EDT (1:1.5) BiC3H6S3
3) CPD 1B-3 Bis-EDT (1:1.5) BiC3H6S3
4) CPD 1C Bis-EDT (soluble Bi prep.) (1:1.5) BiC3H6S3
5) CPD 2A Bis-Bal (1:1) BiC3H6S20
6) CPD 2B Bis-Bal (1:1.5) BiC45H901 533
7) CPD 3A Bis-Pyr (1:1.5) BiC7.51-16N1.501.551 5
8) CPD 3B Bis-Pyr (1:3) BiC15H12N303S3
9) CPD 4 Bis-Ery (1:1.5) BiC6H1203S3
10) CPD 5 Bis-Tol (1:1.5) BiC105H9S3
11) CPD 6 Bis-BDT (1:1.5) BiC6F112S3
12) CPD 7 Bis-PDT (1:1.5) BiC4.51-19S3
13) CPD 8-1 Bis-Pyr/BDT (1:1/1)
14) CPD 8-2 Bis-Pyr/BDT (1:1/0.5)
15) CPD 9 Bis-2hydroxy, propane thiol (1:3)
16) CPD 10 Bis-Pyr/Bal (1:1/0.5)
17) CPD 11 Bis-Pyr/EDT (1:1/0.5)
18) CPD 12 Bis-Pyr/Tol (1:1/0.5)
19) CPD 13 Bis-Pyr/PDT (1:1/0.5)
20) CPD 14 Bis-Pyr/Ery (1:1/0.5)
21) CPD 15 Bis-EDT/2hydroxy, propane thiol (1:1/1)
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Microparticulate bismuth-1,2-ethanedithiol (Bis-EDT, soluble
bismuth preparation) was prepared as follows:
To an excess (11.4 L) of 5% aqueous HNO3 at room temperature
in a 15 L polypropylene carboy was slowly added by dropwise addition 0.331 L
(-0.575 moles) of an aqueous Bi(NO3)3 solution (43% Bi(NO3)3 (w/w), 5% nitric
acid (w/w), 52% water (w/w), Shepherd Chemical Co., Cincinnati, OH, product
no. 2362; 8 -1.6 g/mL) with stirring, followed by slow addition of absolute
ethanol (4 L). Some white precipitate formed but was dissolved by continued
stirring. An ethanolic solution (-1.56 L, -0.55 M) of 1,2-ethanedithiol (CAS
540-
63-6) was separately prepared by adding, to 1.5 L of absolute ethanol, 72.19
mL (0.863 moles) of 1,2-ethanedithiol using a 60 mL syringe, and then stirring

for five minutes. The 1,2-ethanedithiol/ Et0H reagent was then slowly added
by dropwise addition over the course of five hours to the aqueous Bi(NO3)3/
HNO3 solution, with continued stirring overnight. The formed product was
allowed to settle as a precipitate for approximately 15 minutes, after which
the
filtrate was removed at 300 mL/min using a peristaltic pump. The product was
then collected by filtration on fine filter paper in a 15-cm diameter Buchner
funnel, and washed sequentially with three, 500-mL volumes each of ethanol,
USP water, and acetone to obtain BisEDT (694.51 gnn/ mole) as a yellow
amorphous powdered solid. The product was placed in a 500 mL amber glass
bottle and dried over CaCl2 under high vacuum for 48 hours. Recovered
material (yield -200 g) gave off a thiol-characteristic odor. The crude
product
was redissolved in 750 mL of absolute ethanol, stirred for 30 min, then
filtered
and washed sequentially with 3 x 50 mL ethanol, 2 x 50 mL acetone, and
washed again with 500 mL of acetone. The rewashed powder was triturated in
1M NaOH (500 mL), filtered and washed with 3 x 220 mL water, 2 x 50 mL
ethanol, and 1 x 400 mL acetone to afford 156.74 gm of purified BisEDT.
Subsequent batches prepared in essentially the same manner resulted in yields
of about 78-91%.
The product was characterized as having the structure shown
above in formula I by analysis of data from 1H and 13C nuclear magnetic
resonance (NMR), infrared spectroscopy (IR), ultraviolet spectroscopy (UV),
mass spectrometry (MS) and elemental analysis. An HPLC method was
developed to determine chemical purity of BisEDT whereby the sample was
.. prepared in DMSO (0.5mg/mL). The Amax was determined by scanning a
solution of BisEDT in DMSO between 190 and 600nm. lsocratic HPLC elution
at 1 mL/min was performed at ambient temperature in a mobile phase of 0.1%
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formic acid in acetonitrile:water (9:1) on a Waters (Millipore Corp., Milford,
MA)
model 2695 chromatograph with UV detector monitoring at 265 nm (kmax), 2 pL
injection volume, equipped with a YMC Pack PVC Sil NP, 5pm, 250X4.6 mm
inner diameter analytical column (Waters) and a single peak was detected,
reflecting chemical purity of 100 0.1%. Elemental analysis was consistent with
the structure of formula (I).
The dried particulate matter was characterized to assess the
particle size properties. Briefly, microparticles were resuspended in 2%
Pluronic0 F-68 (BASF, Mt. Olive, NJ) and the suspension was sonicated for 10
minutes in a water bath sonicator at standard setting prior to analysis using
a
Nanosizer/Zetasizer Nano-S particle analyzer (model ZEN1600 (without zeta-
potential measuring capacity), Malvern Instruments, Worcestershire, UK)
according to the manufacturer's recommendations. From compiled data of two
measurements, nnicroparticles exhibited a unimodal distribution with all
detectable events between about 0.6 microns and 4 microns in volumetric
mean diameter (VMD) and having a peak VMD at about 1.3 microns. By
contrast, when BisEDT was prepared by prior methods (Domenico et al., 1997
Antimicrob. Agents Chemother. 41(8):1697-1703) the majority of particles were
heterodisperse and of significantly larger size, precluding their
characterization
on the basis of VMD.
EXAMPLE 2
COLONY BIOFILM MODEL OF CHRONIC WOUND INFECTION:
INHIBITION BY BT COMPOUNDS
Because bacteria that exist in chronic wounds adopt a biofilm
lifestyle, BTs were tested against biofilms for effects on bacterial cell
survival
using biofilms prepared essentially according to described methods (Anderl et
al., 2003 Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003
Antimicrob Agents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog.
12:316; Zheng et al., 2002 Antimicrob Agents Chemother 46:900).
Briefly, colony biofilms were grown on 10% tryptic soy agar for 24
hours, and transferred to Mueller Hinton plates containing treatments. After
treatment the biofilms were dispersed into peptone water containing 2% w/v
glutathione (neutralizes the BT), and serially diluted into peptone water
before
being spotted onto plates for counting. Two bacteria isolated from chronic
wounds were used separately in the production of colony biofilms for testing.
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These were Pseudomonas aeruginosa, a gram negative bacterial strain, and
Methicillin Resistant Staphylococcus aureus (MRSA), which is gram positive.
Bacterial biofilm colonies were grown on top of micro porous
membranes resting on an agar plate essentially as described (Anderl et al.,
2003 Antimicrob Agents Chemother 47:1251-56; Walters et al., 2003
Antimicrob Agents Chemother 47:317; Wentland et al., 1996 Biotchnol. Prog.
12:316; Zheng et al., 2002 Antimicrob Agents Chemother 46:900) The colony
biofilms exhibited many of the familiar features of other biofilm models,
e.g.,
they consisted of cells densely aggregated in a highly hydrated matrix. As
also
reported by others (Brown et al., J Surg Res 56:562; Millward et al, 1989
Microbios 58:155; Sutch et al., 1995 J Pharm Pharmacol 47:1094; Thrower et
al., 1997 J Med Microbiol 46:425) it was observed that bacteria in colony
biofilms exhibited the same profoundly reduced anti-microbial susceptibility
that
has been quantified in more sophisticated in vitro biofilm reactors. Colony
biofilms were readily and reproducibly generated in large numbers. According
to non-limiting theory, this colony biofilm model shared some of the features
of
an infected wound: bacteria grew at an air interface with nutrients supplied
from beneath the biofilm and minimal fluid flow. A variety of nutrients
sources
was used to cultivate colony biofilms, including blood agar, which is believed
to
mimic in vivo nutrient conditions.
Colony biofilms were prepared by inoculating 5 pl spots of
planktonic bacterial liquid cultures onto a 25 mm diameter polycarbonate
filter
membrane. The membranes were sterilized prior to inoculation, by exposure to
ultraviolet light for 10 min per side. The inocula were grown overnight in
bacterial medium at 37 C and diluted in fresh medium to an optical density of
0.1 at 600 nm prior to deposition on the membrane. The membranes were then
placed on the agar plate containing growth medium. The plates were then
covered and placed, inverted, in an incubator at 37 C. Every 24 h, the
membrane and colony biofilm were transferred, using sterile forceps, to a
fresh
plate. Colony biofilms were typically used for experimentation after 48 hours
of
growth, at which time there were approximately 109 bacteria per membrane.
The colony biofilm method was successfully employed to culture a wide variety
of single species and mixed species biofilms.
To measure susceptibility to antimicrobial agents (e.g., BT
compounds including combinations of BT compounds; antibiotics; and BT
compound-antibiotic combinations), colony biofilms were transferred to agar
plates supplemented with the candidate antimicrobial treatment agent(s).
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Where the duration of exposure to antimicrobial treatment exceeded 24 hours,
the colony biofilms were moved to fresh treatment plates daily. At the end of
the treatment period, the colony biofilms were placed in tubes containing 10
ml
of buffer and vortexed for 1-2 min to disperse the biofilm. In some cases, it
was
necessary to briefly process the sample with a tissue hom*ogenizer to break up
cell aggregates. The resulting cell suspensions were then serially diluted and

plated to enumerate surviving bacteria, which were reported as colony forming
units (CFU) per unit area. Survival data were analyzed using logio
transformation.
For each type of bacterial biofilm colony cultures (Pseudomonas
aeruginosa, PA; methicilin resistant Staphylococcus aureus, MRSA or SA) five
antibiotics and thirteen BT compounds were tested. Antimicrobial agents tested

against PA included the BTs referred to herein as BisEDT and Compounds 2B,
4, 5, 6, 8-2, 9, 10, 11 and 15 (see Table 1), and the antibiotics tobramycin,
amikacin, imipenim, cefazolin, and ciprofloxacin. Antimicrobial agents tested
against SA included the BTs referred to herein as BisEDT and Compounds 2B,
4, 5, 6, 8-2, 9, 10 and 11 (see Table 1), and the antibiotics rifampicin,
daptomycin, minocycline, ampicillin, and vancomycin. As described above
under "brief descriptions of the drawings", antibiotics were tested at
concentrations of approximately 10-400 times the minimum inhibitory
concentrations (MIC) according to established microbiological methodologies.
Seven BT compounds exhibited pronounced effects on PA
bacterial survival at the concentrations tested, and two BT compounds
demonstrated pronounced effects on MRSA survival at the concentrations
tested; representative results showing BT effects on bacterial survival are
presented in Figure 1 for BisEDT and BT compound 2B (tested against PA) and
in Figure 2 for BT compounds 2B and 8-2 (tested against SA), in both cases,
relative to the effects of the indicated antibiotics. As also shown in Figures
1
and 2, inclusion of the indicated BT compounds in combination with the
indicated antibiotics resulted in a synergistic effect whereby the potency of
reducing bacterial survival was enhanced relative to the anti-bacterial
effects of
either the antibiotic alone or the BT compound alone. In the PA survival
assay,
compound 15 (Bis-EDT/2hydroxy, propane thiol (1:1/1)) at a concentration of 80

pg/mL exhibited an effect (not shown) that was comparable to the effect
obtained using the combination of 1600 pg/mL AMK plus 80 pg/mL BisEDT
(Fig. 1).
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EXAMPLE 3
DRIP FLOW BIOFILM MODEL OF CHRONIC WOUND INFECTION:
INHIBITION BY BT COMPOUNDS
Drip flow biofilms represent an art accepted authentic model for
forming, and testing the effect of candidate anti-bacterial compounds against,
bacterial biofilms. Drip flow biofilms are produced on coupons (substrates)
placed in the channels of a drip flow reactor. Many different types of
materials
can be used as the substrate for bacterial biofilm formation, including
frosted
glass microscope slides. Nutritive liquid media enters the drip flow
bioreactor
cell chamber by dripping into the chamber near the top, and then flows the
length of a coupon down a 10 degree slope.
Biofilms are grown in drip flow bioreactors and exposed to BT
compounds individually or in combinations and/or to antibiotic compounds
individually or in combinations with other antibacterial agents, including BT
compounds, or to other conventional or candidate treatments for chronic
wounds. BT compounds are thus characterized for their effects on bacterial
biofilms in the drip-flow reactor. Biofilms in the drip-flow reactor are
prepared
according to established methodologies (e.g., Stewart et al., 2001 J Appl
Microbiol. 91:525; Xu et al., 1998 App!. Environ. Microbiol. 64:4035). This
design involves cultivating biofilms on inclined polystyrene coupons in a
covered chamber. An exemplary culture medium contains 1 g/I glucose, 0.5 g/I
NH4NO3, 0.25g/I KCI, 0.25 g/I KH2PO4, 0.25 g/I MgSO4-7H20, supplemented
with 5% v/v adult donor bovine serum (ph 6.8) that mimics serum protein-rich,
iron limited conditions that are similar to biofilm growth conditions in vivo,
such
as in chronic wounds. This medium flows drop-wise (50m1/h) over four coupons
contained in four separate parallel chambers, each of which measures 10cnn x
1.9cm by 1.9cm deep. The chambered reactor is fabricated from polysulfone
plastic. Each of the chambers is fitted with an individual removable plastic
lid
that can be tightly sealed. The biofilm reactor is contained in an incubator
at
37 C, and bacterial cell culture medium is warmed by passing it through an
aluminum heat sink kept in the incubator. This method reproduces the
antibiotic tolerant phenotype observed in certain biofilms, mimics the low
fluid
shear environment and proximity to an air interface characteristic of a
chronic
wound while providing continual replenishment of nutrients, and is compatible
with a number of analytical methods for characterizing and monitoring the
effects of introduced candidate antibacterial regimens. The drip-flow reactor
has been successfully employed to culture a wide variety of pure and mixed-
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species biofilms. Biofilms are typically grown for two to five days prior to
application of antimicrobial agents.
To measure the effects of anti-biofilm agents on biofilms grown in
drip-flow reactors, the fluid stream passing over the biofilm is amended or
supplemented with the desired treatment formulation (e.g., one or more BT
compounds and/or one or more antibiotics, or controls, and/or other candidate
agents). Flow is continued for the specified treatment period. The treated
biofilm coupon is then briefly removed from the reactor and the biofilm is
scraped into a beaker containing 10 ml of buffer. This sample is briefly
processed (typically 30s to 1 min) with a tissue hom*ogenizer to disperse
bacterial aggregates. The suspension is serially diluted and plated to
enumerate surviving microorganisms according to standard microbiological
methodologies.
EXAMPLE 4
WOUND BIOFILM INHIBITION OF KERATINOCYTE SCRATCH REPAIR:
BIOFILM SUPPRESSION BY BT COMPOUNDS
This Example describes a modification of established in vitro
keratinocyte scratch models of wound healing, to arrive at a model having
relevance to biofilm-associated wound pathology and wound healing, and in
particular to acute or chronic wounds or wounds containing biofilms as
described herein. According to the keratinocyte scratch model of the effects
of
chronic wound biofilms, cultivation of mammalian (e.g., human) keratinocytes
and bacterial biofilm populations proceeds in separate chambers that are in
fluid contact with one another, to permit assessment of the effects of
conditions
that influence the effects, of soluble components elaborated by biofilms, on
keratinocyte wound healing events.
Newborn human foreskin cells are cultured as nnonolayers in
treated plastic dishes, in which monolayers a controlled "wound" or scratch is

formed by mechanical means (e.g., through physical disruption of the
monolayer such as by scraping an essentially linear cell-free zone between
regions of the monolayer with a suitable implement such as a sterile scalpel,
razor, cell scraper, forceps or other tool). In vitro keratinocyte monolayer
model
systems are known to undergo cellular structural and functional process in
response to the wounding event, in a manner that simulates wound healing in
vivo. According to the herein disclosed embodiments, the influence of the
presence of bacterial biofilms on such processes, for instance, on the healing
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time of the scratch, is observed, and in these and related embodiments the
effects are also assessed of the presence of selected candidate antimicrobial
(e.g., antibacterial and antibiofilm) treatments.
Wounded keratinocyte monolayers cultured in the presence of
biofilms are examined according to morphological, biochemical, molecular
genetic, cell physiologic and other parameters to determine whether
introduction of BT connopunds alters (e.g., increases or decreases in a
statistically significant manner relative to appropriate controls) the
damaging
effects of the biofilms. Wounds are first exposed to each BT compound alone,
and to contemplated combinations of BT compounds, in order to test the
toxicity
of each BT compound treatment prior to assessing the effects of such
treatments on biofilm influences toward the model wound healing process.
In a representative embodiment, a three-day biofilm is cultured on
a membrane (e.g., a TransWell membrane insert or the like) that is maintained
in a tissue culture well above, and in fluid communication with, a
keratinocyte
monolayer that is scratched to initiate the wound healing process. Biofilms
cultured out of authentic acute or chronic wounds are contemplated for use in
these and related embodiments.
Thus, an in vitro system has been developed for evaluating
soluble biofilm component effects on migration and proliferation of human
keratinocytes. The system separates the biofilm and keratinocytes using a
dialysis membrane. Keratinocytes are cultured from newborn foreskin as
previously described (Fleckman et al., 1997 J Invest. Dermatol. 109:36;
Piepkorn et al., 1987 J Invest. Dermatol. 88:215-219) and grown as confluent
monolayers on glass cover slips. The keratinocyte monolayers can then be
scratched to yield "wounds" with a uniform width, followed by monitoring
cellular
repair processes (e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth et al. 2007
Eur. J Cell Biol. 86:747; Phan et al. 2000 Ann. Acad. Med. Singapore 29:27).
The artificial wounds are then placed in the bottom of a sterile double-sided
chamber and the chamber is assembled using aseptic technique. Both sides of
the chamber are filled with keratinocyte growth medium (EpiLife) with or
without
antibiotics and/or bismuth-thiols. Uninoculated systems are used as controls.
The system is inoculated with wound-isolated bacteria and
incubated in static conditions for two hours to enable bacterial attachment to

surfaces in the upper chambers. Following the attachment period, liquid
medium flow is initiated in the upper chamber to remove unattached cells. Flow

of medium is then continued at a rate that minimizes the growth of planktonic
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cells within the upper chamber, by washout of unattached cells. After
incubation periods ranging from 6 to 48 hours, the systems (keratinocyte
monolayers on coverslips and bacterial biofilm on membrane substrate) are
disassembled and the cover slips removed and analyzed. In related
embodiments, mature biofilms are grown in the upper chamber prior to
assembling the chamber. In other related embodiments, the separate co-
culturing of biofilms and scratch-wounded keratinocyte monolayers is
conducted in the absence and presence of one or more BT compounds,
optionally with the inclusion or exclusion of one or more antibiotics, in
order to
determine effects of candidate agents such as BT compounds, or of potentially
synergizing BT compound-plus-antibiotic combinations (e.g., a BT compound
as provided herein such as a BT that is provided in microparticulate form, and

one or more of amikacin, ampicillin, cefazolin, cefepime, chloramphenicol,
ciprofloxacin, clindamycin (or another lincoasamide antibiotic), daptomycin
(Cubicin0),_doxycycline, gatifloxacin, gentamicin, imipenim, levofloxacin,
linezolid (Zyvox0), minocycline, nafcilin, paromomycin, rifampin,
sulphannethoxazole, tobrannycin and vancomycin), on keratinocyte repair of the

scratch wound, e.g., to identify an agent or combination of agents that alters

(e.g., increases or decreases in a statistically significant manner relative
to
appropriate controls) at least one indicator of scratch wound healing, such as
the time elapsing for wound repair to take place or other wound-repair indicia

(e.g., Tao et al., 2007 PLoS ONE 2:e697; Buth et al. 2007 Eur. J Cell Biol.
86:747; Phan et al. 2000 Ann. Acad. Med. Singapore 29:27).
EXAMPLE 5
WOUND BIOFILM INHIBITION OF KERATINOCYTE SCRATCH REPAIR
Isolated human keratinocytes were cultured on glass coverslips
and scratch-wounded according to methodologies described above in Example
4. Wounded cultures were maintained under culture conditions alone or in the
presence of a co-cultured biofilm on a membrane support in fluid
communication with the keratinocyte culture. The scratch closure time interval

during which keratinocyte cell growth and/or migration reestablishes the
keratinocyte monolayer over the scratch zone was then determined. Figure 3
illustrates the effect that the presence in fluid communication (but without
direct
contact) of biofilms had on the healing time of scratched keratinocyte
monolayers.
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Accordingly there are contemplated in certain embodiments a
method of identifying an agent for treating a chronic wound, comprising
culturing a scratch-wounded cell (e.g., keratinocyte or fibroblast) monolayer
in
the presence of a bacterial biofilm with and without a candidate anti-biofilm
agent being present; and assessing an indicator of healing of the scratch-
wounded cell monolayer in the absence and presence of the candidate anti-
biofilm agent, wherein an agent (e.g., a BT compound such as a substantially
monodisperse BT microparticle suspension as described herein, alone or in
synergizing combination with an antibiotic, such as one or more of amikacin,
ampicillin, cefazolin, cefepime, chloramphenicol, ciprofloxacin, clindamycin,
daptomycin (Cubicin0),_doxycycline, gatifloxacin, gentannicin, imipenim,
levofloxacin, linezolid (Zyvox0), minocycline, nafcilin, paromomycin,
rifampin,
sulphamethoxazole, tobramycin and vancomycin) that promotes at least one
indicator of healing is identified as a suitable agent for treating an acute
or
chronic wound or a wound that contains a biofilnn.
EXAMPLE 6
SYNERGIZING BISMUTH-THIOL (BT)-ANTIBIOTIC COMBINATIONS
This example shows instances of demonstrated synergizing
effects by combinations of one or more bismuth-thiol compounds and one or
more antibiotics against a variety of bacterial species and bacterial strains,
including several antibiotic-resistant bacteria.
Materials & Methods. Susceptibility studies were performed by
broth dilution in 96-well tissue culture plates (Nalge Nunc International,
Denmark) in accordance with NCCLS protocols (National Committee for Clinical
Laboratory Standards. (1997). Methods for Dilution Antimicrobial
Susceptibility
Tests for Bacteria that Grow Aerobically: Approved Standard M7-A2 and
Informational Supplement M100-S10. NCCLS, Wayne, PA, USA).
Briefly, overnight bacterial cultures were used to prepare 0.5
McFarland standard suspensions, which were further diluted 1:50 (-2 x 106
cfu/mL) in cation-adjusted Mueller-Hinton broth medium (BBL, co*ckeysville,
MD, USA). BTs (prepared as described above) and antibiotics were added at
incremental concentrations, keeping the final volume constant at 0.2 mL.
Cultures were incubated for 24 h at 37 C and turbidity was assessed by
absorption at 630 nm using an ELISA plate reader (Biotek Instruments,
Winooski, VT, USA) according to the manufacturer's recommendations. The
Minimum Inhibitory Concentration (MIC) was expressed as the lowest drug
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concentration inhibiting growth for 24 h. Viable bacterial counts (cfu/mL)
were
determined by standard plating on nutrient agar. The Minimal Bactericidal
Concentrations (MBC) was expressed as the concentration of drug that
reduced initial viability by 99.9% at 24 h of incubation.
The checkerboard method was used to assess the activity of
antimicrobial combinations. The fractional inhibitory concentration index
(FICI)
and the fractional bactericidal concentration index (FBCI) were calculated,
according to Eliopoulos et al. (Eliopoulos and Moellering, (1996)
Antimicrobial
combinations. In Antibiotics in Laboratory Medicine (Lorian, V., Ed.), pp. 330-

96, Williams and Wilkins, Baltimore, MD, USA). Synergy was defined as an
FICI or FBCI index of 0.5, no interaction at >0.5-4 and antagonism at >4
(Odds, FC (2003) Synergy, antagonism, and what the chequerboard puts
between them. Journal of Antimicrobial Chemotherapy 52:1). Synergy was also
defined conventionally as .4-fold decrease in antibiotic concentration.
Results are presented in Tables 2-17.
TABLE 2
S. aureus Nafcilin resistant
NAF/BE
NAF MIC MIC
Strain (pg/rnI) (pg/ml) A Synergy
60187-2 10.00 0.6 16.7
52446-3 175.00 40.0 4.4
M1978 140.00 50.0 2.8
W54793 130.00 33.3 3.9
S24341 210.00 65.0 3.2
H7544 28.33 15.0 1.9
H72751 145.00 43.3 3.3
W71630 131.67 46.7 2.8
X22831 178.33 75.0 2.4
X23660 123.33 43.3 2.8
036466 191.67 93.3 2.1
BE = 0.2 pg/ml BisEDT; Bacterial strains were obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Nafcillin
was obtained from Sigma (St. Louis, MO).
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TABLE 3
S. aureus Nafcilin resistant
GM/BE
GM MIC MIC
Strain (pg/rnI) (pg/m1) a Synergy
60187-2 0.233 0.004 58.3 +
52446-3 10.667 1.500 7.1 +
M1978 32.500 4.000 8.1 +
W54793 0.250 0.080 3.1
S24341 0.250 0.058 4.3 +
H7544 0.383 0.093 4.1 +
H72751 0.200 0.072 2.8 -
W71630 17.667 3.800 4.6 +
X22831 - 0.085
X23660 22.500 4.000 5.6 +
036466 0.267 0.043 6.2 +
BE = 0.2 pg/ml BisEDT; Bacterial strains were obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Nafcillin was
obtained from Sigma.
TABLE 4
S. aureus
Rifampin/Neomycin/Paromomycin
MIC MIC + BE
ATCC 25923 (pg/m1) (pg/m1) A Synergy
RIF 0.033 0.003 13.0 +
NE0 0.500 0.200 2.5 -
PARO 1.080 0.188 5.7 +
MRSA S2446-3 _
RIF 2.500 2.500 1.0 -
NE0 13.400 8.500 1.6 -
PARO 335.000 183.300 1.8 -
BE = 0.2 pg/ml BisEDT; Strain S2446-3 was obtained from the Clinical
Microbiology Laboratory at Winthrop-University Hospital, Mineola, NY.
Antibiotics
were obtained from Sigma.
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TABLE 5
S. epidermidis - GM resistant
strain ATCC 35984 strain S2400-1
BisEDT MIC MBC MIC MBC
(pg/ml) (pg/ml GM) (pg/nril GM) (pg/ml GM) (pg/ml GM)
0 53.3 384.0 85.3 426.7
0.005 20.0 96.0 96.0 512.0
0.01 37.3 117.3 64.0 256.0
0.02 21.3 26.7 28.0 128.0
0.04 2.0 16.0 2.0 128.0
0.08 2.0 10.7 2.0 53.3
0.16 (MIC) 3.0 10.0
0.32 2.0 4.0
GM = gentamicin; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Gentamicin was
obtained
from the Pharmacy Department at Winthrop; synergy in bold
TABLE 6
S. epidermidis - S2400-1
Biofilm Prevention
BisEDT (pg/ml) A
Antibiotic 0 0.05 0.1 (0.05 BE) Synergy
cefazolin 28 10 1 2.8
vancomycin 3.2 0.9 0.1 3.6 -
gatifloxacin 1.6 0.1 0.1 16.0 ++
rifampicin 0.03 0.04 0.04 0.7 -
nafcillin 48 64 8 0.8
clindamycin 1195 48 12 24.9 ++++
gentamicin 555 144 12 3.9
borderline
minocycline 0.85 0.73 0.08 1.2 -
Data in pg/m1; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
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TABLE 7
S. epidermidis - S2400-1
MIC
BisEDT (pg/ml) A
Antibiotic 0 0.05 0.1 (0.05 BE) Synergy
cefazolin 32 8 1 4.00 +
vancomycin 3.2 2.3 0.3 1.40 -
gatifloxacin 1.7 0.8 0.3 2.13 -
rifampicin 0.03 0.04 0.04 0.75 -
nafcillin 171 192 68 0.89
clindamycin 2048 768 24 2.67 -
gentamicin 2048 320 80 6.40 +
minocycline 1.13 0.43 0.10 2.63 -
Data in pg/ml; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
TABLE 8
S. epidermidis - S2400-1
MBC
BisEDT (pg/ml) A
Antibiotic 0.0 0.1 (0.1 BE) Synergy
cefazolin 48 10 4.80 +
vancomycin 5.4 1.4 3.86 borderline
gatifloxacin 2.8 1.4 2.00 -
rifampicin 0.03 0.07 0.43 -
nafcillin 256 128 2.00 -
clindamycin 2048 768 2.67 -
gentamicin 1536 256 6.00 +
minocycline 1.20 1.20 1.00 -
Data in pg/ml; Strain S2400-1 was obtained from the Clinical Microbiology
Laboratory at Winthrop-University Hospital, Mineola, NY. Antibiotics were
obtained
from the Pharmacy Department at Winthrop.
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TABLE 9
S. epidermidis
ATCC 35984
MIC
BisEDT (pg/ml)
Antibiotic 0.0 0.05 Synergy
Nafcillin 16.00 5.00 3.2
Clindamycin 2048.00 1024.00 2
Gentamicin 213.33 16.00 13.3 ++
Minocycline 0.13 0.04 3.3
Rifampicin 0.021 0.014 1.5
Data in ug/m1; Antibiotics were obtained from the Pharmacy Department at
Winthrop-University Hospital, Mineola, NY.
TABLE 10
E. coli - Ampicillin/Chlorannphenicol resistant
MIC
M IC AB AB/BE MIC BE
Strain (pg/ml) (pg/ml AB) A Synergy
(pg/ml)
MC4100/TN9 (CM) 220 12.7 17.4 + 0.6
MC4100/P9 (AM) 285 49 5.8 + 0.5
MG4100 (AM) 141.7 35 4.0 + 0.6
AB = antibiotic; CM = chloramphenicol; AM = ampicillin; BE = BisEDT at 0.3
ug/m1;
Strains were obtained from the laboratory of Dr. MJ Casadaban, Department of
Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL.
Antibiotics were obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 11
E. coil - Tetracycline-resistant:
Doxycycline + BisEDT
DOX MIC DOX/BE MIC BE MIC
Strain (pg/ml) (pg/ml DOX) A Synergy (pg/ml)
TET M 16.50 4.50 4.0 0.85
TET D 20.50 0.03 820.0 ++++ 0.85
TET A 15.00 10.00 1.5 0.40
TET B 20.13 10.33 2.0 0.60
DOX = doxycycline; BE = BisEDT at 0.3 pg/ml; Strains were obtained from the
laboratory of Dr. I Chopra, Department of Bacteriology, The University of
Bristol,
Bristol, UK. Antibiotics were obtained from the Pharmacy Department at
Winthrop-
University Hospital, Mineola, NY.
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TABLE 12
P. aeruginosa - Tobramycin-resistant:
BisEDT Synergy
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml)
Xen5 0.32 0.19 1.68 - 0.9
Agr PA E 115 70 1.64 - 0.9
Agr PA I 200 73 2.74 - 1
Agr PA K 4.8 3 1.60 0.82
Agr PA 0 130 20.5 6.34 + 0.98
Agr =aminoglycoside resistant; NN = tobramycin; PA = Pseudomonas aeruginosa;
BE = BisEDT, 0.3 pg/ml; Strains were obtained from the laboratory of Dr. K.
Poole,
Department of Microbiology and Immunology, Queens University, Ontario, ON.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 13
B. cepacia
Tobramycin+BE Synergy
MIC
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml)
13945 200 50 4 + 2.4
25416 125 10 12.5 ++ 1.2
H12229 64 8 8 + 0.8
AU 0267 128 2 64 ++++ 0.8
AU 0259 1024 256 4 + 1.6
H12255 64 8 8 + 1.6
AU 0273 512 32 16 ++ 1.6
H12253 64 16 4 + 1.6
H12147 512 8 64 ++++ 1.6
NN = Tobramycin; BE = BisEDT, 0.4 pg/ml; Strains were obtained from the
laboratory of Dr. J.J. LiPuma, Department of Pediatrics and Communicable
Diseases, University of Michigan, Ann Arbor, MI; also Veloira et al. 2003.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
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TABLE 14
B. cepa cia
Tobramycin+BE Synergy
MBC
NN NN+BE BE MIC
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml)
HI 2249 256 8 32 ++ 3.2
HI 2229 128 32 4 + 6.4
AU 0267 256 32 8 + 6.4
AU 0259 1024 1024 1 12.8
HI 2255 128 32 4 + 12.8
HI 2711 512 8 64 ++++ 6.4
AU 0284 1024 64 16 ++ 0.8
AU 0273 512 32 16 ++ 1.6
HI 2253 128 64 2 - 3.2
,
HI 2147 512 128 4 + 6.4
NN = Tobramycin; BE = BisEDT, 0.4 pg/ml; Strains were obtained from the
laboratory of Dr. J.J. LiPuma, Department of Pediatrics and Communicable
Diseases, University of Michigan, Ann Arbor, MI; also Veloira et al. 2003.
Tobramycin was obtained from the Pharmacy Department at Winthrop-University
Hospital, Mineola, NY.
TABLE 15
Tobramycin Resistant Strains
MIC
NN NN+BE Lipo-BE-NN
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml NN)
M13637 512 32 16 ++ 0.25
M13642R 128 64 2 0.25
PA-48913 1024 256 4 + 0.25
PA-48912-2 64 8 8 + 0.25
PA-10145 1 4 0.25 - 0.25
SA-29213 2 1 2 0.25
NN = Tobramycin; BE = BisEDT, 0.8 pg/ml; Lipo-BE-NN = liposomal BE-NN;
Strains were obtained from the laboratory of Dr. A. Omri, Department of
Chemistry
and Biochemistry, Laurentian University, Ontario, CN; (M strains are mucoid B.
cepacia; PA=P. aeruginosa; SA=S. aureus). Tobramycin was obtained from the
Pharmacy Department at Winthrop-University Hospital, Mineola, NY.
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TABLE 16
Tobramycin Resistant Strains
MBC
Lipo-BE-
NN NN+BE NN
Strain (pg/ml) (pg/ml NN) A Synergy
(pg/ml NN)
M13637 1024 64 16 ++ 8
M13642R 256 128 2 16
PA-48913 4096 512 8 4
PA-48912-2 128 32 4 0.5
PA-10145 1 8 0.125 4
SA-29213 2 1 2 0.25
NN = Tobramycin; BE = BisEDT, 0.8 pg/ml; Lipo-BE-NN = liposomal BE-NN;
Strains were obtained from the laboratory of Dr. A. Omri, Department of
Chemistry
and Biochemistry, Laurentian University, Ontario, CN; (M strains are mucoid B.

cepacia; PA=P. aeruginosa; SA=S. aureus). Tobramycin was obtained from the
Pharmacy Department at Winthrop-University Hospital, Mineola, NY.
TABLE 17
BisEDT-Pyrithione Synergy
S. aureus
P. aeruginosa E. con ATCC
NaPYR ATCC 27853 ATCC 25922 25923
(ug/ml) (pg/ml BE) (pg/ml BE) (pg/ml BE)
0 0.25 0.1 0.25
0.025 0.1 0.125
0.05 0.025 0.063
0.1 0.125 0.0125 0.063
0.2 0.125 0.0125 0.031
0.4 0.00625 0
0.8 0.125 0.00625
1.6
(MIC) 0.063 0.00625
3.2 0.063 0
6.4 0.063
12.8 0
BE = BisEDT; NaPYR = sodium pyrithione; Chemicals were obtained from Sigma-
Aldrich; synergy in bold. Indicated bacterial strains were from American Type
Culture Collection (ATCC, Manassas, VA).
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EXAMPLE 7
COMPARATIVE BISMUTH-THIOL (BT) AND ANTIBIOTIC EFFECTS AGAINST GRAM-
POSITIVE AND GRAM-NEGATIVE BACTERIA INCLUDING ANTIBIOTIC-RESISTANT
BACTERIAL STRAINS
In this example the in vitro activities of BisEDT and comparator
agents were assessed against multiple clinical isolates of Gram-positive and ¨

negative bacteria that are responsible for skin and soft tissue infections.
Materials and Methods. Test compounds and test concentration
ranges were as follows: BisEDT (Domenico et al., 1997; Domenico et al.,
Antimicrob. Agents Chemother.45(5):1417-1421. and Example 1), 16-0.015
pg/mL; linezolid (ChemPacifica Inc., #35710), 64-0.06 pg/mL; Daptonnycin
(Cubist Pharmaceuticals #MCB2007), 32-0.03 pg/mL and 16-0.015 pg/nnL;
vancomycin (Sigma-Aldrich, St. Louis, MO, # V2002), 64-0.06 pg/mL;
ceftazidinne, (Sigma #C3809), 64-0.06 pg/mL and 32-0.03 pg/mL; imipenenn
(United States Pharmacopeia, NJ, #1337809) 16-0.015 pg/mL and 8-0.008
pg/mL; ciprofloxacin (United States Pharmacopeia, #10C265), 32-0.03 pg/mL
and 4-0.004 pg/mL; gentamicin (Sigma #G3632) 32-0.03 pg/mL and 16-0.015
pg/mL. All test articles, except gentamicin, were dissolved in DMSO;
gentamicin was dissolved in water. Stock solutions were prepared at 40-fold
the highest concentration in the test plate. The final concentration of DMSO
in
the test system was 2.5%.
Organisms. The test organisms were obtained from clinical
laboratories as follows: CHP, Clarian Health Partners, Indianapolis, IN; UCLA,
University of California Los Angeles Medical Center, Los Angeles, CA; GR
Micro, London, UK; PHRI TB Center, Public Health Research Institute
Tuberculosis Center, New York, NY; ATCC, American Type Culture Collection,
Manassas, VA; Mt Sinai Hosp., Mount Sinai Hospital, New York, NY; UCSF,
University of California San Francisco General Hospital, San Francisco, CA;
Bronson Hospital, Bronson Methodist Hospital, Kalamazoo, MI; quality control
isolates were from the American Type Culture Collection (ATCC, Manassas,
VA). Organisms were streaked for isolation on agar medium appropriate to
each organism. Colonies were picked by swab from the isolation plates and put
into suspension in appropriate broth containing a cryoprotectant. The
suspensions were aliquoted into cryogenic vials and maintained at -80 C.
Abbreviations are: BisEDT, bismuth-1,2-ethanedithiol; LZD, linezolid; DAP,
daptomycin; VA, vancomycin; CAZ, ceftazidime; IPM, imipenem; CIP,
ciprofloxacin; GM, gentamicin; MSSA, methicillin-susceptible Staphylococcus
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aureus; CLSI QC, Clinical and Laboratory Standards Institute quality control
strain; MRSA, methicillin-resistant Staphylococcus aureus; CA-MRSA,
community-acquired methicillin-resistant Staphylococcus aureus; MSSE,
methicillin-susceptible Staphylococcus epidermidis; MRSE, methicillin-
resistant
Staphylococcus epidermidis; VS E, vancomycin-susceptible Enterococcus.
The isolates were streaked from the frozen vials onto appropriate
medium: Trypticase Soy Agar (Becton-Dickinson, Sparks, MD) for most
organisms or Trypticase Soy Agar plus 5% sheep blood (Cleveland Scientific,
Bath, OH) for streptococci. The plates were incubated overnight at 35 C.
Quality control organisms were Included. The medium employed for the MIC
assay was Mueller Hinton II Broth (MHB II- Becton Dickinson, #212322) for
most of the organisms. MHB II was supplemented with 2% lysed horse blood
(Cleveland Scientific Lot # H13913) to accommodate the growth of
Streptococcus pyo genes and Streptococcus agalactiae. The media were
prepared at 102.5% normal weight to offset the dilution created by the
addition
of 5 pL drug solution to each well of the microdilution panels. In addition,
for
tests with daptomycin, the medium was supplemented with an additional
25mg/L Ca24".
The MIC assay method followed the procedure described by the
Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards

Institute. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria
That Grow Aerobically; Approved Standard¨Seventh Edition. Clinical and
Laboratory Standards Institute document M7-A7 [ISBN 1-56238-587-9].
Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA, 2006) and employed automated liquid
handlers to conduct serial dilutions and liquid transfers. Automated liquid
handlers included the Multidrop 384 (Labsystems, Helsinki, Finland), Biomek
2000 and Multimek 96 (Beckman Coulter, Fullerton CA). The wells of Columns
2-12 of standard 96-well microdilution plates (Falcon 3918) were filled with
150pL of DMSO or water for gentamicin on the Multidrop 384. The drugs (300
pL) were dispensed into Column 1 of the appropriate row in these plates.
These would become the mother plates from which the test plates (daughter
plates) were prepared. The Biomek 2000 completed serial transfers through
Column 11 in the mother plates. The wells of Column 12 contained no drug
and were the organism growth control wells in the daughter plates. The
daughter plates were loaded with 185 pL of the appropriate test media
(described above) using the Multidrop 384. The daughter plates were prepared
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on the Multimek 96 instrument which transferred 5 pL of drug solution from
each well of a mother plate to each corresponding well of each daughter plate
in a single step.
Standardized inoculum of each organism was prepared per CLSI
methods (ISBN 1-56238-587-9, cited supra). Suspensions were prepared in
MHB to equal the turbidity of a 0.5 McFarland standard. The suspensions were
diluted 1:9 in broth appropriate to the organism. The inoculum for each
organism was dispensed into sterile reservoirs divided by length (Beckman
Coulter), and the Biomek 2000 was used to inoculate the plates. Daughter
plates were placed on the Biomek 2000 work surface reversed so that
inoculation took place from low to high drug concentration. The Biomek 2000
delivered 10 pL of standardized inoculum into each well. This yielded a final
cell concentration in the daughter plates of approximately 5 x 105 colony-
forming-units/mL. Thus, the wells of the daughter plates ultimately contained
185 pL of broth, 5 pL of drug solution, and 10 pL of bacterial inoculum.
Plates
were stacked 3 high, covered with a lid on the top plate, placed in plastic
bags,
and incubated at 35 C for approximately 18 hours for most of the isolates. The

Streptococcus plates were read after 20 hours incubation. The microplates
were viewed from the bottom using a plate viewer. For each of the test media,
an uninoculated solubility control plate was observed for evidence of drug
precipitation. The MIC was read and recorded as the lowest concentration of
drug that inhibited visible growth of the organism.
Results. All marketed drugs were soluble at all of the test
concentrations in both media. BisEDT exhibited a trace precipitate at 32
pg/mL,
but MIC readings were not affected as the inhibitory concentrations for all
organisms tested were well below that concentration. On each assay day, an
appropriate quality control strain(s) was included in the MIC assays. The MIC
values derived for these strains were compared to the published quality
control
ranges (Clinical and Laboratory Standards Institute. Performance Standards for
Antimicrobial Susceptibility Testing; Eighteenth Informational Supplement.
CLSI
document M100-S18 [ISBN 1-56238-653-0]. Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898
USA, 2008) for each agent, as appropriate.
On each assay day, an appropriate quality control strain(s) was
included in the MIC assays. The MIC values derived for these strains were
compared to the published quality control ranges (Clinical and Laboratory
Standards Institute. Performance Standards for Antimicrobial Susceptibility
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Testing; Eighteenth Informational Supplement. CLSI document M100-S18
[ISBN 1-56238-653-0]) for each agent, as appropriate. Of 141 values for
quality
control strains where quality control ranges are published, 140(99.3%) were
within the specified ranges. The one exception was imipenem versus S. aureus
29213 which yielded one value on a single run 0.008 pg/mL) that was one
dilution below the published QC range. All other quality control results on
that
run were within the specified quality control ranges.
BisEDT demonstrated potent activity against both methicillin-
susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus
(MRSA), and community-acquired MRSA (CA-MRSA), inhibiting all strains
tested at 1 pg/mL or less with an MIC90 values of 0.5 pg/mL for all three
organism groups. BisEDT exhibited activity greater than that of linezolid and
vancomycin and equivalent to that of daptomycin. lmipenem was more potent
than BisEDT against MSSA (MIC90 = 0.03 pg/mL). However, MRSA and
CAMRSA were resistant to imipenem while BisEDT demonstrated activity
equivalent to that shown for MSSA. BisEDT was highly-active against
methicillin-susceptible and rnethicillin¨resistant Staphylococcus epidermidis
(MSSE and MRSE), with MIC90 values of 0.12 and 0.25 pg/mL, respectively.
BisEDT was more active against MSSE than any of the other agents tested
except imipenem. BisEDT was the most active agent tested against MRSE.
BisEDT demonstrated activity equivalent to that of daptomycin,
vancomycin, and imipenem against vancomycin-susceptible Enterococcus
faecalis (VSEfc) with an MIC90 value of 2pg/mL. Significantly, BisEDT was the
most active agent tested against vancomycin-resistant Enterococcus faecalis
(VREfc) with an MIC90 value of 1 pg/mL.
BisEDT was very active against vancomycin-susceptible
Enterococcus faecium (VSEfm) with an MI090 value of 2 pg/mL; its activity was
equivalent to that or similar to that of daptomycin and one-dilution higher
than
that of vancomycin. BisEDT and linezolid were the most active agents tested
against vancomycin-resistant Enterococcus faecium (VREfm), each
demonstrating an MIC90 value of 2 pg/mL. The activity of BisEDT against
Streptococcus pyogenes (MIC90 value of 0.5 pg/mL) was equivalent to that of
vancomycin, greater than that of linezolid, and slightly less than that of
daptomycin and ceftazidime. The compound inhibited all strains tested at 0.5
pg/mL or less. In these studies, the species that was least sensitive to
BisEDT
was Streptococcus agalactiae where the observed MIC90 value was 16 pg/mL.
BisEDT was less active than all of the agents tested except gentamicin.
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The activity of BisEDT and comparators against Gram-negative
bacteria included demonstrated BisEDT potency against Acinetobacter
baumanii (MIC90 value of 2 pg/mL) making BisEDT the most active compound
tested. Elevated MICs for a significant number of test isolates for the
comparator agents resulted in off-scale MIC90 values for these agents.
BisEDT was a potent inhibitor of Escherichia colt, inhibiting all strains at 2

pg/mL or less (MIC90 = 2 pg/mL). The compound was less active than
imipenem, but more active than ceftazidime, ciprofloxacin, and gentamicin.
BisEDT also demonstrated activity against Klebsiella pneumoniae with an
MIC90 value of 8 pg/mL which was equivalent to that of imipenem. The
relatively high MIC90 values exhibited by imipenem, ceftazidime,
ciprofloxacin,
and gentamicin indicated that this was a highly antibiotic-resistant group of
organisms. BisEDT was the most active compound tested against
Pseudomonas aeruginosa with an MIC90 value of 4 pg/mL. There was a high
level of resistance to the comparator agents for this group of test isolates.
In summary, BisEDT demonstrated broad-spectrum potency
against multiple clinical isolates representing multiple species, including
species
commonly involved in acute and chronic skin and skin structure infections in
humans. The activity of BisEDT and key comparator agents was evaluated
against 723 clinical isolates of Gram-positive and Gram¨negative bacteria. The
BT compound demonstrated broad spectrum activity, and for a number of the
test organisms in this study, BisEDT was the most active compound tested in
terms of anti-bacterial activity. BisEDT was most active against MSSA, MRSA,
CA-MRSA, MSSE, MRSE, and S. pyogenes, where the MIC90 value was 0.5
pg/mL or less. Potent activity was also demonstrated for VSEfc, VREfc,VSEfm,
VREfm, A. baumanii, E. coli, and P. aeruginosa where the MIC90 value was in
the range of 1 - 4 pg/mL. MI090 values observed were, for K. pneumoniae
(MIC90 = 8 pg/mL), and for S. agalactiae (MIC90= 16 pg/mL).
EXAMPLE 8
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
This example shows that microparticulate bismuth thiols (BTs)
promote antibiotic activity through enhancing and/or synergizing interactions.
A major complicating factor in treating infections is the emerging
resistance of bacteria to antibiotics. Methicillin resistance in S.
epidermidis
(MRSE) and S. aureus (MRSA) actually reflects multiple drug resistance,
making these pathogens very difficult to eradicate. However, no staphylococci
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from hundreds of strains tested showed resistance to BTs. Furthermore, BTs at
subinhibitory (subMIC) concentrations reduced resistance to several important
antibiotics.
Staphylococcus aureus. A graphic demonstration of the
antibiotic-resensitizing effects of subMIC bismuth ethanedithiol (BisEDT)
against MRSA is provided (Figure 4) showing enhanced antibiotic action of
several classes of antibiotics, including gentamicin, cefazolin, cefepime,
imipenim, sulphamethoxazole, and levofloxacin. Thus, BisEDT nonspecifically
enhanced the activity of most antibiotics.
Broth dilution antimicrobial susceptibility studies were performed
against 12 MRSA strains using several antibiotics combined with subMIC levels
of BisEDT (Table 18). Both the biofilm-prevention concentration (BPC) and the
minimum inhibitory concentration (MIC) were determined in a special biofilm
culture medium (BHIG/X). The MIC and BPC for gentamicin and cefazolin were
reduced by subMIC BisEDT (BisEDT MIC, 0.2-0.4 pg/ml), but not below the
breakpoint for sensitivity. subMIC BisEDT enhanced the sensitivity of MRSA to
gatifloxacin and cefepinne close to the breakpoint for sensitivity. These
strains
were already sensitive to vancomycin, but were made considerably moreso in
the presence of subMIC BisEDT. Generally, the MIC and BPC were reduced 2-
to 5-fold with subMIC BisEDT.
TABLE 18.
Antimicrobial Activity of BT-Antibiotic Combinations against MRSA
MIC Standards
Antibiotic BisEDT (pg/mL) (i.tg/m1)
0 0.025 0.05 0.1
Gentamicin
BPC 81 41 63 30 53 31 33 25
MIC 81 40 60 27 58 30 48 31 16
Cefazolin
BPC 109 86 76 86 76 105 34 28
MIC 93 75 99 76 90 60 45 32 32
Gatifloxacin
BPC 3.6 2.6 2.6 0.9 2.4 1.1 0.9 0.8
MIC 3.6 2.6 4.0 2.8 4.0 2.8 2.4 1.1
Vancomycin
BPC 2.5 1.7 1.5 0.6 1.3 0.5 0.7 0.4
MIC 2.5 1.7 2.5 1.7 1.5 0.6 1.3 0.5 32
Cefepime
BPC 24 37 27 28 18 16 5.0 7.3
MIC 45 32 32 28 37 24 9.3 6.1 32
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12 MRSA clinical isolates were grown in BHIG/X and exposed to serial dilutions
of
antibiotics in the presence of 0-0.1 pg/ml BisEDT. The MIC and BPC, calculated
in
pg/ml, are the means standard deviations from at least three trials. The
right hand
column lists the Standard MIC for antibiotic senstivity (S) and resistance (R)
A broth dilution study of cefepime-resistant MRSA isolates is
shown in Table 19. BisEDT at 0.1 pg/ml significantly enhanced the inhibitory
activity of cefepime in 11 of 12 isolates. In this particular study, the data
indicated synergy between BisEDT and cefepime (FIC < 0.5), with many of the
isolates at the breakpoint for sensitivity.
TABLE 19
Cefepinne-resistant MRSA Sensitized by BisEDT
MIC for Cefepime (ug/mL) in subMIC BisEDT
BE BE BE
0 pg/mL 0.05 pg/mL 0.1 pg/mL
MRSA MIC MIC MIC
Strain #
4 256 256 16
6 256 256 32
7 128 256 32
10 128 32 16
18 256 128 8
24 256 64 8
28 256 128 8
35 256 256 8
37 128 128 8
41 128 256 8
46 256 256 256
47 32 8 8
Twelve cefepime-resistant MRSA were tested in BHIG/X medium in polystyrene
plates for sensitivity to cefepime combined with subMIC BisEDT at 37 C for
48h.
Results for combination studies with nafcillin or gentamicin are
shown in Table 20. Combined with nafcillin, BisEDT (0.2 p.g/m1) reduced the
MIC90 for nafcillin by over 4-fold against MRSA (FIC, 0.74). Combined with
gentamicin, BisEDT reduced the MIC90 for gentamicin over 10-fold against
MRSA (FIC, 0.6). BTs reversed the resistance of all four
gentamicin-resistant
isolates tested to clinically relevant concentrations [Domenico et al., 2002].
The
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MiCS for these antimicrobial agents was reduced substantially, especially for
gentamicin. The broth used in these studies was Trypticase Soy Broth (TSB)
with 2% glucose, which showed results similar to that seen in Mueller-Hinton
II
broth fortified with 1% sheep's blood.
TABLE 20
MRSA: Nafcillin or Gentamicin + BisEDT Synergy
NAF NAF+BE GM GM+BE
Strain MIC MIC A MIC MIC A
60187-2 10.00 0.60 16.67 0.23
0.00 58.33
52446-3 175.00 40.00 4.38 10.67 1.50 7.11
M1978 140.00 50.00 2.80
32.50 4.00 8.13
W54793 130.00 33.33 3.90 0.25 0.08 3.13
S24341 210.00 65.00 3.23 0.25 0.06
4.29
H7544 28.33 15.00 1.89 0.38 0.09
4.11
H72751 145.00 43.33 3.35 0.20 0.07
2.79
W71630 131.67 46.67 2.82 17.67 3.80 4.65
X22831 178.33 75.00 2.38
X23660 123.33 43.33 2.85
22.50 4.00 5.63
036466 191.67 93.33 2.05 0.27 0.04 6.15
AVG A 4.21 AVG A 10.43
NAF or GM in pg/ml; BE at 0.2 pg/ml
Staphylococcus epidermidis. The activities of most classes of
antibiotic were promoted in the presence of BisEDT. With regard to the BPC,
clindamycin and gatifloxacin showed significantly more antibiofilm activity
against S. epidermidis when combined with BisEDT (Figure 5). Stated in
different terms, the BPC for clindamycin, gatifloxacin and gentamicin were
reduced 50-fold, 10-fold and 4-fold, respectively, in the presence of subMIC
BisEDT.
Only modest decreases in the biofilm prevention concentration
(BPC) were noted for minocycline, vancomycin, and cefazolin, while rifampicn
and nafcillin remained unaffected at 0.05 pg/ml BisEDT. At 0.1 ,g/m1 BisEDT
no biofilm was detected, regardless of antibiotic employed, signifying that no
antagonism occurred. This BisEDT concentration was close to the MIC for S.
epidermidis [Domenico et al., 2003] (See Figure 5).
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With regard to growth inhibition, seven of eight antibiotics tested
were significantly enhanced in the presence of 0.1 pg/ml (0.5 pM) BisEDT
against S. epidermidis (Figure 6). The MIC change was most pronounced for
clindamycin and gentamicin, followed by vancomycin, cefazolin, minocycline,
gatifloxacin and nafcillin, with rifampicin unaffected. Of the antibiotics
this strain
was resistant to (NC, CZ, GM, CM), only cefazolin resistance was reversed to
clinically relevant levels by BisEDT.
Minimum bactericidal concentration (MBC) for most antibiotics
tested against S. epidermidis decreased slightly with subMIC BisEDT.
Gentamicin showed the greatest reduction in MBC (4- to 16-fold), followed by
cefazolin (4- to 5-fold), vancomycin and nafcillin (3- to 4-fold), minocycline
and
gatifloxacin (2- to 3-fold), while clindamycin and rifampicin MBC remained
largely unaffected. Clindamycin is a bacteriostatic agent, which explains its
lack of bactericidal activity. Cefazolin resistance was reversed with respect
to
the MBC [Domenico et al., 2003]. These effects were additive.
The potentiation of antimicrobial agents was also demonstrated in
vivo in a graft infection rat model (Table 21). BisEDT levels as low as 0.1
pg/ml
were able to promote the prevention of resistant S. epidermidis biofilm for 7
days.
As summarized in Table 21, implants impregnated with 0.1 pg/m1
BisEDT, 10 g/m1 RIP and 10 g/mIrifampin, alone or combined were
implanted s.c. into rats. Physiological solution (1 ml) containing the MS and
MR
strains at 2x107 cfu/ml was inoculated onto the graft surface using a
tuberculin
syringe. All grafts were explanted at 7 days following implantation and
sonicated for 5 minutes in sterile saline solution to remove the adherent
bacteria. Quantitation of viable bacteria was obtained by culturing dilutions
on
blood agar plates. The limit of detection was approximately 10 cfu/cm2.
TABLE 21
RIP, BTs, and rifampin against S. epidermidis in a graft infection model
Quantitative graft
Group' Graft-bonded culture (cfu/cm2)
drugb
No MSSE <10
Untreated 5.0 x 107 7.7 x 106
MSSE
MS1' RIP 4.3 x 102 1.2 x 102
MS2' BTs 5.8 x 102 0.9 x 102
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Quantitative graft
Group a Graft-bonded culture (cfu/cm2)
drug'
MS3c Rifampin 5.9 x 103 1.8 x 103
mscd RIP plus BTs <10
ms5cc/ RIP plus 2.0 x 101 0.6 x 101
rifampin
MS6ccl BTs plus 1.9 x 101 0.4 x 101
rifampin
No MRSE <10
Untreated 7.8 x 107 2.0 x 107
MRSE
MR1c RIP 6.7 x 102 2.1 x 102
MR2' BTs 6.2 x 102 2.3 x 102
MR3b Rifampin 7.6x 104 2.1 x 104
MR4be RIP plus BTs <10
MR5b RIP plus 4.3 x 101 +1.1 x 101
rifampin
MR6b BTs plus 3.0 x 101 1.1 x 101
rifampin
a Each group had 15 animals; MS, methicillin-susceptible S. epidermidis; MR,
methicillin-resistant S. epidermidis
Dacron graft segments impregnated with 0.1 mg/I of BTs, 10 mg/I of RIP, 10
mg/I of
rifampin
5 C Statistically significant when compared with control groups MS and MR
d Statistically significant when compared with MS3 group
e Statistically significant when compared with MR1, MR2, and MR3 groups
Gram-negative Bacteria. Tobramycin activity against resistant
Pseudomonas aeruginosa was enhanced several-fold with subMIC BisEDT
(Table 22). In these trials, the MIC was defined more precisely as the IC24.
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TABLE 22
Tobramycin-resistant P. aeruginosa: BisEDT Effect
NN MIC BE MIC NN+BE MIC
Strain (pg/ml) (pg/ml) (pg/ml) A
PA Xen5 0.3 0.9 0.2 1.7
Agr PA E 115.0 0.9 70.0 1.6
Agr PA I 200.0 1.0 73.0 2.7
Agr PA K 4.8 0.86 3.0 1.6
Agr PA 0 130.0 0.98 20.5 6.3
Resistant strains of P. aeruginosa were cultured in Mueller-Hinton II broth at
37 C
in the presence of tobramycin (NN) and BisEDT (BE; 0.33 pg/ml). The MIC was
determined as the antibiotic concentration that inhibited growth for 24 1 h.
Against tobramycin-resistant Burkholderia cepacia, 0.4 p.g/ml
BisEDT rendered seven of 10 isolates tobramycin sensitive (mean FIC; 0.48),
and reduced the MIC90 by 10-fold (Table 23). Both the MIC and MBC of
tobrannycin were reduced significantly to achievable levels against 50
clinical
Burkholderia cepacia isolates with subMIC BisEDT [Veloira et al., 2003].
BisEDT and tobramycin in liposomal form have proven highly synergistic
against P. aeruginosa. (Halwani et al., 2008; Halwani et al., 2009).
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TABLE 23
Tobramycin and BisEDT versus B. cepacia
MIC for
Strain Tobramycin BisEDT Tobramycin FIC
(pg/ml) (pg/ml) (BisEDT at 0.4 pg/ml) Index
B. multivorans
HI 2249 256 0.4 a a
HI 2229 64 0.8 8 0.63
AU 0267 128 0.8 2 0.52
AU 0259 1024 1.6 256 0.50
HI 2255 64 1.6 8 0.38
B. cenocepacia
H12711 256 0.4 a a
AU 0284 512 0.4 a a
AU 0273 512 1.6 32 0.31
H12253 64 1.6 16 0.50
HI 2147 512 1.6 8 0.27
a The three strains inhibited by BisEDT at 0.4 pg/ml were excluded from
further
study.
FIC Index 0.5 indicates synergy: FICI >0.5 and <1.0 indicates enhancement.
Chloramphenicol and ampicillin resistant Escherichia co//were
made sensitive to these drugs by the addition of subMIC BisEDT (Table 24).
TABLE 24
Chloramphenicol/Ampicillin Resistant E. coli: BisEDT Effect
Drug + BE
Drug MIC BE MIC MIC
Strain Drug (pg/ml) (pg/ml) (pg/ml) A
MC4100/TN9 CM 220.0 0.6 12.7 17.4
MC4100/P9 AMP 285.0 0.5 49.0 5.8
MC4100 AMP 141.7 0.6 35.0 4.0
Resistant strains of E.coli were cultured in Mueller-Hinton II broth at 37 C
in the
presence of chloramphenicol (CM) or ampicillin (AMP) and BisEDT alone or in
combination (BE; 0.33 pg/ml). The MIC was determined as the antibiotic
concentration that inhibited growth for 24 1 h.
Tetracycline resistant Escherichia coli were made sensitive to
doxycycline by the addition of subMIC BisEDT (Table 25). The combination
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exhibited synergy against the TET M and TET D strains (FIC 0.5), with
additive effects against the TET A and TET B strains.
TABLE 25
Tetracycline Resistant E. Coli: BisEDT Effect
DOX MIC BE MIC DOX+BE MIC
Strain (pg/ml) (pg/ml) (pg/ml) A
TET M 16.5 1.3 0.85 4.5 2.7 4.0
TET D 20.5 1.1 0.85 0.03 0.0 820.0
TET A 15.0 1.8 0.40 10.0 1.0 1.5
TET B 20.1 2.4 0.60 10.3 3.2 2.0
Resistant strains of E.coli were cultured in Mueller-Hinton II broth at 37 C
in the
presence of doxycycline (DOX) and BisEDT alone or in combination (BE; 0.33
pg/ml). The M1C was determined as the antibiotic concentration that inhibited
growth for 24 1 h.
References
Domenico P, R O'Leary, BA Cunha. 1992. Differential effect of
bismuth and salicylate compounds on antibiotic sensitivity of Pseudomonas
aeruginosa. Eur J Clin Microbiol Infec Dis 11:170-175; Domenico P, D Parikh,
BA Cunha. 1994. Bismuth modulation of antibiotic activity against
gastrointestinal bacterial pathogens. Med Microbiol Lett 3:114-119; Domenico
P, Kazzaz JA, Davis JM, Niederman MS. 2002. Subinhibitory bismuth
ethanedithiol (BisEDT) sensitizes resistant Staphylococcus aureus to nafcill
in or
gentamicin. Annual Meeting, ASM, Salt Lake City, UT; Domenico P, Kazzaz JA,
Davis JM. 2003. Combating antibiotic resistance with bismuth-thiols. Research
Advances in Antimicrob Agents Chemother 3:79-85; Domenico P, E Gurzenda,
A Giacometti, 0 Cirioni, R Ghiselli, F Orlando, M Korem, V Saba, G Scalise, N
Balaban. 2004. BisEDT and RIP act in synergy to prevent graft infections by
resistant staphylococci. Peptides 25:2047-2053; Halwani M, Blomme S,
Suntres ZE, Al ipour M, Azghani AO, Kumar A, Omri A. 2008. Liposomal
bismuth-ethanedithiol formulation enhances antimicrobial activity of
tobramycin.
Intl J Pharmaceut 358:278-84; Halwani M, Hebert S, Suntres ZE, Lafrenie RM,
Azghani AO, Omri A. 2009. Bismuth-thiol incorporation enhances biological
activities of liposomal tobramycin against bacterial biofilm and quorum
sensing
molecules production by Pseudomonas aeruginosa. Int J Pharmaceut 373:141-
6; Veloira WG, Gurzenda EM, Domenico P, Davis JM, Kazzaz JA. 2003.
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Synergy of tobramycin and bismuth thiols against Burkholderia cepacia. J
Antimicrob Chemother 52:915-919.
EXAMPLE 9
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
This example shows that the microparticulate bismuth thiol
BisEDT promotes antibiotic activity through enhancing and/or synergizing
interactions with specific antibiotics against specific microbial target
organisms.
Single-point data for each indicated combination in Table 26 were generated
essentially according to the methods used in Example 8.
146

e
CD
CD
CD
0
TABLE 26
FICI Values for single-point BisEDT-antibiotic combinations
0
Anti SA MRSA E Fc SP PRSP EC EC KP PA Bcep Bmult Abau
Msmeg
0_
biotic 100 773 3121 1195 5348 102 2232 1231 1380 1756
5665 2594 817
F>)
0 Oxacillin 1.28 2.28 0.92 1.03
b
Piperacillin 0.57 1.28 1.11
1.11 0.87 1.29 2.23 0.67 1.12 1.12 1.12
Cefuroxime 1.11 4.23 1.11 1.03
Cefotaxime
1.11 2.23 0.73 1.11 1.11 1.37 1.29 0.61 0.64 1.29 1.11 1.29
Cefepime
0.87 0.96 1.11 0.62 1.34 0.96 0.71
Innipenenn
0.67 1.48 0.73 0.92 0.43 1.11 1.29 1.23 1.12 0.73 1.23 0.81
Aztreonam
0.74 1.29 0.73 0.55 0.67 0.96 0.87
.71
Streptomycin 0.95 0.61 0.66
1.29 1.04 1.98 1.37 1.12 2.62 1.13
Tobramycin 0.73 0.78 0.47 0.57
0.96 0.87 1.29 0.91 0.67 1.12
Tetracycline 0.89 1.23 0.92 1.23 0.34 0.62 0.79 1.29 1.29 1.96 1.12 1.12
Minocycline 1.09 1.23 1.11
0.46 1.37 1.04 1.29 0.99 2.23 1.12 1.29
Ciprofloxacin
1.14 1.29 1.29 2.75 2.23 2.29 1.04
Levofloxacin 1.23 1.11 1.08 0.95 0.70
Erythromycin 1.28 0.67 0.92 0.78 1.03
Linezolid 1.23 1.23 1.23 1.01 1.11
Phosphomycin 0.61 1.23 1.45
1.96 1.02 1.86 1.29 1.23 1.12
Capreomycin
0.75
Isoniazid
0.88
0
to

CA 2807993
SA, Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; E Fc, Enterococcus faecalis; SP, Streptococcus
pneumoniae; PRSP, penicillin-resistant Streptococcus pneunnoniae; EC,
Escherichia coli; KP, Klebsiella pneunnoniae; PA, Pseudomonas aeruginosa;
Bcep, Burkholderia cepacia; Bmult, Bukholderia multivorans; Abau,
Acinetobacter baumanii; Msmeg, Mycobacterium snnegmatis.
EXAMPLE 10
MICROPARTICULATE BT-ANTIBIOTIC ENHANCING AND SYNERGIZING ACTIVITIES
The effects of combinations of microparticulate Bis-EDT and four
Bis-EDT analogs prepared as described above, and other agents against
representative strains of several Gram-negative pathogenic bacteria were
tested. A modification of a common laboratory method was used to determine
synergism (FICI <0.5), enhancement (0.5 < FICI < 1.0), antagonism (FICI >
4.0) and indifference (1.0 < FICI <4.0) used fractional inhibitory
concentrations
(FICs) and FIC indices (FICI) (Eliopoulos G and R Moellering. 1991.
Antimicrobial combinations. In Antibiotics in Laboratory Medicine, Third
Edition,
edited by V Lorian. Williams and Wilkins, Baltimore, MD, pp. 432-492; Odds,
2003 J. Antimicrob. Chemother. 52(1):1). The checkerboard technique was
used to determine FIC indices and was employed in this study.
TABLE 27
Test Components
FIC Highest Stock Conc. Range
Test Cpd Lot No. Solvent Concentration Tested in FIC
(pg/mL) (pg/mL)
Bis-EDT MB-1B-3 DMSO 320 0.12-8
Bis-EDT (analog) MB-2B DMSO 320 0.12-8
Bis-EDT (analog) MB-8-2 DMSO 320 0.12-8
Bis-EDT (analog) MB-11 DMSO 320 0.12-8
Bis-EDT (analog) MB-15 DMSO 320 0.12-8
095K1324 10% 0.06-64
Aztreonam 2,560
(Sigma) DMSO
G0D116 0.06-64
Cefepime HCI dH20 2,560
(USP)
084K0674 0.015-16
Cefotaxime (Sigma) dH20 640
014K1362 0.06-64
Piperacillin dH20 2,560
(Sigma)
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Stock solutions of all test articles were prepared at 40X the final
target concentration in the appropriate solvent. All test articles were in
solution
under these conditions. The final drug concentrations in the FIC assay plates
were set to bracket the MIC value of each agent for each test organism, unless
the strain was totally resistant to the test agent. The concentration ranges
tested are displayed in Table 27. The test organisms were originally received
from clinical sources, or from the American Type Culture Collection. Upon
receipt, the isolates were streaked onto Tryptic Soy Agar II (TSA). Colonies
were harvested from these plates and a cell suspension was prepared in an
appropriate broth growth medium containing cryoprotectant. Aliquots were then
frozen at -80 C. The frozen seeds of the organisms to be tested in a given
assay were thawed, streaked for isolation onto TSA plates, and incubated at
35 C. All organisms were tested in Mueller Hinton II Broth (Becton Dickinson,
Lot No.9044411). The broth was prepared at 1.05X normal weight/volume to
offset the 5 % volume of the drugs in the final test plates.
Minimal Inhibitory Concentration (MIC) values were previously
determined using the broth microdilution method for aerobic bacteria (Clinical

and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard¨
Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and
Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne,
Pennsylvania 19087-1898 USA, 2009.).
FIC values were determined using a broth microdilution method
previously described (Sweeney et al., 2003 Antimicrob. Agents Chemother.
47(6):1902-1906). To prepare the test plates, automated liquid handlers
(Multidrop 384, Labsystems, Helsinki, Finland; Biomek 2000 and Multimek 96,
Beckman Coulter, Fullerton CA) were used to conduct serial dilutions and
liquid
transfers.
The appropriate wells of standard 96-well microdilution plates
(Falcon 3918) were filled with 150 pL of the appropriate solvent in columns 2-
12
using the Multidrop 384. Three hundred microliters of each secondary test drug

was added to each well in Column 1 of the plates. These plates were used to
prepare the drug "mother plates" which provided the serial drug dilutions for
the
drug combination plates. The Biomek 2000 was used to transfer 150 pL of
each secondary drug solution (40X) from the wells in Column 1 of the mother
plate and to make eleven 2-fold serial dilutions. Mother plates of Bis-EDT
(and
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analogs) were serial diluted top to bottom by hand, using a multichannel
pipette. Two mother plates, one for each secondary drug and one for Bis-EDT
(or analogs), were combined to form a "checkerboard" pattern by transfer of
equal volumes (using a multi-channel pipette) to the drug combination plate.
Row H and Column 12 each contained serial dilutions of one of the agents
alone for determination of the MIC.
The "daughter plates" were loaded with 180 pL of test medium
using the Multidrop 384. Then, the Multimek 96 was used to transfer 10 pt of
drug solution from each well of the drug combination mother plate to each
.. corresponding well of the daughter plate in a single step. Finally, the
daughter
plates were inoculated with test organism. Standardized inoculum of each
organism was prepared per published guidelines (CLSI, 2009). For all isolates,

the inoculum for each organism was dispensed into sterile reservoirs divided
by
length (Beckman Coulter), and the Bionnek 2000 was used to inoculate the
plates. The instrument delivered 10 ill of standardized inoculum into each
well
to yield a final cell concentration in the daughter plates of approximately 5
x 105
colony-forming-units/mL.
The test format resulted in the creation of an 8 x 12 checkerboard
where each compound was tested alone (Column 12 and Row H) and in
.. combination at varying ratios of drug concentration. All organism plates
were
stacked three high, covered with a lid on the top plate, placed in plastic
bags,
and incubated at 35 C for approximately 20 hours. Following incubation, the
microplates were removed from the incubators and viewed from the bottom
using a ScienceWare plate viewer. Prepared reading sheets were marked for
the MIC of drug 1 (row H), the MIC of drug 2 (column 12) and the wells of the
growth-no growth interface.
An Excel program was used to determine the FIG according to the
formula: (MIC of Compound 1 in combination/MIC of Compound 1 alone) +
(MIC of Compound 2 in combination/MIC of Compound 2 alone). The FICI for
the checkerboard was calculated from the individual FICs by the formula: (FICi
+ FIC2+ FICn)/n, where n = number of individual wells per plate for which

FICs were calculated. In instances where an agent alone yielded an off-scale
MIC result, the next highest concentration was used as the MIC value in the
FIG calculation.
Microparticulate Bis-EDT, the four microparticulate BT analogs,
and all of the other agents (and combinations of agents) were soluble at all
final
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test concentrations. The MIC and FICI values that were determined are
presented in the Tables below.
TABLE 28
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Piperacillin
Compound 1 Compound 2
FICI2
Organism' MIC1 MIC
Name (liginiL) Name (lAgimL)
Alone Alone
P. aeruginosa 1381 1 >64 0.83
P. aeruginosa 1384 1 8 0.96
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-1B-3 0.5 Piperacillin 8 1.12
P. aeruginosa 2566 0.5 32 1.37
P. aeruginosa 2568 1 8 0.71
P. aeruginosa 103 1 8 0.79
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 29
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Aztreonam
Compound 1 Compound 2
MIC1 MIC
Organism' Name
(PgimL) Name (PginiL) FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.04
P. aeruginosa 1384 1 8 0.71
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-1B-3 0.5 Aztreonam 8 0.87
P. aeruginosa 2566 0.5 16 1.37
P. aeruginosa 2568 1 8 0.71
P. aeruginosa 103 1 4 1.29
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 30
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Piperacillin
Compound 1 Compound 2
MIC' MIC
Organisml Name (ug/mL)
Name Clig/ITIL) FICI2
Alone Alone
P. aeruginosa 1381 1 >64 1.29
P. aeruginosa 1384 1 16 0.71
P. aeruginosa 1474 1 8 1.12
P. aeruginosa 1479 MB-15 1 Piperacillin 8 1.29
P. aeruginosa 2566 1 32 1.04
P. aeruginosa 2568 1 8 1.12
P. aeruginosa 103 2 8 0.73
1MI0, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 31
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Aztreonam
Compound 1 Compound 2
MIC' MIC
Organism.' Name
(PginnL) Name (Pgiml-) FICI2
Alone Alone
P. aeruginosa 1381 2 32 1.11
P. aeruginosa 1384 1 8 0.79
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-15 2 Aztreonam 8 0.67
P. aeruginosa 2566 0.5 16 1.12
P. aeruginosa 2568 1 8 0.79
P. aeruginosa 103 2 4 1.23
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 32
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Piperacillin
Compound 1 Compound 2
MIC' MIC
Organism.' Name (ug/mL)
Name (Pgin11-) FICI2
Alone Alone
P. aeruginosa 1381 2 >64 1.23
P. aeruginosa 1384 2 16 0.73
P. aeruginosa 1474 2 8 1.23
P. aeruginosa 1479 MB-8-2 2 Piperacillin 8 1.23

P. aeruginosa 2566 2 32 1.23
P. aeruginosa 2568 2 8 0.98
P. aeruginosa 103 4 8 1.19
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE33
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Aztreonam
Compound 1 Compound 2
MIC1 MIC
Organisml Name
(PginiL) Name (Pgiml-) FICI2
Alone Alone
P. aeruginosa 1381 2 32 1.11
P. aeruginosa 1384 2 8 1.11
P. aeruginosa 1474 2 8 0.73
P. aeruginosa 1479 MB-8-2 2 Aztreonam 8 0.98
P. aeruginosa 2566 2 16 1.23
P. aeruginosa 2568 2 8 0.98
P. aeruginosa 103 4 8 1.19
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 34
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Piperacillin
Compound 1 Compound 2
MIC1 MIC
Organisml Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
P. aeruginosa 1381 1 >64 1.12
P. aeruginosa 1384 1 16 0.71
P. aeruginosa 1474 1 8 1.12
P. aeruginosa 1479 MB-11 1 Piperacillin 8 1.29
P. aeruginosa 2566 0.5 32 1.12
P. aeruginosa 2568 1 8 1.12
P. aeruginosa 103 2 8 1.11
1MI0, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 35
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Aztreonam
Compound 1 Compound 2
MIC' MIC
Organisml Name
(PgimL) Name (Pgiml-) FICI2
Alone Alone
P. aeruginosa 1381 2 32 0.92
P. aeruginosa 1384 1 8 0.96
P. aeruginosa 1474 1 8 0.71
P. aeruginosa 1479 MB-11 1 Aztreonam 8 0.79
P. aeruginosa 2566 0.5 16 1.12
P. aeruginosa 2568 1 8 0.96
P. aeruginosa 103 2 8 1.11
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 36
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Piperacillin
Compound 1 Compound 2
MIC' MIC
Organisml Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
P. aeruginosa 1381 2 >64 1.02
P. aeruginosa 1384 8 16 0.79
P. aeruginosa 1474 8 8 0.91
P. aeruginosa 1479 MB-2B 8 Piperacillin 8 1.08
P. aeruginosa 2566 8 32 1.04
P. aeruginosa 2568 8 8 0.97
P. aeruginosa 103 8 8 1.16
1MI0, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 37
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Aztreonam
Compound 1 Compound 2
MIC' MIC
Organisml Name
(PgimL) Name (Pg/1111-) FICI2
Alone Alone
P. aeruginosa 1381 8 64 0.89
P. aeruginosa 1384 8 8 0.91
P. aeruginosa 1474 8 8 0.54
P. aeruginosa 1479 MB-2B 8 Aztreonam 8 0.87
P. aeruginosa 2566 8 16 0.91
P. aeruginosa 2568 8 8 0.87
P. aeruginosa 103 8 8 1.08
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 38
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1B-3 and Cefotaxime
Compound 1 .. Compound 2
MIC' MIC
Organism.' Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
K. pneumoniae 1346 2 0.06 1.23
K. pneumoniae 1355 1 0.06 2.29
K. pneumoniae 2238 1 16 1.29
K. pneumoniae 2541 MB-1B-3 2 Cefotaxime 0.12 1.23
K. pneumoniae 2546 1 0.25 1.12
K. pneumoniae 2549 1 0.12 0.79
P. aeruginosa 103 1 16 0.96
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 39
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-1 B-3 and Cefepime
Compound 1 Compound 2
MIC' MIC
Organisml Name
(PginiL) Name (PgimL) FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.29
P. aeruginosa 1384 1 2 0.79
P. aeruginosa 1474 1 2 0.79
P. aeruginosa 1479 MB-1B-3 1 Cefepime 4 1.12
P. aeruginosa 2566 0.5 a 1.37
P. aeruginosa 2568 1 2 0.79
P. aeruginosa 103 1 2 0.71
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 40
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Cefotaxime
Compound 1 Compound 2
MIC' MIC
Organism.' Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
K. pneumoniae 1346 2 0.06 1.23
K. pneumoniae 1355 1 0.12 2.37
K. pneumoniae 2238 2 16 1.23
K. pneumoniae 2541 MB-15 2 Cefotaxime 0.12 1.23
K. pneumoniae 2546 2 0.25 0.97
K. pneumoniae 2549 2 0.06 1.23
P. aeruginosa 103 1 16 0.96
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 41
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-15 and Cefepime
Compound 1 Compound 2
MIC' MIC
Organisml Name (PgimL) Name (Pgiml-
) FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.29
P. aeruginosa 1384 1 2 0.79
P. aeruginosa 1474 1 2 1.12
P. aeruginosa 1479 MB-15 1 Cefepime 4 1.12
P. aeruginosa 2566 0.5 8 1.37
P. aeruginosa 2568 1 2 1.12
P. aeruginosa 103 1 1 1.12
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 42
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Cefotaxime
Compound 1 Compound 2
MIC1 MIC
Organism.' Name (pr)
Name (Pgin11-) FICI2
Alone Alone
K. pneumoniae 1346 0.5 0.06 1.37
K. pneumoniae 1355 0.5 0.06 1.37
K. pneumoniae 2238 0.5 16 1.37
K. pneumoniae 2541 MB-8-2 1 Cefotaxime 0.12
1.12
K. pneumoniae 2546 1 0.25 1.29
K. pneumoniae 2549 1 0.06 1.12
P. aeruginosa 103 2 16 1.11
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 43
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-8-2 and Cefepime
Compound 1 Compound 2
MIC1 MIC
Organism' Name (PgimL) Name (PginnL)
FICI2
Alone Alone
P. aeruginosa 1381 2 32 1.23
P. aeruginosa 1384 2 2 0.80
P. aeruginosa 1474 2 2 1.11
P. aeruginosa 1479 MB-8-2 2 Cefepime 4 1.23
P. aeruginosa 2566 2 8 1.23
P. aeruginosa 2568 2 2 0.98
P. aeruginosa 103 2 1 1.11
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 44
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Cefotaxime
Compound 1 Compound 2
MIC1 MIC
Organism.' Name (pg/mL)
Name (pg/mL) Fici2
Alone Alone
K. pneumoniae 1346 0.5 0.06 1.37
K. pneumoniae 1355 0.5 0.06 1.87
K. pneumoniae 2238 0.5 8 1.37
K. pneumoniae 2541 MB-11 0.5 Cefotaxime 0.25 0.73
K. pneumoniae 2546 0.5 0.25 1.37
K. pneumoniae 2549 0.5 0.06 1.37
P. aeruginosa 103 1 16 1.12
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 45
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-11 and Cefepime
Compound 1 Compound 2
MIC' MIC
Organisml Name (PginiL) Name (PginiL) FICI2
Alone Alone
P. aeruginosa 1381 1 32 1.12
P. aeruginosa 1384 1 2 1.12
P. aeruginosa 1474 0.5 2 1.12
P. aeruginosa 1479 MB-11 0.5 Cefepime 8 0.87
P. aeruginosa 2566 0.5 16 0.93
P. aeruginosa 2568 0.5 2 0.87
P. aeruginosa 103 1 1 0.12
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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TABLE 46
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Cefotaxime
Compound 1 Compound 2
MIC' MIC
Organism.' Name (ug/mL)
Name (pg/mL) Fici2
Alone Alone
K. pneumoniae 1346 4 0.06 1.19
K. pneumoniae 1355 4 0.06 1.19
K. pneumoniae 2238 4 8 1.64
K. pneumoniae 2541 MB-2B 8 Cefotaxime 0.25 0.64
K. pneumoniae 2546 8 0.25 1.16
K. pneumoniae 2549 8 0.12 0.83
P. aeruginosa 103 2 16 1.11
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
TABLE 47
Summary of Minimum Inhibitory Concentration and Fractional Inhibitory
Concentration Results for MB-2B and Cefepime
Compound 1 Compound 2
MIC' MIC
Organisml Name (PginiL) Name (PgirilL) FICI2
Alone Alone
P. aeruginosa 1381 4 32 1.09
P. aeruginosa 1384 4 2 0.94
P. aeruginosa 1474 2 2 0.98
P. aeruginosa 1479 MB-2B 2 Cefepinne 4 1.11
P. aeruginosa 2566 2 8 1.23
P. aeruginosa 2568 2 2 1.11
P. aeruginosa 103 2 2 0.61
1MIC, Minimum Inhibitory Concentration
2FICI, Fractional Inhibitory Concentration Index
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EXAMPLE 11
THE EFFECT OF BISMUTH THIOLS ON INFECTION IN A RATTUS NORVEGICUS
FEMUR CRITICAL DEFECT
The current standard of care for open fractures is irrigation,
debridennent and antibiotics; this is intended to reduce the bacterial load in
the
wound to the point that infection does not occur. Despite these treatments,
infections still complicate up to 75% of severe combat open tibia fractures.
Interestingly, even though early infections are often caused by gram negative
bacteria, late infections that are implicated in healing problems and
amputation
are due to gram positive infections, frequently Staphylococci species (Johnson

2007).
One of the reasons that S. aureus are resistant to standard
treatment is their ability to form a biofilm. Bacteria in biofilnns are able
to resist
concentrations of antimicrobial compounds which would kill similar organisms
in
a culture medium (Costerton 1987).
The aim of this study was to determine whether BTs will reduce
infection in a contaminated open fracture model either on their own or with
antibiotics. The contaminated rat femur critical defect model is a well-
accepted
model and was used for the experiments described in this Example. This
model offers a standardized model for comparing various possible treatments
and their effects on reducing infection and/or improving healing.
Compounds (CPD) CPD-8-2 (bismuth pyrithione/ butanedithiol;
Table 1) and CPD-11 (bismuth pyrithione/ ethanedithiol; Table 1) are two
analogues of BIS-Bis that have shown potential against Biofilm secreting
bacteria in vitro, though with a different spectrum of activity than Bis-EDT.
The three BT formulations, Bis-EDT, CPD-11 and CPD-8-2 (see
Table 1) demonstrated inhibitory effects on S. aureus strains in vitro when
used
with and without Tobramicin and Vancomycin in a Poly Methyl Methacrylate
(PM MA) cement bead vehicle. Three formulations of microparticulate BTs were
produced in a clinically useful hydrogel gel form as described herein. These
BTs were tested suspended in a gel at a concentration of 5mg/m1-1 as has been
found to be an appropriate concentration for gel delivery. The gel
formulations
conformed to the wound contours, and did not require removal following
application.
Two treatment arms were used: in the first arm, BT was used
singularly; in the second arm BT was used in conjunction with a systemic
antibiotic (ABx).
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(a) BT Singularly.
Six hours after inoculation with S. aureus, the wound was
debrided, irrigated with saline and lml of BT gel inserted within the defect.
(b) BT with Systemic Antibiotics (ABx).
Six hours after inoculation with S. aureus, the wound was
debrided, irrigated with saline and Imi of BT gel added inserted within the
defect. The antibiotic used was Cefazolin at a dose equivalent to 5nrigKg-1
delivered via sub-cutaneous injection twice daily for a total of 3-days
following
the injury. The first dose was administered immediately prior to debridement.
Previous data suggested that this dose would result in a reduction in bacteria
levels from :z106 to =104 and therefore still allow the relative effect of
different
BTs to be measured.
(c) Control
Six hours after inoculation with S. aureus, the wound was
debrided and irrigated with saline. The control animals were also treated with
Cefazolin as per the regime described above.
PROCEDURE:
The procedure for the in vivo rat injury model was performed as
described by Chen et al. (2002 J. Orthop. Res. 20:142; 2005 J. Orthop. Res.
23:816; 2006 J. Bone Joint Surg. Am. 88:1510; 2007 J. Orthop. Trauma
21:693). The rats were anesthetized and prepped for surgery. The
anterolateral aspect of the femoral shaft was exposed through a 3-cm incision.

The periosteum and attached muscle was stripped from the bone. A polyacetyl
plate (27 x 4 x 4 mm) was placed on the anterolateral surface of the femur.
The
plates were predrilled to accept 0.9-mm diameter threaded Kirschner wires.
The bases of these plates were formed to fit the contour of the femoral shaft.

Pilot holes were drilled through both cortices of the femur using the plate as
a
template and threaded Kirschner wire was inserted through the plate and
femur. The notches that were 6 mm apart on the plate served as a guide for
bone removal. A small oscillating saw was used to create the defect while the
tissue was cooled by continuous irrigation in an effort to prevent thermal
damage.
Several groups of 10 animals each were inoculated with 1x105
CFU of S. aureus and treated with BT alone or in combination with antibiotics
6
hours post-inoculation as described above. The groups were as follows: Bis-
EDT gel; MB-11 gel; MB-8-2 gel; Bis-EDT gel & Abx; MB-11 gel & Abx; MB-8-2
gel & Abx; Control (Abx alone).
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Animals were euthanized 14 days after surgery and bone and
hardware sent for microbiological analysis, the results of which are shown in
Figure 7.
Based on the power analysis, 10 animals per group will give a
power of 80% to detect a 25% difference between the treatment and control
groups. This is with an expected standard deviation of 35% and alpha of 0.05.
As shown in Figure 7, in combination with Bis-EDT, MB-11 and
MB-8-2, Cefazolin antibiotic activity was enhanced as compared to Cefazolin or

any of the Bis compounds alone to reduce S. aureus infection of injured bone.
Cefazolin in combination with MB-11 and MB-8-2 showed enhanced antibiotic
activity as compared to Cefazolin alone to reduce S. aureus infection detected

on hardware. Bis-EDT did not appear to affect Cefazolin activity in this
capacity.
EXAMPLE 12
ACTIVITY OF BISMUTH CONTAINING COMPOUNDS AGAINST MARINE ORGANISMS
This example describes the antimicrobial activity of bismuth
containing compounds. The MIC values of three bismuth containing
compounds, bismuth dinnercaprol (BisBAL), bismuth dimercaptotoluene
(BisTOL), and bismuth ethanedithiol (BisEDT), against three different marine
bacteria were determined using methods routinely practiced by persons skilled
in the art. The data are presented in the following table.
BT Compound (pg/m1) BisBAL BisTOL BisEDT
V. alginolyticus 3.1 1 0.1
H. marina 17.5 7.2 2.6
M. hydrocarbonoclasicus 2 0.4 .28
EXAMPLE 13
EFFECT OF BISMUTH CONTAINING COMPOUNDS ON BARNACLE SETTLEMENT
BEHAVIOR
Compounds, BisBAL and BisTOL were included in an assay to
determine the inhibitory activity of each compound on barnacle larvae
settlement behavior. Methods were performed according to techniques
practiced in the art. BisBAL had an EC50 (the concentration at which 50%
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settlement inhibition occurs) of 1.6 ppm, and BisTOL had an EC50 of 15.4 PPm=
In another experiment, BisEDT was dissolved either directly in natural
seawater
or first dissolved in DMSO and then diluted in natural seawater. The EC50
measurements were not statistically different. BisEDT had an EC50 of 1.5 ppm
when dissolved directly in seawater and had an EC50 of 2.1 ppm when first
dissolved in DMSO. The EC50 of the commercial biocide, SEANINE 211, was
0.5 ppm.
EXAMPLE 14
EFFECT OF BISMUTH CONTAINING COMPOUNDS ON SETTLEMENT OF ALGAE
The effect of three bismuth containing compounds, bismuth
dimercaprol (BisBAL), bismuth dimercaptotoluene (BisTOL), and bismuth
ethanedithiol (BisEDT), on the settlement of algae was determined,
particularly
the ability of each compound to inhibit germination of Enteronnorpha spores.
Each compound was tested at 0.001, 0.01, 0.1, 1.0, and 10.0 pg/ml. BisEDT
was the most effective compound; at 1 ug/nnl BisEDT, germination of
approximately 50% of the algae spore population was inhibited, and at 10
pg/ml, germination of approximately 75% algae spores was inhibited. Up to ten
micrograms per ml of BisBAL and BisTOL had no inhibitory effect on
germination of the spores of this particular algae species.
EXAMPLE 15
EFFECT OF BISMUTH CONTAINING COMPOUNDS ON SETTLEMENT OF ALGAE
The effect of three bismuth containing compounds, bismuth
dimercaprol (BisBAL), bismuth dimercaptotoluene (BisTOL), and bismuth
ethanedithiol (BisEDT), on growth of a marine diatom was determined
according to techniques practiced in the art. Settlement of marine diatoms
(diatoms per toy) was inhibited by increasing concentrations of each of the
three compounds (0.001, 0.01, 0.1, 1.0, and 10.0 pg/ml). Each compound
.. exhibited inhibitory activity at 0.1 pg/ml; BisEDT was the most active,
demonstrating nearly 100% inhibition. Each of BisTOL and BisBAL exhibited
approximately 30% of marine diatom settlement at 0.1 pg/ml.
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The various embodiments described above can be combined to
provide further embodiments. All of the U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign patent
applications and non-patent publications referred to in this specification
and/or
listed in the Application Data Sheet.
Aspects of the embodiments can be modified, if necessary to
employ concepts of the various patents, applications and publications to
provide yet further embodiments.
These and other changes can be made to the embodiments in
light of the above-detailed description. In general, in the following claims,
the
terms used should not be construed to limit the claims to the specific
embodiments disclosed in the specification and the claims, but should be
construed to include all possible embodiments along with the full scope of
equivalents to which such claims are entitled. Accordingly, the claims are not
limited by the disclosure.
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