AND USES THEREOF

FIELD OF INVENTION

[0001] The invention is in the field of anti-mi crobial agents. More specifically, the invention relates to anti-microbial agents derived from Paenibacillus.

BACKGROUND OF THE INVENTION

[0002] In response to the increasing prevalence of antibiotic resistance in pathogenic bacteria, the pharmacokinetic properties and safety profiles of many novel

antimicrobial peptides have been investigated. Bacteriocins are natural proteinaceous antimicrobial compounds produced by bacteria and active against taxonomically related bacteria (Klaenhammer, 1993). Species that produce bacteriocins have been studied extensively in the hope of finding safe and efficient means of inhibiting the growth of pathogenic bacteria, especially in foods (Cleveland et al. 2001).

Bacteriocins produced by Gram-positive staining bacteria, such as lactic acid bacteria, have become a focus of interest as alternatives to conventional antibiotics (Nes et al.

1996) . Nisin, the first bacteriocin ever isolated and now widely used as a food additive, was approved by the World Health Organization for use as a food preservative in 1973. This peptide is generally inactive against Gram-negative staining bacteria, imposing a limitation on its effectiveness when major food-borne pathogens such as Escherichia coli, Salmonella and Yersinia are involved (Du and Shen 1999; Zheng et al. 1999). Davies et al. (1998) reported that nisin produced by Lactococcus lactis was thermostable and remained active after treatment at 121 °C for 15 min at pH 3. Nisin is about 4.4 kDa and is stabilized by disulfide bonds.

[0003] Polymyxins, a class of antimicrobial agents, are synthesized by a non- ribosomal process. The peptide-synthase-directed condensation reactions by which polymyxins are formed in the cell cytoplasm have been reviewed (Marahiel et al.

1997) and their biosynthesis in a cell-free enzyme system reported ( omura et al. 1985).

[0004] Many species within the genus Paenibacillus produce variants of polymyxins, which are generally composed of a cyclic decapeptide with a terminal fatty acid l moiety (Martin et al. 2003). Five chemically distinct compounds, polymyxins A to E, differing in amino acid and fatty acid composition have been identified to date.

Martin et al. (2003) reported that mattacin activity (800 AU ml ^"1 ) produced by P. kobensis M was maximal at 12 h of fermentation. Martin et al. (2003) also reported that mattacin and polymyxin B inhibited all Gram-negative staining species tested including E. coli 0157:H7, Salmonella enterica serovar Rubislaw and Vibrio parahemeolyticus Gl-166 but both failed to inhibit strains of Listeria and Bacillus.

[0005] DeCrescenzo et al. (2007) isolated a new Paenibacillus species (P.

amylolyticus C27) that produces polymyxins El and E2 (colistin A and B). The new antimicrobial peptides were reported to be effective against Gram-negative staining bacteria such as E. coli, Pseudomonas, Salmonella, and Shigella. DeCrescenzo et al. (2007) also reported that polymyxin E produced by P. amylolyticus C27 inhibited Gram-positive staining bacteria such as Staphylococus aureus ATCC 6538,

Enterococcus faecalis ATCC 19433 and Streptococcus pyogenes ATCC 19165. [0006] Zengguo et al. (2007) reported the co-production of polymyxin and lantibiotic by natural isolates of P. polymyxa. The two antimicrobial peptides were reported to display potent activity against many Gram-negative staining bacteria, including E. coli, Pseudomonas aeruginosa and Acinetobacter baumannii, and against Gram- positive food-borne pathogenic bacteria. Zengguo et al. (2007) also reported that polymyxin produced by P. polymyxa OSY-DF is stable from pH 2.0 to 9.0 and retained its activity after a short autoclaving.

[0007] Svetoch et al. (2005) reported the isolation of a new class Ila bacteriocin from P. polymyxa NRRL-B-30509, which has been used for the control of Campylobacter in poultry. SUMMARY OF THE INVENTION

[0008] The present invention provides, in part, an isolated Paenibacillus sp.

bacterium comprising SEQ ID NO: 1.

[0009] In alternative embodiments, the invention provides an isolated Paenibacillus polymyxa (Strain JB05-01-1) bacterium deposited at the ATCC®) under the terms of the Budapest Treaty and designated Accession Number PTA- 10436, or a strain comprising the identifying characteristics thereof. The bacterium may be isolated from a direct-fed microbial product, for example, RE3™.

[0010] The bacterium may include an anti-microbial activity, such as an anti- bacterial activity. The anti-bacterial activity may include inhibiting the growth of a Gram-negative staining bacterium, such as one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RR1, Escherichia coli TBI , or Escherichia coli 0157:H7), Pantoea sp. (e.g., Pantoea agglomerans BCl), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73), Butyrivibrio sp. (e.g., Butyrivibrio fibrisolvens OR85), Fibrobacter sp. (e.g., Fibrobacter succinogenes), Salmonella sp. (e.g.,

Salmonella enteritidis or Salmonella typhi), Shigella sp. (e.g., Shigella dysenteriae), Helicobacter sp. (e.g., Helicobacter pylori), or Campylobacter sp (e.g.,

Campylobacter jejuni). In alternative embodiments, the anti-bacterial activity may include inhibiting the growth of a Gram-positive staining bacterium, such as one or more of a Listeria sp., such as Listeria innocua.

[001 1 ] In alternative embodiments, the bacterium may not inhibit the growth of a Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua. A Gram-positive staining bacterium may include one or more of Pediococcus

acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis or Enterococcus mundtii.

[0012] In alternative embodiments, the anti-microbial activity may be sensitive to an enzyme selected from the group consisting of one or more of proteinase K, trypsin, chymotrypsin or lipase; or may be sensitive to sodium dodecyl sulphate (SDS) or urea; or may be sensitive to a temperature in excess of about 90°C for about 30 minutes; or may be sensitive to a temperature of about 100°C for about 10 minutes; or may be insensitive to a temperature upto about 80°C for about 30 minutes; or may be sensitive to acetonitrile and hexane; or may be insensitive to an organic solvent selected from the group consisting of chloroform, propanol, methanol, ethanol and toluene; or may be insensitive to pH, for example, pH ranging from about 2 to about 9. [0013] In alternative embodiments, the invention provides a cell culture including a bacterium as described herein. The cell culture may be a starter culture.

[0014] In alternative embodiments, the invention provides a cell culture supernatant derived from growing a bacterium as described herein in a cell culture medium. The supernatant may include an anti-microbial activity, such as an anti-bacterial activity.

[0015] In alternative embodiments, the invention provides an anti-microbial agent isolated from a bacterium, cell culture, or cell culture supernatant as described herein. The anti-microbial agent may include a peptide, such as a lipopeptide. The antimicrobial agent may include a molecular weight between about 1000 daltons to about 2500 daltons. The anti-microbial agent may be a polymyxin.

[0016] In alternative embodiments, the invention provides a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.

[0017] In alternative embodiments, the invention provides a pharmaceutical, veterinary, cosmetic or hygiene composition including a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein and a suitable carrier.

[0018] In alternative embodiments, the invention provides a food or feed additive comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.

[0019] In alternative embodiments, the invention provides a packaging material comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein.

[0020] In alternative embodiments, the invention provides a kit comprising a bacterium, cell culture, cell culture supernatant, or anti-microbial agent as described herein together with instructions for use in inhibiting growth of a micro-organism. [0021 ] In alternative embodiments, the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus sp. bacterium comprising SEQ ID NO: 1 ; and culturing the live Paenibacillus sp. bacterium in a cell culture medium, under conditions suitable for production of the anti-microbial agent. VX2523WU

[0022] In alternative embodiments, the invention provides a method of producing an anti-microbial agent, by providing a live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof; and culturing the live Paenibacillus polymyxa (Strain JB05-01-1) bacterium or a strain comprising the identifying characteristics thereof in a cell culture medium, under conditions suitable for production of the anti-microbial agent.

[0023] The methods may further include isolating the anti-microbial agent from the bacterium. The culturing may be performed under conditions suitable for secretion of the anti-microbial agent into the cell culture medium. The methods may further include separating the bacterium from the cell culture medium to provide a cell culture supernatant comprising the anti-microbial agent. The methods may further include isolating the anti-microbial agent from the cell culture supernatant.

[0024] In alternative embodiments, the invention provides an anti-microbial agent produced by the methods as described herein. The anti-microbial agent may include a peptide, such as a lipopeptide. The anti-microbial agent may include a molecular weight between about 1000 daltons to about 2500 daltons. The anti-microbial agent may be a polymyxin.

[0025] The anti-microbial agent may include an anti-microbial activity selected from one or more of: sensitivity to proteinase K, trypsin, chymotrypsin and lipase, sodium dodecyl sulphate (SDS), urea, acetonitrile, or hexane; insensitivity to chloroform, propanol, methanol, ethanol or toluene; insensitivity to pH; sensitivity to a

temperature in excess of about 90°C for about 30 minutes; sensitivity to a temperature of about 100°C for about 10 minutes; insensitivity to a temperature upto about 80°C for about 30 minutes; inhibition of growth of a Gram-negative staining bacterium; inhibition of growth of a Listeria sp. or a Listeria innocua; or no inhibition of growth of a Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua.

[0026] In alternative embodiments, the invention provides a method of inhibiting the growth of a microorganism in a subject or substance in need thereof by administering or applying an effective amount of the bacterium, cell culture, cell culture supernatant or anti-mi crobial agent, as described herein, to the subject or substance. The inhibition of growth may be selective.

[0027] The microorganism may be a bacterium, such as a Gram-positive staining bacterium of Listeria sp. (e.g., Listeria innocua) or Gram-negative staining bacterium, such as one or more of one or more of Escherichia sp. (e.g., Escherichia coli such as Escherichia coli RRl, Escherichia coli TBI, or Escherichia coli 0157:H7), Pantoea sp. (e.g., Pantoea agglomerans BC1), Pseudomonas sp. (e.g., Pseudomonas fluorescens R73), Butyrivibrio sp. (e.g., Butyrivibrio fibrisolvens OR85), Fibrobacter sp. (e.g., Fibrobacter succinogenes), Salmonella sp. (e.g., Salmonella enteritidis or Salmonella typhi), Shigella sp. (e.g., Shigella dysenteriae), Helicobacter sp. (e.g., Helicobacter pylori), or Campylobacter sp (e.g., Campylobacter jejuni).

[0028] The bacterium may be a pathogenic bacterium, such as a food-borne pathogenic bacterium. The microorganism may be a food-borne pathogenic microorganism or a food-spoilage micro-organism. [0029] The subject may be an animal, such as a human or an agricultural animal (e.g., cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, fish, or crustacean). The substance may be a cosmetic, hygiene, feed or food product (e.g., dairy or meat product) or packaging material thereof.

[0030] In alternative embodiments, the invention provides an isolated nucleic acid molecule including SEQ ID NO: 1.

[0031] In alternative embodiments, the invention provides the use of an effective amount of a bacterium, cell culture, cell culture supernatant or anti-microbial agent, as described herein for inhibiting the growth of a microorganism in a subject or substance in need thereof. [0032] This summary of the invention does not necessarily describe all features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0034] FIGURE 1 is a graph showing the kinetics of antimicrobial compound production during stirred batch culture of Paenibacillus polymyxa JB05-01-1 in Luria-Bertani broth at 30°C. Optical density (O.D.6oonm) of Paenibacillus polymyxa JB05-01-1 cultures (♦) and antimicrobial compound concentration expressed as AU ml ^"1 (Δ).

[0035] FIGURE 2 is a graph showing the growth of Escherichia coli RR1 in tryptic soy broth at 30°C in the presence of 16 (□), 32 (Δ) and 96 (♦) AU ml ^"1 of

antimicrobial compound from culture supernatants of Paenibacillus polymyxa JB05-01-1 ; Control (0 AU ml ^"1 ) medium (0).

[0036] FIGURE 3 is a photograph showing SDS- polyacrylamide gel electrophoresis (PAGE) of Paenibacillus polymyxa JB05-01-1 culture supernatant overlaid with tryptic soy broth agar seeded with Escherichia coli RR1 and incubated for 24 h at 30°C (Gel 2); Gel 1 : molecular weight marker.

[0037] FIGURE 4 is a partial sequence of P. polymyxa JB05-01-1 16S rRNA gene (GenBank Accession Number GQ184435; SEQ ID NO: 1).

DETAILED DESCRIPTION [0038] The present invention provides, in part, a Paenibacillus sp. isolate, designated Paenibacillus polymyxa JB05-01-1 (deposited on October 21, 2009, with the

American Type Culture Collection (ATCC®) under the terms of the Budapest Treaty and assigned Accession Number PTA-10436) obtained from an animal feed additive and identified by amplification and sequencing of the 16S rRNA gene.

Characterization of the physical properties and anti-microbial activities of a substance secreted into the culture supernatant of a Paenibacillus polymyxa JB05-01-1 cell culture resulted in the identification of at least one anti-microbial agent. VS2523WU

[0039] Anti-Microbial Agents

[0040] An "anti-microbial agent," as used herein, refers to an agent that exhibits one or more "anti-microbial activity" i.e., any activity that inhibits the growth of a microorganism. By "inhibit," "inhibition" or "inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a microorganism by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent. In alternative embodiments, by "inhibit" "inhibition" or "inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a micro-organism by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of the microorganism in the absence of the anti-microbial agent. In alternative embodiments, the "inhibition" may be more than 100-fold. In alternative embodiments, the "inhibition" may be substantially complete inhibition of growth i.e., the growth rate may be reduced to about zero in the presence of the anti-microbial agent, and the anti- microbial agent may cause death of a micro-organism, when compared to the growth or survival of the micro-organism in the absence of the anti-microbial agent.

Accordingly, an anti-microbial agent may be microbicidal or may be microbistatic.

[0041] In some embodiments, the anti-microbial agent may be an anti-bacterial agent i.e., an agent that exhibits one or more "anti-bacterial activity" i.e., any activity that inhibits the growth of a bacterium. In alternative embodiments, the anti-bacterial agent may be bactericidal or bacteriostatic.

[0042] In some embodiments, an anti-bacterial agent according to the invention may selectively inhibit the growth of a Gram-negative staining bacterium. [0043] In alternative embodiments, an anti -bacterial agent according to the invention may selectively inhibit the growth of a specific Gram-positive staining bacterium, such as Listeria sp. e.g., Listeria innocua.

[0044] By "selectively inhibit" "selective inhibition" or "selectively inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of a Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua. In alternative embodiments, by "selectively inhibit" "selective inhibition" or "selectively inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a Gram-negative staining bacterium by at least about 1-fold or more, for example, about 1.5-fold to about 100- fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of a Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua. In alternative embodiments, the "selective inhibition" may be more than 100- fold. In alternative embodiments, the "selective inhibition" may be substantially complete inhibition of growth of a Gram-negative staining bacterium i.e., the growth rate could be reduced to about zero and the antibacterial agent may cause death of a Gram-negative staining bacterium when compared to the growth or survival of Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua. [0045] In particular embodiments, by "selectively inhibit" "selective inhibition" or "selectively inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a specific Gram-positive staining bacterium, such as a Listeria sp. or a Listeria innocua by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth or survival of a Gram-positive staining V82523WU bacterium other than a Listeria sp. or a Listeria innocua. In alternative embodiments, by "selectively inhibit" "selective inhibition" or "selectively inhibiting" is meant to destroy, prevent, control, decrease, slow or otherwise interfere with the growth or survival of a specific Gram-positive staining bacterium, such as a Listeria sp. or a Listeria innocua by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when compared to the growth or survival of a Gram- positive staining bacterium other than a Listeria sp. or a Listeria innocua. In alternative embodiments, the "selective inhibition" may be more than 100-fold. In alternative embodiments, the "selective inhibition" may be complete inhibition of growth i.e., the growth rate could be reduced to zero and the anti-bacterial agent may cause death of a specific Gram-positive staining bacterium such as a Listeria sp. or a Listeria innocua when compared to the growth or survival of a Gram-positive staining bacterium other than a Listeria sp. or a Listeria innocua.

[0046] Gram-negative staining bacteria include without limitation Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Pseudomonas sp.,

Helicobacter sp., Butyrivibrio sp., Fibrobacter sp. or Campylobacter sp. Examples of Gram-negative staining bacteria species include without limitation Escherichia coli {e.g., Escherichia coli RR1, Escherichia coli TBI, Escherichia coli 0157:H7),

Pantoea agglomerans, Pseudomonas fluorescens, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Butyrivibrio fibrisolvens, Fibrobacter succinogenes or Campylobacter jejuni.

[0047] In alternative embodiments, an anti-microbial agent according to the invention does not substantially inhibit the growth of Gram-positive staining bacteria, such as Pediococcus acidilactici, Paenibacillus polymyxa, Paenibacillus macerans, Bacillus lecheniformis, Bacillus subtilis, Bacillus circulans 9E2, Streptococcus bovis and Enterococcus mundtii.

[0048] In some embodiments, an anti-microbial agent according to the invention may be sensitive or insensitive to various treatments. By "sensitive" or "sensitivity" is meant loss or reduction of anti-microbial activity by at least about 10% to at least about 100%, or any value therebetween for example about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, by "sensitive" or "sensitivity" is meant loss or reduction of anti-microbial activity by at least about 1-fold or more, for example, about 1.5-fold to about 100-fold, or any value therebetween for example about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95-fold when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, the "sensitivity" may include loss or reduction of anti-microbial activity of more than 100-fold.

[0049] It is to be understood that sensitivity may vary with the time of treatment. In alternative embodiments, the time of treatment may range from a few minutes to many hours. For example, the time of treatment may be about 5 minutes to over 25 hours, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 minutes or any value therebetween, or such as 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0 hours, or any value therebetween. Anti-microbial activity may be tested by standard methods such as agar diffusion tests and micro-dilution assay as described herein, or by other standard methods such as disk diffusion, agar dilution, or through the use of automated instrumental testing systems (see, for example, Manual of Clinical

Microbiology. 1995. P. M. Murray (ed). ASM Press, Washington, DC). [0050] By "insensitive" or "insensitivity" is meant no substantial observable effect or sensitivity when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. In alternative embodiments, by "insensitive" or "insensitivity" is meant an observable effect of less than about 10%, for example about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% when an anti-microbial agent is subjected to a particular treatment, when compared to anti-microbial activity in the absence of the treatment. [0051 ] In some embodiments, an anti-microbial agent according to the invention may be sensitive to treatment with proteases such as proteinase K, trypsin, chymotrypsin or with a lipase.

[0052] In some embodiments, an anti-microbial agent according to the invention may be sensitive to treatment with surfactants such as sodium dodecyl sulphate (SDS), chaotropics agents such as urea, or solvents such as acetonitrile or hexane.

[0053] In some embodiments, an anti-microbial agent according to the invention may be insensitive to an organic solvent such as chloroform, propanol, methanol, ethanol or toluene. [0054] In some embodiments, an anti-microbial agent according to the invention may be insensitive to pH, for example, pH ranging from about 2 to about 9.

[0055] In some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 80°C. In some embodiments, an antimicrobial agent according to the invention may be sensitive to a temperature in excess of about 90°C. Accordingly, in some embodiments, an anti-microbial agent according to the invention may be sensitive to a temperature in excess of about 90°C when exposed for at least about 30 minutes. In alternative embodiments, an antimicrobial agent according to the invention may be sensitive to a temperature of about 100°C when exposed for at least about 10 minutes. [0056] In some embodiments, an anti-microbial agent according to the invention may be insensitive to a temperature upto about 80°C when exposed for about 30 minutes.

[0057] In particular embodiments, sensitivity of an anti-microbial agent according to the invention may include: the loss (about 100%) of anti-microbial activity after treatment of a composition including the anti-microbial agent with proteinase for 10 minutes at 100°C; the reduction of anti-microbial activity to about 83% or about 75% by trypsin and chymotrypsin, respectively, or to about 62% by lipase; the reduction of anti-microbial activity to about 66% after SDS treatment and about 58% after Urea treatment. [0058] In some embodiments, the molecular weight of an anti-microbial agent according to the invention may be about 1,000 Da to about 2,500 Da. In some embodiments, an anti-microbial agent according to the invention may include more than one molecule having a molecular weight in the range of about 1 ,000 Da to about 2,500 Da. The agent may be a peptide, for example, a lipopeptide.

[0059] In some embodiments, an anti-microbial agent according to the invention may be a peptidic compound including for example a nonproteinaceous amino acid, such as a D-amino acid or a hydroxy acid and/or may be modified for example by N methylation, acylation, glycosylation, or heterocyclic ring formation. [0060] In some embodiments, an anti-microbial agent according to the invention may be a polymyxin. By "polymyxin" is meant a peptide having anti-microbial activity. In general, the structure of a polymyxin may include a cyclic peptide e.g., a cyclic decapeptide, with a terminal fatty acid moiety, that is capable of inhibiting the growth of a micro-organism such as a Gram-negative staining bacterium. [0061 ] An anti-microbial agent according to the invention may include an antimicrobial agent produced by Paenibacillus polymyxa JB05-01-1, ATCC® Accession Number PTA- 10436, or by a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:l .

[0062] In some embodiments, an anti-microbial agent may include one or more compounds.

[0063] An anti-microbial agent may be present in a cell, or crude extract, cell culture, or cell culture supernatant thereof. The cell may be a Paenibacillus polymyxa JB05- 01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO: l . [0064] Methods of Obtaining and Producing Anti-Microbial Agents

[0065] Anti-microbial agent(s) may be obtained from Paenibacillus polymyxa JB05- 01-1 or from other sources. For example, RE3 (Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada) is a direct-fed microbial product used to V82523WU improve in vitro ruminal fermentation of barley grain/barley silage-based diets and includes a non-sterile liquid formulation containing L. paracasei and L. lactis cultures and their fermentation products. Paenibacillus polymyxa JB05-01-1 was obtained by culturing a sample of RE3™ . Other anti-microbial agents may similarly be found by routine screening for isolates that include the 16S rRNA sequence of SEQ ID NO: 1 as described herein or known in the art.

[0066] Anti-microbial agent(s) may be produced by growing or culturing

Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 in an appropriate cell culture medium under conditions suitable for production of anti-microbial agent(s) as described herein or known in the art. In alternative embodiments, Paenibacillus polymyxa JB05-01-1 or a bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 may be grown in an appropriate cell culture medium under conditions suitable for secretion of antimicrobial agent(s) into the cell culture supernatant as described herein or known in the art.

[0067] The cell culture medium may be a minimal medium or a complete medium. In some embodiments, the cell culture medium may be LB medium (Luria-Bertani medium). The medium may be a liquid medium or may be a solid or semi-solid medium, such as nutrient broth or agar, or tryptic soy broth or agar. In general, the cell culture medium includes a carbon/energy source, NH ₄ -N, and biotin.

[0068] The cell culture conditions {e.g., temperature, time, etc.) may be varied as appropriate to optimize growth and/or production of the anti-microbial agent(s).

[0069] In some embodiments, the temperature may range from about 5°C to about 40°C, such as 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C, or any value therebetween. In alternative embodiments, the temperature may be about 30°C.

[0070] In some embodiments, the time may range from about 5 hours to about 48 hours or any value therebetween. In alternative embodiments, the time may be greater than 48 hours. In alternative embodiments, the time may be about 20 hours.

[0071 ] The cell culture conditions may be aerobic or anaerobic. [0072] Standard separation processes may be used to obtain a substantially pure preparation of an anti-microbial agent. An agent or compound is "substantially pure" or "isolated" when it is separated from the components that naturally accompany it. Typically, an anti-microbial agent or compound is substantially pure when it is at least 10%, 20%, 30%, 40%, 50%, or 60%, more generally 70%, 75%, 80%, or 85%, or over 90%, 95%, or 99% by weight, of the total material in a sample. Thus, for example, a substantially pure preparation or culture of a cell expressing an anti-microbial agent, such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:l, is a preparation of cells or "cell culture" in which contaminating cells that are not a Paenibacillus polymyxa JB05-01-1 cell, or do not have the desired 16S rRNA sequence of SEQ ID NO:l, or do not express an anti-microbial agent as described herein, constitute less than 1%, 5%, 10%, 20%, 30%, 40%, or 50%, of the total number of cells in the preparation. In some embodiments, a substantially pure Paenibacillus polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:l, is a preparation of cells or "cell culture" that contains 100% of such cells.

[0073] In some embodiments, an anti-microbial agent that is isolated by known purification techniques, or isolated as described herein, will be generally be substantially free from its naturally associated components. A substantially pure antimicrobial agent can be obtained, for example, by extraction from a natural source such as a Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO:l .

[0074] In some instances, an anti-microbial agent according to the invention will form part of a composition, for example, a crude extract containing other substances. For example, an anti-microbial agent may be present in a crude extract of a

Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 that may also contain the other naturally occurring components found in such a cell. A crude extract of a

Paenibacillus polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 may be prepared by routine procedures, for example, disruption of the cells using standard mechanical or non- mechanical techniques such as freeze-thaw techniques, osmotic shock, enzyme (e.g., lysozyme) treatment, ultrasonication, liquid extrusion, etc., which may be followed by removal of the cell debris by for example centrifugation. [0075] In alternative embodiments, an anti-microbial agent may be present in a cell culture supernatant, such as a supernatant obtained from growing a Paenibacillns polymyxa JB05-01-1 cell or a naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 in a suitable cell culture medium under conditions suitable for secretion of the anti-microbial agent into the supernatant. The term "culture supernatant" refers to the liquid broth remaining when cells grown in a medium are separated from the culture medium by for example centrifugation, filtration, sedimentation, or other means well known in the art. As an example, if the anti-microbial agent(s) is to be isolated from cell culture supernatant, a salt such as ammonium sulphate may be used at various concentrations, initially. Residual ammonium sulphate may then be removed by dialysis against water. The suspended precipitate containing one or more than one antimicrobial compound may be chromatographed on a column such as an ion exchanger, and the various compounds in the culture supernatant may be separated by monitoring absorbance at 280 nm. Active fractions can be determined from among the compounds thus separated, and selected on the basis of the efficacy with which aliquots thereof kill or inhibit the growth of microbes such as bacterial cells, i.e. the indicator strain, known to be sensitive to the anti-microbial agent(s). Active fractions may then be pooled. Further purification may be carried out by high performance liquid chromatography (HPLC) based on the charge of the compound. The various peaks obtained by monitoring absorbance at 280 nm may be separated and again tested for activity against the indicator strain. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.

[0076] A person skilled in the art would understand that other conventional concentration, purification or fractionation methods may be used to obtain one or more isolated or substantially purified anti-microbial agent(s) or partially purified fractions exhibiting an anti-microbial activity from whole or lysed cells or from cell culture supernatant. Typical methods include, without limitation, size exclusion or ion exchange chromatography, ammonium sulfate, alcohol, or chloroform extraction, or centrifugation with size filters.

[0077] The anti-microbial activity of an anti-microbial agent may be determined by routine methods or as described herein. For example, anti-microbial activity may be detected by agar diffusion tests or micro-dilution assay.

[0078] Pharmaceutical, Veterinary, Nutritional. Cosmetic and Other Uses

[0079] Anti-microbial agent(s) according to the invention may be used in a variety of applications in which inhibition of growth of a micro-organism, such as a bacterium, is desirable. Such applications include, without limitation, pharmaceutical and veterinary applications (e.g., for the treatment of a microbial infection), nutritional supplements and animal feed, personal care (cosmetic or hygiene) applications, etc. In alternative embodiments, anti-microbial agent(s) according to the invention may be used to inhibit the growth of a microorganism (e.g., a bacterium) involved in the spoilage of food or other products.

[0080] Food spoilage micro-organisms include without limitation one or more species of Clostridium, Pseudomonas, Porteus, Chromobacterium,

Chromobacterium, Lactobacillus, Penicillium, Aspergillus, Rhizopus, Micrococcus, Bacillus, Streptococcus, Pediococcus, Leuconostoc, Chromobacterium,

Halobacterium, Alcaigenes, Xanthomonas, Botryitis, Aerobacter, Cornebacterium, Arthrobacter, Microbacterium, Serratia, etc.

[0081 ] The micro-organism may be a pathogenic micro-organism. The bacterium may a pathogenic bacterium, such as food-borne pathogenic bacterium. Food-borne pathogenic bacteria include without limitation one or more species of Staphylococcus, Vibrio, Escherichia, Listeria, Monocytogenes, Salmonella, Streptococcus, Vibrio, Campylobacter, Enterobacter, Shigella, etc.

[0082] The bacterium may include a Gram-negative staining bacterium or a Gram- positive staining bacterium. Gram-positive staining bacteria include without limitation one or more species of Listeria sp. or a Listeria innocua. Gram-negative V82523WU staining bacteria include without limitation one or more species of Escherichia sp., Pantoea sp., Pseudomonas sp., Salmonella sp., Shigella sp., Helicobacter sp., Campylobacter sp. or Butyrivibrio sp., and Fibrobacter sp. Examples of Gram- negative bacteria species include without limitation Escherichia coli (e.g., Escherichia coli RR1 , Escherichia coli TBI, Escherichia coli 0157:H7), Pantoea agglomerans, Pseudomonas fluorescens, Salmonella enteritidis, Salmonella typhi, Shigella dysenteriae, Helicobacter pylori, Campylobacter jejuni, Butyrivibrio fibrisolvens, or Fibrobacter succinogenes.

[0083] Other examples of bacteria include without limitation gram-negative rods such as enteric Gram-negative staining rods, curved Gram-negative staining rods, parvobacteria and Haemophilus, Gram-negative staining cocci such as Neisseria, non- sporing anaerobes, and bacteria such as spirochaetes, rickettsia and chlamydia.

[0084] Examples of microial infections, such as bacterial infections, include without limitation chlamydia, gonorrhea, salmonellosis, shigellosis, tuberculosis, syphilis, bacterial pneumonia, bacterial sepsis (bacteremia), bacterial urinary tract infections, vaginosis, bacterial upper respiratory tract infections, bacterial meningitis, bacterial enteritis, diphtheria, legionellosis, pertussis, scarlet fever, toxic shock syndrome, psittacosis, otitis media, lyme disease, etc.

Pharmaceutical, Veterinary, Nutritional and Other Compositions. Dosages, And Administration

[0085] Anti-microbial agents of the invention can be provided alone or in

combination with other compounds, in the presence of any pharmaceutically, veterinarily or cosmetically acceptable carrier, diluent, and/or excipient in a form suitable for administration to animals, for example, humans, cattle, sheep, pigs, poultry, etc. If desired, administration or application of an anti-microbial agent according to the invention may be combined with more traditional and existing antimicrobial therapies, treatments, supplements, or additives, or with other desirable therapies, treatments, supplements, or additives. [0086] Anti-microbial agents according to the invention may be provided chronically or intermittently. "Chronic" administration refers to administration of the antimicrobial agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.

"Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0087] Conventional pharmaceutical or veterinary practice may be employed to provide suitable formulations or compositions to administer the anti-microbial agent(s) to subjects suffering from or presymptomatic for a microbial infection. Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, intra-anal, intravaginal, aerosol, topical, or oral administration. Therapeutic formulations may be in the form of liquid solutions, syrups, or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols; and topical formulations may come in the form of balms, creams, and lotions.

[0088] Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences" (19 ^th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the anti-microbial agent(s) are administered to a subject in an amount sufficient to inhibit the growth of a micro-organism.

[0089] An "effective amount" of an anti-microbial agent(s) according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as inhibition of the growth of a micro-organism. A therapeutically effective amount of an anti-microbial agent(s) may vary according to factors such as the disease state, age, sex, and weight of the individual or subject, and the ability of the anti- microbial agent(s) to elicit a desired response in the individual or subject. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-microbial agent(s) are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as such as inhibition of the growth of a micro-organism. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. An exemplary range for therapeutically or prophylactically effective amounts of an anti-microbial agent(s) may be any value from about 0.1 nM to about 0.1M, for example about 0.1 nM to about 0.05M, about 0.05 nMto about 15μΜ or about 0.01 nM-to about.

[0090] It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the anti-microbial agent(s). Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical or veterinary practitioners. The amount of active anti-microbial agent(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic or prophylactic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, a subject may be a mammal, an agricultural (e.g., farm) or domestic animal, an experimental animal or any animal that may benefit from the anti-microbial agents as described herein. For example, a subject may include a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, chicken, turkey, duck, goose, dog, cat, fish, crustacean, etc. [0091] In general, anti-microbial agent(s) of the invention should be used without causing substantial toxicity. Toxicity of the anti-microbial agent(s) of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the antimicrobial agent(s).

[0092] In alternative embodiments, an "effective amount" of an anti-microbial agent according to the invention includes an amount effective to inhibit the growth of a micro-organism, such as a bacterium. It is to be understood that such amounts need not be therapeutic or prophylactic amounts, as long as the amount of the antimicrobial agent is capable of inhibiting the growth of a micro-organism, such as a bacterium, in the context in which it is administered or applied, for example, for prevention of food spoilage, etc. [0093] In alternative embodiments, an anti-microbial agent according to the invention may be provided in a cell, for example a substantially pure Paenibacillns polymyxa JB05-01-1 cell or a substantially pure naturally-occurring bacterium that includes the 16S rRNA sequence of SEQ ID NO: 1 , or a cell culture thereof. The cell may be provided in a liquid, or may be frozen or dried, e.g., freeze-dried. The cell culture may be concentrated. The cell culture may be a "starter" culture for example for a dairy product (e.g., milk, cheese, etc.), or for selective media in a laboratory.

[0094] The anti-microbial agent may be provided in a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product. In alternative embodiments, the anti- microbial agent may be provided in the packaging material for, for example, a therapeutic, veterinary, hygiene, cosmetic, food, drink or feed product. The packaging material may include without limitation, plastic, film, styrofoam, etc.

[0095] In alternative embodiments, an anti-microbial agent according to the invention may be provided in a kit that may optionally include additional anti- microbial agents or desirable therapies, treatments, supplements, or additives, optionally with instructions for use thereof.

[0096] In alternative embodiments, an anti-microbial agent according to the invention may be provided as a nutritional or food additive, or feed supplement or additive. [0097] A "nutritional additive" or "food additive" refers to a substance that is added to food, generally to affect the characteristics of the food, such as spoilage. A food additive may be "direct" in that it is directly added to food for example to inhibit growth of a micro-organism. A food additive may be considered "indirect" when it is exposed to food during processing, packaging, or storage but is not present in the final food product. The term "feed additive" or "feed supplement" refers to products used in animal nutrition for purposes of improving the quality of feed, or to improve the animals' performance and health, e.g. providing enhanced digestibility of the feed materials or inhibiting the growth of micro-organisms. An example of an animal feed additive is a direct- fed microbial product which refers to a mono or mixed culture of live micro-organisms, which when applied to a host affects beneficially the host by improving the properties of the indigenous microflora. A non-limiting example of a direct-fed microbial product is RE3™ from Basic Environmental Systems &

Technology Inc. Edmonton, AB, Canada. In some embodiments, RE3™ may be specifically excluded from a feed additive according to the invention. [0098] In alternative embodiments, anti-microbial agents of the invention can be provided in combination with other feed or nutritional supplements or additives. For example, at least one supplement or additive, such as listed herein, can be included for consumption with the anti-microbial agent of the invention and may have, for example, antioxidant, dispersant, antimicrobial, or solubilizing properties.

[0099] A suitable antioxidant is, for example, vitamin C, vitamin E or rosemary extract. A suitable dispersant is, for example, lecithin, an alkyl polyglycoside, polysorbate 80 or sodium lauryl sulfate. A suitable antimicrobial is, for example, sodium sulfite or sodium benzoate. A suitable solubilizing agent is, for example, a vegetable oil such as sunflower oil, coconut oil, and the like, or mono-, di- or triglycerides. Additives include vitamins such as vitamin A (retinol, retinyl palmitate or retinol acetate), vitamin Bl (thiamin, thiamin hydrochloride or thiamin mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinic acid or niacinamide), vitamin B5 (pantothenic acid, calcium pantothenate, d-panthenol or d-calcium pantothenate), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine or pyridoxine hydrochloride), vitamin B12 (cobalamin or cyanocobalamin), folic acid, folate, folacin, vitamin H (biotin), vitamin C (ascorbic acid, sodium ascorbate, calcium ascorbate or ascorbyl palmitate), vitamin D (cholecalciferol, calciferol or ergocalciferol), vitamin E (d- alpha-tocopherol, d-beta-tocopherol, d-gamma-tocopherol, d-delta-tocopherol or d- alpha-tocopheryl acetate) and vitamin K (phylloquinone or phytonadione). Other additives include minerals such as boron (sodium tetraborate decahydrate), calcium (calcium carbonate, calcium caseinate, calcium citrate, calcium gluconate, calcium lactate, calcium phosphate, dibasic calcium phosphate or tribasic calcium phosphate), chromium (GTF chromium from yeast, chromium acetate, chromium chloride, chromium trichloride and chromium picolinate) copper (copper gluconate or copper sulfate), fluorine (fluoride and calcium fluoride), iodine (potassium iodide), iron (ferrous fumarate, ferrous gluconate or ferrous sulfate), magnesium (magnesium carbonate, magnesium gluconate, magnesium hydroxide or magnesium oxide), manganese (manganese gluconate and manganese sulfate), molybdenum (sodium molybdate), phosphorus (dibasic calcium phosphate, sodium phosphate), potassium (potassium aspartate, potassium citrate, potassium chloride or potassium gluconate), VS2>-!3WU selenium (sodium selenite or selenium from yeast), silicon (sodium metasilicate), sodium (sodium chloride), strontium, vanadium (vanadium sulfate) and zinc (zinc acetate, zinc citrate, zinc gluconate or zinc sulfate). Other additives include amino acids, peptides, and related molecules such as alanine, arginine, asparagine, aspartic acid, carnitine, citrulline, cysteine, cystine, dimethylglycine, gamma-aminobutyric acid, glutamic acid, glutamine, glutathione, glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, taurine, threonine, tryptophan, tyrosine and valine. Other additives include animal extracts such as cod liver oil, marine lipids, shark cartilage, oyster shell, bee pollen and d-glucosamine sulfate. Other additives include unsaturated free fatty acids such as γ-linoleic, arachidonic and oc-linolenic acid, which may be in an ester (e.g. ethyl ester or triglyceride) form. Other additives include herbs and plant extracts such as kelp, pectin, Spirulina, fiber, lecithin, wheat germ oil, safflower seed oil, flax seed, evening primrose, borage oil, blackcurrant, pumpkin seed oil, grape extract, grape seed extract, bark extract, pine bark extract, French maritime pine bark extract, muira puama extract, fennel seed extract, dong quai extract, chaste tree berry extract, alfalfa, saw palmetto berry extract, green tea extracts, angelica, catnip, cayenne, comfrey, garlic, ginger, ginseng, goldenseal, juniper berries, licorice, olive oil, parsley, peppermint, rosemary extract, valerian, white willow, yellow dock and yerba mate. Other additives include miscellaneous substances such as menaquinone, choline (choline bitartrate), inositol, carotenoids (beta-carotene, alpha-carotene, zeaxanthin, cryptoxanthin or lutein), para-aminobenzoic acid, betaine HC1, free omega-3 fatty acids and their esters, thiotic acid (alpha-lipoic acid), l,2-dithiolane-3-pentanoic acid, l,2-dithiolane-3-valeric acid, alkyl polyglycosides, polysorbate 80, sodium lauryl sulfate, flavanoids, flavanones, flavones, flavonols, isoflavones, proanthocyanidins, oligomeric proanthocyanidins, vitamin A aldehyde, a mixture of the components of vitamin A2, the D Vitamins D\, D2, D3 and D4) which can be treated as a mixture, ascorbyl palmitate and vitamin K ?.

[00100] The supplement or additive may be packaged for consumption in softgel, capsule, tablet or liquid form. It can be supplied in edible polysaccharide gums, for example carrageenan, locust bean gum, guar, tragacanth, cellulose and carboxymethylcellulose. Cosmetic or hygiene supplements may be provided in for example, shampoos, conditioners, creams, pastes, lotions, lipsticks, lip balms, etc.

[00101] The present invention will be further illustrated in the following examples.

EXAMPLES [00102] The following examples are intended to illustrate embodiments of the invention and should not be construed as limiting.

Example 1: Identification of Antimicrobial Agent Producing Strains

Bacterial strains and growth media

[00103] The bacterial indicator strains used are listed in Table 1. All were maintained at -80°C in appropriate media containing 10% glycerol (w/v). P. polymyxa and all indicator strains except Butyrivibrio fibrisolvens and Fibrobacter succinogenes were propagated aerobically at 30°C in their respective culture media as indicated in Table 1. The media used were: Tryptic soy broth (TSB) (Difco Laboratories, Sparks, MD, USA), de Man, Rogosa and Sharpe broth (MRS) (Rosell Institute, Montreal, PQ, Canada) (de Man et al. 1960) and Luria-Bertani (LB) broth. Liquid or solid (1.2% w/v agar) anaerobic L-10 medium containing glucose, maltose and soluble starch as carbon sources (each at 0.1% w/w) was used for the growth of B. fibrisolvens and F. succinogenes (Caldwell and Bryant 1966). Their growth was carried out at 39°C in a C0 ₂ :H2 atmosphere (95:5 v/v). Before starting the experiments, all strains were sub-cultured at least three times at 24-h intervals using 1% volume transfers.

Isolation and identification of antimicrobial-compound-producing bacteria

[00104] The antimicrobial agent producer P. polymyxa JB05-01-1 was isolated from a direct-fed microbial product (RE3™ Basic Environmental Systems & Technology Inc. Edmonton, AB, Canada). RE3™ was screened for bacteria producing compounds inhibiting the growth of E. coli by a deferred antagonism plating procedure as described by Tagg et al. (1976). Briefly, 100 μΐ of 10 ³ -10 ⁴ dilutions of RE3™ in L-10 or TSB media were spread on L-10 or TSB plates and incubated overnight at 39°C and 37°C. The plates were replicated, and then the bacterial colonies on the original V82523WU plates were washed from the agar surface and the plates were surface-sterilized under Ultraviolet (UV) light at 254 nm for 20 minutes. The plates were then overlaid with 5 ml of melted LB (0.5% agar) containing 50 μΐ of an overnight culture of E. coli RR1 and incubated overnight at 37°C. Colonies producing clearing zones were identified and picked from the replica plates for testing for activity against E. coli 0157:H7.

[00105] Gram staining, motility, catalase and oxidase tests were conducted as a preliminary step in the characterization of the selected colonies. Tentative

identifications were confirmed by amplification and sequencing of 16S ribosomal RNA genes. DNA extraction

[00106] The Paenibacillus strain (JB05-01-1) was grown in 3 ml of TSB at 30°C overnight. The cells were harvested by centrifugation at 5000 x g for 5 min. DNA was extracted using a Power Soil DNA Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. The DNA concentration was measured using the PicoGreen dsDNA quantitation kit (Molecular Probes, Invitrogen, Eugene, OR, USA) in a Multi Detection Microplate Reader (Model SIAFRM, BioTek

Instruments, Winooski, VT, USA) using calf thymus DNA (Sigma-Aldrich, St. Louis, MO, USA) as the standard.

Polymerase Chain Reaction Amplification of 16S rRNA genes [00107] The PCR amplification targeted the approximately 1500 bp of the 16S rRNA gene. The PCR reaction contained 10 ng of template DNA, 2.5 ml of 10 x dilution buffer, 10 pmol of each primer and 1 U of Taq polymerase (Takara Shuzo, Japan) in a final volume of 25 ml. The primers used were the universal bacterial primers 8-27 F (5'-AGA GTT TGA TCC TGG CTC AGA-3 ⁰ ) (Liu et ai. 1997) and 1492R (5'-TAC CTT GTT ACG ACT T-3 ') (Kane et ai, 1993). The amplification conditions involved denaturation at 95°C for 1 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 1.5 min. The nomenclature of the primers used was based on E. coli numbering system.

Cloning and Sequencing of 16S rRNA genes VS^iJWU

[00108] Amplicons from the PCR reaction were electrophoresed on 1% agarose gel and purified by excising the correct sized bands. The DNA was extracted from the gel using QIAquick PCR purification Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. The purified DNA was cloned into TOPO vector (Invitrogen, Carlsbad, CA) and further used to transform electrocompetent E. coli (DH5-a cells) by electroporation. The cells were then plated on LB/Kanamycin (50 mg L) agar plates and incubated overnight at 37°C. Three clones, verified for correct inserts, were grown overnight in LB/Kanamycin (100 mg/L). All clones were sequenced by the University of Calgary Core DNA Services, Calgary, AB, Canada. The 16S rRNA sequence was a consensus from three clones. The 16S rDNA sequences of the isolates were compared with DNA sequences from the National Center for Biotechnology Information (NCBI) database using the standard nucleotide- nucleotide homology search Basic Local Alignment Search Tool (BLAST) (Altschul et ai, 1990). Results

[00109] Sequencing of the 16S rDNA PCR products from the isolate showing the highest activity against E. coli 0157:H7 identified an organism that shared 99% homology with Paenibacillus polymyxa. This isolate was designated as Paenibacillus polymyxa JB05-01-1. The 16S rRNA gene partial sequence of P. polymyxa JB05-01-1 was deposited with GenBank and has been assigned Accession Number GQ184435 (Figure 4, SEQ ID NO: 1).

Table 1. Antimicrobial spectrum of Paenibacillus polymyxa JB05-01-1 20h batch culture supernatant. Results are means of two individual assays

___

*Source **Medium Culture Polymyxin E Nisin A

Supernatant

MRS

Pediococcus acidilactici ATCC LB _ - +++

Paenbacillus polymyxa JB05-01-1 Collection of Ron Teather LB - - +

Paenbacillus macerans LRC culture collection LB - - +

Paenbacillus polymyxa 21551 LRC culture collection LB - - +

Bacillus lecheniformis LRC culture collection LB - - +

Bacillus subtilus 6051 LRC culture collection LB - - +

Bacillus subtilis Collection of Ron Teather LB - - +

Bacillus circulars 9E2 LRC culture collection LB - - +

Streptococcus bovis Collection of Ron Teather LB - - +

Enterococcus mundtii Collection of Ron Teather TSB - - ++

Listeria innocua Collection of Ron Teather TSB + - ++

Escherichia coli RR1 Collection of Ron Teather TSB + + -

Escherichia coli TBI Collection of Ron Teather TSB + + -

Escherichia coli SA1650 LRC culture collection TSB + + -

Pantoea agglomerans BC1 LRC culture collection TSB + + -

Pseudomonas fluorescens R73 LRC culture collection L-10 + + -

Butyrivibrio fibrisolvens OR85 Collection of Ron Teather L-10 + + -

Fibrobacter succinogenes LRC culture collection + + -

- No inhibition detected

+ Diameter of the inhibition zone 10 ± 2 mm

++ Diameter of the inhibition zone 16 ± 2 mm

+++ Diameter of the inhibition zone 28 ± 2 mm

* ATCC: American Type Culture Collection,

LRC : Lethbridge Research Center

V«252JWU

Example 2: Production of the Antimicrobial Agent

[001 10] One litre of LB medium was inoculated with 10 ml of a fresh, overnight culture of P. polymyxa JB05-01-1 and incubated at 30°C with agitation at 200 rpm. The culture optical density at 600 ran was measured every two hours using a

Multi-detection micro-plate reader (Bio-Teck instrument Inc., Winooski, Vermont, USA), and 1 mL of culture was centrifuged (8,000 rpm, 10 min, 4°C) to remove the cells. The supernatant was heated at 70°C for 10 min to inactivate any protease activity, as described by Martin et al. (2003). The agar diffusion assay and

micro-dilution method were used to test the heated supematants for antimicrobial activity as described herein.

[001 11] The determination of soluble protein was done using the Folin phenol reagent method as described by Lowry et al. (1951) with bovine serum albumin as standard. Polymyxin E and Nisin A were used as positive control for antimicrobial activity. Nisin A stock solutions were prepared from pure Nisin obtained from Aplin and Barrett (Beaminster, UK) in the form of Nisaplin™, which contains 2.5% (w/w) Nisin A. Polymyxin E was purchased from Sigma-Aldrich (Oakville, ON, Canada).

[00112] Inhibition of Escherichia coli RR1 by supernatant of a batch culture grown in Luria-Bertani broth, measured by the micro-dilution method, was maximal at 20 h and remained so through 48 h. Thus, based on agar diffusion and micro-dilution tests, the secretion of the antimicrobial agent was shown to start in the exponential phase and reach its maximum in the early stationary phase (Figure 1). Production thus appeared to be growth-associated and activity levels remained stable through 48 h. Inhibition zone diameters at 48 h were approximately 8 ±1 mm and activity was 96 AU ml ^"1 .

Example 3: Spectrum of Activity [001 13] The qualitative antimicrobial spectrum of P. polymyxa culture supernatant was determined using the agar well diffusion method (Wolf and Gibbons 1996). Briefly, a 25-ml volume of molten tryptic soy agar (0.75% agar w/v) was cooled to 47°C and seeded with 1% (v/v) overnight TSB culture of an indicator strain. The seeded agar was then poured into a sterile Petri plate and allowed to solidify at room temperature. Wells (7 mm) were cut in the solidified agar using a sterile metal cork borer and filled with 80 μΐ of supernatant. The plates were left at 5°C for 2 h to allow diffusion of the tested aliquot and then incubated aerobically for 18 h at 30°C.

Absence or presence of inhibition zones as well as their diameters were recorded.

[001 14] The antimicrobial activity was also determined by the micro-dilution method described by Daba et al. (1994). Activity was expressed in arbitrary units per milliliter (AU ml ^"1 ) using the formula (1000/125) ^x (l/£>), where D was the highest dilution causing inhibition of the indicator strains.

[001 15] The minimum inhibitory concentration (MIC) of P. polymyxa JB05-01-1 culture supernatant and pure polymyxin E against E. coli RRl was determined using a Microtest™ polystyrene micro-plate assay (96- well, Becton Dickinson Labware,

Lincoln Park, NJ) as described by Kheadr et al. (2004). To determine the effect of P. polymyxa JB05-01-1 culture supernatant, E. coli RRl cells were cultivated in the presence of 16 AU ml ^"1 , 32 AU ml ^"1 , or 96 AU ml ^"1 , and culture optical density at 600 nm was measured every two hours using a Multi-detection micro-plate reader (Bio-Teck Instrument Inc., Winooski, Vermont, USA) (Naghmouchi et al., 2007).

[001 16] The inhibitory spectrum of the culture supernatant is presented in Table 1. Gram-negative staining bacteria (E. coli RRl, Pantoea agglomerans BC1,

Pseudomonas fluorescens R73, B. fibrisolvens OR85 and F. succinogenes S85) were inhibited while no activity was detected against Gram-positive staining bacteria except for Listeria innocua. The spectrum of activity of the antimicrobial agent produced by P. polymyxa JB05-01-1 was different from that of Nisin A but similar to polymyxin E, except for the inhibition of Listeria innocua.

[00117] E. coli RRl cells were cultivated in the presence of 16 AU ml ^"1 (8h culture supernatant), 32 AU ml ^"1 (16h culture supernatant), or 96 ± 32 AU ml ^"1 (20h culture supernatant) (Figure 2). During the exponential phase, the generation time of E. coli RRl was increased from 90 min in the control culture to 150 min in the presence of 96 AU ml ^"1 , and culture density in early stationary phase was reduced by - 15 %. Nisin A showed no inhibitory effect against E. coli RRl.

Example 4: Characterization of the Antimicrobial Agent [001 18] The sensitivities of the antimicrobial agent to proteases (all from

Sigma-Aldrich, Oakville, ON) or other agents was tested by treating P. polymyxa JB05-01-1 culture supernatant with 2 mg ml ^"1 final concentration of proteinase (Tritirachium album), cc-chymotrypsin (bovine pancreas), lipase (Sigma-Aldrich, Oakville, ON), trypsin (porcine pancreas), urea (Sigma-Aldrich, St. Louis MO), sodium dodecyl sulfate (SDS) (Sigma-Aldrich, St. Louis MO) for 1 h at 37°C (Motta et al. 2007). Thermal stability of the antimicrobial activity was determined by holding aliquots (1000 μΐ) of 20h culture supernatant at temperatures ranging from 50°C to 90°C for 30 min or at 100°C for 10 min. The effect of pH was determined by adjusting the pH of P. polymyxa JB05-01-1 culture supernatant from 2 to 9 using 5 M HC1 or NaOH. The activity of each sample was compared with the activity of untreated P. polymyxa JB05-01 culture supernatant at pH 6.8.

[001 19] The effect of several organic solvents was evaluated by stirring 20 h culture supernatant for 2 h with 10% (v/v) acetonitrile, hexane, propanol, ethanol, toluene, acetone, butanol or methanol (all solvents were obtained from Sigma-Aldrich, St. Louis MO). Residual antimicrobial activities were tested using the agar diffusion assay against E. coli RR1 as described herein, with controls for effects of residual solvent.

[00120] The effect of enzymes, detergents and other compounds on the anti-E. coli activity of the P. polymyxa JB05-01-1 culture supernatant is shown in Table 2.

v w \J

Table 2. Effect of various enzymes, detergents and urea on the antimicrobial activity of Paenibacillus polymyxa JB05-01-1 culture supernatant against Escherichia coli RR1 determined by the agar diffusion test. No antimicrobial activity effect of Nisin A was shown against E. coli RR1.

Agent kesidual Activity (7o) ⁺

Culture supernatant Polymyxin E

None 100 100

Proteinase 0 37.5

Trypsin 83.3 62.5

Chymotrypsin 75 79.5

Lipase 62.5 58.3

SDS 66 70.83

Urea 58 62.5

* Relative to the antimicrobial activity of untreated supernatant. Results are means of two individual assays

[00121] Activity was eliminated after proteinase treatments. Lipase, trypsin, -chymotrypsin, sodium dodecyl sulphate (SDS) and urea reduced the antimicrobial activity by 38%, 17%, 25%, 34% and 42% respectively when compared to untreated activity.

[00122] The antimicrobial activity remained unchanged after heating at 80°C for 30 min. Loss of activity of about 60% was observed after heating at 90°C for 30 min. Heating to 100°C for 10 min completely eliminated the antimicrobial activity.

[00123] Organic solvents such as chloroform, propanol, methanol, ethanol and toluene did not affect the activity of the antimicrobial peptide. Acetonitrile or hexane treatment at the same concentration ( 10%, v/v) reduced the antimicrobial activity by about 5% and 20%, respectively. Activity also remained stable after a two-hour incubation at pH ranging from 2 to 9.

Example 5: Molecular Weight Determination

[00124] P. polymyxa culture supernatant was analysed in duplicate using a NuPAGE 12% Bis-Tris gel kit (Invitrogen, Burlington, ON, Canada) as per manufacturer's instructions at 200 V (constant) for 40 min. The 2.5-200 kDa molecular weight marker kit from Invitrogen was used as a molecular weight standard. After

electrophoresis, one gel was stained with Coomassie Brilliant Blue R250 (Invitrogen). A duplicate gel was used for the plate overlay assay to estimate the molecular weight of the antimicrobial compounds as described by Bhunia et al. (1987). Briefly, a SDS-PAGE gel prewashed with sterile water was placed in a Petri dish and overlaid with 10 ml tryptic soy agar containing growing cells of E. coli RRl at about 10 ⁵ CFU ml ^"1 . The agar was allowed to solidify, held at 4°C for 60 min, and then incubated for 18 h at 30°C. The formation of an inhibition zone indicated the position and size of the active antimicrobial peptide in the gel.

[00125] Coomassie Brilliant Blue staining of SDS-PAGE gels of P. polymyxa JB05-01-1 culture supernatant revealed no distinct protein bands. However, inhibitory activity was detected as a clearly defined zone of inhibition in the region corresponding to a molecular mass of <2.5 kDa (<2500 Da) after gels were overlaid with E. coli RRl -seeded agar (Figure 3). No inhibitory activity was detected when the SDS gels were overlaid with Listeria innocua.

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OTHER EMBODIMENTS

[00151] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Accordingly, although various embodiments of the invention are disclosed herein, many adaptations and

modifications may be made within the spirit and scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range, and of sub-ranges encompassed therein. As used herein, the terms "comprising", "comprises", "having" or "has" are used as an open-ended terms, substantially equivalent to the phrase "including, but not limited to". Terms such as "the," "a," and "an" are to be construed as indicating either the singular or plural. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.