US 20090304783A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0304783 A1 Walsh et al. (43) Pub. Date: Dec. 10, 2009

(54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data TREATING BACTERIA (62) Division of application No. 1 1/545,884, filed on Oct. 11, 2006, now Pat. No. 7,576,054. (75) Inventors: Scott Michael Walsh, Poughguag. (60) Provisional application No. 60/725,193, filed on Oct. NY (US); Mary Catherine 11, 2005, provisional application No. 60/779,034, Pittaway, Poughguag, NY (US); filed on Mar. 3, 2006. James J. Mond, Silver Spring, MD (US) Publication Classification (51) Int. Cl. Correspondence Address: A 6LX 9/27 (2006.01) Casimir Jones, S.C. A638/16 (2006.01) 2275 DEMINGWAY, SUITE 310 (52) U.S. Cl...... 424/450; 514/9 MIDDLETON, WI 53562 (US) (57) ABSTRACT The present invention relates to the field of bacteriology. In (73) Assignee: BIOSYNEXUS particular, the present invention provides compositions (e.g., INCORPORATED, Gaithersburg, comprising a lantibiotic and mupirocin or gentamicin) and MD (US) methods of treating (e.g., killing or inhibiting growth of) bacteria. For example, the present invention provides phar (21) Appl. No.: 12/542,462 maceutical compositions (e.g., comprising a lantibiotic and mupirocin or gentamicin) and methods of using the same in (22) Filed: Aug. 17, 2009 research, therapeutic and drug screening applications. Patent Application Publication Dec. 10, 2009 Sheet 1 of 13 US 2009/0304783 A1

FIGURE 1

Patent Application Publication Dec. 10, 2009 Sheet 2 of 13 US 2009/0304783 A1

FIGURE 2

8.00

7. O O

6. O O

5. OO

4. O O

3. O O

2. O O

1.OO

O.OO 6%. Nisin 2% 6%. Nisin + 6%. Nisin- 5OOU 6%. Nisin- G&W Control Lysostaphin 2% 5OOU Bacitracin G&W Bacitracin Lysostaphin Bacitracin Bacitracin Ointment Ointment (500U) (50OU)

Patent Application Publication Dec. 1 0, 2009 Sheet 4 of 13 US 2009/0304783 A1

FIGURE 4

Patent Application Publication Dec. 10, 2009 Sheet 5 of 13 US 2009/0304783 A1

FIGURES

Baselite --> Number of Animals Treatment Groups Assessment 2-> Days 5-> 7->

1. A: Active X * A?ter Treatment B: Active Y C: Vehicle D. Mupirocin E: Untreated air exposed

Day 0 = Wounding & Inoculation Day 1 = 24 hours after inoculation Day 2 = 48 hour biofilm (Day of Initial Treatment) Day 3 = 2" day of treatment Day 4 = First Assessment Time (48 hours or Day 2 after initial treatment) – 3 day of treatment Day 5 = 4" day of treatment Day 6 = 5" day of treatment Day 7 = Second Assessment Time (168 hours or Day 5 after initial treatment) – 6" day of treatment Day 8 = 7 day of treatment Day 9 - Last Assessment Time (216 hours or Day 7 after initial treatment). Patent Application Publication Dec. 10, 2009 Sheet 6 of 13 US 2009/0304783 A1

FIGURE 6

Day 2- Staphylococcus aureus Recovery

ACtive X ACtive Y Vehicle Mupirocin Control Patent Application Publication Dec. 10, 2009 Sheet 7 of 13 US 2009/0304783 A1

FIGURE 7

Day 5- Staphylococcus aureus Recovery

9 - 8 7 6 5 4 T 3 E m w

Active X ACtive Y Vehicle Mupirocin Control Patent Application Publication Dec. 10, 2009 Sheet 8 of 13 US 2009/0304783 A1

FIGURE 8

Day 7- Staphylococcus aureus Recovery

7 6 SE 5

3 no

2 opo I 1 O Active X Active Y Vehicle Mupirocin Control Patent Application Publication Dec. 10, 2009 Sheet 9 of 13 US 2009/0304783 A1

FIGURE 9

StaphyloCoCCuSaureus Recovery BL E Day 2 E Day 5 P. Day 7

Baseline Active X Active Y Vehicle Mupiroci Control Patent Application Publication Dec. 10, 2009 Sheet 10 of 13 US 2009/0304783 A1

FIGURE 10

Recovered MSSA-24h PT

Y Y B: 1 Application Y - Š2 Application Y Y Y s

Y Y Y s

Y Y Y s

Y Y Y Y

Baseline Active Y Vehicle Mupirocin Control

Recovered MRSA- BD DOS Ing

Baseline Active Y VehicleH_ Mupirocin Control Patent Application Publication Dec. 10, 2009 Sheet 11 of 13 US 2009/0304783 A1

FIGURE 11

Gentamicin and Nisin

0.035 0.035: 0.033: O.035:

&:sis oasis is sis.

Patent Application Publication Dec. 10, 2009 Sheet 13 of 13 US 2009/0304783 A1

FIGURE 13

8

:

x: Untreated Control 6%. Nisin 6%. Nisin + 2% 2% Mupirocin Mupirocin US 2009/0304783 A1 Dec. 10, 2009

COMPOSITIONS AND METHODS FOR 0007. The problem of antibiotic resistance is not unique to TREATING BACTERIA Enterococcus spp. Strains of many other potentially patho genic Gram-positive bacteria displaying antibiotic resistance have been isolated including methicillin-resistant Staphyllo 0001. This invention claims priority to U.S. patent appli coccus aureus (MRSA), Vancomycin-resistant Staphylococ cation Ser. No. 1 1/545,884, filed Oct. 11, 2006, which claims cus aureus (VRSA), glycopeptide intermediate-Susceptible priority to U.S. Provisional Patent Application Ser. Nos. Staphylococcus aureus (GISA), Vancomycin-resistant 60/725,193, filed Oct. 11, 2005, and 60/779,034, filed Mar. 3, MRSA (VR-MRSA) and penicillin-resistant Streptococcus 2006, each of which is hereby incorporated by reference in its pneumoniae (PRSP). Like VRE, therapeutic options for treat entirety. ing infections by these organisms are limited. 0008 Resistance transfer is another complicating factor in the management of antibiotic-resistant infections. Vancomy FIELD OF THE INVENTION cin resistance can transfer from VRE to other Gram-positive 0002 The present invention relates to the field of bacteri bacteria, including S. aureus, in vitro. Thus, the presence of ology. In particular, the present invention provides composi resistant bacteria (e.g., VRE) in a hospital poses not just the tions (e.g., comprising a lantibiotic and mupirocin or gen risk of infection but also the continued evolution of resistant tamicin) and methods of treating (e.g., killing or inhibiting organisms (e.g., creating more Virulent organisms such as growth of) bacteria. For example, the present invention pro VR-MRSA). vides pharmaceutical compositions (e.g., comprising a lanti 0009. A need exists to develop alternative strategies of biotic and mupirocin or gentamicin) and methods of using the antibacterial treatment. For example, there exists a need for same in research, therapeutic and drug screening applica new compositions and methods of treating or preventing bac tions. terial infection (e.g., bacteremia) caused by Strains of bacteria unsusceptible to current forms of antibacterial treatments BACKGROUND OF THE INVENTION (e.g., Gram-positive bacteria such as MRSA and VRE). 0003. As the use of conventional pharmaceutical antibiot SUMMARY OF THE INVENTION ics has increased for medical, veterinary and agricultural 0010. The present invention relates to the field of bacteri purposes, there has been a concurrent emergence of antibi ology. In particular, the present invention provides composi otic-resistant strains of pathogenic bacteria. tions (e.g., comprising a lantibiotic and mupirocin or gen 0004. The emergence of single- or multi-drug resistant tamicin) and methods of treating (e.g., killing or inhibiting bacteria can result from a gene mobilization that responds to growth of) bacteria. For example, the present invention pro selective pressures associated with antibiotic use. Over the vides pharmaceutical compositions (e.g., comprising a lanti last several decades, the increasingly frequent usage of anti biotic and mupirocin or gentamicin) and methods of using the biotics has acted in concert with spontaneous mutations aris same in research, therapeutic and drug screening applica ing in the bacterial gene pool to produce different strains of tions. bacteria not susceptible to current antibacterial treatments. 0011. Accordingly, in some embodiments, the present This repertoire of antibiotic resistant genes can be utilized by invention provides a pharmaceutical composition comprising previously sensitive strains that have access to these genes a lantibiotic and mupirocin. In some embodiments, the lanti (e.g., via conjugative transfer of plasmids or transposons). As biotic is nisin. The present invention is not limited by the type a result, single- and multi-drug resistance genes are com of lantibiotic utilized. Indeed, a variety of lantibiotics find use monly found in a large variety of bacterial plasmids and in the present invention including, but not limited to, Subtilin, conjugative transposons. epidermin, gallidermin, pep 5, cinnamycin, duramycin and 0005 Gram-positive bacteria are a major cause of noso ancovenin. In some embodiments, the pharmaceutical com comial infection. The most common pathogenic isolates in position is formulated as a cream. The present invention is not hospitals include Enterococcus spp., Staphylococcus aureus, limited by any particular formulation of the pharmaceutical coagulase-negative staphylococci, and Streptococcus pneu composition. Indeed, a variety of formulations find use in the moniae (See, e.g., Principles and Practice of Infectious Dis present invention including, but not limited to, spray formu eases, 4th ed. Mandell G. L. Bennett J E. Dolin R, ed. lations and timed-release formulations. A pharmaceutical Churchill Livingstone, New York 1995), many strains of composition comprising a lantibiotic and mupirocin may also which are resistant to one or more antibiotics. Enterococcus be formulated for administering in a particular way. For spp. are part of the normal gut flora in humans. Of the more example, in some embodiments, a pharmaceutical composi than seventeen enterococcal species, only E. faecalis and E. tion of the present invention may be a solution (e.g., colloidal faecium commonly colonize and infect humans in detectable Solution), may be mixed with fibringlue, may be impregnated numbers (E. faecalis is isolated from approximately 80% of onto a wound dressing or bandage, may be applied by a human infections, and E. faecium from most of the rest). controlled-release mechanism; may be impregnated on one or 0006 Vancomycin-resistant enterococcus (VRE) spp. are both sides of anacellular biological matrix, or may beformu becoming increasingly common in hospital settings. In the lated with a liposome or a polymer. first half of 1999, 25.9% of entercoccal isolates from Inten 0012. The present invention also provides a method for sive Care Units were Vancomycin-resistant; an increase from treating bacterial cells comprising: providing a Surface com 16.6% in 1996 and from 0.4% in 1989. VRE are also com prising bacterial cells; and exposing the Surface to a pharma monly resistant to many other commercial antibiotics, includ ceutical composition comprising a lantibiotic and mupirocin. ing beta-lactams and aminoglycosides. Thus, patients who In some embodiments, treating comprises killing bacterial are immunocompromised or those having a prolonged hos cells present within an existing bacterial infection. In some pital stay are at increased risk for acquiring a VRE infection. embodiments, treating comprises prophylactically prevent US 2009/0304783 A1 Dec. 10, 2009

ing a bacterial infection. The present invention is not limited 0025 FIG. 13 shows that nisin and mupirocin function by the type of bacteria present within a bacterial infection synergistically to treat S. aureus in a Suture infection mouse treated via exposure to a composition comprising a lantibiotic model. and mupirocin. In some embodiments, the bacterial cells treated (e.g., killed) and/or prevented from growing comprise DEFINITIONS Staphylococcus aureus. In some embodiments, the bacterial cells treated and/or prevented from growing comprise Sta 0026. As used herein, the term “subject” refers to an indi phylococcus epidermidis. In some embodiments, the Staphy vidual (e.g., human, animal, or other organism) to be treated lococcal bacteria are drug resistant (e.g., methicillin-resis by the methods or compositions of the present invention. tant). In some embodiments, the Surface treated comprises Subjects include, but are not limited to, mammals (e.g., skin of a subject. The present invention is not limited by the murines, simians, equines, bovines, porcines, canines, type of surface treated. Indeed, a variety of surfaces can be felines, and the like), and most preferably includes humans. treated with a pharmaceutical composition of the present In the context of the invention, the term “subject' generally invention including, but not limited to, other types of organic refers to an individual who will receive or who has received Surfaces (e.g., a mucosal Surface, a wound Surface, a food treatment for a condition characterized by the presence of Surface) as well as inorganic Surfaces (e.g., medical devices, bacteria (e.g., pathogenic bacteria Such as MRSA), or in countertops, clothing, etc.). In some embodiments, treating anticipation of possible exposure to bacteria. As used herein, results in a 3 log or greater reduction in the number of bacte the terms “subject' and “patient” are used interchangeably, rial cells present on the Surface. In some embodiments, the unless otherwise noted. reduction occurs within three days of treating. In some 0027. The term “diagnosed, as used herein, refers to the embodiments, the reduction occurs within two days of treat recognition of a disease (e.g., caused by the presence of ing. In some embodiments, treating results in a lack of detect pathogenic bacteria) by its signs and symptoms (e.g., resis able bacteria on the surface. tance to conventional therapies), or genetic analysis, patho logical analysis, histological analysis, and the like. DESCRIPTION OF THE DRAWINGS 0028. As used herein the term, “in vitro” refers to an 0013 FIG. 1 shows the efficacy of nisin alone, mupirocin artificial environment and to processes or reactions that occur alone, and the combination of nisin and mupirocin on S. within an artificial environment. In vitro environments aureus infection in abraded mouse skin. include, but are not limited to, test tubes and cell cultures. The 0014 FIG. 2 shows the efficacy of nisin, lysostaphin, and term “in vivo” refers to the natural environment (e.g., an bacitracin alone and in combination on S. aureus infection in animal or a cell) and to processes or reaction that occur within abraded mouse skin using two different formulations of baci a natural environment. tracin. 0029. As used herein, the terms “attenuate” and “attenua 0015 FIG. 3 shows the efficacy of nisin alone, mupirocin tion used in reference to a feature (e.g. growth) of a bacterial alone, and the combination of nisin and mupirocin on mupi cell or a population of bacterial cells refers to a reduction, rocin-resistant S. aureus infection in abraded mouse skin. inhibition or elimination of that feature, or a reducing of the 0016 FIG. 4 shows the efficacy of nisin and mupirocin in effect(s) of that feature. combination with EDTA on P. aeruginosa infection in 0030. As used herein, the term “effective amount refers to abraded mouse skin. the amount of a composition (e.g., a composition comprising 0017 FIG. 5 depicts the experimental design of using mupirocin and nisin) sufficient to effectabeneficial or desired compositions and methods of the present invention to treat result (e.g., bacterial cell killing). An effective amount can be partial thickness wounds. administered in one or more administrations, applications or 0018 FIG. 6 shows Staphylococcus aureus growth in dosages and is not intended to be limited to a particular inoculated wounds two days after treatment with various formulation or administration route. agents. 0031. As used herein, the term “administration” refers to 0019 FIG. 7 shows Staphylococcus aureus growth in the act of giving a drug, prodrug, or other agent, ortherapeutic inoculated wounds five days after treatment with various treatment (e.g., a composition of the present invention) to a agents. physiological system (e.g., a Subject or in Vivo, in vitro, or ex 0020 FIG. 8 shows Staphylococcus aureus growth in Vivo cells, tissues, and organs). Exemplary routes of admin inoculated wounds seven days after treatment with various istration to the human body can be through the eyes (oph agents. thalmic), mouth (oral), skin (transdermal), nose (nasal), lungs 0021 FIG.9 shows a composite of Staphylococcus aureus (inhalant), mucosal (e.g., oral mucosa or buccal), rectal, ear, growth in inoculated wounds two, five and seven days after by injection (e.g., intravenously, Subcutaneously, intratumor treatment with various agents. ally, intraperitoneally, etc.) and the like. 0022 FIG. 10 shows the mean log(CFU/ml) of bacteria 0032. As used herein, the term “treating a surface” refers recovered after either a first or second treatment (MSSA) or to the act of exposing a Surface to one or more compositions after one and three days (MRSA) of treatment with the of the present invention. Methods of treating a surface reagents indicated. include, but are not limited to, spraying, misting, Submerging, 0023 FIG. 11 shows the minimum inhibitory concentra and coating. The present invention is not limited by the type tion (MIC) of treatment of S. aureus in vitro with nisin alone of Surface treated. In some embodiments, the Surface is an or nisin in combination with either mupirocin, neomycin or organic Surface (e.g., a food product Surface, a subjects (e.g., gentamicin. including, but not limited to, skin, mucosal, and wound (e.g., 0024 FIG. 12 shows that mupirocin does not synergize Superficial wound or non Superficial wound (e.g., a non-Su with or provide additive benefit together with bacitracin in perficial cut)) surface), and inorganic Surfaces (e.g., medical treating skin infection. devices, countertops, clothing, etc.)). US 2009/0304783 A1 Dec. 10, 2009

0033. As used herein, the term “co-administration” refers devices, include, but are not limited to, tampons, sponges, to the administration of at least two agent(s) or therapies to a Surgical and examination gloves, and toothbrushes. Birth Subject. In some embodiments, the co-administration of two control devices include, but are not limited to, intrauterine or more agents or therapies is concurrent. In other embodi devices (IUDs), diaphragms, and condoms. ments, a first agent/therapy is administered prior to a second 0040. As used herein, the term “therapeutic agent” refers agent/therapy. Those of skill in the art understand that the to a composition that decreases the infectivity, morbidity, or formulations and/or routes of administration of the various onset of mortality in a subject contacted by a pathogenic agents ortherapies used may vary. The appropriate dosage for microorganism or that prevent infectivity, morbidity, or onset co-administration can be readily determined by one skilled in of mortality in a host contacted by a pathogenic microorgan the art. In some embodiments, when agents or therapies are ism. Therapeutic agents encompass agents used prophylacti co-administered, the respective agents or therapies are cally (e.g., in the absence of a pathogen) in view of possible administered at lower dosages than appropriate for their future exposure to a pathogen. Such agents may additionally administration alone. Thus, co-administration is especially comprise pharmaceutically acceptable compounds (e.g., desirable in embodiments where the co-administration of the adjuvants, excipients, stabilizers, diluents, and the like). In agents ortherapies lowers the requisite dosage of a potentially Some embodiments, the therapeutic agents of the present harmful (e.g., toxic) agent(s). invention are administered in the form of topical composi 0034. As used herein, the term “toxic' refers to any detri tions, injectable compositions, ingestible compositions, and mental or harmful effects on a Subject, a cell, or a tissue as the like. When the route is topical, the form may be, for compared to the same cell or tissue prior to the administration example, a Solution, cream, ointment, salve or spray. of the toxicant. 0041 As used herein, the term “pathogen refers a bio 0035. As used herein, the term “pharmaceutical composi logical agent that causes a disease state (e.g., infection, sepsis, tion” refers to the combination of an active agent (e.g., a etc.) in a host. “Pathogens' include, but are not limited to, composition comprising mupirocin and nisin) with a carrier, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, inert or active, making the composition especially suitable for prions, and parasitic organisms. diagnostic or therapeutic use in vitro, in vivo or ex vivo. 0042. The terms “bacteria' and “bacterium refer to all 0036. The terms “pharmaceutically acceptable' or “phar prokaryotic organisms, including those within all of the phyla macologically acceptable, as used herein, refer to composi in the Kingdom Procaryotae. It is intended that the term tions that do not substantially produce adverse reactions (e.g., encompass all microorganisms considered to be bacteria toxic, allergic, or immunological reactions) when adminis including Mycoplasma, Chlamydia, Actinomyces, Strepto tered to a subject. myces, and Rickettsia. All forms of bacteria are included 0037. As used herein, the term “topically’ refers to appli within this definition including cocci, bacilli, spirochetes, cation of the compositions of the present invention to the spheroplasts, protoplasts, etc. Also included within this term Surface of the skin or mucosal cells and tissues (e.g., alveolar, are prokaryotic organisms that are Gram-negative or Gram buccal, lingual, masticatory, or nasal mucosa, and other tis positive. “Gram-negative' and “Gram-positive' refer to Sues and cells that line hollow organs or body cavities). staining patterns with the Gram-staining process, which is 0038. As used herein, the term “pharmaceutically accept well known in the art. (See e.g., Finegold and Martin, Diag able carrier refers to any of the standard pharmaceutical nostic Microbiology, 6th Ed., CV Mosby St. Louis, pp. 13-15 carriers including, but not limited to, phosphate buffered (1982)). “Gram-positive bacteria” are bacteria that retain the saline solution, water, emulsions (e.g., Such as an oil/water or primary dye used in the Gram stain, causing the stained cells water/oil emulsions), and various types of wetting agents, any to generally appear dark blue to purple under the microscope. and all solvents, dispersion media, coatings, sodium lauryl “Gram-negative bacteria do not retain the primary dye used Sulfate, isotonic and absorption delaying agents, disintri in the Gram stain, but are stained by the counterstain. Thus, grants (e.g., potato starch or sodium starch glycolate), and the Gram-negative bacteria generally appear red. In some like. The compositions also may include stabilizers and pre embodiments, bacteria are continuously cultured. In some servatives. Examples of carriers, stabilizers, and adjuvants embodiments, bacteria are uncultured and existing in their are described in the art (See e.g., Martin, Remington's Phar natural environment (e.g., at the site of a wound or infection) maceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. or obtained from patient tissues (e.g., via a biopsy). Bacteria (1975), incorporated herein by reference). may exhibit pathological growth or proliferation. Examples 0039. As used herein, the term “medical devices” includes of bacteria include, but are not limited to, bacterial cells of a any material or device that is used on, in, or through a Sub genus of bacteria selected from the group comprising Salmo ject's or patient's body, for example, in the course of medical nella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, treatment (e.g., for a disease or injury). Medical devices Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, include, but are not limited to, such items as medical implants, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Sto wound care devices, drug delivery devices, and body cavity matococcus, Micrococcus, Staphylococcus, Vibrio, Aeromo and personal protection devices. The medical implants nas, Plessiomonas, Haemophilus, Actinobacillus, Pas include, but are not limited to, urinary catheters, intravascular teurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, catheters, dialysis shunts, wound drain tubes, skin Sutures, Rochalimaea, Ehrlichia, Streptococcus, Enterococcus, Aero vascular grafts, implantable meshes, intraocular devices, coccus, Gemella, Lactococcus, Leuconostoc, Pedicoccus, heart valves, and the like. Wound care devices include, but are Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, not limited to, general wound dressings, biologic graft mate Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Neis rials, tape closures and dressings, and Surgical incise drapes. seria, Campylobacter, Arcobacter, Wolinella, Helicobacter, Drug delivery devices include, but are not limited to, needles, Achronobacter, Acinetobacter; Agrobacterium, Alcaligenes, drug delivery skin patches, drug delivery mucosal patches Chryseomonas, Comamonas, Eikenella, Flavimonas, Fla and medical Sponges. Body cavity and personal protection vobacterium, Moraxella, Oligella, Pseudomonas, US 2009/0304783 A1 Dec. 10, 2009

Shewanella, Weeksella, Xanthomonas, Bordetella, Francie aureus expresses resistance to antiseptics and disinfectants, sella, Brucella, Legionella, Afipia, Bartonella, Calymmato Such as quaternary ammonium compounds, that may aid its bacterium, Cardiobacterium, Streptobacillus, Spirillum, survival in the hospital environment. Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, 0048 For serious hospital infections with multi-drug Ruminococcus, Propionibacterium, Mobiluncus, Bifidobac resistant S. aureus, Vancomycin had existed as the only effec terium, Eubacterium, Lactobacillus, Rothia, Clostridium, tive antibiotic. Vancomycin resistance is carried by conjuga Bacteroides, Porphyromonas, Prevotella, Fusobacterium, tive plasmids that can be transferred to S. aureus in a labora Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megas tory setting (Noble et al., 1992) and has appeared naturally in phaera, Veilonella, NorCardia, Actinomadura, NorCardiop enterococci (See, e.g., Arthur et al., 1993). However, the sis, Streptomyces, Micropolysporas, Thermoactinomycetes, appearance of Vancomycin resistant S. aureus has been Mycobacterium, Treponema, Borrelia, Leptospira, and reported (See, e.g., Lowry, 1998; Mathews et al., J Acquir Chlamydiae. Immune Defic Syndr. 2005 Oct. 1:40(2):155-160) in the U.S. 0043. As used herein, the term “microorganism” refers to and abroad. Vaccines have been developed targeting the any species or type of microorganism, including but not lim organism or exotoxins it produces, but these approaches have ited to, bacteria, archaea, fungi, protozoans, mycoplasma, met with little success (See, e.g., Mamo et al., 1994), under and parasitic organisms. scoring the need to develop new methods to control Staphy 0044 As used herein, the term “non-human animals’ lococcal infections. refers to all non-human animals including, but not limited to, 0049 Similar to other gram positive bacteria, S. aureus Vertebrates such as rodents, non-human primates, Ovines, causes disease chiefly through the production of virulence bovines, ruminants, lagomorphs, porcines, caprines, equines, factors such as hemolysins, enterotoxins and toxic shock canines, felines, aves, etc. syndrome toxin, which facilitate the survival, multiplication 0045. As used herein, the term "kit' refers to any delivery and spread of the organism in infected tissue (See, e.g., system for delivering materials. In the context of reaction Mekalanos, 1992). The synthesis of most virulence factors in materials (e.g., compositions comprising mupirocin and S. aureus is controlled by the accessory global regulon (agr) nisin), such delivery systems include systems that allow for locus, which is activated by secreted autoinducing peptides the storage, transport, or delivery of reaction reagents and/or (AIPs) (See, e.g., Novicket al., 1993; Novicket al., 1995). Supporting materials (e.g., written instructions for using the 0050. The presence of agr and regulation of virulence by materials, etc.) from one location to another. For example, RNAIII has been demonstrated in all strains of S. aureus kits include one or more enclosures (e.g., boxes) containing tested to date as well as several other species of staphylococci the relevant reaction reagents and/or Supporting materials. As including S. epidermidis, S. lugdunesis, S. hemolyticus (See, used herein, the term “fragmented kit' refers to delivery e.g., Vandenesch et al., 1993), and S. warneri (See, e.g., systems comprising two or more separate containers that each Tegmarket al., 1998). contain a Subportion of the total kit components. The contain 0051 Staphylococcus aureus infection remains one of the ers may be delivered to the intended recipient together or most common nosocomial and community-acquired infec separately. For example, a first container may contain a com tions. With the continuing emergence of methicillin-resistant position comprising mupirocin and nisin for a particular use, S. aureus (MRSA) and strains of S. aureus that are interme while a second container contains a second agent (e.g., an diately resistant to glycopeptides and the isolation of clinical antibiotic or spray applicator). Indeed, any delivery system strain of S. aureus that are fully Vancomycin resistant, S. comprising two or more separate containers that each con aureus is becoming an even more difficult health problem to tains a subportion of the total kit components are included in address, particularly in settings such as hospitals and nursing the term “fragmented kit.” In contrast, a “combined kit' refers homes. to a delivery system containing all of the components of a 0052 Currently, BACTROBAN (2%mupirocinointment: reaction materials needed for a particular use in a single SmithKline Beecham, Bristol, Tenn.) is the most widely pre container (e.g., in a single box housing each of the desired scribed and effective antibacterial agent for treatment of S. components). The term "kit' includes both fragmented and aureus skin infections. Mupirocin is an antibacterial agent combined kits. produced by fermentation using the organism Pseudomonas fluorescens. It is active against a wide range of gram-positive DETAILED DESCRIPTION OF THE INVENTION bacteria including most strains of S. aureus, including methi 0046 Staphylococci are gram positive bacterial pathogens cillin-resistant S. aureus (MRSA), most strains of S. epider that cause a wide variety of diseases ranging from Superficial midis, S. saprophyticus, and Streptococcus. Mupirocin inhib abscesses (e.g., boils, styes, furuncles and other localized its bacterial protein synthesis by reversibly and specifically abscesses) to deeper infections (e.g., osteomyelitis, pneumo binding to bacterial isoleucyl transfer-RNA synthetase. Due nia, endocarditis, urinary tract infections, septic arthritis, to this unique mode of action, mupirocin demonstrates no in meningitis, post-operative wound infections, septicemia and vitro cross-resistance with other classes of anti-microbial food poisoning). S. aureus is a major cause of hospital agents. Although BACTROBAN (2% mupirocin) is a com acquired (nosocomial) infection of Surgical wounds and S. mon treatment, a variety of compounds similar to mupirocin epidermidis causes infections associated with indwelling (e.g., derivatives and functional equivalents) are in use or in medical devices. (See, e.g., Silverstein et al., 1990; Pattietal. development and are known in the art. 1994; Dann et al., 1994.) 0053. Unfortunately, as with many antimicrobials, mupi 0047 Multiple antibiotic resistance is increasingly com rocin resistance is emerging in S. aureus and coagulase-nega mon in S. aureus and S. epidermidis. Hospital strains of tive staphylococci. Furthermore, this antibiotic does not rou Staphylococcus are often resistant to many different antibiot tinely eliminate all infectious organisms in all patients. ics. S epidermidis nosocomial isolates are also often resistant 0054 Nisin is an antimicrobial substance produced by to several antibiotics including methicillin. In addition, S. Lactococcus lactis belonging to the Lancefield serological US 2009/0304783 A1 Dec. 10, 2009

group. It is a member of a group of similar Substances referred present invention and the present invention is not limited to to as lantibiotics, which include Subtilin, epidermin, gallider any particular mechanism of action, it is contemplated that, in min, pep 5, cinnamycin, duramycin and ancovenin. Nisin is a Some embodiments, a composition comprising an anti-infec peptide comprised of 34-amino acid residues and contains tive peptide is capable of generating pores in bacteria thereby five ring structures cross-linked by thioether bridges that form enhancing the entry of compounds (e.g., Small molecule anti lanthionine or B-methyllanthionine. Formulations ofnisin are biotic or metabolism antibiotic) into bacteria. Although an described in U.S. Pat. Nos. 5,135,910 and 5,753,614, each of understanding of the mechanism is not necessary to practice which is herein incorporated by reference in its entirety. Vari the present invention and the present invention is not limited ants of nisin are described in U.S. Pat. No. 6,448,034, herein incorporated by reference in its entirety. Additional lantibiot to any particular mechanism of action, it is contemplated that, ics similar to nisin are described in U.S. Pat. Nos. 5,594,103 in some embodiments, the compositions of the present inven and 5,928,146, each of which is herein incorporated by ref tion may stimulate (e.g., when in contact with the skin) a host erence in its entirety. defense response (e.g., an innate immune response) thereby 0055 Nisin has broad-spectrum activity against gram decreasing the likelihood of or reducing bacterial infection. positive bacteria and some activity against gram negative 0058. In other preferred embodiments, the present inven bacteria. Nisin has been used as an antimicrobial food pre tion provides a composition comprising nisin and mupirocin. servative and is generally accepted as safe. Blackburn et al. In some embodiments, a composition comprising nisin and (U.S. Pat. No. 5,866.539, the contents of which are incorpo mupirocin is administered to a subject under conditions such rated in their entirety by reference) generally describes use of that pathogenic bacteria are killed. In some embodiments, a nisin along with anti-bacterial agents to treat skin infections. composition comprising nisin and mupirocin is administered 0056 Despite impressive successes in controlling or to a subject under conditions such that pathogenic bacteria eliminating bacterial infections by antibiotics, the wide growth is prohibited and/or attenuated. In some embodi spread use of antibiotics both in human medicine and as a feed ments, greater than 90% of (e.g., greater than 95%, 98%, Supplement in poultry and livestock production has led to 99%, all detectable) bacteria are killed. In some embodi drug resistance in many pathogenic bacteria. The emergence ments, there is greater than 2 log(e.g., greater than 3 log, 4 log, of pan-resistant Strains of Staphylococci (e.g., Strains that are 5 log, or more) reduction in bacteria. In some embodiments, unsusceptible to current forms of antibacterial treatment) the reduction is observed in two days or less following initial makes the need to control Staphylococcal infection an impor treatment (e.g., less than 24 hours, less than 20 hours, 18 tant medical concern. Thus, it is desirable to provide new hours or less). In some embodiments, the reduction is compositions and methods of treatment that display efficacy observed in three days or less, four days or less, or five days in reducing the incidence and severity of Staphylococcal or less. infection. Specifically, there is a need for new treatments for 0059. The present invention demonstrates that a composi resistant strains of bacteria, in particular, S. aureus strains that tion comprising a lantibiotic (e.g., nisin) and mupirocin is are resistant to mupirocin and other antibiotics. There is also more efficacious (e.g., in Some embodiments, provides an a need for anti-invectives that have a broader range of activity additive effect, and in other embodiments, provides a syner against gram negative bacteria, and for more effective anti gistic effect) at treating skin infections (e.g., killing bacteria invectives. Additionally, it would be beneficial for such treat or prohibiting bacterial growth (e.g., of Subcutaneous skin ments to be well tolerated by patients with minimal or no side infections or deep wound infections)) when compared to effects. either agent alone (See Example 1). When nisin was com 0057 Accordingly, the present invention provides compo bined with two other anti-bacterial agents separately (lysos sitions and methods for the treatment of bacteria (e.g., bacte taphin and bacitracin), no additive or synergistic effect was rial infection). In some embodiments, the present invention observed. Thus, in Some embodiments, the present invention provides a combinatorial treatment for bacteria (e.g., a treat provides a composition comprising mupirocin and a lantibi ment that inhibits growth of and/or that kills bacteria). For otic (e.g., nisin). example, in some embodiments, the present invention pro 0060. The compositions comprising mupirocin and a lan vides a composition comprising an anti-infective peptide tibiotic (e.g., nisin) of the present invention can be adminis (i.e., a peptide that inhibits growth or that is capable of killing tered to a subject (e.g., to the skin or other Surface of a Subject) bacteria (e.g., Gram positive bacteria)) and other type of as a therapeutic or as a prophylactic to prevent bacterial anti-infective pharmaceutical or compound (e.g., non peptide infection. It is contemplated that a composition comprising anti-infective). In some embodiments, the anti-infective pep mupirocinandalantibiotic (e.g., nisin) can be administered to tide is a cationic peptide. In some embodiments, the anti a Subject via a number of delivery mechanisms. infective peptide is a defensin. In some embodiments, the 0061 For example, the compositions of the present inven anti-infective peptide is a lantibiotic (e.g., nisin). In some tion can be administered to a Subject (e.g., to a skin burn or embodiments, the lantibiotic is any one of subtilin, epider wound Surface) by multiple methods, including, but not lim min, gallidermin, pep 5, cinnamycin, duramycin and ancov ited to:being Suspended in a solution (e.g., colloidal solution) enin. In some embodiments, the second anti-infective agentis and applied to a surface; being Suspended in a solution and a small molecule antibiotic. In some embodiments, the sec sprayed onto a Surface using a spray applicator; being mixed ond anti-infective agent is an antibiotic that alters bacterial with fibrin glue and applied (e.g., sprayed) onto a surface metabolism (e.g., an antibiotic that interferes with cellular (e.g., skin burn or wound); being impregnated onto a wound metabolism and/or RNA and/or protein synthesis). In some dressing or bandage and applying the bandage to a surface embodiments, the second anti-infective agent is an amino (e.g., an infection or wound); being applied by a controlled glycoside (e.g., gentimycin). In some embodiments, the sec release mechanism; being impregnated on one or both sides ond anti-infective agent is mupirocin. Although an under of an acellular biological matrix that can then be placed on a standing of the mechanism is not necessary to practice the Surface (e.g., skin wound or burn) thereby protecting at both US 2009/0304783 A1 Dec. 10, 2009

the wound and graft interfaces; being applied as a liposome; sulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); or being applied on a polymer. and other pharmaceutically acceptable Surfactants. 0062. While an understanding of the mechanism is not 0070. In certain embodiments of the invention, the formu necessary to practice the present invention and while the lations may further comprise one or more alcohols, zinc present invention is not limited to any particular mechanism containing compounds, emollients, humectants, thickening of action, it is contemplated that, in Some embodiments, once and/or gelling agents, neutralizing agents, and Surfactants. administered to a site comprising bacteria (e.g., pathogenic Water used in the formulations is preferably deionized water bacteria Such as MRSA), compositions comprising mupiro having a neutral pH. Additional additives in the topical for cin and nisin come into contact with the pathogenic bacteria mulations include, but are not limited to, silicone fluids, dyes, thereby killing the pathogens. fragrances, pH adjusters, and vitamins. 0063. In other embodiments, the compositions and meth 0071. The topical formulations may also contain compat ods of the present invention find application in the treatment ible conventional carriers, such as cream or ointment bases of surfaces for the attenuation or growth inhibition of and ethanol or oleyl alcohol for lotions. Such carriers may be unwanted bacteria (e.g., pathogens). For example, Surfaces present as from about 1% up to about 98% of the formulation. that may be used in invasive treatments such as Surgery, The ointment base can comprises one or more of petrolatum, catheterization and the like may be treated to prevent infec mineral oil, ceresin, lanolin alcohol, panthenol, glycerin, bis tion of a Subject by bacterial contaminants on the Surface. It is abolol, cocoa butter and the like. contemplated that the methods and compositions of the 0072. In some embodiments of the present invention the present invention may be used to treat numerous Surfaces, pharmaceutical compositions may be formulated and used as objects, materials and the like (e.g., medical or first aid equip foams. Pharmaceutical foams include formulations such as, ment, nursery and kitchen equipment and Surfaces) in order to but not limited to, emulsions, microemulsions, creams, jellies control and/or prevent bacterial contamination thereon. and liposomes. While basically similar in nature these formu 0064. In other embodiments, the compositions may be lations vary in the components and the consistency of the final impregnated into absorptive materials, such as Sutures, ban product. dages, and gauze, or coated onto the Surface of Solid phase 0073. The compositions of the present invention may materials, such as Surgical staples, Zippers and catheters to additionally contain other adjunct components convention deliver the compositions to a site for the prevention of micro ally found in pharmaceutical compositions. Thus, for bial infection. Other delivery systems of this type will be example, the compositions may contain additional, compat readily apparent to those skilled in the art. ible, pharmaceutically-active materials such as, for example, 0065 Other uses for a composition comprising mupirocin antipruritics, astringents, local anesthetics or anti-inflamma and nisin of the invention are also contemplated. These tory agents, or may contain additional materials useful in include a variety of agricultural, horticultural, environmental physically formulating various dosage forms of the compo and food processing applications. For example, in agriculture sitions of the present invention, such as dyes, flavoring agents, and horticulture, various plant pathogenic bacteria may be preservatives, antioxidants, opacifiers, thickening agents and targeted in order to minimize plant disease. One example of a stabilizers. However, such materials, when added, preferably plant pathogen Suitable for targeting is Erwinia amylovora, do not unduly interfere with the biological activities of the the causal agent of fire blight. components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with 0066. The compositions of the invention may be formu auxiliary agents (e.g., lubricants, preservatives, stabilizers, lated for administration by any route, Such as oral, topical or parenteral. The compositions may be in the form of tablets, wetting agents, emulsifiers, salts for influencing osmotic capsules, powders, granules, lozenges, creams or liquid pressure, buffers, colorings, flavorings and/or aromatic Sub preparations, such as oral or sterile parenteral Solutions or stances and the like) that do not deleteriously interact with Suspensions. mupirocin and nisin of the formulation. 0074. In some embodiments, the invention provide phar 0067. The topical formulations of the present invention maceutical compositions containing (a) a composition com may be presented as, for instance, ointments, creams or prising mupirocin and a lantibiotic (e.g., nisin); and (b) one or lotions, foams, eye ointments and eye or ear drops, impreg more other agents (e.g., an antibiotic). Examples of other nated dressings and aerosols, and may contain appropriate types of antibiotics include, but are not limited to, almecillin, conventional additives such as preservatives, solvents to amdinocillin, amikacin, amoxicillin, amphomycin, amphot assist drug penetration, and emollients in ointments and ericin B, amplicillin, azacitidine, azaserine, azithromycin, CCS. aZlocillin, aztreonam, bacampicillin, bacitracin, benzyl peni 0068. The topical formulations may also include agents cilloyl-polylysine, bleomycin, candicidin, capreomycin, car that enhance penetration of the active ingredients through the benicillin, cefaclor, cefadroxil, cefamandole, cefazoline, cef skin. Exemplary agents include a binary combination of dinir, cefepime, cefixime, cefinenoXime, cefinetazole, N-(hydroxyethyl)pyrrolidone and a cell-envelope disorder cefodizime, cefonicid, cefoperaZone, ceforanide, cefotaxime, ing compound, a Sugar ester in combination with a Sulfoxide cefotetan, cefotiam, cefoxitin, cefpiramide, cefpodoxime, or phosphine oxide, and Sucrose monooleate, decyl methyl cefprozil, cefsulodin, ceftazidime, ceftibuten, ceftizoxime, Sulfoxide, and alcohol. ceftriaxone, cefuroxime, cephacetrile, cephalexin, cepha 0069. Other exemplary materials that increase skin pen loglycin, cephaloridine, cephalothin, cephapirin, cephradine, etration include Surfactants or wetting agents including, but chloramphenicol, chlortetracycline, cilastatin, cinnamycin, not limited to, polyoxyethylene Sorbitan mono-oleoate ciprofloxacin, clarithromycin, clavulanic acid, clindamycin, (Polysorbate 80); sorbitan mono-oleate (Span 80): p-isooctyl clioquinol, cloxacillin, colistimethate, colistin, cyclacillin, polyoxyethylene-phenol polymer (Triton WR-1330); poly cycloserine, cyclosporine, cyclo-(Leu-Pro), dactinomycin, oxyethylene sorbitan tri-oleate (Tween 85); dioctyl sodium dalbavancin, dalfopristin, daptomycin, daunorubicin, deme US 2009/0304783 A1 Dec. 10, 2009

clocycline, detorubicin, dicloxacillin, dihydrostreptomycin, described herein sensitize target cells to known agents (and dirithromycin, doxorubicin, doxycycline, epirubicin, eryth Vice versa) and, accordingly, less of these agents are needed to romycin, eveminomycin, floxacillin, fosfomycin, fusidic achieve a therapeutic benefit. acid, gemifloxacin, gentamycin, gramicidin, griseofulvin, 007.9 The sensitizing function of the claimed composi hetacillin, idarubicin, imipenem, iseganan, ivermectin, kana tions also addresses the problems associated with toxic mycin, laspartomycin, lineZolid, linocomycin, loracarbef, effects of known therapeutics. In instances where the known magainin, meclocycline, meropenem, methacycline, methi agent is toxic, it is desirable to limit the dosages administered cillin, mezlocillin, minocycline, mitomycin, moenomycin, in all cases, and particularly in those cases were drug resis moxalactam, moxifloxacin, mycophenolic acid, nafcillin, tance has increased the requisite dosage. Thus, in some natamycin, neomycin, netilmicin, niphimycin, nitrofuran embodiments, when the claimed compounds are co-adminis toin, novobiocin, oleandomycin, oritavancin, oxacillin, tered with the known agent, they reduce the dosage required oxytetracycline, paromomycin, penicillamine, penicillin G, which, in turn, reduces the deleterious effects. Further, penicillin V, phenethicillin, piperacillin, plicamycin, poly because the claimed compounds are themselves both effec tive and non-toxic in moderate doses, co-administration of myxin B, pristinamycin, quinupristin, rifabutin, rifampin, proportionally more of these compounds than known toxic rifamycin, rollitetracycline, Sisomicin, spectrinomycin, Strep therapeutics will achieve the desired effects while minimiz tomycin, streptozocin, Sulbactam, Sultamicillin, tacrolimus, ing toxic effects. taZobactam, teicoplanin, tellithromycin, tetracycline, ticarcil 0080. In some embodiments, pharmaceutical preparations lin, tigecycline, tobramycin, troleandomycin, tunicamycin, comprising compositions comprising mupirocin and nisin are tyrthricin, Vancomycin, Vidarabine, viomycin, Virginiamy formulated in dosage unit form for ease of administration and cin, BMS-284,756, L-749,345, ER-35,786, S-4661, L-786, uniformity of dosage. Dosage unit form, as used herein, refers 392, MC-02479, Pep5, RP 5.9500, and TD-6424. In some to a physically discrete unit of the pharmaceutical preparation embodiments, two or more combined agents (e.g., a compo appropriate for the patient undergoing treatment. Each dos sition comprising mupirocin and nisin and another antibiotic) age should contain a quantity of the compositions comprising may be used together or sequentially. In some embodiments, mupirocin and a lantibiotic (e.g., nisin) calculated to produce another antibiotic may comprise bacteriocins, type Alantibi the desired antibacterial (e.g., killing or growth attenuation of otics, type Blantibiotics, liposidomycins, mureidomycins, bacteria) effect in association with the selected pharmaceuti alanoylcholines, quinolines, eveninomycins, glycylcyclines, cal carrier. Procedures for determining the appropriate dos carbapenems, cephalosporins, streptogramins, oxazoli age unit are well known to those skilled in the art. donones, tetracyclines, cyclothialidines, bioxalomycins, cat I0081 Dosage units may be proportionately increased or ionic peptides, and/or protegrins. In some embodiments, the decreased based on the weight of the patient. Appropriate composition comprises lysostaphin. concentrations for achieving eradication of pathogenic bac 0075. The present invention also includes methods involv teria in a target cell population or tissue may be determined by ing co-administration of compounds comprising mupirocin dosage concentration curve calculations, as known in the art. I0082 In some embodiments, the composition comprises and a lantibiotic (e.g., nisin) with one or more additional from 0.1 to 2000 ug/mL of lantibiotic (e.g., nisin) and 0.1 to active agents (e.g., an antibiotic, anti-oxidant, etc.). Indeed, it 2000 g/mL mupirocin (e.g., 1-1000 ug/mL, 1-500 lug/mL, is a further aspect of this invention to provide methods for 5-200 g/mL etc.). In some embodiments, the composition is enhancing existing therapies and/or pharmaceutical compo from 0.01 to 15% (e.g., 0.1-10%, 0.5-5%, 1-3%, 2%) by sitions by co-administering a composition comprising mupi weight lantibiotic (e.g., nisin) and/or mupirocin. In some rocin and nisin of this invention. In co-administration proce embodiments, the amount of lantibiotic (e.g., nisin) and/or dures, the agents may be administered concurrently or mupriocin delivered to a subject is from 0.1 to 1000 mg/kg/ sequentially. In one embodiment, the compounds described day (e.g., 1 to 500 mg/kg/day, 5 to 250 mg/kg/day, 10-100 herein are administered prior to the other active agent(s). The mg/kg/day, etc.). In some embodiments, the ratio of lantibi pharmaceutical formulations and modes of administration otic (e.g., nisin) concentration to mupirocin concentration is may be any of those described herein. In addition, the two or 10:1, 5:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:10, etc. more co-administered agents may each be administered using I0083. It is contemplated that the compositions and meth different modes or different formulations. ods of the present invention will find use in various settings, 0076. The additional agents to be co-administered, such as including research settings. For example, compositions and other antibiotics, can be any of the well-known agents in the methods of the present invention also find use in studies of art, including, but not limited to, those that are currently in antibiotic resistance (e.g., via analysis of proteins and phar clinical use. maceuticals capable of altering antibiotic resistance) and in in 0.077 Treatment of the various diseases and disorders vivo studies to observe susceptibility of bacterial cells to described herein are often generally limited by the following antibacterial treatments. Uses of the compositions and meth two major factors: (1) the development of drug resistance and ods provided by the present invention encompass human and (2) the toxicity of known therapeutic agents. Some therapeu non-human Subjects and samples from those subjects, and tic agents have deleterious side effects, including non-spe also encompass research applications using these subjects. cific toxicity. Thus, it is not intended that the present invention be limited to 0078. The methods described herein address both these any particular Subject and/or application setting. problems. Drug resistance, where increasing dosages are I0084. The compositions of the present invention find use required to achieve therapeutic benefit, is overcome by co where the nature of the infection present or to be avoided is administering the compounds comprising mupirocin and a known, as well as where the nature of the infection is lantibiotic (e.g., nisin) described herein with or without a unknown. For example, the present invention contemplates known agent. In some embodiments, the compounds use of the compositions of the present invention in treatment US 2009/0304783 A1 Dec. 10, 2009

of or prevention of infections associated with any topical alternating 5 seconds at 3W and 5 seconds at 0 WinaVirsonic application involving ailments of the skin, including, but not 600 with microtip (VirTis). After mixing by vortex, 50 uL of limited to, skin lesions, wounds, ulcers, bed Sores, diaper each sample was streaked onto blood agar plates, incubated at rash, blisters, acne, psoriasis, and warts. 37° C. overnight, and the colonies were counted and com pared to untreated controls. Experimental (0090. For the following data, except where noted in FIG. 0085. The following examples are provided in order to 2, all antibiotic formulations were in a common polyethylene demonstrate and further illustrate certain preferred embodi glycol (PEG) ointment (PEG 400 plus PEG 3350). ments and aspects of the present invention and are not to be (0091 FIG. 1 shows that the efficacy of nisin alone construed as limiting the scope thereof. increases with increasing nisin doses and decreases infectious CFUS by 2 logs, compared to a 3 log drop with mupirocin Example 1 alone (on a molar bases, 2% mupirocin is twice the dose of 6% nisin). Neither drug alone was able to completely clear the Compositions Comprising Mupirocin and Nisin infection. In contrast, 3 of 10 animals in the 2% nisin/2% Clear Mouse Skin Infection mupirocin combination group and 5 of 11 in the 6% nisin/2% I0086 Materials: Lysostaphin was made by Biosynexus, mupirocin combination group were cleared of infection and Inc. and Nisin (AMBICINN) was obtained from AMBI, Inc. the infectious CFUs in the residual infections were reduced Mupirocin (BACTROBAN Ointment), bacitracin (Sigma), by 5 logs, 100-fold more than either therapy alone. and Bacitracin Ointment (G&W Laboratories Inc) were all 0092 FIG. 2 shows the efficacy of nisin, lysostaphin, and obtained commercially. Polyethylene glycol (PEG) 400 and bacitracin alone and in combination on S. aureus infection in PEG 3350 were purchased from Spectrum Chemicals. abraded mouse skin using two different formulations of baci 0087 Mouse Skin Infection Model: An overnight culture tracin: PEG ointment and a commercially available formula of S. aureus grown in tryptic soy broth (SA8; range from 1 to tion from G&W Laboratories Inc (petrolatum base). None of 6x10 CFUs/mL) was centrifuged at 4000xg for 10 minutes these antibacterial agents administered independently, or and resuspended in an equal Volume of phosphate buffered administered in a composition comprising nisin, were signifi saline (PBS). The bacteria were diluted to a percent transmit cantly more efficacious than the independent administration tance of 40 (Spectronic 20D+) and diluted a subsequent of nisin (about 1.5 log reduction). 1:1000 in PBS for a final bacteria concentration of about 0093. As shown in FIG.3, mupirocin has minimal activity 3x10. SKH1 (hrhr) hairless mice (Charles River) were against an S. aureus strain resistant to mupirocin. However, sedated with 0.2 mL of ketamine (80 mg/kg) and xylazine (32 nisin is capable of reducing infectious CFUs by 3 logs. Mupi mg/kg) delivered intraperitoneally. The upper back of the rocin has previously been shown to have poor activity against mice were scrubbed with a 70% alcohol wipe and allowed to Paeruginosa. However, nisin's spectrum of activity includes dry. Fine abrasions were made on the backs of the mice gram negative bacteria when formulated in the presence of between the shoulders using 150 grit sandpaper. Bacteria chelators, Surfactants, or essential oils. were swabbed over the abraded area with a sterile, cotton 0094. As shown in FIG. 4, nisin plus EDTA has more tipped applicator until the area was saturated with the solu activity than nisin alone in this formulation and reduced tion. infectious CFUs by 1.5 logs. Mupirocin does not add any I0088 Topical Ointment: 74 g of PEG 400 and 24 g of PEG additional activity to the nisinformulation. 3350 were added to a 250 mL. glass beaker and heated until all of the PEG 3350 was melted. The Solution was stirred well Example 2 and allowed to cool to room temperature, which results in a Smooth, opaque ointment. Powdered nisin and bacitracin Compositions Comprising Mupirosin and Nisin were added to the ointment on a w/w basis and stirred until Eliminate Inoculated Staphylococcus aureus from homogenously mixed. For formulations containing mupiro Partial Thickness Wounds cin, 1 g of Bactroban Ointment was weighed into a container and powdered nisin added to 2% or 6% (w/w) and mixed well. 0.095 Experimental animals. A young female specific Lysostaphin was first dissolve to a concentration of about 150 pathogen free (SPF: Looper Farms, North Carolina) pig mg/mL in DI water before mixing into the ointment to give a weighing 25-30 kg was kept in house for two weeks prior to final concentration of 2% (w/w). initiating the experiment. The animal was fed a basal diet ad I0089 Treat ent of Infected Skin with Topical Ointment: libitum and housed individually in animal facilities (meeting Treatments aimed to eradicate S. aureus skin infections in the American Association for Accreditation of Laboratory Ani mouse model were started on the morning after infection mal) with controlled temperature (19-21°C.) and lights (12 (Day 1). About 0.1 g of the topical ointments containing nisin h/12 h LD). The experimental animal protocols used for this (0, 2, or 6% w/w), mupirocin (0 or 2%), bacitracin (0 or 500 study followed the federal guidelines for the care and use of U), and lysostaphin (0 or 2% w/w) were swabbed over the laboratory animals (U.S. Department of Health and Human infected area 3 times a day on Days 1 and 2 using a sterile, Services, U.S. Department of Agriculture). Animals were polyester-tipped applicator. The mice were sacrificed by CO monitored daily for any observable signs of pain or discom asphyxiation on the morning of Day 3 and a 0.5-cm patch of fort. In order to help minimize possible discomfort, an anal skin around the infected area was excise. The skin sample was gesic buprenorphine 0.03 mg/kg (Buprenex injectable; Rec disected and placed into a test tube containing 1 mL of 5 kitt Benckiser Hull, England) was given to each animal on the mg/mL proteinase K. 20 mg/mL esterase, and 20 mg/mL first day, and every third day thereafter, while under anesthe activated charcoal in PBS to neutralize the activity of the sia; a fentanyltransdermal system: 25ug/hr (Duragesic; Alza antibacterial agents. Bacteria were dislodged from the skin Corp. Mountain View, Calif.) were used during the entire sample by Sonication: 2 minute treatments per sample with experiment. US 2009/0304783 A1 Dec. 10, 2009

0096 Animal Preparation, Wounding and Treatment. forming units per mL (CFU/ml) calculated. The presumptive Each animal was anesthetized with Telazol HCl (1.4 mg/kg), identification test for the pathogenis the ability of S. aureus to Xylazine (2 mg/kg), Atropine (0.05 mg/kg) I.M. and inhala coagulate rabbit plasma. tion of an isofluorane and oxygen combination. Hair on the 0100 Three wounds per treatment group were cultured 2. back of the pig was clipped with standard animal clippers. 5 and 7 days post treatment. They were serially diluted, plated Skin on both sides of the animal was prepared by washing on Mannitol-salt agar, and incubated for 24 hours. After the with a non-antibiotic soap (NEUTROGENA) and sterile incubation period colonies were counted and the Log colony water. The animal was blotted dry with sterile gauze. Forty forming units/mL determined. The geometric mean of the eight partial thickness wounds measuring (10 mmx7 mmx0.3 Log(CFU/mL) and standard deviation were calculated for mm deep) were made in the paravertebral and thoracic area of each time and treatment. The initial inoculum size used for each animal with a specialized electrokeratome fitted with a 7 this experiment was 6.13 log cfu/ml. The baseline counts after mm blade. The wounds were separated from one another by at 24 hours of inoculation in the wound environment were least 7 cm of unwounded skin. Each wound was then inocu 7.48+1.0 log cfu/ml. The combined raw data for the total lated with a known amount of Staphylococcus aureus (10 experiment is shown in Table 1. Active X is a composition Suspension). The Suspension was lightly scrubbed into the having nisin (6%). Active Y is a composition having nisin test site for ten seconds using a sterile TEFLON spatula. After (6%) and mupirocin (2%). inoculation wounds were covered with TEGADERM poly urethane film dressing (3M, Inc.) for 24 hours before the TABLE 1 initiation of the treatment in order to give the bacteria time to colonize the wounds and develop the biofilm. Treatment Av. Count 0097 Wound Inoculation. A fresh culture of pathogenic Active X 2.292.O isolate obtained directly from American Type Culture Col Active Y O Vehicle 6.580.4 lection (ATCC), Rockville, Md., was used (Staphylococcus Mupirocin 4.96 - 0.8 aureus ATCC #6538). The freeze-dried bacteria culture was Control 6.9 O.9 recovered per ATCC standard recovering protocol. All inocu Active X O.77 1.3 lum Suspensions were made by Scraping the overnightgrowth Active Y O Vehicle 1.5 - 1.3 from a culture plate into 4.5 ml of sterile water and making up Mupirocin O.77 1.3 the Suspension until the turbidity of the Suspension was Control 4.57 - 1.2 equivalent to that of a McFarland #8 Turbidity Standard. This ActiveX 1.OS 1.8 resulted in a suspension concentration of approximately 10 Active Y O colony forming units/ml (CFU/ml). The 10 suspension was Vehicle 1.771.57 serially diluted to make an inoculum Suspension with a con Mupirocin 1.63 - 142 centration of 10 CFU/ml in 35 mls of Tryptic Soy Broth Control 3.73 1.70 (TSB). A small amount of the inoculum suspension was plated onto culture media to quantitate the exact concentra 0101 The data comparing treatments day by day is as tion of viable organisms prior to the experiment. The inocu follows: lum suspension was used directly to inoculate each site. A 25 0102) Two days after initial treatment of inoculated ul aliquot of the Suspension was deposited into a sterile glass wounds, a composition comprising mupirocin and nisin was cylinder (22 mm diameter) in the center of each wound site. observed to completely eliminate Staphylococcus aureus The suspension was lightly scrubbed into the test site for ten from the wounds (See FIG. 6). The next most effective treat seconds using a sterile TEFLON spatula. After inoculation ments were nisin, and Mupirocin, which yielded 2.29+2.0 the wounds were covered with a polyurethane dressing for 48 and 4.96+0.8 Log CFU/ml, respectively. The Vehicle-treated hours to allow the bacteria to develop a biofilm on the wounds and the wounds left air-exposed yielded similar num wounds. Dressings were then removed to culture the baseline bers on this day (6.5 Log cfu/ml and 6.9 Log cfu/ml, respec wounds and to treat the rest according to the experimental tively) (See FIG. 6). design described in FIG. 5. Wounds were treated twice per 0103) On Day 5 after treatment, the same trend was day. observed as the prior assessment point. Bacteria cultured 0098 Recovery Methods. Three wounds were cultured 48 from wounds treated with a composition comprising mupiro hours post inoculation to quantitate the biofilm baseline, and cin and nisinyielded no Staphylococcus aureus (See FIG. 7), two wounds from each treatment group on Days 2, 5 and 7 with nisin being the next most effective treatment yielding a post treatment. At each sampling time, sites were cultured less than 1 Log cfu/ml. Mupirocin had a similar outcome. quantitatively. Each site was cultured only once. The Vehicle-treated wounds yielded about 3 log cfu/ml of bacteria wounded area was encompassed by a sterile glass cylinder less than the negative control group, which had about 4.5 log (22 mm outside diameter) held in place by two handles. One cfu/mlbacteria (See FIG. 7). mL of scrub solution was pipetted into the glass cylinder and 0104 Day 7 post treatment, the final day of assessment, the site was scrubbed with a sterile TEFLON spatula for 30 the trend observed in the previous two time points remained. seconds. Wounds treated with a composition comprising mupirocin 0099 Serial dilutions were made and scrub solutions were and nisin yielded no Staphylococcus aureus (See FIG. 8), quantitated using the Spiral Plater System that deposits a while nisin yielded 1.05+1.8 Log cfu/ml. Mupirocin yielded small defined amount (50 ul) of suspension over the surface of 1.63 Log cfu/ml, which was close to the Vehicle results a rotating agar plate. The selective media for Staphylococcus (1.77+1.6 Log CFU/ml). The negative control resulted in a aureus was Mannitol Salt Agar. All samples were incubated little less than 4 log cfu/ml (3.7) (See FIG. 8). A summary of aerobically for 24 hours at 37°C. After the incubation period the data from all three treatment timepoints is depicted in (24 hrs), colonies on the plates were counted and the colony FIG. 9. US 2009/0304783 A1 Dec. 10, 2009

Example 3 (e.g., kill and/or inhibit growth of) bacteria in a Superficial 0105 Compositions Comprising Gentimycin and Nisin wound model (See Example 2), nisin or gallidermin were Eliminates Staphylococcus aureus used in combination with either gentamicin and mupirocin, 0106 Mice provided a superficial skin abrasion and inocu respectively, and tested. Nisin did synergize with gentamicin lated with S. aureus as described in Example 2 were treated and gallidermin did synergize with mupirocin to reduce bac with a composition comprising a combination of 6% nisin terial counts in the skin infection model. and 0.1% gentamicin. This combination eliminated Substan tially all detectable S. aureus. Example 8 Mupirocin does not Synergize with Bacitracin in Example 4 Treating Skin Infection A Composition Comprising Nisin and Mupirocin is 0112. In order to determine if mupirocin could function in Superior to a Composition Comprising Only Mupi a synergistic way with bacitracinto treat (e.g., kill and/or rocin in Treating Methicillin Sensitive and Methicil inhibit growth of) bacteria in a superficial wound model (See lin Resistant S. Aureus Example 2), mupirocin was used in combination with baci 0107 Mice were provided a superficial skin abrasion as tracin and tested for the ability to treat bacteria. As shown in described in Example 2 and were infected with either methi FIG. 12, mupirocin did not synergize with bacitracin, nor was cillin sensitive S. aureus (MSSA) or methicillin resistant S. there an additive benefit when the two were used together in aureus (MRSA) and then treated with a composition com the reduction of S. aureus in the infected wound model. prising: vehicle (PEG cream) alone, mupirocin (2%), or mupirocin (2%) and nisin (6%) (Active Y). FIG. 10 shows the Example 9 mean log(CFU/ml) of bacteria recovered after either a first or A Composition Comprising Nisin and Mupirocin second treatment (MSSA) or after one and three days Provide a Synergistic Ability to Treat S. Aureus in a (MRSA). The combination of nisin and mupirocin was supe rior in treating (e.g., killing and/or inhibiting growth of) both Suture Infection Model MSSA and MRSA compared to controls and either treatment 0113. It was determined whether nisin and mupirocin, alone. either alone or in combination with each other, would be able to treat S. aureus in a deep tissue infection model. A Suture Example 5 skin infection model was generated in which a deep cut (e.g., Nisin does not Synergize with Mupirocin, Neomy one needing Sutures to close) was made in a mouse and S. aureus introduced into the incision. As shown in FIG. 13, cin, or Gentamicin In Vitro nisin and mupirocin alone show very little efficacy in treating 0108. In order to determine if nisin could function in a the S. aureus. However, the combination of nisin plus mupi synergistic way with other antimicrobials to treat (e.g., kill rocin nearly eradicated the infection. Thus, a combination of and/or inhibit growth of) bacteria in vitro, the minimum nisin and mupirocin can be used to treat a deep tissue infec inhibitory concentration (MIC) of nisin alone or nisin in tion as well as a Subcutaneous infection. combination with either mupirocin, neomycin or gentamicin 0114 All publications and patents mentioned in the above were tested. The MIC was determined by calculating the log specification are herein incorporated by reference. Various reduction of viable S. aureus following incubation with the modifications and variations of the described method and amounts ofnisin and other antimicrobials as indicated in FIG. system of the invention will be apparent to those skilled in the 11. art without departing from the scope and spirit of the inven 0109 As documented in FIG. 11, none of the antimicro tion. Although the invention has been described in connection bials tested (i.e., mupirocin, neomycin, and gentamicin) dis with specific preferred embodiments, it should be understood played the ability to synergize in vitro with nisin. that the invention as claimed should not be unduly limited to Such specific embodiments. Indeed, various modifications of Example 6 the described modes for carrying out the invention that are Nisin does not Synergize with Neomycin or Galli obvious to those skilled in the relevant fields are intended to dermin in Treating Skin Infection be within the scope of the following claims. 0110. In order to determine if nisin could function in a We claim: synergistic way with other antimicrobials to treat (e.g., kill 1. A composition comprising nisin and mupirocin. and/or inhibit growth of) bacteria in a superficial wound 2. The composition of claim 1, comprising 6% nisin and model (See Example 2), nisin was used in combination with 2% mupirocin. either neomycin or gallidermin and tested. Nisin did not 3. The composition of claim 2, wherein said formulation synergize with neomycin nor with gallidermin in the skin provides a more than additive killing and/or growth inhibition infection model. of bacterial cells administered the formulation compared to individual administration of either nisin or mupirocin. Example 7 4. The composition of claim3, wherein said killing and/or inhibiting bacterial cells occurs within an existing bacterial Nisin does Synergize with Gentamicin and Gallider infection. min does Synergize with Mupirocin in Treating Skin 5. The composition of claim3, wherein said bacterial cells Infection comprise bacteria of the genus Staphylococcus. 0111. In order to determine if nisin or gallidermin could 6. The composition of claim 5, wherein said bacterial cells functionina synergistic way with otherantimicrobials to treat comprise Staphylococcus aureus. US 2009/0304783 A1 Dec. 10, 2009

7. The composition of claim 6, wherein said Staphylococ 15. The composition of claim 1, further comprising an cus aureus are pathogenic acellular biological matrix. 8. The composition of claim 6, wherein said Staphylococ 16. The composition of claim 1, further comprising a lipo cus aureus comprise drug resistant Staphylococcus aureus. SO. 9. The composition of claim 6, wherein said Staphylococ 17. The composition of claim 1, further comprising a solid cus aureus are methicillin resistant. phase material selected from the group consisting of Surgical 10. The composition of claim 1, further comprising a phar Staple, Zipper and catheter. maceutically acceptable carrier. 18. The composition of claim 1, further comprising an 11. The composition of claim 10, wherein said carrier agent selected from the group consisting of polyoxyethylene comprises polyethylene glycol. sorbitan mono-oleoate (Polysorbate 80), sorbitan mono-ole 12. The composition of claim 10, wherein said carrier is an ate (Span 80), p-isooctyl polyoxyethylene-phenol polymer emulsion. (Triton WR-1330), polyoxyethylene sorbitan tri-oleate 13. The composition of claim 10, formulated for adminis (Tween 85), dioctyl sodium sulfo Succinate, and Sodium sar tration to a wound. cosinate (Sarcosyl NL-97). 14. The composition of claim 1, further comprising a wound dressing or bandage. c c c c c