EP1229923A1 - Inhibitions d'agents pathog nes l'aide de bact ries probiotiques - Google Patents

Inhibitions d'agents pathog nes l'aide de bact ries probiotiques

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Publication number
EP1229923A1
EP1229923A1 EP00978435A EP00978435A EP1229923A1 EP 1229923 A1 EP1229923 A1 EP 1229923A1 EP 00978435 A EP00978435 A EP 00978435A EP 00978435 A EP00978435 A EP 00978435A EP 1229923 A1 EP1229923 A1 EP 1229923A1
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EP
European Patent Office
Prior art keywords
bacillus
composition
bacillus coagulans
strain
lactic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00978435A
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German (de)
English (en)
Inventor
Sean Farmer
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Ganeden Biotech Inc
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Ganeden Biotech Inc
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Publication of EP1229923A1 publication Critical patent/EP1229923A1/fr
Ceased legal-status Critical Current

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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 microflora has been shown to play a number of vital roles in maintaining gastrointestinal tract function and overall physiological health. For example, 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 al, 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 bactenal species including, but not limited to.
  • novel lactic acid-producing bacterial strains e.g., the novel strains of Bacillus coagulans disclosed herein
  • 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 bactenal species including, but not limited to.
  • 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 oi ganisms 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
  • the novel strains of Bacillus coagulans disclosed herein possess biochemical and physiological characteristics which include, but are not limited to (/) the production of the (L)+ optical isomer of lactic acid (propiomc acid), (n) have an optimal growth temperature of between 20-44°C, (in) 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 en ⁇ ironmental factors), (a ) 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
  • pu ⁇ fied or isolated preparation of a bactenal strain is meant that the preparation does not contain another bactenal species or strain in a quantity sufficient to interfere with the replication of the preparation at a particular temperature
  • a punfied or isolated preparation of a bactenal strain is made using standard methods, e g , plating at limiting dilution and temperature selection
  • a therapeutic composition compnsmg 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 dned or lyophihzed cell mass
  • An embodiment of the present invention involves the administration of from approximately 1 ⁇ 10 to 1 x 10 CFU of viable.
  • Bacillus coagulans vegetative bacteria or spore per day more preferably from approximately 1 ⁇ 10 to 1x10" , and preferably from approximately 5x 10 to 1 x 10 CFU of viable, vegetative bacteria or spores per day
  • the typical dosage is approximately 1 x10 to 1 x10 CrU of viable, vegetative bacteria or spores per day, preferably from approximately l xl O 8 to l x lO 10 , and more preferably from approximately 2 5x10 to 1 ⁇ 10 CFU of viable, vegetative bactena or spores per day
  • a composition compnsmg an extracellular product of Bacillus coagulans in a pharmaceutically-acceptable carrier suitable for oral administration to a human or animal is disclosed
  • 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, lyophihzed 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 compnsmg a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 1 % to 90% extracellular product with the remainder comprising the earner or delivery component
  • the subject is preferably a mammal, e g , a human
  • the bactena and/or products derived from the bacteria are also suitable for veterinary use, e g , to treat animals such as dogs and cats
  • a prefened embodiment comp ⁇ ses 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 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 va
  • 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.
  • 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 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 lxl O 12 CFU/gram, whereas in other preferred embodiments the concentrations are approximately lxlO 9 to lxl O 13 CFU/gram, approximately lxlO 5 to lxl0 7 CFU/g, or approximately 1 x 10 8 to 1 x 10 9 CFU/gram
  • 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 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 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-Pomt 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-Pomt 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,
  • 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 1 1 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.
  • 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.
  • Lactobacilli Previously, numerous species of Lactobacilli have been examined including, but not limited to, Lactobacillus bulgai icus Lactobacillus b diis 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 In view of this fact, the World Health Organization (WHO) has recommended restricted intake of D(-) lactic acid for adults and total avoidance in infant nutrition
  • WHO World Health Organization
  • probiotic bactena mitigate or prevent the growth of putrefactive or pathogenic microorganisms by the process of competitive inhibition, through the generation of a non-physiologically conducive acidic environment (i.e., through the production of lactic or other biological acids) and/or by the production of antibiotic-like substances (i.e., bacteriocins), which are responsible for the bacteria's anti-microbial effects.
  • bacteriocins antibiotic-like substances
  • Lactobacillus strains which produce antibiotics
  • lactobacillus reuteri has been shown to produce antibiotics which possess anti-microbial 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 abenant 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: 65-11. 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.
  • 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: 1 157-1 159; Solis-Pereyra and
  • 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. Immunopliarmacol. 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 bactena 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, Gai dnerella ⁇ ag ⁇ nal ⁇ s and various non-specific bacteria, which are responsible for v aginal infections
  • pathogens such as Candida albicans, Gai dnerella ⁇ ag ⁇ nal ⁇ s and various non-specific bacteria, which are responsible for v aginal infections
  • Antibiotics are widely used to control pathogenic microorganisms in both humans and animals
  • anti-microbial agents especially broad-spectrum antibiotics
  • e flora beneficial, non-pathogenic microorganisms
  • 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 ithin the gastrointestinal tract
  • VRE Vancomycin-resistant enterococci
  • enterococcal infections are caused by Enterococcus faecahs, 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 Enteiococci, the environment, and humans, (//) far more prudent antibiotic use, (in) 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
  • Two types of Enterococci cause infections (/) those originating from patients' native flor
  • 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 ( ) those originating from patients' native flora, which are unlikely to possess resistance beyond that intnnsic to the genus and are unlikely to be spread from bed to bed, and (u) 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 ammoglycoside resistance in environmental isolates of enterococci Appl Environ Microbiol 61 374-376 The predominant species inhabiting the intestine vanes In Europe, the United States, and the Far East, Enterococcus faeca s predominates in some instances, and Enterococcus faecium in others Moreover, of the 4 or more known enterococcal species (see, e g , Devnese, 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 f
  • VanA resistance to vancomycin and teicoplamn
  • VanB resistance to vancomycin alone
  • enterococci have naturally occurnng or inherent resistance to various drugs, including cephalosponns and the semisynthetic pemcihinase-resistant penicillins (e g oxacilhn) and clinically-achievable concentrations of clindamycin and aminoglycosides Compared v ⁇ ith sti eptococci, most enterococci are relatively resistant to penicillin, ampicillin, and the pseudopeniciUins Many enterococci are also tolerant to the killing effects of cell- ll active agents (e g ampicillin, vancomycin, etc ), although recent data suggest that this property may not be inherent, but rather acquired after exposure to antibiotics For example, the inherent in vivo resistance of Enterococcus faecahs to tnmethopnm- sulfamethoxazole, may explain the lack of efficacy in animal models Moreover, bactencidal activity against Enterococcus faecahs seems
  • Another system of conjugation involves broad host-range plasmids that can transfer among species of enterococci and other gram-positiv e organisms such as sti eptococci and staphvlococci 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 staphvlococci, 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 A third type of conjugation, which involves 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 Microbial 40.
  • conjugative transposons As opposed to ordinary transposons, which can jump withm a cell from one DNA location to another, conjugative transposons also encode the ability to bring about conjugation between different bacterial cells Since plasmids typically require rather complex machinery for replication (often depending on successful interactions with host proteins) and must face additional problems of surface exclusion and incompatibility, conjugative transposons (which do not replicate, but instead insert into the chromosome or into a plasmid of the new host) 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 memngococci, as well as into mycoplasma and ureaplasma See,, e g , Roberts, 1990 Charactenzation of the TetM determinants in urogenital and respiratory bacteria Antimicrob Agents Chemother 34 476-478 Other resistance genes, including
  • 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 staphvlococci)
  • 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 staphvlococci)
  • TSN Database Data collected by the TSN Database between 1995 and September 1 , 1997 were analyzed to determine whether the earlier increase in vancomycin resistance was unique to vancomycin, w hether it represented a continuing trend, and whether speciation is quantifiably important in analyzing this trend Enterococci faecahs resistance to ampicillin and vancomycin is uncommon Little change in resistance prevalence occ
  • Enterococcus faecahs would significantly confound the current therapeutic dilemma There is little reason to suspect that vancomycin and ampicillin resistances only provide selective advantage for the species faecium and not faecahs The relative absence of these resistances in Enterococcus faecahs may simply reflect a momentary lack of penetrance and equilibration of the traits Because of these important differences between the two species, meaningful surveillance of Enterococcal resistance must include species identification
  • enterococci account for approximately 1 10.000 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 Entei ococcal infection-related deaths have been difficult to ascertain, due to the fact that severe co-morbid illnesses are common
  • enterococcal sepsis is implicated in up to 50% of fatal cases
  • 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 ould be more effective if they included prudent prescnbing of all antibiotics, not just single agents (e g vancomycin) For example, use of this approach substantially decreased intestinal colonization with VRE in one
  • Vancomycin had been in clinical use since the 1950s, although it was not heavily used until the late- 1970s and particularly the 1980s Because multiple bactenal genes arc involved in the generation of vancomycin resistance, the development of such resistance was neither easy nor recent
  • Vancomycin resistance in enterococci is heterogeneous on many levels
  • Three phenotypes of vancomycin resistance (designated VanA, VanB, and VanC), each associated with a different hgase, are now well-described, a fourth, type VanD, has been recently reported See,, e g , Noble, et al , 1992 Co-transfer of vancomycin and other resistance genes from Enterococcus faecahs NCTC 12201 to Staphlococcus aureus F EMS Microbiol Lett 93 195- 198 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 mducible (i e , exposure of bactena to vancomycin results
  • 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 Tnl 547)
  • the VanB-cotnaimng 64-kb transposon is part of a 250-kb mobile element shown to move from the chromosome of one Entei ococcus and insert into the chromosome of another
  • circula ⁇ zation 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 Entei ococcus and that plasmid can then transfer by conjugation
  • VanC l and VanC2 are normally occumng genes that are endogenous species characteristics of F gannarum and F cassehfiavus, 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 Combinations of penicillin with vancomycin, ciprofloxacin with ampicillin, or novobiocin with doxycychne, among others, have been used, but can be unpredictable and remain clinically unproven
  • the substantial drawback of the broad spectrum approach is that the more organisms affected (i e , both protective commensals as vv ell 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 return 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, Cainpylobacter, Peptococcus, Heliobacter, Hemophylus, Staphvlococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, and Rotovirus species.
  • pathogens including, but not limited to, Candida, Clostridium, Escherichia, Klebsiella, Cainpylobacter, Peptococcus, Heliobacter, Hemophylus, Staphvlococcus, Yersinia, Vibrio, Shigella, Salmonella, Streptococcus, Proteus, Pseudomonas, Toxoplasmosis, and Rotovirus species.
  • non-antibiotic, probiotic bacteria-based therapeutic regimen include, but are not limited to: ( ) 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; (/ ' / ' ) 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 inco ⁇ orated 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 bactena 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 (/) their superior colonization abilities, (n) parasitism of undesirable microorganisms, (in) the production of acid (e g , lactic, acetic, and other acidic compounds) and/or other extracellular products possessing anti-microbial activity, and (a ) various combinations thereof
  • acid e g , lactic, acetic, and other acidic compounds
  • 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 (c g , lactic acid, acetic acid, etc ), (n) demonstrates beneficial function within the gastrointestinal tract, and (in) is non-pathogenic
  • acidic compounds c g , lactic acid, acetic acid, etc
  • n demonstrates beneficial function within the gastrointestinal tract
  • non-pathogenic By way of example and not of limitation, 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 cunently-classificd 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 (/ e , increases acidity) within the local micro-floral environment and does not contnbute to the growth of many undesirable,
  • Typical lactic acid-producing bactena useful as a probiotic of this invention are efficient lactic acid producers, which include non-pathogenic members of the Bacillus genus which produce bactenocins or other compounds which inhibit the growth of pathogenic organisms
  • the 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 Pnor to the invention, it was generally accepted that the various "classic" Lactobacillus and/or Bifidiobactei ium 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 low er than standard known strains of Bacillus coagulans
  • probiotic Bacillus coagulans is non-pathogenic and is generally legarded as safe (/ e GRAS classification) by the U S Federal Drug Administration (FDA) and the U S Department of Agriculture (USDA), 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 earner are particularly pretened for making and using compositions disclosed in the present invention
  • the Gram positive rods of 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 bactena 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 , Bergev's Manual of Systemic Bacteriology, Vol. 2, Sneath, P.H A. et al, eds., Williams & Wilkins, Baltimore, MD, 1986).
  • Punfied 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-dextrm glucano-transferase (U S. Pat. No. 5,102,800).
  • Bacillus coagulans has also been utilized to produce lactic acid (U S Pat. No.
  • Bacillus coagulans also referred to as Lactobacillus sporogenes; Sakaguti & Nakayama, ATCC No 31284
  • other lactic acid producing bacteria and Bacillus natto to produce a fermented food product from steamed soybeans (U.S. Pat. No 4,1 10,477).
  • 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 941 1492)
  • Bacillus coagulans strains have been used as general nutntional supplements and agents to control constipation and dianhea in humans and animals.
  • Bacillus coagulans cultures have been deposited with the following primary international culture collections. Agricultural Research Service Culture Collection; Russian Collection of Microorganisms, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, VKM DSMZ), American Type Culture Collection (ATCC), Finnish Microorganism Collection (University of Goteborg, Sweden); Japan Collection of Microorganisms (JCM), and Japan Federation for Culture Collection From the aforementioned deposits there are a total of eight 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 (u) deposited as another closely related species but, have recently been re-classified as Bacillus coagulans.
  • Bacillus coagulans Bacillus stereothermophilus Bacillus thermoacidurans, Lactobacillus sporogenes, Bacillus smitliu, Bacillus dextrolacticus,
  • 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 Amencan Type Culture Collection (ATCC, Rockville, MD) include the following accession numbers: Bacillus coagulans Hammer NRS 727 (ATCC No. 1 1014), Bacillus coagulans Hammer strain C (ATCC No 11369); Bacillus coagulans Hammer (ATCC No 31284), and Bacillus coagulans Hammer NCA 4259 (ATCC No 15949) Punfied 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, corresponds to ATCC No 1 1014), Bacillus coagulans Hammer (DSM No 2384, corresponds to ATCC No 11369), and Bacillus coagulans Hammer (DSM No 2385, conesponds to ATCC No 15949) Bacillus coagulans
  • 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 spoi ogenes (See Nakamura et al 1988 hit J S ⁇ st Bacteno
  • 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 prev ent the growth of numerous bacterial and fungal pathogens
  • Table 1 below, is a comparativ e chart showing the biochemical attributes of lactic acid-producing bacteria and their similanties Table 1
  • 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:
  • Bacillus coagulans possesses the ability to grow well in either environments that have free-oxygen or in strictly anaerobic environments. This is important due to the fact that Lactobacilli and Bifidobacteria are not aero-tolerant Thus, in essence, these aforementioned bactenal species are strictly anaerobic and do not proliferate well in environments containing free-oxygen Because 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 stei eothermoph ⁇ us, is used for quality control purposes in autoclaves This fact is crucial due to the frailty of all Lactobacilli and Bifidobactei ui
  • 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-positiv e spore- forming rods approximately 0 9 ⁇ m x 3 0-5 0 ⁇ m in size, aerobic to microaerophihc, produce L(+) (dextrorotatory) lactic acid in a homo fermentative manner Due to the fact that Bacillus coagulans exhibits characteristics typical of both genera Lactobacillus and Bacillus its taxonomic position between the families Lactobacillaceae and Bacillaceae has often been discussed
  • 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.
  • Micro-aerophilic exhibit fermentative metabolism and are facultatively aerobic.
  • 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.
  • 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 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 tnals that had been compromised due to pathogenesis by lactic acid-producing bactena In view of these facts, which are not disputed within the relevant scientific fields, Bacillus coagulans is safe as a therapeutic compositions
  • Bacte ⁇ ocins are proteins or protein-particulate complexes with bactencidal activities directed against species, which are closely related to the producer bacte ⁇ um
  • the inhibitory activity of lactic acid-producmg bacteria (e g , Bacillus coagulans) towards putrefactive organisms is thought to be partially due to the production of bactenocins
  • Table 4 lists some of the various bacterocins, which have been isolated and charactenzed from lactic acid-producing bactenal species Table 4
  • 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 bactena were ineffectual as probiotics due to vanous 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), ( ) have an optimal growth temperature of less than 45°C, (in) the production ot 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), (n ) the production of one or more extracellular products exhibiting probiotic activity which inhibits the growth of bacteria, yeast, fungi, v irus, or any combinations thereof, and/or ( ⁇ ) the ability to utilize a wide spectrum of substrates for proliferation
  • 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 (i.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 i.e., vegetative bacterial cells
  • spores i.e., 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.
  • 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.
  • An embodiment of the present invention involves the administration of from approximately l xl0 to lxlO 14 CFU of viable, vegetative bacteria or spore per day, more preferably from approximately l xl O 5 to l xl O 10 , and most preferably from approximately 5xl 0 8 to l x l O 9 CFU of viable, vegetative bacteria or spores per day.
  • the typical dosage is approximately lxl O 2 to l xl O 14 CFU of viable, vegetative bacteria or spores per day, preferably o 1 1 ⁇ R I O from approximately 1 x 10 to 1 x 10 , and more preferably from approximately 2.5x10 to 1x10 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 earner 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 ohgosacchandes, flavorings, earners, and the like components
  • One particularly prefened 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
  • pathogen and/or antibiotic-resistant pathogenic organisms in the gastrointestinal tract of animals
  • the te ⁇ s "pathogen” and “parasite” are used interchangeably in the context of a deletenous organism growing in the gastrointestinal tract and/or feces of an animal, although it appreciated that these terms have distinctive meanings
  • the present invention describes compositions and methods of use for inhibiting or preventing growth of a pathogen in the gastrointestinal tract of an animal comprising the step of administering a composition of the invention to the gastrointestinal tract of the animal one or more of the following Bacillus coagulans bacterial cells (i e vegetative bacterial cells), spores, and/or isolated Bacillus coagulans extracellular products (which contains a metabolite possessing antibiotic-like properties) to the animal, so as to treat or prevent the colonization of antibiotic-resistant pathogens with the gastrointestinal tract
  • 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 b 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
  • 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 compnsmg about 0 1 g to 50 g per dosage, for one to seven days are sufficient to achieve the desired result
  • a preferred method involves the administration into the digestive tract of from lxlO 2 to lxlO 10 viable bactenum or spore per day, in some embodiments from lxlO 3 to lxlO 6 , in other embodiments from lxlO 6 to lxl O 9 , and more preferably about from 5xl0 8 to lxl O 9 viable bacterium or spore per day
  • Exemplary dosages range from about 1 x10 to lxl 0 6 viable bacterium per day, or alternati v ely range from about l xl O 6 to 1x10 viable bactenum 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 prefened embodiment comprises a composition a total concentration ratio of Bacillus coagulans extracellular product ranging from approximately 10% to 75% extracellular product ith the remainder comprising the earner 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 w here 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 pu ⁇ oses are readily apparent to one skilled in the arts of nutrient abso ⁇ tion, feed utilization and bioavailabihty
  • the present invention further contemplates a therapeutic svstem 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 compnse 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 inst ctions 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 prefened 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.
  • gastrointestinal parasites infect a v ariety of animals and can include Spii ocei ca species such as S lupi that cause esopheageal worms in canines and Pln soloptera species that cause stomach worms in canines and felines
  • the composition can be included in the pelletized or granular food, or can comprise a mixture of the pelletized food combined with a pelletized composition of this invention
  • Mixing pelletized food w ith a pelletized formulation of a composition of this inv ention is a particularly prefe ⁇ cd method for practicing the present invention, insofar as it provides a conv enient system for using commercial feeds and simultaneously regulating the amounts of a composition of this invention to be administered
  • 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
  • the present invention further contemplates a system for inhibiting growth of parasites and/or pathogens in the gastrointestinal tract of an animal or in animal feces compnsmg a container comprising label and a composition according to the present invention, wherein said label comprises instructions for use of the composition for inhibiting pathogen/parasite growth
  • the system is present in the form of a package containing a composition of this invention, or in combination with packaging material
  • the packaging mate ⁇ al 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 pre-selected food stuffs, and the like information.
  • Bacillus coagulans is aerobic and facultative, and is typically cultured at pH 5.7 to 6.8, in a nutrient broth containing up to 2% (by wt) NaCI, although neither NaCI, nor KC1 are required for growth. A pH of approximately 4.0 to 7.5, is optimum for initiation of sporulation (i.e., the formation of spores).
  • the novel strains of Bacillus coagulans disclosed herein are optimally grown at 20°C to 40°C, and the spores can withstand pasteurization. Additionally, the bacteria exhibit facultative and heterotrophic growth by utilizing a nitrate or sulfate source.
  • 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 MgS0 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 preferred, 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 preferred.
  • a preferred 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 (CockeyesviUe, MD); Advanced Microbial Systems (Shakopee, MN); and Troy Biologicals (Troy, MD).
  • An efficacious growth medium for Bacillus coagulans is a Glucose Yeast extract (GYE) medium. The formulation for GYE is shown below in Table 6.
  • the pH of the medium was then adjusted to approximately 6.3 followed by sterilization with steam at 1.2 kg/cm ' pressure at 120°C for 15 minutes.
  • 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 1 x10 to 1x10 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.
  • Bacillus coagulans bacterial cells or spores were collected using standard methods (e.g., filtration, centrifugation) and the collected cells and spores may subsequently be lyophihzed, spray dried, air dried or frozen.
  • the supernatant from the cell culture can be collected and used as an extracellular agent secreted by Bacillus coagulans which possesses anti-microbial activity useful in a formulation of this invention.
  • a typical yield obtained from the aforementioned culture methodology is in the range of approximately 1 x 10 - 1 x 10 viable spores and, more typically, approximately 10-15 x 10 10 cells/spores per gram prior to being dried. It should also be noted that the Bacillus coagulans spores, following a drying step, maintain at least 90% viability for up to 7 years when stored at room temperature. Hence, the effective shelf-life of a composition containing Bacillus coagulans Hammer spores at room temperature is approximately 10 years.
  • 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.
  • Batch fermentation comprises the following procedure.
  • a single colony of Bacillus coagulans was selected with sterile loop from a petri-dish colony. This single colony was then used to inoculate a two-liter Erlenmeyer flask containing GYE media, dextrose, and minerals. The culture was incubated for approximately 18 hours in an orbital shaker (possessing a 2" orbit) at 35°C. This 2 liter culture was used to inoculate a sterilized, 500 liter Batch Fermenter containing GYE media, dextrose, and minerals. The Batch Fermenter was run at 35°C for approximately 30 hours under high aeration (36-38 LPM).
  • the Batch Fermenter aeration was turned off and temperature reduced to 20°C for 4 hours to facilitate settling of the bacterial cells therein.
  • the fermentation broth was harvested using Alpha-Laval Sha ⁇ les continuous-feed centrifuge at 12,000 ⁇ m at 10°C and the bacterial solids were removed for subsequent lyophilization.
  • a culture of dried Bacillus coagulans spores may be prepared, e.g., as follows. Approximately 1x10 spores were inoculated into one liter of culture medium containing: 30 g (wt./vol GYE media, dextrose, and minerals. The culture was maintained for 72 hours under a high oxygen environment at 37°C so as to produce a culture having approximately 15xl ⁇ ' cells/gram of culture. The culture was then filtered to remove the liquid culture medium and the resulting bacterial pellet was resuspended in water and lyophihzed. The lyophihzed bacteria were ground to a fine "powder" by use of standard good manufacturing practice (cGMP) methodologies.
  • cGMP standard good manufacturing practice
  • the culture was maintained for 5 days as described.
  • the culture was first autoclaved for 30 minutesat 250 F, and then centrifuged at 4000 r.p.m. for 15 mm.
  • the resulting supernatant was collected and subjected to sub-micron filtration by the initial use of a Buchner funnel with a 0.8 ⁇ im filter.
  • the filtrate was then collected and further filtered through a 0.2 ⁇ m Nalge vacuum filter.
  • the resulting filtrate was then collected (an approximate volume of 900 ml/liter of culture medium) and comprised a liquid containing an extracellular product, which was to be quantitatively analyzed and utilized in the subsequent inhibition studies.
  • Electrophoresis was performed by the method of Laemmh (see Laemmh, 1970 Nature 227 680-685) and the acrylamide gels were poured in
  • the liquid containing the extracellular product may be formulated into a liquid ointment composition for use in direct application onto dermal, cuticular, or mucous membrane tissues
  • the liquid ointment was prepared by combining the liquid extracellular product produced above with, e g Emu Oil in a ratio of approximately 8 2
  • Plating Glucose Yeast Extract agar medium was liquefied and then cooled to 45 C in a water-bath A total of 5 petn dishes per sample were utilized 1 ml from heat-treated final dilution tube was added into each petn dish, followed by the addition of 5 ml of the above- identified liquefied GYE agar medium into the petri dishes and thorough mixing. When solidified, the plates were incubated in an inverted position at 40°C for a total of 48 hours.
  • the plates showing 30-300 colonies were selected for counting. Plates possessing a very nanow 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.
  • 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 the 28 total flasks, representing 7 flasks for each strain. These inoculated flasks were incubated on a rotary environmental shaker at 45"C for 28 hours. Every 4 hours, the shaker was stopped, and the new culture removed for evaluations.
  • OD readings in % Optical Transmittance, pH, and Total Heterotrophic Plate Counts by 3M Petrifilm spread plate method performed to monitor bacterial cell density and pH changes at these different time intervals.
  • the results of the pH evaluations, OD in % Transmittance, and Total Heterotrophic Plate Counts are shown below in Table 9, Table 10, and Table 1 1 , respectively.
  • FIG 2 and FIG 3 show the End-Pomt Kinetics of both the 1 % Bacillus coagulans strain (GBI-1 ) and the ATCC- 99% Bacillus coagulans strain, respectively
  • Bacillus coagulans Isolates Two cultures of Bacillus coagulans strains were analyzed with Growth/End-Point Kinetic
  • each microplate well also contained a tetrazolium dye/redox indicator system.
  • Bacterial growth i.e., metabolic respiration or oxidation of carbon sources
  • 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.
  • CFU colony forming units
  • 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 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.
  • characterization of a given microbial isolate is performed by streaking the organism onto a nutrient medium (e.g., GYE or TSA) that will support vigorous microbial proliferation and growth.
  • a nutrient medium e.g., GYE or TSA
  • the more fastidious organisms may require chocolate or BIER agar for growth, whereas many "environmental" were found to grow better in the more minimal media.
  • the culture plates were incubated at 28 C to 35 C for 4-18 hours.
  • the bacterial colonies were removed from the culture plate by use of a saline-moistened, cotton swab. A suspension of uniform turbidity was then prepared in 0.85% saline by comparison with a known turbidimetric standard. The bacterial suspension was inoculated into the microplate wells (150 ⁇ l/well) and the plate was covered with the associated microplate lid. The covered plates were then incubated at 28 C to 35 C for 4 hours or overnight ( 16-24 hours).
  • microplates were then read using a microplate reader at 590 nm.
  • the absorbence or transmittance (i.e., color) in each well was referenced against the negative control well (A-1) so that any pu ⁇ le color recorded above this control level was read as a positive utilization of that particular carbon source.
  • the data were reported as the Percent Color Change as compared to well A-1 utilizing the following formula:
  • the reaction within the given well was considered to be "positive". However, this value must be empirically dete ⁇ nined, as the parameters for each substrate may be different and the positive test below a value of 40 may be possible.
  • the computer algorithms employed provide standardization of settings ensuring repeatability and avoidance of operator bias. Names of all carbon source substrates employed are provided in the results regardless of response.
  • Table 12 illustrates the Total Heterotrophic Plate Count using Trypticase Soy Agar (TSA) for the novel Bacillus coagulans strains disclosed herein. Table 12
  • 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 w as subsequently examined against both the Acrobc (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
  • 16S rRNA gene was PCR amplified from genomic DNA isolated from bacterial colonies.
  • the primers which were utilized for the amplification correspond to E. coli positions 005 and 531 for the 500 bp package.
  • Excess primers and dNTPs were subsequently removed from the amplification products by use of a Microcon 100 (Amicon) molecular weight cut-off membranes.
  • the PCR amplification products were then subjected to agarose gel electrophoretic analysis to ascertain both quality and quantity of these products.
  • Cycle sequencing of the 16S rRNA amplification products was performed using AmpliTaq FS DNA polymerase and dRhodamine dye terminators. Excess dye-labeled 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" 1 (RMC) polyacrylamide/urea gel for approximately 6 hours. The resulting sequence data was analyzed using PE/ Applied Biosystems DNA editing and assembly software.
  • RMC Long Ranger" 1
  • FIG. 6 through FIG. 8 provides alignment of the novel Bacillus coagulans strains disclosed in the present invention with various other Bacillus species, as well as the results obtained by Neighbor Joining Tree and Concise Alignment analysis. The 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. //;/. 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.
  • GBI-1 Bacillus coagulans 1.68% difference Genus level ID
  • 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 recent development of a time-resolved, 96-well plate fluorometer provides a rapid and highly sensitive method to obtain peptidase profiles for microbial identification. See, Mossman, et al, 1997. Aminopepetidase profiling using a time-resolved, 96-well plate filter fluorometer. Appl. Spectroscopy 51 : 1443-1446.
  • Aminopeptidase profiling was shown to be an effective procedure for the differentiation of the novel strains of Bacillus coagulans disclosed herein, from those previously known and characterized (e.g., the ATCC type strain).
  • the cell densities were adjusted to 2.5 x 10 6 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),
  • FIG 9 represents a histogram plot of the of the fluorescence intensity for each the aminopeptidase enzvme activities for the Bacillus coagulans 99% ATCC isolate
  • FIG 10 represents a histogram plot of the oi the fluorescence intensity for each the aminopeptidase enzyme activities for the Bacillus coagulans GBI-1 isolate
  • FIG 1 1 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
  • GLU 7 L-glutamic acid
  • TRP tryptophan
  • VRE Vancomycin-Resistant Enterococci
  • mice 4 6.6 log 10 CFU/g stool
  • VRE VRE-VRE
  • 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 6.4 log ⁇ 0 CFU/gram of stool) and all of these mice still had low levels of Bacillus coagulans detectable in their stool 4 days after completion of therapy.
  • Bacillus coagulans in the form of both vegetative bacteria and spores
  • results which were obtained with the use of this murine model correlate well with the findings in various studies which were examined VRE-colonized human patients.
  • this established mouse model provides a means to study the efficacy of agents designed to eliminate VRE colonization.
  • Thirty-five percent of mice receiving Bacillus coagulans were found to have undetectable levels of VRE four days after completing therapy. In comparison, none of the mice receiving saline were VRE-free. On average, a 25- to 28-fold reduction in the level of VRE was observed in the Bacillus coagulans-treated mice in comparison with the saline-treated mice.
  • sixty- five percent of mice receiving Bacillus coagulans had a reduction of VRE equal to approximately 50-times the original inoculation.
  • Micro-Environment Modification which usually serves to alter the physiological or biochemical properties or activities of bacteria's cell membrane by the production of acid (e.g., lactic, acetic, etc.) or other agents possessing anti-microbial properties.
  • acid e.g., lactic, acetic, etc.
  • Enterococcus faecium which is the Enterococcus species responsible for most, if not all, VRE carriage and infections
  • Enterococcus faecium is used as a probiotic in the animal production industry.
  • 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 1x10 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 lyophihzed.
  • 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 (i.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 anti-microbial properties are also currently under development.
  • a preferred 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.
EP00978435A 1999-11-08 2000-11-08 Inhibitions d'agents pathog nes l'aide de bact ries probiotiques Ceased EP1229923A1 (fr)

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Families Citing this family (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU785159B2 (en) * 1997-04-18 2006-10-05 Ganeden Biotech, Inc. Inhibition of pathogens by bacillus coagulans strains
US7767203B2 (en) 1998-08-07 2010-08-03 Ganeden Biotech, Inc. Methods for the dietary management of irritable bowel syndrome and carbohydrate malabsorption
US6849256B1 (en) 1999-11-08 2005-02-01 Ganeden Biotech Incorporated Inhibition of pathogens by probiotic bacteria
FR2828407B1 (fr) * 2001-08-10 2005-06-24 Cobiotex Compositions a base de complexes bacteriens et leurs applications pour la prevention des infections nosocomiales
JP2004067599A (ja) * 2002-08-07 2004-03-04 Kunihiko Tominaga 腟内洗浄剤
US7854927B2 (en) * 2004-05-11 2010-12-21 Ganeden Biotech, Inc. Methods and compositions for the dietary management of autoimmune disorders
US8092793B2 (en) 2004-12-15 2012-01-10 Qingdao East Sea Pharmaceuticals, Ltd. Treating inflammatory bowel disease with live bacteria
CN101095698B (zh) 2006-06-26 2010-12-01 青岛东海药业有限公司 酪酸梭菌防治便臭毒素引起的相关症状和疾病的用途
PL2211626T3 (pl) * 2007-08-29 2020-03-31 Ganeden Biotech, Inc. Wyroby wypiekane
ES2700855T3 (es) * 2007-10-16 2019-02-19 Ganeden Biotech Inc Composiciones bebibles
US20090305387A1 (en) * 2008-01-30 2009-12-10 Ganeden Biotech, Inc. Compositions and methods for cleaning surfaces
DK2348888T3 (en) 2008-10-16 2016-09-12 Ganeden Biotech Inc PROBIOTIC CORN-BASED COMPOSITIONS
WO2010127132A1 (fr) 2009-04-29 2010-11-04 Ganeden Biotech, Inc. Composition cellulaire bactérienne activée
WO2012135499A1 (fr) 2011-03-31 2012-10-04 Ganeden Biotech, Inc. Compositions nutritionnelles probiotiques pour l'activité sportive
KR101769444B1 (ko) 2011-06-10 2017-08-22 (주)아모레퍼시픽 차나무 잎에서 분리한 신규 락토바실러스 플란타룸
KR101769445B1 (ko) 2011-06-10 2017-08-22 (주)아모레퍼시픽 차나무 잎에서 분리한 신규 락토바실러스 플란타룸
JP6033290B2 (ja) * 2011-06-10 2016-11-30 株式会社アモーレパシフィックＡｍｏｒｅｐａｃｉｆｉｃ Ｃｏｒｐｏｒａｔｉｏｎ チャノキ葉から分離した新規なラクトバチルス・プランタラム
KR101769446B1 (ko) 2011-06-10 2017-08-22 (주)아모레퍼시픽 차나무 잎에서 분리한 신규 락토바실러스 플란타룸
KR101719197B1 (ko) * 2011-06-10 2017-03-24 (주)아모레퍼시픽 차나무 잎에서 분리한 신규 락토바실러스 플란타룸
JP5912610B2 (ja) 2012-02-07 2016-04-27 矢崎総業株式会社 ねじ締めブロックの係止構造
US9596861B2 (en) * 2013-12-24 2017-03-21 Sami Labs Limited Method of producing partially purified extracellular metabolite products from Bacillus coagulans and biological applications thereof
JP2020500860A (ja) * 2016-11-23 2020-01-16 ゴジョ・インダストリーズ・インコーポレイテッド プロバイオティック／プレバイオティックな有効成分を含む消毒薬組成物
AU2018282018B2 (en) * 2017-06-09 2021-11-04 Sami Labs Limited Compositions and methods for reducing flatulence
CN115322931B (zh) * 2022-08-19 2023-04-25 华南农业大学 一株能够抑制α-葡萄糖苷酶和/或α-淀粉酶活性的凝结魏兹曼杆菌及其应用
CN115354002B (zh) * 2022-09-20 2023-12-19 微康益生菌(苏州)股份有限公司 一种凝结芽孢杆菌bc07、其用途及其抑菌剂、药物、食品和抑菌方法
CN116463258B (zh) * 2023-04-12 2024-01-09 山东威曼宠物食品有限公司 一种用于预防宠物腹泻的凝结芽孢杆菌King27及菌剂、制备方法和应用
CN117143770B (zh) * 2023-08-30 2024-03-08 广州格拉姆生物科技有限公司 一株凝结魏茨曼氏菌glm336及其应用

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1262927B (it) * 1992-01-14 1996-07-22 Consiglio Nazionale Ricerche Batteri sporgenti e loro impiego come probiotici
WO1994011492A1 (fr) * 1992-11-12 1994-05-26 Chr. Hansen's Laboratory, Inc. Procede modifiant de maniere avantageuse la microflore intestinale de la volaille
JP3381010B2 (ja) * 1994-05-10 2003-02-24 株式会社林原生物化学研究所 プロポリスエキスを含有する発酵物とその製造方法並びに用途
US5785990A (en) * 1995-07-10 1998-07-28 Merrick's, Inc. Feed fortifier and enhancer for preruminant calves and method of using same
JPH09194384A (ja) * 1996-01-19 1997-07-29 Snow Brand Milk Prod Co Ltd ミネラル吸収促進剤
DE69707413T3 (de) * 1997-01-09 2009-07-02 Société des Produits Nestlé S.A. Probiotik enthaltendes Getreideprodukt
AU785159B2 (en) * 1997-04-18 2006-10-05 Ganeden Biotech, Inc. Inhibition of pathogens by bacillus coagulans strains
DE69832267T2 (de) * 1997-04-18 2006-08-10 Ganeden Biotech, Inc., La Jolla Oberflächige verwendung von probiotischen bacillussporen zur verhinderung oder bekämpfung von mikrobiellen infektionen
ATE407687T1 (de) * 1997-06-03 2008-09-15 Ganeden Biotech Inc Probiotisches milchsäurebakterium zur behandlung bakterieller infektionen in zusammenhang mit sids
ES2258841T3 (es) * 1998-04-01 2006-09-01 Ganeden Biotech, Inc. Procedimientos para reducir el colesterol con esporas de bacillus coagulans, sistemas y composiciones asociados.
EP1102595A2 (fr) * 1998-08-07 2001-05-30 Ganeden Biotech, Inc. Procedes mettant en oeuvre des bacteries probiotiques non productrices d'acide lactique pour augmenter la solubilite de substances nutritives
US6461607B1 (en) * 1998-08-24 2002-10-08 Ganeden Biotech, Inc. Probiotic, lactic acid-producing bacteria and uses thereof
EP1173233A1 (fr) * 1999-04-14 2002-01-23 Ganeden Biotech, Inc. Procedes visant a inhiber des infections microbiennes associees a des produits hygieniques
US20030124104A1 (en) * 2001-11-05 2003-07-03 Sean Farmer Probiotic compositions
JP5232404B2 (ja) * 2006-06-07 2013-07-10 第一三共ヘルスケア株式会社 有胞子性乳酸菌含有抗感冒ウイルス又は抗インフルエンザウイルス用組成物
DK2348888T3 (en) * 2008-10-16 2016-09-12 Ganeden Biotech Inc PROBIOTIC CORN-BASED COMPOSITIONS

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0134168A1 *

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JP2012024092A (ja) 2012-02-09
CA2389982C (fr) 2015-06-16
AU785159B2 (en) 2006-10-05
WO2001034168A9 (fr) 2002-08-15
AU1590001A (en) 2001-06-06
JP2003513649A (ja) 2003-04-15
WO2001034168A1 (fr) 2001-05-17
JP2014001245A (ja) 2014-01-09
CA2389982A1 (fr) 2001-05-17

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