BACKGROUND OF THE INVENTION

Field of the Invention

[0001] This invention relates to the control of disease in animals, especially poultry, through the use of a novel bacteriocin-producing strain of Lactobacillus salivarius and/or a novel bacteriocin produced by this strain. It also relates to a novel bacteriocin, amino acid sequence for the novel bacteriocin, and to the strain Lactobacillus salivarius 1077 NRRL B-50053 producing the novel bacteriocin. It also relates to therapeutic compositions containing the novel bacteriocin and/or strain of Lactobacillus salivarius 1077 producing them and to uses of the therapeutic compositions.

Description of the Related Art

[0002] Microorganisms produce a variety of compounds which demonstrate antibacterial properties. One group of these compounds, the bacteriocins, consists of bacteriocidal proteins with a mechanism of action similar to ionophore antibiotics.

Bacteriocins are often active against species which are closely related to the producer. Their widespread occurrence in bacterial species isolated from complex microbial communities such as the intestinal tract, the oral or other epithelial surfaces, suggests that bacteriocins may have a regulatory role in terms of population dynamics within bacterial ecosystems. Bacteriocins are defined as compounds produced by bacteria that have a biologically active protein moiety and bactericidal action (Tagg et al., Bacteriological Reviews, Volume 40, 722-726, 1976). Other characteristics may include: (1) a narrow inhibitory spectrum of activity centered about closely related species; (2) attachment to specific cell receptors; and (3) plamid-borne genetic determinants of bacteriocin production and of host cell bacteriocin immunity. Incompletely defined antagonistic substances have been termed "bacteriocin-like substances". Some bacteriocins effective against Gram-positive bacteria, in contrast to Gram-negative bacteria, have wider spectrum of activity. It has been suggested that the term bacteriocin, when used to describe inhibitory agents produced by Gram-positive bacteria, should meet the minimum criteria of (1) being a peptide; and (2) possessing bactericidal activity (Tagg et al, supra).

[0003] Bacteriocins (BCN) are low molecular weight proteins, which are produced ribosomally in bacteria and posses antimicrobial properties (Klaenhammer, 1993; Nissen- Meyer and Nes, 1997; Cleveland et al, 2001; Eijsink et al, 2002; Riley and Wertz, 2002). These proteins are primarily cationic, hydrophobic or amphiphilic peptides, with molecular weights of 5-6 kDa. Mature BCN antimicrobials are comprised of 20-60 amino acids (Nissen-Meyer and Nes, 1997; Riley and Wertz, 2002). BCN are produced by widely diverse microorganisms that belong to a variety of systematic groups and occupy various ecological niches. According to a widely accepted classification

(Klaenhammer, 1993), sub-class Ila carries, pediocin-like anti-Listeria peptides, which have a conserved N-terminus sequence of YGNGV (SEQ ID NO 2) (Tyr-Gly-Asn-Gly- Val) and two cysteine bridges.

[0004] Numerous aspects of BCN distinguish them from antibiotics (Cleveland et al, 2001): a) BCN are produced on the surface of ribosomes in microbial cells, while antibiotics are secondary metabolites of the cell; b) unlike producers of antibiotics, BCN producers are insensitive to the bactericidal effects; c) BCN molecules may attach to the target cell wall anywhere on the surface as no specific receptors on the target cell wall apparently exist; d) the mechanism of BCN for lethality is versatile and is associated with the process of pore-formation in the outer cell membrane. BCN bind with targets on cell walls of susceptible microbes, cause ionic imbalances and generate pores. Inorganic ions leak through the pores, thereby killing the target cell. Antibiotics, on the other hand can inhibit synthesis of the sub-cellular processes (cell wall synthesis, intracellular protein production, DNA and RNA replication); e) limited resistance of a target-cell to a BCN develops when the target alters its cell membrane chemical composition.

[0005] BCN have advantages over antibiotics: a) there is no information suggesting that BCN are toxic for animals or accumulate in tissues. This is explained by the fact that BCN are susceptible to proteases, and should degrade in the host; b) BCN are effective against antibiotic-resistant pathogens (Svetoch et al, 2009); c) BCN are already produced in animal hosts by normal microflora and likely have an important role in the kinetics of the microbial ecosystem; d) consumers desire naturally grown and processed foods. An EU-import ban on poultry treated with chlorinated water has been in place since 1997, and has limited imports of United States poultry meat which is generally treated by this process. Application of BCN would provide an alternative to the chemical disinfection approaches that are presently employed.

[0006] The consumption of improperly prepared poultry products has resulted in human intestinal diseases. It has long been recognized the Salmonella spp. are causative agents of such disease and more recently, Campylobacter spp., especially Campylobacter jejuni, has also been implicated. Both microorganisms may colonize poultry gastrointestinal tracts without any deleterious effects on the birds, and although some colonized birds can be detected, asymptomatic carriers can freely spread the microorganisms during production and processing, resulting in further contamination of both live birds and carcasses. Poultry serves as the primary reservoir for Salmonella and Campylobacter in the food supply (Jones et al, Journal of Food Production, Volume 54(7), 502-507, July 1991). Prevention of colonization in live poultry during grow out production may diminish the problem of poultry contamination.

[0007] A number of factors contribute to the colonization and continued presence of bacteria within the digestive tract of animals. These factors have been extensively reviewed by Savage (Progress in Food and Nuitrition Science, Volume 7, 65-74, 1983). Included among these factors are (1) Gastric acidity (Gilliland, Journal of Food

Production, Volume 42, 164-167, 1979); (2) bile salts (Sharpe & Mattick,

Milchwisenschaft, Volume 12, 348-349, 1967; Floch et al., American Journal of Clinical Nutrition , Volume 25, 1418-1426, 1972; Lewis & Gorbach, Archives of Internal Medicine, Volume 130, 545-549, 1972; Gilliland and Speck, Journal of Food Protection, Volume 40, 820-823, 1977; Hugkahl et al, Infection and Immunity, Volume 56, 1560- 1566, 1988); (3) peristalsis; (4) digestive enzymes (Marmur, Journal of Molecular Biology, Volume 3, 208-218, 1961); (5) immune response; and (6) indigenous microorganisms and the antibacterial compounds which they produce. The first four factors are dependent on the phenotype of the host and may not be practically

controllable variables. The immune response in the gastrointestinal (GI) tract is not easily modulated. The factors involving indigenous microorganisms and their metabolites are dependent on the normal flora of the GI tract.

[0008] One potential approach is to control Campylobacter and/or Salmonella colonization through the use of competitive exclusion (CE). Numi and Rantala (Nature, Volume 241, 210-211, 1973) demonstrated effective control of Salmonella infection by gavaging bacteria from healthy poultry intestinal tract materials into young chicks whose microflora had not yet been established, against Salmonella colonization.

Administration of undefined CE preparations to chicks speeds the maturation of gut flora in newly-hatched birds and provides a substitute for the natural process of transmission of microflora from the adult hen to its offspring. Results from laboratory and field investigations provide evidence of benefits in Campylobacter control through

administering normal microflora to chickens ; decreased frequency of Camplyobacter- infected flocks (Mulder and Bolder, IN: Colonization Control of human bacterial enteropathogens in poultry; L.C. Blankenship (ed), Academic Press, San Diego, Calif, 359-363 1991) and reduced levels of Campylobacter jejuni (C. jejuni) in the feces of colonized birds has been reported (Stern, Poultry Science, Volume 73, 402-407, 1994).

[0009] Schoeni and Wong (U.S. Patent Number 4,335,107, June, 1982) reported a significant reduction in broiler colonization by C. jejuni through the application of carbohydrate supplements together with three identified antagonists: Citrobacter diversus 22, Kelbsiella pneumonia 23, and Escherichia coli 25. There is also evidence of a significant decrease of C. jejuni in intestinal samples from infected broilers after treatment with poultry-isolated cultures of Lactobacillus acidophilus and Streptococcus faecium (Morishita et al, Avian Diseases, Volume 41, 850-855, 1997).

[0010] Synoeyenbos et al. (U.S. Patent 4,335,107, June 1982) developed a competitive exclusion (CE) microflora technique for preventing Salmonella colonization by lyophilized fecal droppings and culturing this preparation anaerobically. Mikola et al. (U.S. Patent 4,657,762, April, 1987) used intestinal fecal and cecal contents as a source of CE microflora for preventing Salmonella colonization. Stern et al. (U.S. Patent 5,451,400, September 1995 and U.S. Patent No. 6,241,335, April 2001) disclose a mucosal CE composition for protection of poultry and livestock against colonizations by Salmonella and Campylobacter where the mucin layer of prewashed caeca is scraped and the scrapings, kept in an oxygen-free environment, are cultured anaerobically. Nisbet et al. (U.S. Patent No. 5,478,557, December 1996) disclose a defined probiotic that can be obtained from a variety of domestic animals which is obtained by continuous culture of a batch culture produced directly from fecal droppings, cecal and/or large intestine contents of the adult target animal.

[0011] Lactic acid bacteria are among the most important probiotic microorganisms. They are Gram-positive, nonsporing, catalase negative organisms devoid of cytochromes. They are anaerobic but aerotolerant, fastidious, acid-tolerant, and strictly fermentative with lactic acid as the major end-product of sugar fermentation. Lactic acid producing bacteria include Lactobacillus species, Bifidobacterium species, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici, Sporolactobacillus inulinus, Streptococcus thermophilus, etc. These species are of particular interest in terms of widespread occurrence of bacteriocins within the group and are also in wide use throughout the fermented dairy, food and meat processing industries. Their role in the preservation and flavor characteristics of foods has been well documented. Most of the bacteriocins produced by this group are active only against other lactic acid bacteria, but several display anti-bacterial activity towards more phylogenetically distant Gram-positive bacteria and, under certain condition, gram- negative bacteria.

[0012] Lactobacilli have been extensively studied for production of antagonists. These include the well characterized bacteriocins (DeKlerk, Nature, Volume 214, 609, 1967; Upreti and Hinsdill, Antimicrob, Agents, Chemother., Volume 7, 139-145, 1975;

Barefoot and Klaenhammer, Antimicrob, Agents, Chemother., Volume 45, 1808-1815, 1983; Joerger and Klaenhammer, Journal of Bacteriology, Volume 167, 439-446, 1986) and potential bacteriocin-like substances (Vincent et al., Journal of Bacterioll, Proa, 9, 1965; Hamdan and Milcolajeik, Journal of Antibiotics, Volume 8, 631-636, 1974;

Mikolajeik and Hmadan, Cultured Dairy Freducts, Page 10, 1975; and Shahani et al., Cultured Dairy Products Journal, Volume 11, 14-17, 1976).

[0013] Klaenhammer (FEMS, Microbiol, Rev., Volume 12, 39-86, 1993) has classified the lactic acid bacteria bacteriocins known to date into four major groups:

[0014] Group I: Lantibiotics which are small peptides of <5 kDa containing the unusual amino acids lanthionine and β-methyl lantionine. These are of particular interest in that they have a very broad spectrum of activity relative to other bacteriocins. Examples include nisin, nisin Z, carnocin U 149, lacticin 81 and lactocin 5.

[0015] Group II: Small non-lanthionine containing peptides. A heterogenous group of small peptides of <10 kDa. This group includes peptides active against Listeria spp.

[0016] Group III: Large heat labile proteins of <30 kDa. An example is Helveticin.

[0017] Group IV: Complex bacteriocins-proteins containing additional moieties such as lipids and carbohydrates.

[0018] Stern et al, United States Patent No. 7,132,102, issued November 7, 2006, describe a Lactobacillus salivarius which produces bacteriocin OR-7 having a 54 amino acid sequence and a molecular weight of about 6 kDa and a pi of about 9.0.

[0019] The present invention provides novel compositions containing at least one novel strain of a lactic acid-producing bacterial strain and/or novel bacteriocin produced by the at least one novel strain; a method of using the strain or bacteriocin, the novel strain and method of using, all of which are different from related art strains, bacteriocins, and methods of using. Summary of the Invention

[0020] It is therefore an object of the present invention to provide a novel strain of Lactobacillus that produces a novel bacteriocin.

[0021] Another object of the present invention is to provide a novel Lactobacillus salivarius strain 1077 as represented by the deposited strain NR L B-50053.

[0022] A further object of the present invention is to provide a novel bacteriocin produced by a novel strain of Lactobacillus salivarius.

[0023] A still further object of the present invention is to provide a novel bacteriocin BCN L-1077 having an amino acid sequence as set forth by SEQ ID NO 1.

[0024] Another object of the present invention is to provide a method for at least reducing the levels of colonization of at least one target bacteria in animals by administering to the animal a therapeutic composition including at least one novel strain of Lactobacillus salivarius that produces a novel bacteriocin, at least one novel bacterioicn produced by a novel strain of Lactobacillus salivarius or a combination of the novel strain and novel bacteriocin.

[0025] A further object of the present invention is to provide a method for at least reducing levels of colonization by at least one target bacteria in animals by administering to the animal a therapeutic composition including at least a novel strain of Lactobacillus salivarius represented by NRRL B-50053.

[0026] A still further object of the present invention is to provide a method for at least reducing levels of colonization by at least one target bacteria in animals by administering to the animal a therapeutic composition including at least a novel strain of Lactobacillus salivarius represented by NRRL B-50053; at least one bacteriocin having an amino acid sequence as set forth in SEQ ID NO 1 , or a combination of the novel strain and novel bacteriocin. [0027] A still further object of the present invention is to provide a method for at least reducing levels of colonization by at least one target bacteria in animals by administering to the animal a therapeutic composition including at least a novel bacteriocin having an amino acid sequence as set forth in SEQ ID NO 1.

[0028] A still further object of the present invention is to provide a method for at least reducing levels of colonization by at least one target bacteria in animals by administering to the animal a therapeutic composition including at least a novel bacteriocin produced by a novel strain of Lactobacillus salivarius represented by NRRL B-50053.

[0029] Further objects and advantages of the present invention will become apparent from the following description.

Deposit of the Microorganisms

[0030] Lactobacillus salivarius, designated NRRL B-50053 was deposited on August 6, 2007 . It was deposited under the provisions of the Budapest Treaty, on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations there under (Budapest Treaty) with the U.S.D.A. Agricultural Research Service Patent Culture Collection, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, Illinois 61604. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant(s) will maintain and will make this deposit available to the public pursuant to the Budapest Treaty.

Detailed Description of the Invention

[0031] The importance of enteric infections in humans has been increasingly well recognized and the relationship of poultry contamination and human infection is well documented. The ability to diminish this health hazard by interventions at poultry processing plants is also well known. During broiler production and processing, fecal materials containing pathogens are transferred into meat and persist in the food processing kitchens.

[0032] Metabolites of competing organisms may contribute to the control of pathogens such as Campylobacter jejuni and Salmonella. The novel antagonistic strain of the present invention was isolated from cecal content of healthy commercial broiler chickens. The native components of the characterized antagonist are low modular weight peptide, bacteriocin, which has a wide spectrum of antagonistic activity.

[0033] The present invention provides a novel Lactobacillus salivarius strain, an amino acid sequence of said bacteriocin, a therapeutic composition containing the novel bacteriocin and /or strain producing the bacteriocin, and methods for using the novel therapeutic composition.

[0034] The Lactobacillus salivarius L-1077 strain is a facultative anaerobe

(microaerophile), Gram-positive, catalase negative, nonmotile, pleomorphic rods. The colonies produce circular to regular shaped, smooth, convex colonies with sharp margins that are about 1 mm to about 3 mm in diameter after microaerophile incubation for about 18-24 hours at about 37 degrees centigrade. The strains produce lactic acid and H ₂ 0 ₂ .

[0035] Screening of isolated Lactobacillus for the production of bacteriocin activity is performed on nutrient agar on cultures seeded with different target bacteria of interest. Other test strains are cultured under aerobic conditions at about 37 degrees centigrade for about 14-24 hours. The isolates found to be antagonistic are evaluated for bacteriocin production. Crude antimicrobial preparations (CAPS) are prepared by ammonium sulfate precipitation only from cell-free cultures of antagonistic strains grown on starvation media: K2HPO4 6.0 grams

KH2PO4 0.2 grams

(NH ₄ ) ₂ S0 ₄ 0.2 grams

MgS0 ₄ 0.1 grams

Glucose 9.0 grams

Histidine 0.08 grams

Arginine 0.02 grams

Distilled water was added to approximately 1000 ml, pH about 7.2 at about 37 degrees centigrade for about 18 hours under aerobic conditions. The culture fluids were then centrifuged at about 12,000 X g for about 10 minutes. The resulting supernatants were then adjusted to pH of about 6.2 by adding IN NaOH and about 130 U/ml catalase to remove organic acids and hydrogen peroxide, and inhibiting factors. Antagonistic peptides were isolated from supernatant by a combination of ammonium sulfate precipitation, desalting chromatography and gel filtration to produce a crude

antimicrobial preparation (CAP). CAP samples are filtered through 0.22 micron filters (Milllipore, Bedford, Mass., USA).

[0036] Molecular weights of all of the peptides were determined by SDS-PAGE electrophoresis, pis of the peptides were determined by isoelectric focusing. Amino acid sequences were determined by Edman degradation using, for example, a 491 cLC Automatic Sequencer (Applied Biosystems, Inc.).

[0037] For purposes of the present invention the term "peptide" is defined as a compound of at least two or more amino acids or amino acid analogs. The amino acids or amino acid analogs may be linked by peptide bonds. In another embodiment, the amino acids may be linked by other bonds, e.g., ester, ether, etc. Peptides can be in any structural configuration including linear, branched, or cyclic configurations. As used herein, the term "amino acids" refers to either natural or synthetic amino aicds including both the D or L optical isomers, and amino acid analogs.

[0038] Peptide derivatives and analogs of the present invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in conservative amino acid substitution.

[0039] For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to significantly affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.

[0040] Non-conservative amino acid substituitions may also be introduced to substitute an amino acid with a particularly preferable property. For example, Cys may be introduced at a potential site for disulfide bridges with another Cys. Pro may be introduced because of its particularly planar structure.

[0041] The peptide of the present invention can be chemically synthesized. Synthetic peptides can be prepared using well known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, and can include natural and/or synthetic amino acids. Amino acids used for peptide synthesis may be standard Boc(N -amino protected N - t-butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and washing protocols of the original solid phase procedure of Merrifield (J. Am. Chem. Soc, Volume 85, 2149-2154,1963), or the base- labile N -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acid (Carpino and Han, J. org. Chem., Volume 37, 3403-3409, 1972). In addition, the method of the present invention can be used with other N-protecting groups that are familiar to those skilled in the art. Solid phase peptide synthesis may be accomplished by techniques within the ordinary skill in the art (see for example Stewart and Young, Solid Phase Synthesis, Second Edition, Pierce Chemical Co., Rockford, III, 1984; Fields and Noble, Int. J. Pept. Protein Research, Volume 35, 161-214, 1990) or by using automated synthesizers.

[0042] In accordance with the present invention, the peptides and/or the novel bacterial strains can be administered in a therapeutically acceptable carrier topically, parenterally, transmucosally, such as for example, orally, nasally or rectally or transdermally. The peptides of the present invention can be modified if necessary to increase the ability of the peptide to cross cellular membranes such as by increasing the hydrophobic nature of the peptide, introducing the peptide as a conjugate to a carrier, such as a ligand to a specific receptor, etc.

[0043] The present invention also provides for conjugating a targeting molecule to the peptide of the present invention. Targeting molecules for purposes of the present invention mean a molecule which when administered in vivo, localizes to a desired location or locations. In various embodiments of the present invention, the targeting molecule can be a peptide or protein, antibody, lectin, carbohydrate, or steroid. The targeting molecule can be a peptide ligand of a receptor on the target cell or an antibody such as a monoclonal antibody. To facilitate cross-linking, the antibody can be reduced to two heavy and light chain heterodimers, or the F(ab') ₂ fragment can be reduced and crosslinked to the peptide via the reduced sulfhydryl.

[0044] Another aspect of the present invention is to provide therapeutic compositions. The compositions may be for oral, nasal, pulmonary administration, injection, etc. The therapeutic compositions include effective amounts of at least one bacteriocin of the present invention and their derivatives and/or at least one novel strain to at least reduce the levels of colonization by at least one target bacteria together with acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and/or carriers. Diluents can include buffers such as Tris-HCl, acetate, phosphate, for example; additives can include detergents and solubilizing agents such as Tween 80, Polysorbate 80, etc., for example; antioxidants include for example, ascorbic acid, sodium metabisulfite, etc.; preservatives can include, for example, thimersol, benzyl alcohol, etc.; and bulking substances include, for example, lactose, mannitol, etc.

[0045] The therapeutic composition of the present invention can be incorporated into a particulate preparation of polymeric compounds such as polyvinylpyrrolidone, polylactic acid, polyglycolic acid, etc., or into liposomes. Liposomal encapsulation includes encapsulation by various polymers. A wide variety of polymeric carriers may be utilized to contain and/or deliver one or more of the therapeutic agents discussed above, including for example both biodegradable and non-biodegradable compositions. Representative examples of biodegradable compositions include albumin, collagen, gelatin, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose,

hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate, casein, dextrans, polysaccharides, fibrinogen, poly (d,L lactide), poly (D, L-lactide-co-glycolide), poly (glycolide) poly (hydroxybutyrate), poly(alkylcarbonate), and poly(orthoesters), polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene therphthalate), poly(malic acid), poly(amino acids), and their copolymers, (see generally, Ilium, L., Davids, SS (eds) "Polymers in Controlled Drug Delivery" Wright, Bristol, 1987;

Arshady, J. Controlled Release, Vol. 17, 1-22, 1991; Pitt, Int. J. Pharm., Volume 59, 173- 196, 1990; Holland et al, J. Controlled Release, Volume 4, 155-180, 1986).

[0046] Representative examples of non-degradable polymers include poly(ethylene-vinyl acetate) (EVA) copolymers, silicone rubber, acrylic polymers (polyacrylic acid, polymethylacrylic acid, polymethylmethacrylate, polyalkylcynoacrylate), polyethylene, polypropylene, polyamides (nylon 6,6), polyurethane, poly (ester urethanes), poly(ester urea), polyethers (poly (ethylene oxide), poly(propylene oxide), pluronics and

poly(tetramethyleneglycol), silicone rubbers and vinyl polymers such as

polyvinylpyrrolidone, poly(vinyl alcohol), poly(vinyl acetate phthalate). Polymers may also be developed which are either anionic (e.g. alginate, carrageenin, carboxymethyl cellulose and poly(acrylic acid), or cationic (e.g. chitosan, poly-L-lysine,

polyethyleneimine, and poly(allyl amine) (see generally, Dunn et al., J. Applied Polymer Sci., Volume 50, 353-365, 1993; Cascone et al, J. Materials Sci.: Materials in Medicine. Vol. 5, 770-774,m 1994; Shiraishi et al, Biol. Pharm. Bull, Volume 16(11), 1164-1168, 1993; Thacharodi and Rao, Int'l J. Pharm., Volume 120, 115-118m 1995; Miyazaki et al, Int'l J. Pharm., Volume 118, 257-263, 1995).

[0047] Polymeric carriers can be fashioned in a variety of forms, with desired release characteristics and/or specific desired properties. For example, polymeric carriers may be fashioned to release a therapeutic agent upon exposure to a specific triggering event such as pH (See e.g., Heller et al, Chemically Self-Regulated Drug Delivery Systems, in Polymers in Medicine III, Elsevier Science Publishers B.V., Amsterda,., 1988, 175-188; Kang et al, J. Applied Polymer Sci., Volume 48, 343-354, 1993; Dong et al, J. Controlled Release, Volume 19, 171-178, 1992; Dong and Hoffman, J. Controlled Release, Volume 15, 141-152, 1991; Kim et al, J. Controlled Release, Volume 28, 143- 152, 1994; Cornejo-Bravo et al, J. Controlled Release, Volume 33, 223-229, 1995; Wu and Lee, Pharm. Res., Voluem 10(10), 1544-1547, 1993; Seres et al, Pharm. Res., Volume 13(2), 196-201, 1996; Peppas, Fundamentals of pH and Termperature-Sensitive Drug Delivery Systems, in Gurny et al. (eds), Pulsatile Drug Delivery, Wissenschaftliche Verlags-gesellschaft mbH, Stuttgart, 193, 41-55; Doelker, Cellulose Derivatives, 1993, in Peppas and Langer (eds), Biopolymers I, Springer- Verlag, Berlin). Representative examples of pH-sensitive polymers include polyacrylic acid and its derivatives including for example, homopolymers such as poly(aminocarboxylic acid); poly(acrylic acid); poly(methyl acrylic acid), copolymers of such homopolymers, and copolymers of poly(acrylic acid) and acrylmonomers such as those discussed above. Other pH-sensitive polymers include polysaccharides such as cellulose acetate phthalate; hydroxypropylmethylcellulose phthalate; hydroxypropylmethylcellulose acetate succinate; celluloseacetate trimellilate; and chitosan. Yet other pH-sensitive polymers include any mixture of a pH sensitive polymer and a water soluble polymer.

[0048] Likewise, polymeric carriers can be fashioned which are temperature sensitive (see e.g., Chen et al, "Novel Hydrogels of a Temperature-Sensitive Pluronic Grafted to a Bioadhesive Polyacrylic Acid Backbone for Vaginal Drug Delivery," in Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22: 167 168, Controlled Release Society, Inc., 1995; Okano, "Molecular Design of Stimuli-Responsive Hydrogels for Temporal Controlled Drug Delivery," in Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22: 111 112, Controlled Release Society, Inc., 1995; Johnston et al, Pharm. Res. 9(3): 425 433, 1992; Tung, Int'l J. Pharm. 107: 85 90, 1994; Harsh and Gehrke, J. Controlled Release 17: 175 186, 1991; Bae et al, Pharm. Res. 8(4): 531 537, 1991; Dinarvand and D'Emanuele, J. Controlled Release 36: 221 227, 1995; Yu and Grainger, "Novel Thermo-sensitive Amphiphilic Gels: Poly N-isopropylacrylamide-co-sodium acrylate-co-n-N- alkylacrylamide Network Synthesis and Physicochemical Characterization," Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, Oreg., pp. 820 821; Zhou and Smid, "Physical Hydrogels of Associative Star Polymers," Polymer Research Institute, Dept. of Chemistry, College of Environmental Science and Forestry, State Univ. of New York, Syracuse, N.Y., pp. 822 823; Hoffman et al, "Characterizing Pore Sizes and Water ^" Structure ^" in Stimuli-Responsive Hydrogels," Center for Bioengineering, Univ. of Washington, Seattle, Wash., p. 828; Yu and Grainger, "Thermo-sensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic Hydrogels," Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, Oreg., pp. 829 830; Kim et al, Pharm. Res. 9(3): 283 290, 1992; Bae et al, Pharm. Res. 8(5): 624 628, 1991; Kono et al, J. Controlled Release 30: 69 75, 1994; Yoshida et al, J. Controlled Release 32: 97 102, 1994; Okano et al, J. Controlled Release 36: 125 133, 1995; Chun and Kim, J. Controlled Release 38: 39 47, 1996; D'Emanuele and Dinarvand, Int'l J. Pharm. 118: 237 242, 1995; Katono et al, J. Controlled Release 16: 215 228, 1991; Hoffman, "Thermally Reversible Hydrogels Containing Biologically Active Species," in Migliaresi et al. (eds.), Polymers in Medicine III, Elsevier Science Publishers B. V., Amsterdam, 1988, pp. 161 167; Hoffman, "Applications of Thermally Reversible Polymers and Hydrogels in Therapeutics and Diagnostics," in Third International Symposium on Recent Advances in Drug Delivery Systems, Salt Lake City, Utah, Feb. 24 27, 1987, pp. 297 305; Gutowska et al, J. Controlled Release 22: 95 104, 1992; Palasis and Gehrke, J. Controlled Release 18: 1 12, 1992; Paavola et al., Pharm. Res. 12(12): 1997 2002, 1995).

[0049] Representative examples of thermogelling polymers, and their gelatin temperature (LCST (.degree. C.)) include homopolymers such as poly(N-methyl-N-n- propylacrylamide), 19.8; poly(N-n-propylacrylamide), 21.5; poly(N-methyl-N- isopropylacrylamide), 22.3; poly(N-n-propylmethacrylamide), 28.0; poly(N- isopropylacrylamide), 30.9; poly(N, n-diethylacrylamide), 32.0; poly(N- isopropylmethacrylamide), 44.0; poly(N-cyclopropylacrylamide), 45.5; poly(N- ethylmethyacrylamide), 50.0; poly(N-methyl-N-ethylacrylamide), 56.0; poly(N- cyclopropylmethacrylamide), 59.0; poly(N-ethylacrylamide), 72.0. Moreover thermogelling polymers may be made by preparing copolymers between (among) monomers of the above, or by combining such homopolymers with other water soluble polymers such as acrylmonomers (e.g., acrylic acid and derivatives thereof such as methylacrylic acid, acrylate and derivatives thereof such as butyl methacrylate, acrylamide, and N-n-butyl acrylamide). Other representative examples of thermogelling polymers include cellulose ether derivatives such as hydroxypropyl cellulose, 41. degree. C; methyl cellulose, 55. degree. C; hydroxypropylmethyl cellulose, 66. degree. C; and ethylhydroxyethyl cellulose, and Pluronics such as F-127, 10-15 degree. C; L-122, 19.degree. C; L-92, 26.degree. C; L-81, 20.degree. C; and L-61, 24 degree. C.

[0050] A wide variety of forms may be fashioned by the polymeric carriers of the present invention, including for example, rod-shaped devices, pellets, slabs, or capsules (see e.g., Goodell et al, Am. J. Hosp. Pharm. 43: 1454 1461, 1986; Langer et al,

"Controlled release of macromolecules from polymers", in Biomedical Polymers, Polymeric Materials and Pharmaceuticals for Biomedical Use, Goldberg, E. P., Nakagim, A. (eds.) Academic Press, pp. 113 137, 1980; Rhine et al, J. Pharm. Sci. 69: 265 270, 1980; Brown et al, J. Pharm. Sci. 72: 1181 1185, 1983; and Bawa et al, J. Controlled Release 1 : 259 267, 1985).

[0051] Therapeutic agents may be linked by occlusion in the matrices of the polymer, bound by covalent linkages, or encapsulated in microcapsules. Within certain preferred embodiments of the invention, therapeutic compositions are provided in non-capsular formulations such as microspheres (ranging from nanometers to micrometers in size), pastes, threads of various size, films and sprays.

[0052] Another aspect of the present invention is to provide a therapeutic composition and animal feed. The therapeutic composition of the present invention can be encapsulated using a polymeric carrier as described above and then added to a feed by any known means of applying it to feed such as for example, by mechanical mixing, spraying, etc. The therapeutic composition includes, for example, an amount of at least one bacteriocin and/or antagonistic bacteria effective to at least reduce the levels of colonization by at least one target bacteria in an animal, such as for example

approximately 0.5 grams each of the novel bacteriocin/ 1000 grams, approximately 1.25 grams of a polymeric carrier such as polyvinylpyrrolidone/1000 grams, and about 8.6% of a diluent such as water/ 1000 grams mixed with any granular component that is digestible, such as for example, milled maize grain; ground grains such as for example oats, wheat, buckwheat; ground fruits such as for example, pears, etc. The therapeutic composition is then added to any type of animal feed in amounts effective to at least reduce the levels of colonization of at least one target bacteria such as for example in ratios of bacteriocin to feed of about 1 : 10 to about 1 : 100. For purposes of the present invention, examples of animal feed include green fodder, silages, dried green fodder, roots, tubers, fleshy fruits, grains, seeds, brewer's grains, pomace, brewer's yeast, distillation residues, milling byproducts, byproducts of the production of sugar, starch or oil production, and various food wastes. The product can be added to the animal feedstuffs for cattle, poultry, rabbit, pig, or sheep rearing, etc. It can be used mixed with other feed additives for this stock. [0053] The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

EXAMPLE 1

[0054] Healthy commercial broiler chickens served as donors of cecal content providing antagonistic flora. These materials were streaked onto MRS agar and incubated within an anaerobic atmosphere overnight at 37°C. Lactobacillus spp. colonies were isolated and identified with the API 50 CHL micro-test system (bioMerieux, France). Isolates were grown anaerobically at 37°C overnight to confluence on MRS agar. Agar blocks (-0.5 cm ³ ) containing the growth were aseptically cut and transferred to brucella agar supplemented with 5% lysed blood seeded with C. jejuni NCTC 11168. Plates were incubated at 42°C under microaerobic atmosphere for 24 to 48 h. Zones of growth inhibition were noted and the diameters were measured.

[0055] The isolate produced a zone of inhibition, and Lactobacillus salivarius L-1077 (NRRL B-50053) was selected for further study. Consistent with methods previously described in United States Patent No. 7,132,102, November 07, 2006; herein incorporated by reference in its entirety, the bacterocin was produced and purified to homogeneity. The isolate was fermented overnight in a minimal broth medium under quiescent aerobic conditions held at 37°C. The spent culture was centrifuged to remove the cells. The supernatant was adjusted to pH 6.2 by adding 1 N NaOH and 130 U/ml catalase to remove the influence of organic acids and hydrogen peroxide. Proteins in the cell-free supernatant were precipitated with ammonium sulfate, and then dialyzed. The crude preparation was filtered through a 0.22 μιη filter (Millipore, Bedford, MA) and further purified by cation exchange and hydrophobic interaction chromatographic separations.

Activities of the chromatographic fractions were initially screened against C. jejuni NCTC 11168. An additional 32 widely diverse, pathogenic bacterial isolates (Table 3) were tested for minimum inhibitory concentrations (MIC; μg/ml). Protein concentrations of the fractions were determined (Lowry et al, 1951). Doubling dilutions of the preparations were performed and 10 μΐ of each dilution was spotted onto plates of blood- supplemented brucella agar previously seeded with cells of C. jejuni NCTC 11168 or the additional bacterial isolates, as described by Zheng and Slavik (1999). All assays were conducted in duplicate.

[0056] The amino acid sequence of the purified BCN was determined by Edman degradation using a 491 cLC automatic sequencer (Garneau et al, 2002) (Applied Biosystems). The BCN molecular weight was determined by mass spectrometry using a Voyager-DERP (Perkin-Elmer). The MALDI-TOF analysis was determined as per manufacturer's instructions. After the biochemical determination of the primary amino acid sequence, the physical characteristics were analyzed utilizing Protean of the

DNASTAR (Madison, WI) (Nishikawa et al., 1987). The primary amino acid sequence was entered into BLAST (Altschul et al., 1997) to search for proteins with similar sequences. After ammonium sulfate protein precipitation of the fermentation culture supernatant, BCN L-1077 activity was low and the protein concentration was relatively high (Table 1). Each succeeding purification step increased the specific activity against C. jejuni and reduced the contaminating non-BCN proteins. A 95.7% purity of the BCN was ultimately achieved having a specific activity of 229,000 AU/mg of protein. This purified material was subjected to polyacrylamide gel electrophoresis (PAGE; data not shown) to further purify the molecule. The peptide band was then subjected to Edman degradation and the characteristic "YGNGV" consensus sequence typical of class Ila BCN was observed for BCN L-1077 (Table 2). The MALDI TOF MS analysis indicated the molecular weight of BCN L-1077 as approximately 3,454 Da with an iso-electric point of approximately pi = 9.1. The chemically determined molecular weight and pi are in relatively close agreement with the predicted values of approximately 4,000 Da and approximately 9.8 respectively based on amino acid sequence of BCN L-1077. The bacteriocin is composed of 15 hydrophobic residues (A, I, L, F, V) and 9 polar residues (N, Q, T, Y) with one acidic (D) and four basic (K, R) amino acids. BCN L-1077 is predicted to have a charge of approximately 2.91 at a pH of approximately 7. The interior of the peptide from residues 9 through 25 are hydrophobic while the N-terminal and C- terminal regions are hydrophilic. No peptides similar to BCN L-1077 were discovered following BLAST analyses.

[0057] The MIC values observed for the 32 pathogenic bacteria tested ranged from approximately 0.09 to approximately 1.5 μg/ml (Table 3). The wide genetic panorama among the isolates tested is significant. Many of the Gram-negative isolates tested approached the lower MIC values which are reported but, the Gram-positive Listeria monocytogenes, Staphylococcus spp. and Clostridium perfringens isolates also manifested similar sensitivities to the BCN L-1077. Each of the isolates tested represent serious etio logic agents causing a variety of human disease or pathological poultry and livestock infections (Lennette et al., 1980; Saif et al., 2003).

[0058] The MIC values listed in Table 3 indicated the high potency of BCN L-1077. Campylobacter jejuni L-4 was the most sensitive isolate tested, with an MIC of 0.09 μg/ml and Proteus vulgaris 1 A was the least sensitive of the isolates tested at a highly respectable MIC of 1.5 μg/ml. The pathogens tested manifest their virulence in both gastrointestinal and systemic infections. The shiga-like toxin expressed by the isolates of E. coli 0157:H7 might initially establish within the intestinal tract and, the toxin will subsequently cause a more disseminated pathology. Salmonella enteritidis and

Salmonella choleraesuis will initially colonize the GI tract of poultry and swine, respectively, but then translocate to infect the parenchymal organs of the host. The other pathogens have their own unique mechanisms and sites of infection. Therefore, as observed with the S. enteritidis 92 Rif ¹ in our chicken experiments, the BCN treatment was effective in dramatically reducing extra-intestinal infections associated with the low MIC values. There appears to be an association of low MIC values with in- vivo therapeutic control of bacterial infections with BCN L-1077. Table 1. Biochemical purification of bacteriocin L-1077 using specified procedures.

Table 2. Characterization of bacteriocin L-1077 by Edman degradation, MALDI TOF MS anal sis, and iso-electric oint anal sis.

Table 3. Antimicrobial activities (minimum inhibitory concentrations μ§/ι 1; MIC) of bacteriocin L-1077 against selected pathogens, as determined with a 10 μΐ spot test. Test strains were surface plated onto agar plates, successive one-half concentration dilutions were spotted onto the plates and the plates were incubated.

Example 2

[0059] Purified BCN was diluted in tap water to provide approximately 25, 12.5, 6.25 mg or no BCN L-1077/L in the experimental bird drinking waters. We obtained commercial age birds which had already been colonized by C. jejuni through field exposure. The approximately 43 d-old birds were challenged with 0.2 ml suspension of -10 ^10"11 cfu Salmonella enteritidis 92 Ri . The rifampicin resistant S. enteritidis isolate had been created by sequential passages on media containing increasing levels of rifampicin. Strain S. enteritidis 92 Ri was cultured overnight in Endo's medium containing 100 μg/ml rifampicin. The adult birds were provided feed and water ad- libitum. The three treated groups of broilers were provided one of the three levels of BCN L-1077/L in drinking water for three days. In the second therapeutic trial, groups of 40 d-old broilers, commercially colonized with C. jejuni and challenged with S.

enteritidis 92 Rii*, were treated for one, two or three days with 12.5 mg BCN L-1077/L in their drinking water.

[0060] Microbiological sampling. At designated times (after days 1, 2 or 3 of treatment) groups of broilers were sacrificed and weighed. The average weights of all the birds within each group together with the amount of water they consumed were used to estimate the total average amount of BCN provided to the groups of broilers. The birds were dissected to aseptically remove the ceca, liver and spleen of the animals. The chilled tissues were returned directly to the laboratory and were subjected to ten- fold serial dilutions and plated onto the following media and conditions: 1) MRS agar 24 h at about 37°C an-aerobically, 2) Campylobacter-Cefex agar (Stern et al, 1992) 48 h at about 42°C micro-aerobically, or 3) Endo agar + 100 μg rifampicin/ml-24 h at about 37°C aerobically, to estimate levels of lactic acid bacteria, C. jejuni and, S. enteritidis, respectively.

[0061] The data in Table 4 is derived from an experiment in which we provided different concentrations of BCN L-1077 to infected broilers over a 3-d treatment. Table 5 provides data for a similar experiment in which a single fixed quantity of BCN

(approximately 25 mg/L) was administered in drinking waters. These birds were sacrificed at daily intervals after 1 to 3 days of treatment. All levels of treatment greatly reduced the systemic S. enteritidis infections (Table 4), as did all durations of treatment (Table 5). These observations are notable because BCN was able to survive the acidic GI tract environment, protease activities within the intestinal tract and, penetrate the intestinal wall following oral ingestion. The BCN was then transported (likely through the blood) to the spleen and liver where the S. enteritidis infection was prominent. The orally administered BCN was then able to induce its bactericidal activity in those two organs as well as in the ceca. The bactericidal influence upon C. jejuni within the GI tract was less pronounced than what was seen for both C. jejuni L-4 and NCTC 11168 isolates under in- vitro conditions. The commercially acquired C. jejuni infections contained isolates more resistant than the laboratory isolates. Thus, we observed only 4 logio reductions as reported in Table 5 while the birds enrolled in the trial reported in Table 4 showed reductions to non detectable levels of C. jejuni. Reductions of 3 logio on processed poultry are postulated to reduce human health risks for Campylobacter substantially (Rosenquist et al, 2003).

Table 4. The therapeutic effect of 3-d bacteriocin (BCN) L-1077 treatments, of various concentrations, against a mixed infection of Campylobacter jejuni and Salmonella enteritidis 92rif in 43 day-old broilers. All birds commercially colonized with non-specified C. jejuni.

ND ¹ None detected; detection limit >100 cells Table 5. The therapeutic effects of 1 to 3 d of bacteriocin (BCN) L-1077 (12.5 mg/L) treatment against a mixed infection of Campylobacter jejuni and Salmonella enteritidis 92rif in 40 to 42 d old broilers. Birds challenged and colonized with 1.3 x 10 ¹⁰ CFU S. enteritidis 92rif. All birds commercially colonized with non-specified C. jejuni.

ND None detected; detection limit >100 cells

[0062] The foregoing detailed description is for the purpose of illustration. Such detail is solely for that purpose and those skilled in the art can make variations without departing from the spirit and scope of the invention.