WO2001034168A9 - Inhibitions d'agents pathogènes à l'aide de bactéries probiotiques - Google Patents

Inhibitions d'agents pathogènes à l'aide de bactéries probiotiques

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Publication number
WO2001034168A9
WO2001034168A9 PCT/US2000/030737 US0030737W WO0134168A9 WO 2001034168 A9 WO2001034168 A9 WO 2001034168A9 US 0030737 W US0030737 W US 0030737W WO 0134168 A9 WO0134168 A9 WO 0134168A9
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WIPO (PCT)
Prior art keywords
bacillus
composition
bacillus coagulans
strain
lactic acid
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PCT/US2000/030737
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English (en)
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WO2001034168A1 (fr
Inventor
Sean Farmer
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Ganeden Biotech Inc
Sean Farmer
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Application filed by Ganeden Biotech Inc, Sean Farmer filed Critical Ganeden Biotech Inc
Priority to CA2389982A priority Critical patent/CA2389982C/fr
Priority to JP2001536165A priority patent/JP2003513649A/ja
Priority to EP00978435A priority patent/EP1229923A1/fr
Priority to AU15900/01A priority patent/AU785159B2/en
Publication of WO2001034168A1 publication Critical patent/WO2001034168A1/fr
Publication of WO2001034168A9 publication Critical patent/WO2001034168A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants

Definitions

  • the present invention relates to methods of treatment and compositions using novel stains of probiotic organisms and/or their extracellular products in therapeutic compositions. More specifically, the present invention relates to the utilization of one or more species or strains of probiotic bacteria and/or their extracellular products for the control of gastrointestinal pathogens, including antibiotic-resistant species.
  • the gastrointestinal microfiora has been shown to play a number of vital roles in maintaining gastrointestinal tract function and overall physiological health.
  • the growth and metabolism of the many individual bacterial species inhabiting the gastrointestinal tract depend primarily upon the substrates available to them, most of which are derived from the diet. See, e.g., Gibson et al., 1995. Gastroenterology 106: 975-982; Christl, et ai., 1992. Gut 33: 1234-1238; Gorbach, 1990. Ann. Med. 22: 37-41; Reid et al, 1990. Clin. Microbiol. Rev. 3: 335-344.
  • probiotics which are live microbial food supplements.
  • the best-known probiotics are the lactic acid-producing bacteria (i.e., Lactobacilli and Bifidobacteria) , which are widely utilized in yogurts and other dairy products. These probiotic organisms are non-pathogenic and non-toxigenic, retain viability during storage, and survive passage through the stomach and small intestine. Since probiotics do not permanently colonize the host, they need to be ingested regularly for any health promoting properties to persist.
  • Commercial probiotic preparations are generally comprised of mixtures of Lactobacilli and Bifidobacteria, although yeast species such as Saccharomyces have also been utilized.
  • the invention provides compositions, therapeutic systems, and methods of use which exploit the discovery that novel lactic acid-producing bacterial strains (e.g., the novel strains of Bacillus coagulans disclosed herein), or extracellular products thereof, possess the ability to exhibit inhibitory activity in mitigating and preventing the growth and/or colonization rates of pathogenic bacterial, particularly gastrointestinal pathogens such as antibiotic-resistant pathogenic bacterial species including, but not limited to, Enter ococccus, Clostridium, Escherichia, Klebsiella, Campylobacter, Peptococcus, Heliobacter, Hemophylus, Staphylococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, and Rotovirus species, as well as mitigating the deleterious physiological effects of the infection by the pathogen(s).
  • novel lactic acid-producing bacterial strains e.g.
  • the bacteria are probiotic.
  • probiotic microorganisms are those, which confer a benefit when grow in a particular microenvironment by, e.g., directly inhibiting or preventing the growth of other biological organisms within the same microenvironment.
  • probiotic organisms include, but are not limited to, bacteria, which possess the ability to grow within the gastrointestinal tract, at least temporarily, to displace or destroy pathogenic organisms, as well as providing other benefits to the host. See, e.g., Salminen et al, 1996. Antonie Van Leeuwenhoek 70: 347- 358; Elmer et al, 1996. JAMA 275: 870-876; Rafter, 1995. Scand. J. Gastroenterol. 30: 497-502; Perdigon et al, 1995. J. Dairy Sci. 78: 1597-1606; Gandi, Townsend Lett.
  • novel strains of Bacillus coagulans disclosed herein possess biochemical and physiological characteristics which include, but are not limited to: (0 the production of the (L)+ optical isomer of lactic acid (propionic acid); (ii) have an optimal growth temperature of between 20-44°C; (iii) produces spores resistant to temperatures of up to approximately 90°C which are able to germinate in a human or animal body without specific inducement (e.g., heat-shock or other environmental factors); (iv) the production of one or more extracellular products exhibiting probiotic activity which inhibits the growth of bacteria, yeast, fungi, virus, or any combinations thereof; and/or (v) the ability to utilize a wide spectrum of substrates for proliferation.
  • the purified population of Bacillus coagulans has an optimal growth temperature of less than 45 degrees C.
  • the isolated population of Bacillus coagulans has an optimal growth temperature of 20 degrees C, more preferably 30 degrees C, more preferably 35 degrees C, more preferably 36 degrees C, and most preferably 37 degrees C.
  • previously identified populations of Bacillus coagulans have an optimal growth temperature of greater than 37 degrees C, e.g., an optimal growth temperature of 45 degrees C.
  • the strain grows at low pH such as pH conditions found in the gastrointestinal tract of a mammal, e.g., pH 2-5.
  • purified or isolated preparation of a bacterial strain is meant that the preparation does not contain another bacterial species or strain in a quantity sufficient to interfere with the replication of the preparation at a particular temperature.
  • a purified or isolated preparation of a bacterial strain is made using standard methods, e.g., plating at limiting dilution and temperature selection.
  • a therapeutic composition comprising Bacillus coagulans in a pharmaceutically-acceptable carrier suitable for oral administration to the gastrointestinal tract of a human or animal, is disclosed.
  • a Bacillus coagulans strain is included in the therapeutic composition in the form of spores.
  • a Bacillus coagulans strain is included in the composition in the form of a dried or lyophilized cell mass.
  • An embodiment of the present invention involves the administration of from approximately lxlO 3 to lxlO 14 CFU of viable, Bacillus coagulans vegetative bacteria or spore per day, more preferably from approximately lxlO 5 to lxlO 1 ', and preferably from approximately 5x10 to 1x10 CFU of viable, vegetative bacteria or spores per day.
  • the typical dosage is approximately lxlO 2 to lxlO 14 CFU of viable, vegetative bacteria or spores per day, preferably from approximately lxlO 8 to lxlO 10 , and more preferably from approximately 2.5xl0 8 to lxlO 10 CFU of viable, vegetative bacteria or spores per day.
  • a composition comprising an extracellular product of Bacillus coagulans in a pharmaceutically-acceptable carrier suitable for oral administration to a human or animal.
  • the extracellular product is a supernatant or filtrate of a culture of an isolated Bacillus coagulans strain.
  • the extracellular product is a semi-purified or purified, lyophilized supernatant or filtrate of a culture of an isolated Bacillus coagulans strain.
  • the extracellular product is the active agent(s) possessing the anti-microbial activity, which are isolated and purified from a supernatant or filtrate of a culture of an isolated Bacillus coagulans strain.
  • the extracellular product is administered to a subject in a composition comprising a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 1% to 90% extracellular product with the remainder comprising the carrier or delivery component.
  • the subject is preferably a mammal, e.g., a human.
  • the bacteria and/or products derived from the bacteria are also suitable for veterinary use, e.g., to treat animals such as dogs and cats.
  • a preferred embodiment comprises a composition a total concentration ratio oi Bacillus coagulans extracellular product ranging from approximately 10% to 75% extracellular product with the remainder comprising the carrier or delivery component.
  • the present invention is not limited solely to oral administration of the therapeutic compounds disclosed herein.
  • Skin and or mucous membranes are treated using compositions containing Bacillus coagulans vegetative cells, spores, or extracellular products produced by vegetative cells.
  • the administration of the Bacillus coagulans strains, and/or the extracellular products thereof aid in the mitigation of vaginal pathogens, as well as reducing the incidence of relapse by re- population of the vagina with these probiotic, lactic acid-producing bacteria.
  • the compositions are used to treat a condition characterized by a reduction or absence of lactic acid-producing bacteria within the vagina, which condition is the a common etiology of both vaginal yeast infections and bacterial vaginosis.
  • compositions are administered vaginally, intra-ocularly, intra-nasally, intra-otically, or buccally.
  • a further embodiment of the present invention involves the utilization of probiotic organisms in livestock production, in which antibiotics such as Vancomycin and Gentamicin are commonly used to stimulate health and weight gain.
  • antibiotics such as Vancomycin and Gentamicin are commonly used to stimulate health and weight gain.
  • Most, if not all, probiotic organisms are sensitive to these two antibiotics and this fact has limited the potential use of such microorganisms in the livestock industry.
  • there are many environmentally-related problems associated with the use of antibiotics in livestock production For example, antibiotic laden animal waste degrades very slowly and the antibiotic residue can persist, further slowing biodegradation. With the addition of species of bacteria that are resistant to Vancomycin, Gentamicin, and other antibiotics, biodegradation is enhanced.
  • compositions, therapeutic systems, and methods of use for inhibiting pathogen and/or parasite growth in the gastrointestinal tract and feces of animals According to the invention, there is provided a composition comprising Bacillus coagulans vegetative cells or spores in a pharmaceutically- or nutritionally-acceptable carrier suitable for oral administration to the digestive tract of an animal.
  • the extracellular product from a Bacillus coagulans culture is utilized, with or without Bacillus coagulans vegetative cells or spores.
  • the bacteria is present in the composition at a concentration of approximately lxlO 3 to lxlO 14 colony forming units (CFU)/gram, preferably approximately lxlO 5 to lxlO 12 CFU/gram, whereas in other preferred embodiments the concentrations are approximately lxlO 9 to lxl 0 13 CFU/gram, approximately lxlO 5 to 1 x 10 7 CFU/g, or approximately 1 x 10 8 to 1 x 10 9 CFU/gram.
  • CFU colony forming units
  • the bacteria is in a pharmaceutically acceptable carrier suitable for oral administration to an animal, preferably, as a powdered food supplement, a variety of pelletized formulations, or a liquid formulation.
  • the invention also describes a therapeutic system for inhibiting pathogen and/or parasite growth in the gastrointestinal tract and/or feces of an animal comprising a container comprising a label and a composition as described herein, wherein said label comprises instructions for use of the composition for inhibiting pathogen and/or parasite growth.
  • non-antibiotic, probiotic bacteria-based therapeutic regimen include, but are not limited to: (/) the administration of the composition will result in the reduction of the colonization rate of enter ococci in the gastrointestinal tract; (ii) no contribution to the development of antibiotic resistance; (iii) the composition can be used prophylactically to reduce the reservoir of enterococci in hospitals, which will concomitantly reduce the chances of high-risk patients from acquiring VRE; (iv) the dosage of the composition can be varied according to patient age, condition, etc; and (v) the composition may be utilized in food animal to reduce the development of further antibiotic resistance.
  • FIG. 1 is a bar graph showing the minimal and optimal culture temperatures for the Bacillus coagulans 1 % isolate (GBI- 1 ); ATCC- 99% isolate (ATCC #31284); the 5937-20°C isolate (GBI-20); and the 5937-30°C isolate (GBI-30), in either Trypticase Soy Broth (TSA) or Glucose Yeast Extract (GYE) media.
  • FIG. 2 is a bar graph showing the End-Point Kinetics of the 1% Bacillus coagulans strain (GBI-1).
  • FIG. 3 is a bar graph showing the End-Point Kinetics of the ATCC- 99%
  • Bacillus coagulans strain (ATCC #31284).
  • FIG. 4 is a bar graph showing the End-Point Kinetics of the 5937- 20°C Bacillus coagulans strain (GBI-20) and the 5937- 30°C Bacillus coagulans strain (GBI-30) with TSB and GYE media.
  • FIG. 5 is a bar graph showing the End-Point Kinetics of the 5937- 20°C Bacillus coagulans strain (GBI-20) and the 5937- 30°C Bacillus coagulans strain (GBI-30) with NB and BUGMB media.
  • FIG. 6 is a diagram showing the results from Alignment with other Bacillus species, Neighbor Joining Tree, and Concise Alignment analysis for the Bacillus coagulans ATCC- 99% isolate (ATCC #31284).
  • FIG. 7 is a diagram showing the results from Alignment with other Bacillus species, Neighbor Joining Tree, and Concise Alignment analysis for the Bacillus coagulans 20°C isolate (GBI-20).
  • FIG. 8 is a diagram showing the results from Alignment with other Bacillus species, Neighbor Joining Tree, and Concise Alignment analysis for the Bacillus coagulans 30°C isolate (GBI-30).
  • FIG. 9 is a bar graph showing the results of the Aminopeptidase profile for the Bacillus coagulans ATCC- 99% isolate (ATCC#31284).
  • FIG. 10 is a bar graph showing the results of the Aminopeptidase profile for the Bacillus coagulans ATCC- 1 % isolate (GBI- 1 ).
  • FIG. 11 is a bar graph showing the results of the Aminopeptidase profile for the Bacillus coagulans ATCC- 30°C isolate (GBI-30).
  • FIG. 12 is a bar graph showing the results of the Aminopeptidase profile for the Bacillus coagulans ATCC- 20°C isolate (GBI-20).
  • Lactic acid-producing bacterial species e.g., Lactobacillus, Bifidiobacterium, and the majority of Bacillus species have generally been thought to be unsuitable for colonization of the gut due to their instability in the harsh (i.e., acidic) pH environment of the bile, particularly human bile.
  • Bacillus coagulans including the novel strains disclosed herein, was found to survive and colonize the gastrointestinal tract such as a bile environment and grown in this low pH range.
  • the human bile environment is different from the bile environment of animal models, and heretofore there has not been any accurate descriptions of Bacillus coagulans growth in human gastrointestinal tract models.
  • Lactic acid producing bacteria are gram positive and vary in morphology from long, slender rods to short coccobacilli, which frequently form "chains". Their metabolism is fermentative; with some species being aerotolerant (i.e., may utilize oxygen through the enzyme flavoprotein oxidase) while others are strictly anaerobic. Spore-forming lactic acid-producing bacteria are facultative anaerobes, whereas the rest are strictly anaerobic. The growth of these bacteria is optimum at pH 5.5-5.8, and the organisms have complex nutritional requirements for amino acids, peptides, nucleotide bases, vitamins, minerals, fatty acids, and carbohydrates. The lactic acid bacteria have the property of producing lactic acid from fermentable sugars.
  • the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus are important members of this group.
  • the taxonomy of lactic acid-producing bacteria has been based on the gram reaction and the production of lactic acid from various fermentable carbohydrates. These groups include:
  • Homofermentative produce more than 85% lactic acid from glucose.
  • Hetero fermentative produce only 50% lactic acid and considerable amounts of ethanol, acetic acid and carbon dioxide.
  • Well-known are the hetero-fermentative species, which produce DL-lactic acid, acetic acid and carbon dioxide. These species, which have been used therapeutically, include: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus brevis, Lactobacillus delbruekii, and Lactobacillus lactis.
  • probiotic preparations were initially systematically evaluated for their effect on health and longevity in the early-1900's (see e.g., Metchinikoff, Prolongation of Life, Willaim Heinermann, London 1910), their utilization has been markedly limited since the advent of antibiotics in the 1950's to treat pathological microbes. See, e.g., Winberg, et al, 1993. Pediatr. Nephrol. 7: 509-514; Malin et al, Ann. Nutr. Metab. 40: 137-145; and U.S. Patent No. 5,176,911. Unfortunately, the majority of these early studies on probiosis were observational rather than mechanistic in nature, and thus the processes responsible for many probiotic phenomena were not quantitatively elucidated.
  • the ecosystem of the human gastrointestinal tract is colonized by more than 500 species of bacteria and represents an extremely complex microenvironment.
  • the composition of the intestinal micro flora is constantly changing, being influenced by such factors as: diet, stress, age, and treatment with antibiotics and other drugs.
  • Successful utilization depends upon the following factors: (/) a high count of viable organisms retaining their viability during manufacturing into dosage forms and subsequent storage; (ii) survival of these lactic acid producing bacteria, once ingested, in the acidic gastric secretions and their passage into the intestine; and (iii) the production of a sufficient quantity of metabolites antagonistic to pathogens (e.g., L(+) (dextrorotatory) lactic acid and bacteriocins).
  • pathogens e.g., L(+) (dextrorotatory) lactic acid and bacteriocins
  • Lactobacilli Previously, numerous species of Lactobacilli have been examined including, but not limited to, Lactobacillus bulgaricus, Lactobacillus bifidus, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus brevis. Interestingly, however, Lactobacillus acidophilus, long regarded as the best candidate for therapeutic use, has been subsequently shown to be highly ineffective as a probiotic organism for the re- colonization of the gastrointestinal tract and in the alleviation of gastrointestinal disorders. Moreover, this bacterial strain produces D(-) (levorotatory) lactic acid, which is not an effective antagonistic agent and may potentially introduce metabolic disturbances.
  • Lactobacillus strains which produce antibiotics, have been demonstrated as effective for the treatment of infections, sinusitis, hemorrhoids, dental inflammations, and various other inflammatory conditions. See, e.g., U.S. Patent No. 5,439,995.
  • Lactobacillus reuteri has been shown to produce antibiotics which possess antimicrobial activity against Gram negative and Gram positive bacteria, yeast, and various protozoan. See, e.g., U.S. Patent Nos. 5,413,960 and 5,439,678.
  • proteolytic, lipolytic, and ⁇ -galactosidase activities of probiotic bacteria have also been shown to improve the digestibility and assimilation of ingested nutrients, thereby rendering them valuable in convalescent /geriatric nutrition and as adjuncts to antibiotic therapy.
  • Probiotics have also been shown to possess anti-mutagenic properties.
  • Gram positive and Gram negative bacteria have been demonstrated to bind mutagenic pyrolysates which are produced during cooking at a high temperature.
  • Studies performed with lactic acid producing bacteria has shown that these bacteria may be either living or dead, due to the fact that the process occurs by adsorption of mutagenic pyrolysates to the carbohydrate polymers present in the bacterial cell wall. See, e.g., Zang, et al, 1990. J. Dairy Sci. 73: 2702-2710.
  • Lactobacilli have also been shown to degrade carcinogens (e.g., N-nitrosamines), which may serve an important role if the process is subsequently found to occur at the level of the mucosal surface. See, e.g., Rowland and Grasso, 1986. Appl. Microbiol. 29: 7-12. Additionally, the co- administration of lactulose and Bifidobacteria longum to rats injected with the carcinogen azoxymethane was demonstrated to reduce intestinal aberrant crypt foci, which are generally considered to be pre-neoplastic markers. See, e.g., Challa, et al., 1997. Carcinogenesis 18: 5175-21.
  • carcinogens e.g., N-nitrosamines
  • Purified cell walls of Bifidobacteria may also possess anti-tumorigenic activities in that the cell wall of Bifidobacteria infantis induces the activation of phagocytes to destroy growing tumor cells. See, e.g., Sekine, et al., 1994. Bifidobacteria and Microflora 13: 65-77. Bifidobacteria probiotics have also been shown to reduce colon carcinogenesis induced by 1,2-dimethylhydrazine in mice when concomitantly administered with fructo-oligosaccharides(FOS; see e.g., Koo and Rao, 1991. Nutrit. Rev.
  • microbiota of the gastrointestinal tract affects both mucosal and systemic immunity within the host. See, e.g., Famularo, et al, Stimulation of Immunity by Probiotics. In: Probiotics: Therapeutic and Other Beneficial Effects, pg. 133-161. (Fuller, R., ed. Chapman and Hall, 1997).
  • B- and T-lymphocytes, and accessory cells of the immune system have all been implicated in the aforementioned immunity. See, e.g., Schiffrin, et al, 1997. Am. J. Clin. Nutr. 66(suppl): 5-20S.
  • Other bacterial metabolic products, which possess immunomodulatory properties include: endotoxic lipopolysaccharide, peptidoglycans, and lipoteichoic acids. See, e.g., Standiford, 1994. Infect. Linmun. 62: 119-125.
  • probiotic organisms are thought to interact with the immune system at many levels including, but not limited to: cytokine production, mononuclear cell proliferation, macrophage phagocytosis and killing, modulation of autoimmunity, immunity to bacterial and protozoan pathogens, and the like. See, e.g., Matsumara, et al, 1992. Animal Sci. Technol. (Jpn) 63: 1157-1159; Solis-Pereyra and Lemmonier, 1993. Nutr. Res. 13: 1127-1140.
  • Lactobacillus strains have also been found to markedly effect changes in inflammatory and immunological responses including, but not limited to, a reduction in colonic inflammatory infiltration without eliciting a similar reduction in the numbers of B- and T-lymphocytes. See, e.g., De Simone, et al, 1992. Immunopharmacol. Immunotoxicol. 14: 331-340.
  • Bifidobacteria are known to be involved in resisting the colonization of pathogens in the large intestine. See, e.g., Yamazaki, et al, 1982. Bifidobacteria and Microflora 1: 55-60.
  • lactic acid producing bacteria also are able to colonize the skin and mucus membranes, and may be used either prophylactically or therapeutically to control bacterial infections.
  • lactic acid producing bacteria are able to utilize glycogen in the vaginal epithelial cells to produce lactic acid, which keeps the pH of this environment in the range 4.0 to 4.5.
  • This acidic environment is not conducive for the growth of pathogens such as Candida albicans, Gardnerella vaginalis, and various non-specific bacteria, which are responsible for vaginal infections.
  • pathogens such as Candida albicans, Gardnerella vaginalis, and various non-specific bacteria, which are responsible for vaginal infections.
  • Antibiotics are widely used to control pathogenic microorganisms in both humans and animals.
  • anti-microbial agents especially broad-spectrum antibiotics
  • antibiotics often kill beneficial, non-pathogenic microorganisms (i.e., flora) within the gastrointestinal tract, which contribute to digestive function and health.
  • relapse the return of infections and their associated symptoms
  • secondary opportunistic infections often result from the depletion of lactic acid producing and other beneficial flora within the gastrointestinal tract.
  • MRS A meticillin-resistant Staphylococcus aurous
  • VRE vancomycin-resistant Enterococci
  • VRE Vancomycin-resistant enterococci
  • Intestinal colonization with VRE is the most important source for spread of these organisms. Most patients harboring VRE have a-symptomatic intestinal colonization that may persist for months. These patients are at risk to develop VRE infection and are a potential source for spread to healthcare workers, the environment, and to other patients. The infection control measures that are implemented to minimize the spread of VRE are expensive and inconvenient for patients, family members, and healthcare workers. Recent studies have demonstrated a profound potential for lactic acid producing
  • Bacillus coagulans species especially the novel strains of Bacillus coagulans disclosed herein, for use in bio-rational therapies for the prophylactic or therapeutic treatment of antibiotic-resistant digestive pathogens.
  • bio-rational therapies for the prophylactic or therapeutic treatment of antibiotic-resistant digestive pathogens.
  • new therapies and new ways of thinking about controlling pathogens are required.
  • Antibiotics in some applications, have outlived their usefulness when considering the massive reservoir of new and antibiotic resistant strains that have resulted from the misuse of antibiotics in the healthcare setting and "growth factors" in production animal operations.
  • Enterococci leading causes of nosocomial bacteremia, surgical wound infection, and urinary tract infection, are becoming resistant to many and sometimes all standard therapies. New rapid surveillance methods are highlighting the importance of examining enterococcal isolates at the species level. Most enterococcal infections are caused by Enterococcus faecalis, which are more likely to express traits related to overt virulence but, at least for the moment, also more likely to retain sensitivity to at least one effective antibiotic. The remaining infections are mostly caused by Enterococcus faecium, a species virtually devoid of known overt pathogenic traits but more likely to be resistant to even antibiotics of last resort.
  • Effective control of multiple drug- resistant Enterococci will require: (i) better understanding of the interaction between Enterococci, the environment, and humans; (ii) far more prudent antibiotic use; (iii) better contact isolation in hospitals and other patient care environments; (iv) improved surveillance; and, most importantly, (v) the development of new therapeutic paradigms (e.g., non-antibiotic-based) which are less vulnerable to the cycle of drug introduction and drug resistance.
  • new therapeutic paradigms e.g., non-antibiotic-based
  • MDR multiple-drug resistant Enterococci
  • enterococci have become recognized as leading causes of nosocomial bacteremia, surgical wound infection, and urinary tract infection.
  • Two types of enterococci are generally found to be associated with causing infections: (i) those originating from patients' native flora, which are unlikely to possess resistance beyond that intrinsic to the genus and are unlikely to be spread from bed to bed; and (ii) isolates that possess multiple antibiotic resistance traits and are capable of nosocomial transmission.
  • MDR multiple-drug resistant enterococci
  • Enterococci normally inhabit the bowel and may be found in the intestine of nearly all animals, from cockroaches to humans. In humans, typical concentrations of enterococci in stool are up tol x 10 8 CFU per gram. See, e.g., Rice, et al, 1995. Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci. Appl. Environ. Microbiol. 61: 374-376. The predominant species inhabiting the intestine varies. In Europe, the United States, and the Far East, Enterococcus faecalis predominates in some instances, and Enterococcus faecium in others.
  • enterococcal species see, e.g., Devriese, et al, 1993. Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J. Appl. Bacteriol. 75: 399-408), only Enterococcus faecalis and Enterococcus faecium commonly colonize and infect humans in detectable numbers with Enterococcus faecalis being isolated from approximately 80% of human infections, and Enterococcus faecium from the remaining individuals.
  • Enterococci are exceedingly hardy and tolerate a wide variety of growth conditions, including temperatures of 10°C to 45°C, and hypotonic, hypertonic, acidic, or alkaline environments.
  • Sodium azide and concentrated bile salts which inhibit or kill most microorganisms, are tolerated by Enterococci and are actually used as selective agents in agar-based media.
  • enterococci grow under reduced or oxygenated conditions, although enterococci are usually considered strict fermenters because they lack a Kreb's Cycle and respiratory chain.
  • enterococci grow under reduced or oxygenated conditions, although enterococci are usually considered strict fermenters because they lack a Kreb's Cycle and respiratory chain.
  • Enterococcus faecalis is an exception since exogenous hemin can be used to produce d, b, and o type cytochromes. Enterococcus faecalis cytochromes are only expressed under aerobic conditions in the presence of exogenous hemin and, therefore, may promote the colonization of inappropriate sites. Enterococci are also intrinsically resistant to many antibiotics. Unlike acquired resistance and virulence traits, which are usually transposon or plasmid encoded, intrinsic resistance is based in chromosomal genes, which typically are non- transferable.
  • Penicillin, ampicillin, piperacillin, imipenem, and vancomycin are among the few antibiotics that show consistent inhibitory, but not bactericidal, activity against Enterococcus faecalis.
  • Enterococcus faecium is less susceptible to ⁇ -lactam antibiotics than Enterococcus faecalis because the penicillin-binding proteins of the former have markedly lower affinities for the antibiotics.
  • the first reports of strains highly resistant to penicillin began to initially appear in the 1980s. See, e.g., Bush, et al, 1989. High- level penicillin resistance among isolates of enterococci: implications for treatment of enterococcal infections. Ann. Intern. Med. 110: 515-520; Sapico, et al, 1989.
  • Enterococci often acquire antibiotic- resistance through exchange of resistance-encoding genes carried on conjugative transposons, pheromone-responsive plasmids, and other broad-host-range plasmids.
  • the past two decades have witnessed the rapid emergence of MDR enterococci.
  • High- level gentamicin resistance was initially reported in 1979 (see, e.g., Horodniceanu, et al, 1979. High-level, plasmid-borne resistance to gentamicin in Streptococcus faecalis sub-sp. zymogenes. Antimicrob. Agents Chemother.
  • VanA resistance to vancomycin and teicoplanin
  • VanB resistance to vancomycin alone
  • VRE vancomycin-resistant Enterococci
  • Staphylococcus aureus has been rendered vancomycin-resistant through apparent transfer of resistance from Enterococcus faecalis.
  • enterococci have naturally occurring or inherent resistance to various drugs, including cephalosporins and the semisynthetic penicihinase-resistant penicillins (e.g., oxacillin) and clinically-achievable concentrations of clindamycin and aminoglycosides.
  • penicillin e.g., ampicillin
  • pseudopenicillins e.g., ampicillin, vancomycin, etc.
  • cell-wall active agents e.g., ampicillin, vancomycin, etc.
  • enterococci In addition to natural resistance to many agents, enterococci have also developed plasmid-and transposon-mediated resistance to the tetracyclines (e.g., minocycline and doxycycline); erythromycin (e.g., azithromycin and clarithromycin); chloramphenicol; high levels of trimethoprim; and high levels of clindamycin.
  • tetracyclines e.g., minocycline and doxycycline
  • erythromycin e.g., azithromycin and clarithromycin
  • chloramphenicol high levels of trimethoprim
  • clindamycin high levels of trimethoprim
  • the best-characterized system of conjugation involves pheromone oligopeptides and pheromone-responsive plasmids. See,, e.g., Clewell and Keith, 1989. Sex pheromones and plasmid transferin Enterococcus faecalis. Plasmid 21: 175-184.
  • strains of Enterococcus faecalis typically secrete into the culture medium a number of different small, oligopeptide sex pheromones which are specific for different types of plasmids.
  • a cell containing a pheromone- responsive plasmid i.e., the potential donor cell
  • transcription of a gene on the plasmid is turned on, resulting in the synthesis of an aggregation substance on the surface of its cell membrane.
  • the donor cell comes in contact with another Enterococcus faecalis bacterium
  • the aggregation substance which contains two Arg-Gly-Asp motifs adheres to the binding substance on the surface of most Enterococcus faecalis cells, causing them to aggregate.
  • the pheromone-responsive plasmid can then transfer from the donor bacterium to the other (recipient) bacterium. Once the recipient cell has acquired this particular plasmid, the synthesis of the corresponding sex pheromone is shut-off to prevent self-aggregation.
  • This system of conjugation which occurs primarily in Enterococcus faecalis, is a highly efficient means of plasmid transfer.
  • Another system of conjugation involves broad host-range plasmids that can transfer among species of enterococci and other gram- positive organisms such as streptococci and st ⁇ phylococci. See,, e.g., Clewell, 1981. Plasmids, drug resistance, and gene transfer in the genus Streptococcus. Microbiol Rev. 45: 409-436. The transfer frequency is generally much lower than with the pheromone system. Since staphylococci, streptococci, and enterococci share a number of resistance genes, these broad host-range plasmids may be a mechanism by which some of these resistance genes have spread among different genera.
  • conjugative transposons may also explain the spread of resistance genes to many different species. See,, e.g., Clewell, 1986. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu. Rev. Microbiol. 40: 635-659. As opposed to ordinary transposons, which can jump within a cell from one DNA location to another, conjugative transposons also encode the ability to bring about conjugation between different bacterial cells.
  • conjugative transposons which do not replicate, but instead insert into the chromosome or into a plasmid of the new host
  • conjugative transposons appear to be an even more efficient and far-reaching way of disseminating a resistance gene. This may explain why the tetM gene of the conjugative transposon Tn916 has spread beyond the gram-positive species into gram- negative organisms, including gonococci and meningococci, as well as into mycoplasma and ureaplasma. See,, e.g., Roberts, 1990.
  • TSN Database collects and compiles data daily from more than 100 clinical laboratories within the United States, identifies potential laboratory testing errors, and detects emergence of resistance profiles and mechanisms that pose a public health threat (e.g., vancomycin-resistant staphylococci).
  • Enterococci faecium resistance notwithstanding, Enterococci faecalis remained by far the most commonly encountered of the two Enterococcal species in TSN Database. Enterococci faecalis to Enterococci faecium total isolates were approximately 4:1; blood isolates 3: 1; and urine isolates 5:1. This observation underscores important differences in the survival strategies and likelihood of therapeutic success, critical factors usually obscured by lumping the organisms together as Enterococcus species or enterococci. Widespread emergence and dissemination of ampicillin and vancomycin resistance in Enterococcus faecalis would significantly confound the current therapeutic dilemma.
  • enterococci account for approximately 110,000 urinary tract infections, 25,000 cases of bacteremia, 40,000 wound infections, and 1 , 100 cases of endocarditis annually in the United States, with most of these infections occurring in hospitals. Enterococcal infection-related deaths have been difficult to ascertain, due to the fact that severe co-morbid illnesses are common. However, enterococcal sepsis is implicated in up to 50% of fatal cases. Moreover, several recent case-control and historical cohort studies have shown that death risk associated with antibiotic-resistant enterococcal bacteremia is markedly higher than with susceptible enterococcal bacteremia. This trend is predicted to increase, as MDR isolates become more prevalent.
  • Antibiotics may promote colonization and infection with MDR Enterococci by at least two mechanisms.
  • Antibiotic-induced alterations in the protective flora of the intestine serve as a catalyst for colonization with exogenous pathogens such as MDR Enterococci.
  • Antibiotic restriction programs would be more effective if they included prudent prescribing of all antibiotics, not just single agents (e.g., vancomycin).
  • use of this approach substantially decreased intestinal colonization with VRE in one hospital pharmacy that restricted vancomycin, cefotaxime, and clindamycin use. See,, e.g., Quale, et al, 1996. Manipulation of a hospital antimicrobial formulary t control an outbreak of vancomycin-resistant enterococci. Clin. Infect. Dis. 23:1020-1025.
  • Vancomycin resistance in enterococci is heterogeneous on many levels.
  • Three phenotypes of vancomycin resistance (designated VanA, VanB, and VanC), each associated with a different ligase, are now well-described; a fourth, type VanD, has been recently reported.
  • VanA- and VanB-type resistance is encoded by gene clusters that are acquired (i.e., not part of the normal genome of enterococci) and are often transferable.
  • VanA-type strains are typically highly resistant to vancomycin and moderately to highly resistant to teicoplanin. This phenotype is often plasmid or transposon mediated and is inducible (i.e., exposure of bacteria to .. vancomycin results in the induction of the synthesis of several proteins that together confer resistance). See,, e.g., Hiramatsu, et ai, 1997. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J. Antimicrob. Chemother. 40: 135-146.
  • the VanA gene cluster has been found in a small r «3-like transposon,7>t/546, and in elements that appearto be closely related (e.g., Tn5488, which has an insertion sequence [IS 1251] within Tnl546. See,, e.g., Eliopoulos, et al, 1994. In vitro activities of two glycylcyclines against gram-positive bacteria. Antimicrob. Agents Chemother. 38: 534-541. These elements have, in turn, been found on both transferable and nontransferable plasmids, as well as on the chromosome of the host strain.
  • VanB type resistance was initially not found to be transferable, but at least in some instances, the VanBgene cluster has been found on large (i.e., 90 kb to 250 kb) chromosomally- located transferable elements, one of which contains within it a 64-kb composite transposon (i.e., Tnl547).
  • the VanB-cotnaining 64-kb transposon is part of a 250-kb mobile element shown to move from the chromosome of one Enterococcus and insert into the chromosome of another.
  • circularization of the vanB containing large mobile elements resembles the mechanism described for conjugative transposons that can excise from the chromosome of one strain, circularize, transfer from one Enterococcus to another, and reinsert into the chromosome of the recipient.
  • the 64-kb transposon can also jump to another plasmid within the host
  • VanCl and VanC2 are normally occurring genes that are endogenous species characteristics of E. gaiinarum and F. casseliflavus, respectively, and are not transferable.
  • Suitable antibiotics often are not available to treat MDR enterococcal infections (e.g., endocarditis or bacteremia), in the presence of neutropenia.
  • the substantial drawback of the broad spectrum approach is that the more organisms affected (i.e., both protective commensals as well as pathogens), the more opportunities for resistance to evolve.
  • Broad spectrum antibiotics permit empiric therapy in the absence of a specific diagnosis and generate a more substantial retum on investment in the short-term.
  • broad-spectrum antibiotics affect not only disease-causing organisms but also commensals present in numbers large enough to generate resistance by otherwise rare mutational or genetic exchange events.
  • the probiotic composition of the present invention is effective against other common or antibiotic-resistant strains of pathogens including, but not limited to, Candida, Clostridium, Escherichia, Klebsiella, Campylobacter, Peptococcus, Heliobacter, Hemophylus, Staphylococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, and Rotovirus species.
  • pathogens including, but not limited to, Candida, Clostridium, Escherichia, Klebsiella, Campylobacter, Peptococcus, Heliobacter, Hemophylus, Staphylococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, and Rotovirus species.
  • non-antibiotic, probiotic bacteria-based therapeutic regimen include, but are not limited to: (i) the administration of the composition will result reduction of the colonization rate of enterococci in the gastrointestinal tract; (// ' ) no contribution to the development of antibiotic resistance; (iii) the composition can be used prophylactically to reduce the reservoir of enterococci in hospitals, which will concomitantly reduce the chances of high-risk patients from acquiring VRE; (iv) the dosage of the composition can be varied according to patient age, condition, etc; and (v) the composition may be utilized in a food animal to reduce the development of further antibiotic resistance.
  • skin creams, lotions, gels, and the like which contain the novel stains of Bacillus coagulans disclosed herein, and/or the extracellular products thereof, would be effective in the mitigation or prevention of pathogenic organisms on the skin, mucus membrane, and cuticular tissues and further reduce the emergence of antibiotic resistant pathogens.
  • the cells, spores, and/or extracellular products from these novel Bacillus coagulans strains could be incorporated into these skin products for this express purpose.
  • pathogenic antibiotic-resistant strains of Pseudomonas, Staphylococcus, and/or Enterococcus are frequently associated with infections of severe burns.
  • the salves, lotions, gels, and the like, combined with the novel Bacillus coagulans strains, and/or their extracellular products, as disclosed in the present invention, would be effective in mitigating or preventing these pathogenic organisms. Additionally, administration of these probiotic bacteria would help to achieve a state of proper biodiversity to the skin in burn cases, as, generally, such biodiversity is not associated with pathogenic overgrowth.
  • probiotic refers to microorganisms that form at least a part of the transient or endogenous flora and thereby exhibit a beneficial prophylactic and/or therapeutic effect on the host organism. Probiotics are generally known to be clinically safe (i.e., non-pathogenic) by those individuals skilled in the art.
  • the prophylactic and/or therapeutic effect of an acid-producing bacteria of the present invention results, in part, from a competitive inhibition of the growth of pathogens due to: (i) their superior colonization abilities; (ii) parasitism of undesirable microorganisms; (iii) the production of acid (e.g., lactic, acetic, and other acidic compounds) and/or other extracellular products possessing anti-microbial activity; and (iv) various combinations thereof.
  • acid e.g., lactic, acetic, and other acidic compounds
  • the aforementioned products and activities of the acid-producing bacteria of the present invention act synergistically to produce the beneficial probiotic effect disclosed herein.
  • a probiotic bacteria which is suitable for use in the methods and compositions of the present invention (/) possesses the ability to produce and excrete acidic compounds (e.g., lactic acid, acetic acid, etc.); (ii) demonstrates beneficial function within the gastrointestinal tract; and (iii) is non-pathogenic.
  • acidic compounds e.g., lactic acid, acetic acid, etc.
  • demonstrates beneficial function within the gastrointestinal tract e.g., lactic acid, acetic acid, etc.
  • iii) demonstrates beneficial function within the gastrointestinal tract
  • (iii) is non-pathogenic.
  • many suitable bacteria have been identified and are described herein, although it should be noted that the present invention is not to be limited to cu ⁇ ently- classified bacterial species insofar as the purposes and objectives as disclosed.
  • the physiochemical results from the in vivo production of lactic acid is key to the effectiveness of the probiotic lactic acid-producing bacteria of the present invention.
  • Lactic acid production markedly decreases the pH (i.e., increases acidity) within the local micro-floral environment and does not contribute to the growth of many undesirable, physiologically-deleterious bacteria and fungi.
  • the probiotic inhibits growth of competing pathogenic bacteria.
  • Typical lactic acid-producing bacteria useful as a probiotic of this invention are efficient lactic acid producers, which include non-pathogenic members of the Bacillus genus which produce bacteriocins or other compounds which inhibit the growth of pathogenic organisms.
  • Bacillus species particularly those species having the ability to form spores (e.g., Bacillus coagulans), are a preferred embodiment of the present invention.
  • the ability to sporulate makes these bacterial species relatively resistant to heat and other conditions, provides for a long shelf-life in product formulations, and is deal for survival and colonization of tissues under conditions of pH, salinity, and the like within the gastrointestinal tract.
  • additional useful properties of many Bacillus species include being non-pathogenic, aerobic, facultative and heterotrophic, thus rendering these bacterial species safe and able to readily colonize the gastrointestinal tract.
  • Preferred methods and compositions disclosed herein utilize novel strains of Bacillus coagulans and/or extracellular products thereof as a probiotic.
  • the various "classic" Lactobacillus and/or Bifidiobacterium species are unsuitable for colonization of the gut due to their instability in the highly acidic environment of the gastrointestinal tract, particularly the human gastrointestinal tract.
  • the purified Bacillus coagulans strains of the present invention are able to survive and colonize the gastrointestinal tract because the optimal temperature for growth is lower than standard known strains of Bacillus coagulans..
  • probiotic Bacillus coagulans is non-pathogenic and is generally regarded as safe (i.e., GRAS classification) by the U.S. Federal Drug Administration (FDA) and the U.S. Department of Agriculture (USD A), and by those individuals skilled within the art.
  • Bacillus coagulans possesses the ability to produce heat-resistant spores, it is particularly useful for making pharmaceutical compositions, which require heat and pressure in their manufacture. Accordingly, formulations that include the utilization viable Bacillus coagulans spores in a pharmaceutically-acceptable carrier are particularly prefe ⁇ ed for making and using compositions disclosed in the present invention.
  • Bacillus coagulans have a cell diameter of greater than 1.0 ⁇ m with variable swelling of the sporangium, without parasporal crystal production.
  • Bacillus coagulans is a non-pathogenic, Gram positive, spore- forming bacteria that produces L(+) lactic acid (dextrorotatory) under homo-fermentation conditions. It has been isolated from natural sources, such as heat-treated soil samples inoculated into nutrient medium (see e.g., Bergey's Manual of Systemic Bacteriology, Vol. 2, Sneath, P.H.A. et al, eds., Williams & Wilkins, Baltimore, MD, 1986).
  • Bacillus coagulans strains have served as a source of enzymes including endonucleases (e.g., U.S. Pat. No. 5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S. Pat. No. 4,323,651) and cyclo-malto-dextrin glucano-transferase (U.S. Pat. No. 5,102,800).
  • Bacillus coagulans has also been utilized to produce lactic acid (U.S. Pat. No. 5,079,164).
  • a strain of Bacillus coagulans also referred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No.
  • Bacillus coagulans strains have also been used as animal feeds additives for poultry and livestock to reduce disease and improve feed utilization and, therefore, to increase growth rate in the animals (International PCT Pat. Applications No. WO 9314187 and No. WO 9411492). In particular, Bacillus coagulans strains have been used as general nutritional supplements and agents to control constipation and diarrhea in humans and animals.
  • Bacillus coagulans cultures have been deposited with the following primary international culture collections: Agricultural Research Service Culture Collection;
  • lactic acid-producing bacterial species which have either been: ( ) classified and deposited as Bacillus coagulans in the past but, have been re-classified as another related Bacillus species; or (ii) deposited as another closely related species but, have recently been re-classified as Bacillus coagulans.
  • These related species include, but are not limited to, Bacillus coagulans, Bacillus stereothermophilus, Bacillus thermoacidurans, Lactobacillus sporogenes, Bacillus smithii, Bacillus dextrolacticus, Lactobacillus cereale, and Bacillus recemilacticus.
  • Bacillus stereothermophilus is a Bacillus strain known to have an optimum growth of approximately 55°C.
  • Bacillus coagulans bacterial strains which are currently commercially available from the American Type Culture Collection (ATCC, Rockville, MD) include the following accession numbers: Bacillus coagulans Hammer NRS 727 (ATCC No. 11014); Bacillus coagulans Hammer strain C (ATCC No. 11369); Bacillus coagulans Hammer (ATCC No. 31284); and Bacillus coagulans Hammer NCA 4259 (ATCC No. 15949).
  • Purified Bacillus coagulans bacteria are also available from the Deutsche Sarumlung von Mikroorganismen und Zellkuturen GmbH (Braunschweig, Germany) using the following accession numbers: Bacillus coagulans Hammer 1915 (DSM No. 2356); Bacillus coagulans Hammer 1915 (DSM No. 2383, co ⁇ esponds to ATCC No. 11014); Bacillus coagulans Hammer (DSM No. 2384, corresponds to ATCC No.
  • Bacillus coagulans bacteria can also be obtained from commercial suppliers such as Sabinsa Corporation (Piscataway, NJ) or K.K. Fermentation (Kyoto, Japan).
  • Bacillus coagulans strains and their growth requirements have been described previously (see e.g., Baker, D. et al, 1960. Can. J. Microbiol. 6: 557-563; Nakamura, H. et al, 1988. Int. J. Svst. Bacteriol 38: 63-73.
  • various strains of Bacillus coagulans can also be isolated from natural sources (e.g., heat-treated soil samples) using well-known procedures (see e.g., Bergey 's Manual of Systemic Bacteriology, Vol. 2, p. 1117, Sneath, P.H.A. et al, eds., Williams & Wilkins, Baltimore, MD, 1986).
  • Bacillus coagulans had originally been mis-characterized as a Lactobacillus in view of the fact that, as originally described, this bacterium was labeled as Lactobacillus sporogenes (See, Nakamura et al. 1988. Int. J. Syst. Bacteriol. 38: 63- 73). However, initial classification was inco ⁇ ect due to the fact that Bacillus coagulans produces spores and through metabolism excretes L(+)-lactic acid, both aspects which provide key features to its utility. Instead, these developmental and metabolic aspects required that the bacterium be classified as a lactic acid Bacillus, and therefore it was re-designated.
  • Bacillus coagulans being a member of the Bacillus genus, is spore-forming which upon activation in the acidic environment of the stomach, can germinate and proliferate in the intestine, produce the favored L(+) optical isomer of lactic acid, and effectively prevent the growth of numerous bacterial and fungal pathogens.
  • a Lactobacillus plantarum may be motile and contains m-A PMc in Its cell wall
  • Lactobacillus species are generally believed to be unsuitable for colonization of the gut due to their instability in the harsh (i.e., acidic) pH environment of the digestive tract, e.g., in the presence of bile, particularly human bile. This instability is one of the primary reasons why the use of lactic acid-producing bacterial strains as probiotics has not been more vigorously explored.
  • Bacillus coagulans is able to survive, colonize, and grow in the gastrointestinal tract.
  • human bile environment is different from the bile environment of animal models, and growth of Bacillus coagulans in human gastrointestinal tract models has not been described.
  • the following proliferative attributes illustrate the strengths of Bacillus coagulans over other species of lactic acid-producing bacteria include, but are not limited to: Facultative Aerobe: Bacillus coagulans possesses the ability to grow well in either environments that have free-oxygen or in st ⁇ ctly anaerobic environments This is important due to the fact that Lactobacilli and Bifidobacteria are not aero- tolerant. Thus, in essence, these aforementioned bacterial species are strictly anaerobic and do not proliferate well in environments containing free-oxygen.
  • Bacillus coagulans is viable in a free-oxygen environment, it can be used in surface-active formulations (e g , skin powders, creams, ointments, etc) to act prophylactically against the overgrowth of pathogens.
  • surface-active formulations e g , skin powders, creams, ointments, etc
  • Thermo-Tolerant The vegetative cells of Bacillus coagulans possess the ability to grow at temperatures as high as 65°C, whereas the endospores can withstand temperatures in excess of 100°C
  • Bacillus coagulans, along with Bacillus stereothermophilus, is used for quality control purposes in autoclaves. This fact is crucial due to the frailty of all Lactobacilli and Bifidobacteria For a bacterium to have commercial viability it must be stabile and viable at the time of packaging. This viability must be retained in order to deliver an efficacious product to the consumer.
  • Halo-Tolerant Bacillus coagulans possesses the ability to grow in highly alkaline environments including 7% NaCI or 10% caustic soda.
  • Bacillus coagulans as cited in Bergey's Manual (Seventh Edition), include: Gram-positive spore-forming rods approximately 0.9 ⁇ m x 3.0-5.0 ⁇ m in size; aerobic to microaerophilic; produce L(+) (dextrorotatory) lactic acid in a homofermentative manner. Due to the fact that Bacillus coagulans exhibits characte ⁇ stics typical of both genera Lactobacillus and Bacillus, its taxonomic position between the families Lactobacillaceae and Bacillaceae has often been discussed. It is often very difficult to distinguish between two species of bacte ⁇ a, which are morphologically similar and possess similar physiological and biochemical characteristics.
  • DNA homology analysis is a useful technique in resolving this difficulty.
  • the base composition (i.e , % GC content) and the specific nucleotide sequence of the bacte ⁇ al DNA generally differs between bacterial species and sub- species. Additionally, DNA from closely related bacte ⁇ a hybndize with each other more efficiently. It the present invention, these aforementioned methodologies have been effectively employed to differentiate, as well as to recognize the innate resemblance between Bacillus coagulans and members of the genus Lactobacillus and to validate it's taxonomical placement under genus Bacillus.
  • Table 2 discusses the colony mo ⁇ hology of Bacillus coagulans.
  • Cells are long and slender (0.3 to O. ⁇ m), some are bent and all the cells have rounded ends Motile with peritrichous flagellas.
  • Colonies are usually 2.5 mm in diameter, convex, smooth, glistening and do not produce any pigment.
  • MRS medium supplemented with tomato juice, manganese, acetate and Tween-80 is a suitable medium for growth.
  • Bacillus coagulans Bacillus coagulans:
  • Bacillus coagulans enjoys a longer safe history of use than most of the common Lactobacillus and Bifidobacterium species that are commonly sold as "nutritional supplements" at health food stores, or used in the production of cultured dairy products.
  • General recognition of biological safety may be based only upon the views of experts qualified by scientific training and experience to evaluate the safety of substances directly or indirectly added to food. The basis of such views may be derived through:
  • General recognition of safety through experience based on common use in food prior to January 1, 1958 may be determined without the quantity or quality of scientific procedures required for approval of a food additive regulation.
  • General recognition of safety through experience based on common use in food prior to January 1, 1958 shall be based solely on food use of the substance prior to January 1, 1958, and shall ordinarily be based upon generally available data and information.
  • Lactic acid-producing bacteria are a necessary component in fermented dairy products. Due to the fact that Bacillus coagulans was first isolated in 1932, has been used in the production of food products prior to January 1, 1958, and has not been implicated in any pathogenic or opportunistic diseases since its isolation, it qualifies under as many as 9 sections and subsections of the United States Federal Registry for GRAS (Generally-Regarded as Safe) listing. The GRAS list simply indicates that a food additive is not thought to illicit any toxigenic or pathogenic response and is considered safe by those skilled in the art of food science, biochemistry, and microbiology. Bacillus coagulans, subspecies Hammer (ATCC-31284), was first isolated as a soil isolate at Yamanashi University in 1933 by Nakayama.
  • Bacillus coagulans species are usually soil isolate. With the exception of Bacillus cereus and Bacillus anthraices, Bacillus species are known to be benign in the environment. To date, there have been no references of any species of Bacillus coagulans being involved in a pathogenic or opportunistic illness. Similarly, in an analysis of published data, there have also been no clinical trials that had been compromised due to pathogenesis by lactic acid- producing bacteria. In view of these facts, which are not disputed within the relevant scientific fields, Bacillus coagulans is safe as a therapeutic compositions.
  • Bacillus coagulans was found to be susceptible to: penicillin; vancomycin; gentamicin (500 ⁇ g/ml); streptomycin(2,000 ⁇ g/ml); nitrofurantoin; norfloxacin; chloramphenicol, and was resistant to tetracycline. Additionally, Nitrocefin testing was performed and indicated Bacillus coagulans was positive for low-level ⁇ -lactamase production.
  • Bacteriocins are proteins or protein-particulate complexes with bactericidal activities directed against species, which are closely related to the producer bacterium.
  • the inhibitory activity of lactic acid-producing bacteria (e.g., Bacillus coagulans) towards putrefactive organisms is thought to be partially due to the production of bacteriocins.
  • Table 4 lists some of the various bacterocins, which have been isolated and characterized from lactic acid-producing bacterial species.
  • lactic acid-producing bacteria also inhibit the growth of pathogenic/putrefactive microorganisms through other metabolic products such as hydrogen peroxide, carbon dioxide, and diacetyl.
  • Previously-available strains of lactic acid-producing bacteria were ineffectual as probiotics due to various factors including, but not limited to, their high optimal growth temperature (i.e., >40°C) requirement and their requirement for an 80°C "spore shock" for spore germination. These requirements were incompatible with the use of these previously-available strains of Bacillus coagulans as probiotics, in therapeutic compositions (e.g., in the treatment of antibiotic-resistant gastrointestinal pathogens), and the like.
  • Bacillus coagulans described herein possess biochemical and physiological characteristics which include, but are not limited to: (i) the production of the (L)+ optical isomer of lactic acid (propionic acid); (ii) have an optimal growth temperature of less than 45°C; (iii) the production of spores resistant to temperatures of up to approximately 90°C which are able to germinate in a human or animal body without specific inducement (e.g., spore-shock or other environmental factors); (iv) the production of one or more extracellular products exhibiting probiotic activity which inhibits the growth of bacteria, yeast, fungi, virus, or any combinations thereof; and/or (v) the ability to utilize a wide spectrum of substrates for proliferation.
  • novel strains will be more fully discussed, below.
  • ATCC-31284 ATCC-31284
  • ATCC-99% a mixed microbial community of Bacillus coagulans colonies where they exhibited differences in both colony morphology and optimal growth temperature from that of the Bacillus coagulans ATCC-type strain
  • the present invention contemplates a method for treating, reducing or controlling antibiotic-resistant bacterial gastrointestinal infections using the therapeutic composition or therapeutic system disclosed herein.
  • the disclosed methods of treatment function so as to inhibit the growth of the pathogenic bacteria which are associated with gastrointestinal infections, as well as to concomitantly mitigate the deleterious physiological effects/symptoms of these pathogenic infections.
  • the novel strains of Bacillus coagulans disclosed herein are generally regarded as safe by those skilled within the art (i.e., GRAS Certified by the FDA) and, therefore, suitable for direct ingestion in food stuffs or as a food supplement.
  • the methods of the present invention comprise administration of a therapeutic composition containing one or more Bacillus coagulans strains and/or the extracellular products thereof, to the gastrointestinal tract of a human or animal, to treat or prevent bacterial infection.
  • Administration is preferably made using a liquid, powder, solid food and the like formulation compatible with oral administration, all formulated to contain a therapeutic composition of the present invention by use of methods well-known within the art.
  • the methods of the present invention includes administration of a composition containing one or more of the following: Bacillus coagulans bacterial cells (t.e., vegetative bacterial cells); spores; and/or isolated Bacillus coagulans extracellular products (which contains a metabolite possessing antibiotic-like properties) to a human or animal, so as to treat or prevent the colonization of antibiotic-resistant pathogens with the gastrointestinal tract.
  • Bacillus coagulans bacterial cells t.e., vegetative bacterial cells
  • spores spores
  • isolated Bacillus coagulans extracellular products which contains a metabolite possessing antibiotic-like properties
  • the methods includes administering to the patient, for example, Bacillus coagulans in food or as a food supplement.
  • Oral administration is preferably in an aqueous suspension, emulsion, powder or solid, either already formulated into a food, or as a composition which is added to food by the user prior to consumption.
  • Administration to the gastrointestinal tract may also be in the form of an anal suppository (e.g., in a gel or semi-solid formulation). All such formulations are made using standard methodologies.
  • Administration of a therapeutic composition is preferably to the gastrointestinal tract using a gel, suspension, aerosol spray, capsule, tablet, powder or semi-solid formulation (e.g., a suppository) containing a therapeutic composition of the present invention, all formulated using methods well-known within the art.
  • Administration of the compositions containing the active probiotic lactic acid-producing bacterium which is effective in preventing or treating a pathogenic bacterial infection generally consist of one to ten dosages of approximately 10 mg to 10 g of the therapeutic composition per dosage, for a time period ranging from one day to one month. Administrations are (generally) once every twelve hours and up to once every four hours. In the preferred embodiment, two to four administrations of the therapeutic composition per day, of approximately 0.1 g to 5 g per dose, for one to seven days. This preferred dose is sufficient to prevent or treat a pathogenic bacterial infection.
  • the specific route, dosage and timing of the administration will depend, in part, upon the particular pathogen and/or condition being treated, as well as the extent of said condition.
  • the typical dosage is approximately lxlO 2 to lxlO 14 CFU of viable, vegetative bacteria or spores per day, preferably from approximately 1x10 to lxlO 10 , and more preferably from approximately 2.5xl0 8 to lxlO 10 CFU of viable, vegetative bacteria or spores per day.
  • Another embodiment of the present invention discloses the administration of a composition comprising a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 1% to 90% extracellular product with the remainder comprising the carrier or delivery component.
  • a preferred embodiment comprises a composition a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 10% to 75% extracellular product with the remainder comprising the carrier or delivery component.
  • the present invention further contemplates a therapeutic system for treating, reducing and/or controlling pathogenic bacterial infections.
  • the system is in the form of a package containing a therapeutic composition of the present invention, or in combination with packaging material.
  • the packaging material includes a label or instructions for use of the components of the package.
  • the instructions indicate the contemplated use of the packaged component as described herein for the methods or compositions of the invention.
  • a system can comprise one or more unit dosages of a therapeutic composition according to the present invention.
  • the system can alternately contain bulk quantities of a therapeutic composition.
  • the label contains instructions for using the therapeutic composition in either unit dose or in bulk forms as appropriate, and may also include information regarding storage of the composition, disease indications, dosages, routes and modes of administration and the like information.
  • the system may optionally contain either combined or in separate packages one or more of the following components: bifidogenic oligosaccharides, flavorings, carriers, and the like components.
  • One particularly preferred embodiment comprises unit dose packages of Bacillus coagulans spores for use in combination with a conventional liquid product, together with instructions for combining the probiotic with the formula for use in a therapeutic method.
  • the present invention also discloses compositions and methods of use for inhibiting growth of parasites and/or antibiotic-resistant pathogenic organisms in the gastrointestinal tract of animals.
  • Oral administration is preferably in an aqueous suspension, emulsion, powder or solid, either already formulated into a food, or as a composition which is added to food by the user prior to consumption.
  • Administration to the gastrointestinal tract may also be in the form of an anal suppository (e.g., in a gel or semi-solid formulation). All such formulations are made using standard methodologies.
  • the method comprises administration of a composition of this invention containing the active ingredients to an animal in various dosage regimens as described herein to achieve the nutritional result.
  • Administration of the compositions containing the active ingredients effective in inhibiting parasite growth in the intestine and in feces generally consist of one to ten unit dosages of 10 mg to 10 g per dosage of the composition for one day up to one month for an animal of approximately 100 kg body weight. Unit dosages are generally given once every twelve hours and up to once every four hours. Preferably two to four dosages of the composition per day, each comprising about 0.1 g to 50 g per dosage, for one to seven days are sufficient to achieve the desired result.
  • Another embodiment of the present invention discloses the administration of a composition comprising a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 1% to 90% extracellular product with the remainder comprising the carrier or delivery component.
  • a prefe ⁇ ed embodiment comprises a composition a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 10% to 75% extracellular product with the remainder comprising the carrier or delivery component.
  • the method is typically practiced on any animal where inhibiting pathogen or parasites is desired.
  • the animal can be any livestock or zoological specimen where such inhibition of parasites/pathogens provides economic and health benefits.
  • Any animal can benefit by the claimed methods, including birds, reptiles, mammals such as horses, cows, sheep, goats, pigs, and the like domesticated animals, or any of a variety of animals of zoological interest.
  • Other ptuposes are readily apparent to one skilled in the arts of nutrient abso ⁇ tion, feed utilization and bioavailability.
  • a system can comprise one or more unit dosages of a therapeutic composition according to the present invention.
  • the system can alternately contain bulk quantities of a therapeutic composition.
  • the label contains instructions for using the therapeutic composition in either unit dose or in bulk forms as appropriate, and may also include information regarding storage of the composition, disease indications, dosages, routes and modes of administration and the like information.
  • the system may optionally contain either combined or in separate packages one or more of the following components: bifidogenic oligosaccharides, flavorings, carriers, and the like components.
  • One particularly preferred embodiment comprises unit dose packages of Bacillus coagulans spores for use in combination with a conventional liquid product, together with instructions for combining the probiotic with the formula for use in a therapeutic method.
  • feces provide growth and breeding grounds for undesirable organisms
  • controlling and/or inhibiting growth of parasites and pathogenic organisms in feces inhibits growth and reproduction of these undesirable organisms in areas where feces is produced, deposited and/or stored.
  • feces provide growth and breeding grounds for undesirable organisms
  • controlling and/or inhibiting growth of parasites and pathogenic organisms in feces inhibits growth and reproduction of these undesirable organisms in areas where feces is produced, deposited and/or stored.
  • parasites/pathogens to irritate, spread, reproduce and/or infect other hosts.
  • the invention contemplates a method for reducing and/or controlling flying insect populations in animal cages/pens/enclosures where animals are maintained comprising administering a composition of the present invention to the gastrointestinal tract of the caged animals.
  • the present invention is useful at controlling a large variety of parasites and pathogenic organisms, and therefore the invention need not be limited to inhibiting any particular genus or species of organism.
  • the invention need not be limited to inhibiting any particular genus or species of organism.
  • all insect varieties which can act as an animal parasite can be targeted by the methods of the present invention.
  • Parasites can infect any of a variety of animals, including mammals, reptiles, birds and the like, and therefore the invention is deemed to not be limited to any particular animal. Examples of well-known or important parasites are described herein for illustration of the invention, but are not to be viewed as limiting the invention.
  • Stroncrvlus species such as S. vulcraris, S. epuinus or S. edentatus, small strongyles of the cecum and colon caused by Stroncrvlus species such as S. vulcraris, S. epuinus or S. edentatus, small strongyles of the cecum and colon caused by Stroncrvlus species such as S. vulcraris, S. epuinus or S. edentatus, small strongyles of the cecum and colon caused by Stroncrvlus species such as S. vulcraris, S. epuinus or S. edentatus, small strongyles of the cecum and colon caused by Stroncrvlus species such as S. vulcraris, S. epuinus or S. edentatus, small strongyles of the cecum and colon caused by Stroncrvlus species such as S. vulcraris, S. epuinus
  • Triodontophorus species such as T. tenuicollis, pinworms caused by Oxvuris species such as 0. eaui, strongyloides infections of the intestine caused by Stroncivloides westeri, tapeworms caused by Anonlocephala species such as A. macma and A. perfoliata, and caused by Par anonlocephala mamillana.
  • Parasites caused in ruminants, typically swine include stomach worms caused by Hvostroncmulus species.
  • gastrointestinal parasites infect a variety of animals and can include Spirocerca species such as S. lupi that cause esopheageal worms in canines and Physoloptera species that cause stomach worms in canines and felines.
  • Administration of a therapeutic composition is preferably to the gut using a gel, suspension, aerosol spray, capsule, tablet, granule, pellet, wafer, powder or semi — solid formulation (e.g., a suppository) containing a nutritional composition of this invention, all formulated using methods well known in the art.
  • a gel, suspension, aerosol spray, capsule, tablet, granule, pellet, wafer, powder or semi — solid formulation e.g., a suppository
  • the system is present in the form of a package containing a composition of this invention, or in combination with packaging material.
  • the packaging material includes a label or instructions for use of the components of the package.
  • the instructions indicate the contemplated use of the package component as described herein for the methods or compositions of the invention.
  • a system can comprise one or more unit dosages of a therapeutic composition according to the invention.
  • the system can contain bulk quantities of a composition.
  • the label contains instructions for using the composition in either unit dose or in bulk forms as appropriate, and may include information regarding storage of the composition, feeding instruction, health and diet indications, dosages, routes of administration, methods for blending the composition with preselected food stuffs, and the like information.
  • Bacillus coagulans can be cultured in a variety of media, although it has been demonstrated that certain growth conditions are more efficacious at producing a culture which yields a high level of sporulation. For example, sporulation is demonstrated to be enhanced if the culture medium includes 10 mg/1 of MgSO 4 sulfate, yielding a ratio of spores to vegetative cells of approximately 80:20.
  • certain culture conditions produce a bacterial spore which contains a spectrum of metabolic enzymes particularly suited for the present invention (i.e., production of lactic acid and enzymes for the enhanced probiotic activity and biodegradation).
  • the spores produced by these aforementioned culture conditions are prefe ⁇ ed, various other compatible culture conditions which produce viable Bacillus coagulans spores may be utilized in the practice of the present invention.
  • Suitable media for the culture of Bacillus coagulans include: PDB (potato dextrose broth); TSB (tryptic soy broth); and NB (nutrient broth), which are all well- known within the field and available from a variety of sources.
  • media supplements which contain enzymatic digests of poultry and/or fish tissue, and containing food yeast are particularly prefe ⁇ ed.
  • a prefe ⁇ ed media supplement produces a media containing at least 60% protein, approximately 20% complex carbohydrates, and approximately 6% lipids.
  • Media can be obtained from a variety of commercial sources, notably DIFCO (Newark, NJ); BBL
  • Small-scale culture of Bacillus coagulans may be accomplished by use of the aforementioned Glucose Yeast extract (GYE) medium.
  • the medium was inoculated and grown to a cell density of approximately lxlO 8 to lxlO 9 cells/ml.
  • the bacteria were cultured by utilization of a standard airlift fermentation vessel at 30°C.
  • the range of MnSO acceptable for sporulation was found to be 1.0 mg/1 to 1.0 g/1.
  • the vegetative bacterial cells can actively reproduce up to 65°C, and the spores are stable up to 90°C.
  • the fermentation vessel may include: a 500 liter 314 series stainless airlift fermentation vessel with 60 psi pressure rating; Hanna duel set-point pH control system with in-process electrode; High pressure turbine blower with 0.2 ⁇ m in-line filters for sterile air feed; a lOkw process temperature controller; and appropriate high burst-pressure stainless steel sanitary hose and fittings.
  • Ultraviolet and Visible Spectroscopy Differential absorbance spectra were determined between 200 and 600 nm wavelengths in 1 cm quartz cuvettes using a Uvikon 930 scanning spectrophotometer (Kontron Instruments). The baseline was determined with water or culture media.
  • the electrophoretic results demonstrated a significant number of proteinaceous bands in the ⁇ 4,000 to 30,000 Dalton range for Bacillus coagulans.
  • the plates showing 30-300 colonies were selected for counting. Plates possessing a very na ⁇ ow variation in total colony count were counted and then an average count per plate was calculated. The number of viable cells per gram of sample was obtained by multiplying the average number of colonies counted per plate by the reciprocal of the dilution factor (e.g., if the average number of colonies per plate was 90 and final dilution factor was 2 x 10 "6 , then viable spore count was 90 x (2 x 10 6 ) or 1.8 x 10 10 viable spores per gram.
  • the dilution factor e.g., if the average number of colonies per plate was 90 and final dilution factor was 2 x 10 "6 , then viable spore count was 90 x (2 x 10 6 ) or 1.8 x 10 10 viable spores per gram.
  • GYE Yeast Extract
  • TLB Trypticase Soy Broth
  • FIG. 1 illustrates, in histogram form, the minimal and optimal culture temperatures for the Bacillus coagulans 1% isolate (GBI-1); ATCC- 99% isolate; the 5937-20°C isolate (GBI-20); and the 5937-30°C isolate (GBI-30), in either Trypticase Soy Broth (TSA) or Glucose Yeast Extract (GYE) media.
  • a total of four cultures of Bacillus coagulans strains were analyzed with pH Kinetic Testing, Heterotrophic Plate Counts, and Optical Density (OD) in % Optical Transmittance of culture growth at 4 hour intervals for 28 hours in tryptic soy broth (TSB) media.
  • These stains included: the 20°C Bacillus coagulans isolate (GBI-20); 30°C Bacillus coagulans isolate (GBI-30); the ATTC 99% Bacillus coagulans isolate (ATCC- 99%>); and the 1% Bacillus coagulans isolate (GBI-1).
  • Each of the aforementioned bacterial stains were placed in 50 ml Erlenmeyer flasks containing 20ml of TSB media. Seven flasks were prepared for each of the four isolates, one for each 4hour interval of the 28 hour study.
  • Initial seed cultures were broth cultures in test tubes, which had a % transmittance of 10%. 1.0ml of this culture was then place into each of
  • diluted cultures were placed into the wells of a 96-well microtiter plate which contained a specific growth medium which comprised one of the following: TSB, Glucose Yeast Extract (GYE) medium, or, either with or without additional oxygenation.
  • a specific growth medium which comprised one of the following: TSB, Glucose Yeast Extract (GYE) medium, or, either with or without additional oxygenation.
  • GYE Glucose Yeast Extract
  • each microplate well also contained a tetrazolium dye/redox indicator system. Bacterial growth (i.e., metabolic respiration or oxidation of carbon sources) was monitored by tetrazolium reduction as measured at 590 nm in a spectrophotometric microplate reader.
  • Bacterial growth was measured every 20 minutes during a total incubation of 22 hours at 32°C. The kinetic data was processed and the background blank values subtracted.
  • the diluted cultures were placed into the wells of a 96-well microtiter plate which contained a specific growth medium which comprised one of the following: GYE or Trypticase Soy Broth, Nutrient Broth (NB), or Biolog Universal Growth Medium (BUGMB),either with or without additional oxygenation.
  • a specific growth medium which comprised one of the following: GYE or Trypticase Soy Broth, Nutrient Broth (NB), or Biolog Universal Growth Medium (BUGMB),either with or without additional oxygenation.
  • GYE or Trypticase Soy Broth Nutrient Broth (NB), or Biolog Universal Growth Medium (BUGMB)
  • NB Nutrient Broth
  • BUGMB Biolog Universal Growth Medium
  • Bacterial growth was measured every 20 minutes during a total incubation of 18 hours at 32 C. The kinetic data was processed and the background blank values subtracted.
  • FIG. 4 and FIG. 5 represent histograms of the End-Point Kinetics of the 5937-20°C Bacillus coagulans isolate (GBI-20) and 5937-30°C Bacillus coagulans isolate (GBI-30), respectively.
  • the Biolog Microplate System was utilized for microbial identification and characterization by carbon source pattern recognition of the Bacillus coagulans strains disclosed in the present invention.
  • the aforementioned microplate technique allows for microbial characterization by use of 95 different analytical methods, thus yielding a total of 4 x 10 28 possible patterns generated from a single microplate.
  • Each strain of microorganism yields a distinct pattern, and the different species of bacteria will give different "families" of patterns which can be recognized and differentiated by the Biolog Microlog software.
  • Analytical microplates for the Biolog Microlog system are available for gram-negative bacteria, gram-positive bacteria, yeast, lactic acid- producing bacteria, and E. colil Salmonella analysis. In addition, further analyses may also be performed by use of additional selective media.
  • Table 13 illustrates the approximate percentages of aerobic strain types in each of samples comprising the novel strains of Bacillus coagulans disclosed herein.
  • the bacterial strains were streaked onto Trypticase Soy Agar (TSA) plates.
  • TSA plates were then prepared for Gas Chromatography Fatty Acid Methyl Ester (GC- FAME) analysis following a 24 hour incubation by standard, published GC-FAME methodologies.
  • GC- FAME Gas Chromatography Fatty Acid Methyl Ester
  • the bacterial strain was subsequently examined against both the Aerobe (TSBA) and the Clinical Aerobe (CLIN) computer databases. The results of the GC-FAME analysis is shown below, in Table 14.
  • rRNA 16S Ribosomal RNA
  • AmpliTaq FS TM DNA polymerase and dRhodamine dye terminators were removed from the sequencing reactions using a Sephadex G- 50 spin column. The amplification products were then collected by centrifugation, dried under vacuum, and stored at -20°C until use. The products were resuspended in a solution of formamide/blue dextrin/EDTA, and heat-denatured prior to electrophoresis. The samples were electrophoresed on a ABI Prism 377 DNA Sequencer using a pre- poured, 5% Long Ranger TM (RMC) polyacrylamide/urea gel for approximately 6 hours.
  • RMC Long Ranger TM
  • results for the ATCC- 99%> isolate are shown in FIG. 6; results for GBI-20 are shown in FIG. 7; and results for GBI-30 are shown in FIG. 8.
  • Species Level This indicates a species level match.
  • a 16S rRNA sequence homology of greater than 99% is indicative of a species level match (see, Staekebrandt and Goebel, 1994. Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology. Int. J. Syst. Bacteriol. 44: 846-849).
  • Genus Level This indicates that the sample appears to group within a particular genus but the alignment did not produce a species level match. A genus level match indicates that the sample species is not included in the MicroSeq database.
  • GenBank and Ribosomal Database Project (RDP) databases with the sample sequence was subsequently performed to try to provide a closer match. If the sample sequence does not match well with either of these databases, it may represent a new species or a species whose 165 rRNA gene sequence is not present in any of the databases.
  • Aminopeptidase profiling or activity has been used to differentiate bacteria and fungi to species and sub-species (see, e.g., Hughes, et ⁇ l, 1988. LacZY gene modified peptidase activity in Pseudomonas aureofaciens. Phytopathology 78: 1502; Hughes, et al, 1989. Identification of immobilized bacteria by aminopeptidase profiling. Anal. Chem. 61: 1656-1660), as well as to define ecological niches of parasites and develop media for fastidious organisms.
  • the cell densities were adjusted to 2.5 x 10° cells/ml by spectrophotometry at 540 nm (85% transmittance) before placing 0.5 ml into each cell of a 96-well, flat bottom, black, polystyrene plate (FluoroNunc; Nalge-Nunc, Naperville, IL).
  • Each well contained one of 20 different non-fluorescent, L-amino acid- ⁇ -naphthylamide substrates (Sigma Chemical Co., St. Louis, MI) at a final concentration of 1 x 10 "4 M.
  • the balance of the microplate well volume of 300 ⁇ l consisted of 250 ⁇ l of the 10 mM phosphate buffer.
  • the 20 different peptidase substrates used to produce the profiles included ⁇ -naphthylamides of the following amino acids: L-alanine (ALA), L-arginine (ARG), L-asparagine (ASN), L-aspartic acid (ASP), L-cysteine (CYS), glycine (GLY), L- glutamic acid (GLU), L-histidine (HIS), L-isoleucine (I LE), L-leucine (LEU), L-lysine (LYS), DL-methionine (MET), L-phenylalanine (PHE), L-proline (PRO), L-serine (SER), trans hydroxy-L-prolme (HPR), L-tryptophan (TRP), L-tyrosine (TYR), and L- valine (VAL).
  • L-alanine ALA
  • L-arginine ARG
  • ASN L-asparagine
  • ASP L-aspartic acid
  • a 389 nm cut-on filter was used to select the desired emission wavelength before detection with a 931 A photomultiplier tube.
  • a total of 25 fluorescent decays were averaged by a Tektronix DSA 602 digital oscilloscope and transfe ⁇ ed to a PC computer via an IEEE-488 interface card to provide a readout of relative fluorescence after blank subtraction.
  • FIG. 9 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans 99% ATCC isolate
  • FIG. 10 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans GBI-1 isolate
  • FIG. 9 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans GBI-1 isolate
  • FIG. 11 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans GBI-30 isolate
  • FIG. 12 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans GBI-20 isolate.
  • Each of the specific Aminopeptidases and controls, as set forth in FIG. 9 through FIG. 12, are identified using numbers 1-24. These numbers are as follows:
  • Kirby-Bauer Antibiotic Susceptibility Testing Susceptible to: ampicillin, ciprofloxacin, trimethoprim- sulfamethoxazole, rifampin, erythromycin, vancomycin, gentamicin, and oxacillin
  • mice 4 6.6 log 10 CFU/g stool
  • VRE In comparison to the saline controls, the level of VRE declined more rapidly in the mice receiving Bacillus coagulans. Five days after clindamcin was discontinued (after 4 days of Bacillus coagulans therapy), the mean level of VRE was found to be
  • VRE levels 25-fold reduction in VRE levels (p ⁇ 0.05).
  • Eight days after clindamycin was discontinued (4 days after Bacillus coagulans therapy was completed), the mean level of VRE was found to be 2.9 log ⁇ 0 VRE/gram of stool compared with 4.3 logioVRE/gram of stool in the saline controls. This represented a 28-fold reduction (p ⁇ 0.05).
  • Thirty- five percent (6/17 animals) of Bacillus coagulans treated mice had undetectable levels of VRE eight days after clindamycin was discontinued, whereas none of the saline controls had undetectable levels of VRE at that time point (p ⁇ 0.05).
  • the mean level of VRE present in the stool of the 5 mice receiving Bacillus coagulans spores was also significantly lower than the level in the saline control mice (p ⁇ 0.05), however none of these five mice had undetectable VRE levels.
  • mice receiving Bacillus coagulans had detectable levels of Bacillus coagulans in their stool one day after completion of four days of therapy (range 3.1 to
  • Enterococci are not inhibited by changes in the pH of its micro-environment.
  • Enterococcus faecium which is the Enterococcus species responsible for most, if not all, VRE carriage and infections
  • This organism itself, produces a D-optical isomer of lactic acid and is generally co-administered with Lactobacillus and Bifidiobacterium, which produce the L-optical isomer of lactic acid. Therefore, Enterococcus faecium is not affected by lactic acid-producing organisms, regardless of optical isomer of lactic acid produced.
  • the second method used by probiotic bacteria does not appear to play a role in the inhibition of VRE by Bacillus coagulans. Due to the aforementioned experimental results, it is believed that the amelioration of VRE by Bacillus coagulans is due to the production of one or more anti-microbial agents by the Bacillus.
  • This anti-microbial agent may be an organic molecule(s) and/or an thermo- tolerant protein(s).
  • a composition for inhibiting VRE growth contains a large concentration (i.e., lxlO 9 to lxlO 11 CFU) of Bacillus coagulans vegetative bacteria and/or spores in combination with the culture medium (supernatant) in either an unpurified or semi- purified form.
  • the culture medium has also been designated a GRAS classification by the FDA.
  • the medium may be partially- or fully lyophilized.
  • the concomitant administration of both the vegetative bacteria/spores and a supernatant component of some type would serve to ensure that all possible probiotic inhibitory mechanisms (t.e., antibiosis, parasitism, competitive inhibition and microenvironment/pH modification) were covered by the administration of the aforementioned therapeutic composition.
  • Bacillus coagulans culture medium has been shown to contain extracellular product(s), produced and secreted by the bacteria, which possess marked anti-microbial properties against bacteria, fungus, yeast, and virus. Methodologies for the purification of the one or more agents responsible for these antimicrobial properties are also cu ⁇ ently under development.
  • a prefe ⁇ ed embodiment of the present invention would, accordingly, comprise a large concentration (i.e., lxlO 9 to lxlO 11 CFU) of Bacillus coagulans vegetative bacteria and/or spores in combination with the either a purified or semi-purified form of these extracellular product(s).
  • Bacillus coagulans therapy is also useful to inhibit other strains of VRE.
  • Bacillus coagulans is used to prevent or ameliorate the level of colonization of other pathogenic organisms such as Candida species, Salmonella, coagulase-negative Staphylococci, and multi-resistant gram-negative rods such as Klebsiella species and Escherichia coli.

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Abstract

Cette invention a trait à des compositions contenant une souche bactérienne produisant de l'acide lactique, notamment Bacillus coagulans, aux fins de l'inhibition d'infections causées par des bactéries pathogènes. Les spores ou les produits extracellulaires générés par ces souches bactériennes se révèlent également des plus utiles en tant qu'agents inhibiteurs.
PCT/US2000/030737 1997-04-18 2000-11-08 Inhibitions d'agents pathogènes à l'aide de bactéries probiotiques WO2001034168A1 (fr)

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CA2389982A CA2389982C (fr) 1999-11-08 2000-11-08 Inhibitions d'agents pathogenes a l'aide de bacteries probiotiques
JP2001536165A JP2003513649A (ja) 1999-11-08 2000-11-08 Ｂａｃｉｌｌｕｓｃｏａｇｕｌａｎｓによる病原体の阻害
EP00978435A EP1229923A1 (fr) 1999-11-08 2000-11-08 Inhibitions d'agents pathog nes l'aide de bact ries probiotiques
AU15900/01A AU785159B2 (en) 1997-04-18 2000-11-08 Inhibition of pathogens by bacillus coagulans strains

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AU785159B2 (en) 2006-10-05
JP2012024092A (ja) 2012-02-09
JP2003513649A (ja) 2003-04-15
JP2014001245A (ja) 2014-01-09
AU1590001A (en) 2001-06-06
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