Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/19—Dairy proteins
• • 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
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/10—Laxatives
• • 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
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli

Definitions

• the present invention relates to novel bacterial strains, compositions including same and methods of using such strains in probiotic treatment of gastrointestinal disorders.
• Probiotics are defined as living organisms, which exert a positive effect on a host gastro-intestinal (GI) system.
• the most commonly used probiotics are strains of the lactic acid bacteria (LAB), particularly those classified to the Lactobacillus, Laclococcus, and Enterococcus genera.
• LAB lactic acid bacteria
• the goal of probiotic therapy is to increase the number and activity of health- promoting microorganisms until normal GI flora can be reestablished.
• inhibitory substances e.g., antibiotics, organic acids, hydrogen peroxide and bacteriocins
• H blocking of adhesion sites by competitive inhibition of bacterial adhesion sites on intestinal epithelial surfaces
• Saccharomyces fungemia Thirteen cases of Saccharomyces fungemia were caused by vascular catheter contamination [Hennequin (2000) Eur. J. Clin. Microbiol. Infect. Dis. 19:16-20] and Bacillus infections linked to probiotic consumption all in patients with underlying disease [Spinosa (2000) Microb. Ecol. Health Dis. 12:99-101; Oggioni (1998) J. Clin. Microbiol. 36:325-326].
• Enterococcus is emerging as an important cause of nosocomial infections and isolates are increasingly vancomycin resistant.
• Non-pathogenic lactose-positive E. coli comprise the main group of healthy aerobic microflora in the intestine of humans and animals, providing microbiological balance and playing an important role in alimentation and immunity.
• E. coli BU-230-98 ATCC Deposit No. 202226 (DSM 12799) (E. coli M 17), which is capable of restoring normal GI flora of a variety of mammals and avian.
• a probiotic composition comprising this probiotic organism suspended in the formulation was found to be effective in the treatment and prevention of various gastrointestinal disorders.
• the probiotic formulation per se was also found effective as a body weight gain enhancer and as an immuno-slimulator in mammals and avian (See U.S. Pat. Nos. 6,500,423 assigned to "The Bio Balance Corp.” and related applications each of which is incorporated herein by reference).
• a biologically pure culture of an E. coli Ml 7 bacterial strain exhibiting nalidixic acid resistance there is provided a biologically pure culture of an E. coli Ml 7 bacterial strain exhibiting nalidixic acid resistance.
• a probiotic composition comprising, as an active ingredient, the bacterial strain and a carrier or diluent.
• the composition comprising 10 3 -10 10 of bacterial cells of the bacterial strain per gram of the composition.
• the carrier comprises a formulation for maintaining viability of the bacterial strain.
• the formulation comprises a volatile fraction of a plant extract.
• composition further comprises an antifungal agent.
• compositions further comprises an antibiotic.
• composition further comprises a probiotic microorganism selected from the group consisting of a yeast cell, a mold and a bacterial cell.
• the carrier is a colonization carrier.
• a food additive comprising as an active ingredient, the bacterial strain and a carrier suitable for human consumption.
• the colonization carrier is selected from the group consisting of a saccharide, a modified saccharide and a combination thereof.
• a feed additive comprising as an active ingredient, the bacterial strain and a carrier suitable for animal consumption.
• the carrier is selected from the group consisting of limestone, saccharides and wheat midds.
• a foodstuff comprising the bacterial strain.
• the foodstuff is a milk product.
• a method of treating a gastrointestinal disorder comprising administering to a subject in need thereof a therapeutically effective amount of the bacterial strain, thereby treating the gastrointestinal disorder.
• the gastrointestinal disorder is selected from the group consisting of pouchitis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, small bowel bacterial overgrowth, gastroesophageal reflux disease, diarrhea, Clostridium difficile colitis and/or antibiotic associated diarrhea, irritable bowel syndrome, irritiable pouch syndrome, acute diarrhea, traveller's diarrhea, lactose intolerance, HIV-associated diarrhea, sucrose isomaltase deficiency, carcinogenesis, enteral feeding associated diarrhea, and disorders which are associated with enteropathogens, non-erosive esophageal reflux disease (NERD) and associated small bowel bacterial overgrowth, functional dyspesia, necrotizing enterocolitis,
• E. coli M17 is selected from the group consisting of BU-239, BU-230-98, BU230-01 and ATCC Deposit No. 202226 (DSM 12799).
• the bacterial strain comprises a genomic nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-9.
• the bacterial strain is capable of proliferating and colonizing in a mammalian gastrointestinal tract.
• a method of detecting presence of the bacterial strain in a fecal sample comprising detecting bacterial growth in the presence of nalidixic acid, thereby detecting presence of the bacterial strain in the fecal sample.
• the present invention successfully addresses the shortcomings of the presently known configurations by providing novel bacterial strains, compositions including same and probiotic use thereof.
• FIG. 1 shows Pulsed Field Gel Electrophoresis of the Ml 7 parental strain and the nalidixic acid-resistanf (M17 S NAR) derivatives generated according to the teachings of the present invention.
• Each strain was digested with Xbal.
• the Markers consist of lambda concatamers.
• MC 1061 which is a K- 12 derivative of E. coli and is genetically unrelated to Ml 7, was run as a negative control. The strains are indicated above the relevant lane of the image.
• FIG. 2 shows Amplified Fragment Length Polymorphism (AFLP) analyses of the M 17 parental and M 17 S NAR strains.
• DNA from M 17, M 17 S NAR, and ECOR strains was subjected to AFLP analysis using the EcoRI-A + Msel GA primer combination.
• the reaction products were resolved by denaturing gel polyarcylamide gel electrophoresis on a Li-Cor/NEN 4200 global analyzer. The strain is indicated above the appropriate lane containing the resolved reaction products.
• FIGS. 3a-d are graphs showing the shedding of total coliforms and M17 SNAR in fecal samples of canines dosed with M17 SNAR - Animals [dogs 3028 ( Figure 3a) and 3029 ( Figure 3b) were male; dogs 3032 ( Figure 3c) and 3033 ( Figure 3d) were female] were dosed with 1 X 10 13 colony forming units of M17 $NAR on fourteen consecutive days after day 0 (the high dose intake in the Ricerca study). Fecal samples were collected and total coliforms enumerated in duplicate on VRBA media while M17 S NAR was enumerated on VRBA + 25 ⁇ g/ml nalidixic acid. Nalidixic acid- resistant colonies were confirmed with the Contig 127 PCR assay. Results are shown separately for each animal.
• FIG. 4 is a scheme showing whole genome shotgun sequencing approach for E. coli Ml 7sNAR-
• the present invention is of bacterial strains, which can be used in the treatment of gastrointestinal and immune-related disorders.
• the present inventors have previously found a single species of a nonpathogenic probiotic microorganism, E. coli BU-230-98, ATCC Deposit No. 202226 (DSM 12799), which is capable of restoring normal GI flora of a variety of mammals and avian.
• a probiotic composition comprising this probiotic organism was found to be effective in the treatment and prevention of various gastrointestinal disorders.
• the probiotic composition was also found effective as a body weight gain enhancer and as an immuno-stimulator in mammals and avian (See U.S. Pat. Nos. 6,500,423 assigned to "The Bio Balance Corp.” and related applications each of which is incorporated herein by reference).
• DSM 12799 (M 17) render it a favorable therapeutic tool for the treatment of a myriad of gastrointestinal disorders and related disorders (e.g., immune related), indicating that antibiotic resistant strains of this bacterial species may be of regulatory importance and used in combination with antibiotic treatment.
• the present inventors have enriched and selected from a parental stock of E. coli BU-230- 98, ATCC Deposit No. 202226 (DSM 12799), naturally-occurring, nalidixic acid- resistant mutants.
• the nalidixic acid-resistant strain termed M 17 SNAR ATCC Deposit No. 7295 is indistinguishable from the parental strain by PFGE and AFLP analyses.
• Unique nucleic acid sequences of the M17 SN AR genome were then identified by whole genome sequencing and computational analysis followed by PCR analysis against a panel of E. coli strains.
• M17SNAR into composite fecal samples, followed by selective plating on VRBA and confirmation by nucleic acid analysis showed that the M17 SNAR could be specifically quantified in spiked fecal samples.
• the limit of detection of the assay was estimated to be 33 CFU/ml of M17 SNAR in a fecal sample. Given the combination of selective plating on VRBA + nalidixic acid and the confirmation by nucleic acid analysis, the assay appears to be highly specific for detecting and quantifying E. coli M17 S NAR in fecal samples.
• E. coli Ml 7 bacterial strain refers to the strain per se and non-pathogenic derived strains which maintain a probiotic activity and biochemical characteristics as listed in Tables 1-3, below.
• Probiotic activity refers to the property of inhibiting the growth of at least one pathogen. Testing the inhibition of pathogen growth may be effected on solid medium in which culture supernatants of candidate isolated bacteria are observed for their property of inhibiting the growth of a pathogen when applied to the surface of the solid medium.
• a paper disc impregnated with the culture supernatant of a candidate probiotic strain is placed on the surface of an agar plate seeded with the pathogen.
• Probiotic bacterial supernatants cause a ring of clear agar or of reduce growth density indicating inhibition of the pathogen in the vicinity of the disc.
• the E. coli M 17 bacterial strain is BU-239, BU-230-98, BU-230-01 and ATCC Deposit No. 202226 (DSM 12799).
• E. coli BU-239 bacterial strain of the present invention exhibits nalidixic acid resistance.
• nalidixic acid resistance refers to the ability of the culture of this aspect of the present invention to multiply even in the presence of the quinolone antibiotic, nalidixic acid (NegGram).
• Cultures of the present invention are preferably resistant to at least 5 ⁇ g/ml nalidixic acid, more preferably at least 15 ⁇ g/ml nalidixic acid, even more preferably 25 ⁇ g/ml nalidixic acid, even more preferably 50 ⁇ g/ml nalidixic acid, even more preferably 100 ⁇ g/ml nalidixic acid.
• the isolation, identification and culturing of the bacterial strains of the present invention can be effected using standard microbiological techniques. Examples of such techniques may be found in Gerhardt,
• Isolation of the bacterial strains of the present invention is preferably effected by streaking the specimen (e.g., E. coli BU-230-98) on a solid medium (e.g., nutrient agar plates) to obtain a single colony which is characterized by the phenotypic traits described hereinabove (e.g., Gram negative, capable of lactose fermentation and nalidixic acid resistance) and to reduce the likelihood of working with a culture which has become contaminated and/or has accumulated mutations.
• a solid medium e.g., nutrient agar plates
• the bacterial strains of the present invention can be propagated in a liquid medium under conditions which are described in the Examples section.
• Medium for growing the bacterial strains of the present invention includes a carbon source, a nitrogen source and inorganic salts as well as specially required substances such as vitamins, amino acids, nucleic acids and the like (Examples 1 of the Examples section which follows describes embodiments of medium compositions which can be used in accordance with the present invention).
• suitable carbon sources which can be used for growing the bacterial strains of the present invention include, but are not limited to, starch, peptone, yeast extract, . amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol and glycerol and the like; oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil.
• amino acids sugars such as glucose, arabinose, mannose, glucosamine, maltose, and the like
• salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like
• the amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 gram per liter medium.
• glucose, starch, and/or peptone is contained in the medium as a major carbon source, at a concentration of 0.1-5% (W/V).
• suitable nitrogen sources which can be used for growing the bacterial strains of the present invention include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia or combinations thereof.
• the amount of nitrogen source varies according the nitrogen source, typically between 0.1 to 30 gram per liter medium.
• potassium dihydrogen phosphate, di potassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate can be used alone or in combination.
• the amount of inorganic acid varies according to the kind of the inorganic salt, typically between 0.001 to 10 gram per liter medium.
• specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dried yeast and combinations thereof. Cultivation is effected at a temperature, which allows the growth of the probiotic bacterial strains of the present invention, essentially, between 28 0 C and 46 0 C. A preferred temperature range is 30-37 0 C.
• the medium is preferably adjusted to pH 7.0 — 7.4. It will be appreciated that commercially available media may also be used to culture the bacterial strains of the present invention, such as Luria Broth available from Difco, Detroit, MI.
• Bacterial cells thus obtained are isolated using methods, which are well known - in the art. Examples include, but are not limited to, membrane filtration and centrifugal separation.
• the pH may be adjusted using sodium hydroxide and the like and the culture may be air dried or dried using a freeze dryer, until the water content becomes equal to 4 % or less.
• Such qualification may include testing resistance to nalidixic acid, lactose fermentation, resistance to gastric acidity, gastrointestinal tract colonization, resistance to bile acid, which correlates with gastric survival in vivo, adherence to mucus and/or human epithelial cells and cell lines, antimicrobial activity against potentially pathogenic bacteria, ability to reduce pathogen adhesion to surfaces and bile salt hydrolase activity [Conway (1987) J. Dairy Sci. 70:1-12].
• Newly isolated strains are preferably further characterized as being molecularly indistinguishable from the parental strain while still exhibiting nalidixic acid resistance. This may be attributed to the presence of genomic sequences such as set forth in SEQ ID NO: 1-9 or homologous sequences (e.g., above about 80 %, 90 % or 95 % identity).
• the strain has all identifying characteristics of the strain deposited under the Budapest Treaty in the American Type Culture Collection (ATCC) on December 22, 2005, as strain PTA - 7295 (referred to herein as M17 SNAR )-
• the probiotic bacterial strains of the present invention exhibit antibiotic resistance and as such may be adventitiously used, from a regulatory point of view, for the treatment of a variety of gastrointestinal disorders, simply because tracking of same in fecal samples is now allowed. Additionally, co-treatment with the strains of the present invention with nalidixic acid is now allowed, thus maintaining a viable gastrointestinal flora while practicing antibiotic treatment such as for urinary tract infection.
• a method of treating or preventing a gastrointestinal disorder in a subject is effected by administering to a subject in need thereof a therapeutically effective amount of the probiotic bacterial strains of the present invention.
• treating refers to alleviating or diminishing a symptom associated with a disorder.
• treating cures, e.g., substantially eliminates, the symptoms associated with the disorder.
• Subjects which may be treated with the bacterial cultures of the present invention include humans and animals which may benefit from probiotic treatment. Examples include but are not limited to mammals, reptiles, birds, fish and the like.
• Escherichia coli Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Pseudomonas aeruginosa and Pseudomonas maltophilia, Salmonella sp.
• Viruses such as rotavirus and fungi such as Candida albicans and Aspergillus fumigatus, and combinations of these species.
• Additional disorders which may be treated using the strains of the present invention include, but are not limited to, non-erosive esophageal reflux disease (NERD) and associated small bowel bacterial overgrowth, functional dyspesia, necrotizing enterocolitis, diabetes gastropathy, constipation (associated with the changes in gastrointestinal microflora), uro-genital tract associated diseases, urinary bladder infections (uterine infections and infections of the cervix, vagina and vulva commonly occur in human beings and domestic animals, especially following birth).
• NAND non-erosive esophageal reflux disease
• CAD non-erosive esophageal reflux disease
• functional dyspesia necrotizing enterocolitis
• diabetes gastropathy associated with the changes in gastrointestinal microflora
• constipation associated with the changes in gastrointestinal microflora
• uro-genital tract associated diseases urinary bladder infections (uterine infections and infections of the cervix, vagina and vulva commonly occur in human beings and domestic
• Typical infecting organisms of the endometrium (i.e., uterine mucosa) and contiguous mucosal surfaces in the lower genital tract include, for example, ⁇ -hemolytic streptococci, Candida albicans, Klebsiella pneumoniae, coliform bacteria including Escherichia coli, Corynebacterium pyogenes and C. vaginale, various Campylobacter or Trichomonas species such as T. vaginalis, and the like (see U.S. Pat. No. 5,667,817).
• urogenital pathogens include but are not limited to Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus, HIV, papillomavirus and Treponema pallidum.], respiratory diseases associated with pathogenic bacteria including but not limited to, Staphylococcus aureus, Streptococcus pneumoniae, beta- hemolytic streptococci and Haemophilus influenza; rheumatoid arthritis [Malin (1996) Br J Rheumatol;35:689— 94], allergies associated with reduced microbial stimulation associated with the western world lifestyle (i.e., improvement of hygiene and reduced family size).
• Atopic diseases such as associated with the decrease in Lactobacillus and Eubacterium combined with higher counts of Clostridium ssp [Biorksten et al. Clin. Exp. Allergy (1999) 29:342-346].
• the bacterial strains of the present invention may be used to treat other diseases or disorders (i.e., extraintestinal), which may be treated by probiotics.
• the ability of the bacterial strains of the present invention to treat bacterial, fungal or viral infections in other organs is an outcome of stimulating multiple defense mechanisms [reviewed by Isolauri (2001) Am. J. Clin. Nut. 73:444S-450S] including promotion of a nonimmunologic gut defense barrier which may inhibit translocation of potential pathogens and thus prevent infections of the blood stream and other tissues or organs.
• Another defense mechanism is improvement of the intestine's immunologic barrier, particularly through intestinal immunoglobulins A responses and alleviation of intestinal inflammatory responses which produce a gut stabilizing effect.
• immune regulation particularly through balance control of proinflammatory and anti-inflammatory cytokines.
• extraintestinal diseases which can be treated with the probiotic cultures of the present invention include, but are not limited to appendicitis, autoimmune disorders, multiple sclerosis, rheumatoid arthritis, coeliac disease, small bowel or gastric overgrowth associated with diabetic gastropathy, organ transplantation, periodontal disease, urogenital diseases (vaginal, urethral and perineal), surgical associated trauma, surgical-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disorders, rhinovirus-associated diseases (e.g., otitis media, sinusitis, asthma and pulmonary diseases), hepatic diseases (e.g., hepatic encephalopathy) constipation, nutritional disorders, epidermal disorders, psoriasis, anthrax and/or acne vulgaris [see Examples 5-8, U.S. Pat. Application No. 20030113306, Rolfe (2000) Journal of Nutrition 130:396
• Typical concentration range of probiotic microorganisms administered is 10 3 to 10 13 cells per day.
• at least about 10 6 , at least about 10 7 , at least about 10 10 cells per day are used in probiotic administration (see U.S. Pat. Nos. 6,221,350 and 6,410,305).
• the amount of bacteria to be administered will vary according to a number of parameters including subject's size, type of disorder and severity of symptoms.
• the bacterial cultures of the present invention can be formulated in a nutritional composition (e.g., foodstuff, food additive or feed additive).
• a nutritional composition e.g., foodstuff, food additive or feed additive.
• the bacterial strains of the present invention may be included in fermented milk products (i.e., nutraceuticals), such as described in U.S. Pat. No. 6,156,320.
• the bacterial strain of the present invention may be formulated in a pharmaceutical composition, where it is mixed with a pharmaceutically acceptable carrier preferably for oral or enteral administration route, selected according to the intended use.
• active ingredient refers to the bacterial preparation accountable for the biological effect.
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
• the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in
• compositions or nutritional compositions of the present invention may also include, colonization carriers, formulations for maintaining viability of the bacterial strain (e.g., volatile fraction of a plant extract, see U.S. Pat. No. 6,500,423), nutrients, antibiotics, anti-fungal agents, antioxidants, plant extracts, buffering agents, coloring agents, flavorings, vitamins and minerals, which are selected according to the intended use and the route of administration employed.
• colonization carriers formulations for maintaining viability of the bacterial strain (e.g., volatile fraction of a plant extract, see U.S. Pat. No. 6,500,423), nutrients, antibiotics, anti-fungal agents, antioxidants, plant extracts, buffering agents, coloring agents, flavorings, vitamins and minerals, which are selected according to the intended use and the route of administration employed.
• the compositions of the present invention may include a colonization carrier which transports the probiotic microorganisms to the large bowel or other regions of the gastrointestinal tract.
• the carrier is a saccharide such as amylose, inulin, pectin, guar gum, chitosan, dextrans, cyclodextrins, alginate and chondroitin sulphate [Chourasia and Jain (2003) J. Pharm. Pharmaceut. Sci. 6:33-66].
• modified and/or unmodified resistant starches are used as colonization carriers (see U.S. Pat. No. 6,221,350).
• resistant starch refers to starch forms defined as RSl, RS2, RS3 and RS4 as defined in Brown, McNaught and Moloney (1995) Food Australia 47: 272-275.
• a resistant starch is used in a probiotic composition since it is essentially not degraded until it reaches the large bowel. Therefore it provides a readily available substrate for fermentation by the probiotic microorganisms once they reach the large bowel.
• the resistant starch is a high amylose starch, including but not limited to maize starch having an amylose content of 50 % w/w or more, particularly 80 % w/w or more, rice and wheat starch having an amylose content of 27 % w/w or more and; particular granular size ranges of starches having an amylose content of 50 % or more and enhanced resistant starch content, these starches including maize, barley, wheat and legumes.
• Other forms of resistant starch derived from sources such as bananas or other fruit types, tubers such as potatoes, and mixtures or combinations thereof can also be used in accordance with the present invention.
• the colonizing carrier may also be an oligosaccharide. Oligosaccharides are known to increase the number of probiotic microorganisms in the gastrointestinal tract.
• oligosaccharides which can be used as colonizing carriers include but are not limited fructo-, galacto-, malto-, isomalto-, gentio-, xylo-, palatinose-, soybean- (including raffinose and stachyose), chito-, agaro-, neoagaro-, ⁇ -gluco-, ⁇ -gluco-, cyclo-inulo-, glycosylsucrose, lactulose, lactosucrose and xylsucrose.
• the oligosaccharide can be used in the composition in a concentration of about 0.01 to 10 % (w/w). Preferably the concentration of the oligosaccharide is about 0.05 to 5 %.
• a combination of starch and an oligosaccharide is used as the colonizing agent of this aspect of the present invention.
• compositions of the present invention may include nalidixic acid preferably at a range selected from 0.1-10 " ⁇ g/mL.
• Anti-fungal agents The compositions of the present invention may include a therapeutically-effective amount of an anti-fungal agent. Typical anti-fungal agents which may be utilized include, but are not limited to: Clotrimazole, Fluconazole, Itraconazole, Ketoconazole, Miconazole, Nystatin, Terbinafine, Terconazole, Tioconazole, and the like.
• compositions of the present invention may include antioxidants, buffering agents, plant extracts and other agents such as coloring agents, flavorings, vitamins or minerals.
• the composition of the present invention may contain one or more of the following minerals: calcium citrate (15-350 mg); potassium gluconate (5-150 mg); magnesium citrate (5-15 mg); and chromium picollinate (5-200 ⁇ g).
• a variety of salts may be utilized, including calcium citrate, potassium gluconate, magnesium citrate and chromium picollinate. Chemicals are commercially available from Spectrum Quality Products, Inc (Gardena, Calif.), Sigma Chemicals (St.
• Carriers - The active agents (e.g., bacterial cells) of the compositions of the present invention are combined with a carrier, which is physiologically compatible with the tissue of the species to which it is administered (i.e., suitable for human consumption or animal consumption).
• the carriers can be solid-based, dry materials for formulation into tablet, capsule or powdered form.
• the carrier can be of liquid or gel-based materials for formulations into liquid or gel forms.
• the specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration.
• Typical carriers for dry formulations include, but are not limited to: alginate
• composition e.g., calcium alginate), trehalose, malto-dextrin, rice flour, micro-crystalline cellulose (MCC), magnesium stearate, inositol, fructo-oligosaccharides (FOS), gluco— oligosaccharide (GOS), dextrose, sucrose, and the like.
• MCC micro-crystalline cellulose
• FOS fructo-oligosaccharides
• GOS gluco— oligosaccharide
• dextrose sucrose
• dry fillers which distribute the components and prevent caking.
• Exemplary anti-caking agents include MCC, talc, diatomaceous earth, amorphous silica, gelatin, saccharose, skimmed dry milk powder, starch and the like, which are typically added in an amount of from approximately 1% to 95% by weight.
• dry formulations which are subsequently rehydrated (e.g., liquid formula) or given in the dry state (e.g., chewable wafers, pellets or tablets) are preferred to initially hydrated formulations.
• Dry formulations e.g., powders
• may be added to supplement commercially available foods e.g., liquid formulas, strained foods, or drinking water supplies).
• Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like).
• water-based carriers have a neutral pH value (i.e., pH 7.0).
• composition of the carrier can be varied so long as it does not interfere significantly with the pharmacological activity of the active ingredients or the viability of the bacterial strains of the present invention.
• Other types of carriers, which can be used according to this aspect of the present invention are described hereinbelow.
• Nutrient supplements - A nutrient supplement component of the compositions of the present invention can include any of a variety of nutritional agents, which are well known in the art, including vitamins, minerals, essential and non-essential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements, and the like.
• the compositions of the present invention can include fiber, enzymes and other nutrients.
• Preferred fibers include, but are not limited to: psyllium, rice bran, oat bran, corn bran, wheat bran, fruit fiber and the like. Dietary or supplementary enzymes such as lactase, amylase, glucanase, catalase and the like can also be included. Vitamins for use in the compositions of the present invention include vitamins B, C, D, E, folic acid, K, niacin, and the like. Typical vitamins are those, recommended for daily consumption and in the recommended daily amount (RDA).
• RDA recommended daily amount
• composition of the present invention is formulated according to the intended use.
• a review of conventional formulation techniques can be found in e.g. "The Theory and Practice of Industrial Pharmacy” (Ed. Lachman L. et al, 1986) or Laulund (1994).
• compositions of the present invention are formulated for enteral administration.
• enteral administration refers to administration of a pharmacological agent through any part of the gastro-intestinal tract, such as rectal administration, colonic administration, intestinal administration (proximal or distal) and gastric administration.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as alginate (e.g., calcium alginate), sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions of the present invention can be encapsulated into an enterically-coated, time-released capsule or tablet.
• the enteric coating allows the capsule/tablet to remain intact (i.e., undissolved) as it passes through the gastrointestinal tract, until such time as it reaches the small intestine.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• parenteral administration of live bacteria cells are known in the art [see for example, Tjuvajev (2001) J. Control Release 74(l-3):313-5. Rosenberg (2002) J. Immunother. 25:218-25; Sheil (2004) Gut 53(5):694-700; and Matsuzaki (2000) Immunol. Cell Biol. 78(l):67-73].
• bacteria cells of the present invention may also be administered in an attenuated form so as to modulate immune responses [Matsuzaki (2000) Immunol. Cell Biol. 78(l):67-73].
• the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• rectal compositions such as suppositories or retention enemas
• conventional suppository bases such as cocoa butter or other glycerides.
• Formulations suitable for genital application include solutions.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, jelly, foams or sprays or aqueous or oily suspensions, solutions or emulsions (i.e., liquid formulations), or films containing carriers as are known in the art to be appropriate (described in details in U.S. Pat. No. 5,756,681).
• Compositions suitable for application to the vagina are disclosed in U.S. Pat.
• the composition contains one or more selected carriers excipients, such as water, silicone, waxes, petroleum jelly, polyethylene glycol (PEG), propylene glycol (PG), liposomes, sugars such as mannitol and lactose, and/or a variety of other materials, with polyethylene glycol and derivatives thereof. It is preferred that the pharmaceutical compositions contain one or more transurethral permeation enhancers, i.e., compounds which act to increase the rate at which the selected drug permeates through the urethral membrane.
• transurethral permeation enhancers i.e., compounds which act to increase the rate at which the selected drug permeates through the urethral membrane.
• Suitable permeation enhancers include dimethylsulfoxide (DMSO), dimethyl formamide (DMF), N,N-dimethylacetamide (DMA), decylmethylsulfoxide, polyethylene glycol monolaurate (PEGML), glycerol monolaurate, lecithin, the 1 -substituted azacycloheptan-2-ones, particularly l-n-dodecylcyclaza-cycloheptan-2-one (available under the trademark Azone R TM from Nelson Research & Development Co., Irvine, Calif.), SEP A R TM (available from Macrochem Co., Lexington, Mass.), alcohols (e.g., ethanol), surfactants including, for example, Tergitol R TM, Nonoxynol-9 R1 M and TWEEN-80 R TM, and lower alkanols such as ethanol.
• DMSO dimethylsulfoxide
• DMF dimethyl formamide
• DMA N,N-di
• transurethral administration of an agent can be carried out in a number of different ways.
• the agent can be introduced into the urethra from a flexible tube, squeeze bottle, pump or aerosol spray.
• the agent may also be contained in coatings, pellets or suppositories, which are absorbed, melted or bioeroded in the urethra.
• the agent is included in a coating on the exterior surface of a penile insert.
• Formulations of the present invention are selected so as to maintain bacterial viability. However, when the use of attenuated bacteria is desired, formulations of the present invention may be selected of a broader range.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
• bacteria species of the present invention may constitute 1-90 %, more preferably 5-90 %, even more preferably 10-90 % by weight of the final composition and still more preferably 15-88% % by weight contained within a formulation suitable for administration.
• the composition of the present invention may contain at least 10 6 , more preferably at least 10 8 , even more preferably at least 10 10 viable bacteria per one dose of composition.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals (see Examples 1-4 of the Examples section which follows).
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
• Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• bacterial cells are preferably encapsulated.
• Methods of encapsulating live bacterial cells are well known in the art (see e.g., U.S. Patent to General Mills Inc. such as U.S. Pat. No. 6,723,358).
• micro-encapsulation with alginate and and Hi-MaizeTM starch followed by freeze- drying has been proved successful in prolonging shelf-life of bacterial cells in dairy products [see e.g., Kailasapathy et al. Curr Issues Intest Microbiol.
• the probiotic compositions of the present invention can be provided to animals using methods, which are well known in the art.
• the probiotic composition is introduced into the animal's gastrointestinal tract via a feed additive, which is added to a feed diet.
• feed additive which is added to a feed diet.
• Alternative methods of administration are liquid ingestion, paste or gel ingestion,- boles, powder dusting surface of animal and the like.
• the feed additive may include, for example, carrier materials such as, limestone and wheat midds (see U.S. Pat. No. 6,410,305).
• carrier materials such as, limestone and wheat midds (see U.S. Pat. No. 6,410,305).
• the feed additive can be added to the animal's regular diet at a rate of 0.01 to 10 and preferably about 0.5 to 2.5 pounds of additive per ton of animal feed.
• the feed additive may contain about 0.3% to about 20% by weight of probiotic bacterial cells.
• the feed additive contains 7 % to 15 % by weight probiotic premix and most preferably about 10 % to 13 % by weight.
• the probiotic microorganisms of the present invention may not adhere to the intestinal epithelium.
• the bacteria remain in the gastrointestinal tract for maximal time of approximately 3-5 days and are considered to be a transient flora (see Figures 3a-d).
• the relatively rapid gastrointestinal-clearance time and inability to adhere to the gastrointestinal epithelium of the strains of the present invention has the advantage of preventing the later development of bacteremia in, for example, immunocompromised individuals.
• Fecal shedding assay as shown in Example 6 of the Examples section may be used to assess removal of the bacteria from the treated subject.
• the bacterial strains and or compositions of the present invention can be included in a product identified for treating a particular disorder such as described above.
• the product is in the form of a package containing the bacterial cells or compositions including same, or in combination with packaging material.
• the packaging material is selected to retain bacterial viability and includes a label or instructions for, for example, 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, contents (e.g., genus, species, strain designation), minimum numbers of viable bacteria at end of shelf-life, proper storage conditions and corporate contact details for consumer information.
• the label may also provide information related to the freshness of the product.
• This information may include a date of manufacture, a "sell be” date or a "best before date”.
• a "sell by” date specifies by which date the product should have been sold to the consumer.
• a "best before” date specifies by when the product should be disposed of by vendor or consumer.
• active labeling may be used.
• U.S. Pat. Nos. 4,292,916, 5,053,339 5,446,705 and 5,633,835 describe color changing devices for monitoring the shelf-life of perishable products. These devices are initiated by physically bringing into contact reactive layers so that the reaction will start, and this action can only conveniently be performed at the time of packaging. This approach is suitable for monitoring the degradation of foodstuffs which lose freshness throughout the entire distribution chain.
• U.S. Pat. No. 5,555,223 describes a process for attaching timing indicators to packaging, including the step of setting the timer clock at the exact time of production.
• the product may optionally contain either combined or in separate packages one or more of the following components: colonization carriers, flavorings, carriers, and the like components.
• the product can include the probiotic of the present invention 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 bacterial strains of the present invention can also be used as pharmaceutical delivery systems. It will be appreciated that such delivery systems are inherently safer than the use of attenuated pathogens in humans, including infants, the elderly and individuals whose immune function is impaired [Grangette (2001) Infect. Immun. 69:1547-1553].
• the bacterial strains of the present invention can also be modified to express heterologous expression products using expression systems, which are well known in the art. This approach was used to reduce colitis in mice intragastrically administered with the IL-10-secreting L. lactis strain [Steidler (2000) Science 289:1352-1355].
• Ml 7 strain of Escherichia coli in fecal samples collected from mammalian sources.
• the approach used was to first isolate a spontaneously occurring nalidixic acid-resistant mutant derivative of Ml 7 and second to develop a method for specific enumeration and confirmation of this strain from fecal samples.
• Materials Materials Vendors of chemicals are listed in Table 4 below. Table 4
• deoxyribonucleotide triphosphates dATP, dCTP, dGTP, dTTP
• Automated DNA Sequencers Li-Cor/NEN model 4200 Global IR 2 automated DNA sequencer (item No. 9942-155). Dual laser/detector (685 nm and 785 nm diode lasers with silicon avalanche photodiode detectors), complete with Netwinder server (80 Gb storage, server software, operating system). Operating software: e-Seq Version (Item No. 9942-154).
• Electrophoresis system + chiller system (item No. 170-3725). 100/120 V electrophoresis cell with drive module, control module, variable-speed pump, 14 x 13 cm casting stand with frame and platform, comb holder, 15-well, 1.5 mm thick comb, screened cap, disposable plug molds, and 12 ft Tygon tubing.
• Imaging system Syngene Ingenious imaging system, 8 bit monochrome imaging camera with 768 x 582 pixel resolution, manual zoom lens, darkroom + 20 X 30 cm UV2 (302 nm/365 nm wavelength) transilluminator, GeneSnap image capture software and GeneTools image analysis software.
• the Ml 7 strain of Escherichia coli is known to be sensitive to the antibiotic nalidixic acid. Only a small number of E. coli strains isolated from human clinical samples are known to be resistant to nalidixic acid and other quinilone antibiotics (4, 8, 9). Because nalidixic acid resistance, which is associated with mutations in gyrA or parC, occurs spontaneously in vitro and is only observed at low frequencies in clinical samples, it is well suited as a simple means for marking the M 17 strain.
• an Ml 7 E. coli culture was grown for 16 hours at 37 0 C in Luria Broth and 0.1 ttiL portions of the culture were then spread onto the surface of Luria agar supplemented with 15 ⁇ g/mL Nalidixic acid. The agar plates were then incubated for 16 hours at 37 0 C.
• Ml 7 50-1 thru M17 50-4 from M17 15-1 parent
• M17 50-5 thru M17 50-8 from the Ml 7 15-2 parent
• the Ml 7 50-1 thru M 1750-8 colonies were then streaked onto Luria agar with 50 ⁇ g/mL nalidixic acid, inoculated into Luria broth supplemented with 50 ⁇ g/mL nalidixic acid, and incubated for 16 hours at 37 0 C.
• Portions of each Luria broth culture were then spread onto the surface of Luria agar plates supplemented with 100 ⁇ g/mL nalidixic acid. The plates were incubated overnight at 37 0 C. A single colony from the Luria agar with 100 ⁇ g/mL nalidixic acid plates derived from each parental strain (M 17 50-1 thru Ml 7 50-8) was chosen, and labeled M17 100-1 thru M 17 100-8, streaked onto Luria agar supplemented with 100 ⁇ g/mL nalidixic acid and grown for 16 hours at 37°C.
• Pulsed Field Gel Electrophoresis Confirmation of Nalidixic acid- resistant M17 derivatives - PFGE was performed to confirm nalidixic acid-resistant derivatives of M17 are indeed Ml 7 derivatives. PFGE was performed using the CDC protocol, "Laboratory Protocol for Molecular Subtyping of Escherichia coli O157. ⁇ 7 by Pulsed Field Gel Electrophoresis.” see www.cdc.gov/pulsenet/protocols.htm Centers for Disease Control and Prevention. Standardized molecular subtyping of foodborne bacterial pathogens by pulsed-field gel electrophoresis: a manual. Atlanta: National Center for Infectious Diseases " ; 1996 (updated 2000).
• a Luria agar plate was streaked from frozen stock cultures of the Ml 7 parent strain and the Ml 7 100-1 thru Ml 7 100-8 nalidixic acid resistant derivatives onto Luria agar or Luria agar supplemented with 10 ⁇ g/mL Nalidixic acid and grown for 16 hours at 37 0 C.
• a sterile microbiological loop about 20 ⁇ L of cells were transferred from each plate to a tube containing 1 mL of Suspension Buffer (10OmM Tris, 10OmM EDTA, pH 8.0). The suspension was adjusted to an absorbance value at 610 run of 1.35 using additional cells or suspension buffer as necessary.
• the cell suspension (0.4 mL) from each strain was then mixed with 20 ⁇ L of Proteinase K (stock concentration 20 mg/mL), and inverted several times.
• 0.4 mL of molten agarose was then mixed with the cells and immediately dispensed into the plug molds of the CHEF H-DR PFGE apparatus. After solidification, the plugs were then removed into 50 mL screw cap tubes using a spatula and 5 mL of Cell Lysis Buffer [5OmM tris, 5OmM EDTA, 1% Sarkosyl (sodium lauryl sarcosine) pH 8.0].
• Restriction digests were performed by placing 2 mm slices of the plug into a sterile 1.5 mL microcentrifuge tube and adding 0.1 mL of Restriction Buffer.
• Restriction Buffer for Xbal restriction enzyme included I OmM Tris, 1OmM MgCI2,
• Xbal restriction enzyme was added. The samples were then incubated for 2 hours at
• the analytical agarose gel was cast by mixing a slurry of 1 % SKG agarose in 0.5X TBE and melting the agarose. After cooling in a 60 0 C water bath, the agarose was then poured into the CHEF II-DR gel form and the comb carefully placed into the molten gel. After 1 hour of solidification ' at room temperature, the comb was removed and the restriction-digested plugs placed into the appropriate wells, being sure to push the gel slice to the front of the well and removing any bubbles. A small volume of molten agarose was then added to fill the remaining area in the wells. The gel was then placed into the CHEF DRII PFGE tank, being sure to place the gel within the gel frame of the chamber. The chamber was then filled with 0.5 X TBE (1 M Tris, 1 M Boric Acid, 20 mM EDTA, pH 8.3 diluted to 0.5X concentration with water for electrophoresis).and electrophoresed under the following conditions:
• the gel was stained for 15 minutes in a solution of 0.02 ⁇ g/ml Ethidium bromide and destained for 30 minutes in water.
• the stained DNA was visualized by placing the gel onto a 302 tun UV lightbox and imaged with CCD camera.
• Amplified Fragment Length Polymorphism (AFLP) - AFLP reactions were performed according to Li-Cor, Inc. Document #988-07304, Rev. 1. Template DNA was prepared by standard methods and redissolved in 10 mM Tris, 1 mM EDTA pH 7.5. Genomic DNA was extracted from the bacterial strains by standard methods (5). The DNA samples were dissolved in 10 mM Tris-0.1 mM EDTA pH 8.0. A total of 100 ng of DNA from each sample was digested with EcoRI and Msel. Double- stranded, synthetic DNA adapters, containing short single strand sequences complementary to the EcoRI and Msel overhangs were then ligated to the digested DNA fragments.
• Fragments with ligated adapters were then diluted and amplified by PCR in pre-amplification reactions using PCR primers specific for the adapters.
• the amplified fragments then served as templates for selective amplification in which fluorescently labeled primers are used in conjunction with unlabeled primers.
• the selective amplification uses primers that are complementary to the adapter sequences with an additional 2 bases at the 3'end. This selectively amplified (and labels) DNA fragments in which the 2 bases immediately adjacent to the original EcoRI or Msel site are complementary to the * 3' base of the primers.
• the labeled selective amplification products were resolved by denaturing polyacrylamide gel electrophoresis on a Li-Cor/NEN 4200 global analyzer (automated DNA sequencer).
• M17 SNAR Sudden-positive, Nalidixic Acid Resistant
• Genomic DNA for DNA sequence analysis, total genomic DNA from the Ml 7100-8 SNAR isolate was prepared. This clone was grown overnight on Luria agar with 100 ⁇ g/mL nalidixic acid. Following 16 hours of growth at 37 0 C, a loopful of cells was transferred to 500 mL of Luria broth supplemented with 100 ⁇ g/mL nalidixic acid and grown for 18 hours at 37 0 C with shaking at 200 rpm. The cells were then harvested by centrifugation in a Backman J2 high speed centrifuge at 6,000 rpm for 10 minutes using 250 mL bottles in a JAlO rotor.
• the cells were then resuspended in 25 mL of 1OmM Tris-lmM EDTA (pH 7.5) with 2 mg/mL lysozyme. After 10 minutes of incubation at room temperature, 2.5 mL of 1% Sodium Dodecyl-Sulfate supplemented with 5 mg/mL Proteinase K and incubated at 5O 0 C for 90 minutes. 25 mL of phenol (saturated with 1OmM tris-lmM EDTA pH 8.0) was then added and the bottles were rocked for 3 hours on high speed.
• the phases were then separated by centrifugation at 8,000 rpm for 15 minutes in a Beckman J2 High-speed centrifuge using a JAlO rotor.
• the aqueous phase (20 mL) was then removed to a beaker and 2 mL of 3 M Sodium Acetate (pH 5.2) was added and mixed with swirling.
• the DNA was then precipitated by the addition of 40 mL of ethanol. Precipitated DNA was spooled onto a glass from the interface and the spooled DNA washed by submerging several times into 70% ethanol.
• the DNA was finally dissolved in 1 mL of 1OmM Tris-lmM EDTA.
• PCR primers should produce products between 500 bases and 3 kilobases in length. PCR primers should be positioned within the region of unique sequence. PCR primer pairs should have melting points that are within 2 0 C of each other.
• PCR primers from each unique region should give products that are of distinct size so they can be multiplexed. Potential primer combinations are listed in Table 1 1 below.
• M17sNAR-specific genome segments M17SSGS
• a single PCR primer combination from the list of candidates was tested against M 17 SNAR chromosomal DNA to determine if a specific product of the predicted size was produced.
• the reactions were run at different melting temperatures using the TGradient thermocycler (Biometra). The gradients were centered at 59 0 C with 15 0 C variance on the low and high ends.
• the reactions were run using 1 ng of M17 SNAR DNA in a 20 ⁇ L reaction volume with 1 X PCR buffer (containing 2.5 mM MgCl 2 final concentration), 250 ⁇ M dNTPs, 1 unit of Taq DNA polymerase, and 1 ⁇ M each primer.
• the reaction volume was made to 20 ⁇ L for each reaction using sterile water. Reactions were heated to 95 0 C for 2.5 minutes and then 30 cycles of 95 0 C for 30 seconds, melting temperature (56 or 63 degrees depending on primer set) for 45 seconds, 45 seconds at 72 0 C. After 30 cycles, the reaction was extended for 5 minutes at 72 0 C and held at 4 0 C until ready for gel electrophoresis.
• nalidixic acid resistance where the trait of nalidixic acid resistance is used to selectively grow resistant bacteria — i.e. identify only those bacteria in the feces which can grow in the presence of nalidixic acid 1 — and then confirm their identity as M17 SNAR using specific genetic tests
• nalidixic acid-resistant bacteria from fecal samples and confirm their identity as M17 SNAR using a genetic test for a DNA segment that is unique to the M 17 parent and M17 SNAR derivative.
• Genomic DNA was extracted from M17 SNAR - The DNA was subsequently physically sheared into different size fragment lengths and three different clone libraries were generated, each having different average insert sizes (4 Kilobases, 10 Kilobases, and 40 Kilobases). Each of these libraries was then subjected to high-throughput shotgun DNA sequence analysis and the sequence reads were assembled into large contiguous DNA sequences based on their overlap.
• the shotgun DNA sequencing phase included DNA sequence analysis of enough clones such that each segment of the entire genome would be sequenced multiple times in independent overlapping clones.
• the 10kb and Fosmid libraries provided larger physical links from paired-end 'reads' and facilitated ordering and orienting contiguous sequence 'blocks' in the assembly process.
• Phred20 bases Bases which receive a quality score of 20 or more when subjected to Phred analysis, a base-calling program developed at the University of Washington Genome Center.
• Contig building software which builds contigs based on the "quality" of each base in a 'read', was used to ultimately build the DNA sequence into large contiguous stretches of sequence. Any gaps, discrepancies or ambiguities in the sequence were also identified. Contigs were then ordered and linked together into larger supercontigs by using paired 'reads' lying in different contigs. Using this approach, a total of 464 contigs were assembled and the details are listed in Table 9 below. Whole genome assembly was performed using the Paracel Genome AssemblerTM, version 2.6.2, coupled with the Agencourt's LIMS system while, Paracel's scaffold viewer and Consed (version 13.0) were used to finish the assembly. Table 9. Summary of the se uence assembl
• a pairwise BLAST analysis was consequently done of entire contigs against the E. coli CFT073 genome sequence and subsequently identified the non-aligning regions from the Ml 7 contigs. These non- aligned sequences were then used in pairwise BLAST to identify any sequence homology against the E. coli MG1655 (K-12) and E. coli EDL933 (O157:H7) and Sakai (O157:H7) genome sequences. Segments not showing significant alignment (sequence homology) to either the E. coli CFT073, K-12, or O157:H7 genomes were then used in a BLAST search against the entire nr NCBI database (www.ncbi.nlm.nih.gov/BLAST/).
• BLAST analysis was effected with each of the 464 contigs first split up into 200 bp (base pair) fragments and then individually blasted against two specialized databases.
• the first database constructed was an ""E.coW database, in which four strains of E. coli (www.ncbi.nlm.nih.gov/genomes/lproks.cgi) were included to create a database named NCBIrefseq_ecoli.dna (Escherichia coli strains included were CFT073, K12, O157:H7 Sakai and O157:H7 EDL933).
• NCBIrefseq_bacteria.dna For the second database, all bacterial genomes present in the NCBI database were consolidated to create a 'Bacterial' database and named NCBIrefseq_bacteria.dna. Next, each of the 200 bp fragments was subjected to a blastn analysis against both consolidated NCBI databases of both E. coli and bacteria, described above. Sequences with 'no hits,' were identified to be unique sequences.
• PCR primers were designed to amplify segments within several of the unique regions. The PCR primers were then used in PCR reactions performed on the Ml 7 and M17 SNAR strains to first optimize the PCR reactions. Optimizations were performed using temperature gradients centered at 59 0 C and +/- 7.5 0 C above and below this temperature. The PCR products from each reaction were run alongside one another on an analytical agarose gel, stained with ethidium bromide, and imaged over a 302 nm UV lightbox using a CCD camera.
• the M17 SNAR strains of the present invention are molecularly distinct from other E. coli strains Experimental Procedures Once the reactions were optimized for each PCR primer combination, the combinations were confirmed by testing them in PCR reactions against a panel of E. coli strains designed to represent the genetic diversity of the total E. coli population (ECOR collection) and to represent common serotype O2 strains.
• the collection consists of the 72 ECOR strains, described originally by Ochman and Selander in 1984 (6), which has been extensively studied and is generally regarded as representative of the population structure of the species.
• a set of five different strains having an O2 serotype was also used to test for the uniqueness of the M17S NAR segment in genetically unrelated E.
• the reaction mixtures each contained 2 ⁇ L of the diluted DNA, I ⁇ L each of the relevant primers (final concentration IuM) 2 ⁇ L of 1OX PCR buffer (Takara), 2 ⁇ L of 25OmM dNTP mixture (Takara), 1 unit of Taq DNA polymerase (Takara) and 11 ⁇ L of sterile water.
• the reactions were then cycled in a Biometra thermocycler using the following cycling conditions: 2.5 minutes at 95 0 C, followed by 30 cycles of 30 seconds at 95 0 C, 45 seconds at 53 0 C (Primer combination C 127) or 56 0 C (Primer combination C291), 45 seconds at 72 0 C.
• a final extension of 5 minutes at 72 0 C was performed after the thirty cycles and the reactions were held at 4°C until ready for gel electrophoresis.
• PCR primers were designed to amplify segments within several of the unique regions.
• the PCR primers were then used in PCR reactions performed on the Ml 7 and M17 SNAR strains to first optimize the PCR reactions. Optimizations were performed using temperature gradients centered at 59°C and +/- 7.5°C above and below this temperature.
• the PCR products from each reaction were run alongside one another on an analytical agarose gel, stained with ethidium bromide, and imaged over a 302 nm UV lightbox using a CCD camera. The relative intensity of fluorescence from the stained DNA bands was quantified using GcncTools software (Syngene) software.
• the optimized Contig 127 PCR reactions were next tested against genomic DNA from a panel of E. coli strains representing the genetic diversity of naturally occurring E. coli populations (ECOR collection) as well as additional serotype O2 strains (AOS strains) that are genetically unrelated to the serotype O2 M17 SNAR strains.
• ECOR collection E. coli populations
• AOS strains additional serotype O2 strains
• Purified genomic DNA from each of the 72 ECOR strains, each AOS strain, the M17 S NAR strain, and the M 17 parental strain were subjected to PCR in individual reactions with the Contig 127 primers using the following conditions:
• the PCR reactions were heated for 2.5 minutes at 95 0 C followed by 30 cycles of 95°C for 30 seconds, 63 0 C for 45 seconds, 72 0 C for 45 seconds. The reactions were extended for 5 minutes at 72°C and finally held at 4°C until electrophoretic separation.
• loading dye was added and the reactions were loaded into a 0.8% agarose gel. After electrophoresis, the gels imaged over a
• Table 12 summarizes the results of contig 127 analysis in the strains of the present invention and other E. coli strains tested. Table 12 - Validation of Contig_127 PCR reaction on Ml 7 S N AR , ECOR strains, and additional serot e O2 strains.
• a combination of selective plating of on Violet Red Bile Agar (VRBA) and PCR confirmation was used to determine if M17 SNAR could be selectively enumerated in a spiked fecal sample.
• a composite fecal sample was prepared by mixing 10 gram samples from 100 independent human fecal samples into a single composite. The composite was mixed for three 1 -minute pulses in a Waring blender and the resulting slurry was distributed in approximately 25 mL aliquots into sterile 50 mL conical tubes.
• these samples are referred to as the fecal composite sample.
• aliquots of composite sample were stored at -80 0 C-
• aliquots of fecal samples were thawed and diluted in triplicate by serial 10-fold dilutions into sterile 0.1 % peptone.
• 0.1 mL samples of each dilution were then plated in duplicate onto the surface of Luria agar, Luria agar + 75 ⁇ g/mL nalidixic acid, VRBA, VRBA + 25 ⁇ g/mL nalidixic acid.
• M17 SN AR cells were spiked into the 10-1 dilution of two independent fecal composite aliquots.
• a suspension of M17 SNAR cells was prepared by scraping a single colony of M17 SNAR cells from a culture that had been streaked onto Luria agar + 75 ⁇ g/mL nalidixic acid.
• the colony was resuspended in 5 ml of sterile 0.1 % peptone by vortex mixing for 30 seconds using a vortex mixer.
• the M17 SNA R cells present in the M17 SNAR suspension were enumerated by performing serial 10-fold dilutions of the suspension into sterile 0.1 % peptone and plating 0.1 mL portions of each dilution onto Luria agar, Luria agar + 75 ⁇ g/mL nalidixic acid, VRBA, VRBA + 25 ⁇ g/mL nalidixic acid.
• the M17 SN ⁇ R cell suspension was used to spike 10-1 dilutions of the fecal composite samples at two different concentrations by adding 0.1 mL of a 10-fold or 1000-fold dilution of M17 SN ⁇ R suspension into the 10-1 dilution of independent fecal composite aliquots.
• the spiked 10-1 dilutions were then diluted by serial 10-fold dilutions into 0.1% peptone. 0.ImL aliquots of each dilution were then plated in duplicate onto the surface of Luria agar, Luria agar + 75 ⁇ g/mL nalidixic acid, VRBA, VRBA + 25 ⁇ g/mL nalidixic acid.
• the cells were then heated to 95 0 C for 10 minutes in the thermocycler. From the heated cell suspensions, 2 ⁇ L was removed and distributed into a fresh 96-well PCR plate and 18 ⁇ L of PCR mix was added.
• the PCR mix consists of IX PCR buffer (Takara), 250 mM dNTPs (Takara), 1.4 units of Taq DNA polymerase, IuM of each primer (Contig 127 primers, above).
• the plate was then covered with a 96-well Hd and placed into the thermocycler. Reactions were heated to 95 0 C for 2.5 minutes followed by 40 cycles of 95 0 C for 30 seconds, 63 0 C for 45 seconds, 72 0 C for 45 seconds. A final cycle of 72°C for 5 minutes was then conducted and the samples held at 4 0 C until ready for gel electrophoresis.
• a composite fecal sample was prepared from 10-gram samples of 100 human fecal samples. Samples of the composite were then mixed with measured quantities of the M17 SNAR cells and subjected to serial dilution followed by plating of portions of each dilution onto VRBA + 25 ⁇ g/mL nalidixic acid as well as control media (VRBA, Luria agar, and Luria agar with 75 ⁇ g/mL nalidixic acid). After incubation, the number of colonies was averaged from duplicate plates of each dilution.
• the VRBA + 25 ⁇ g/mL nalidixic acid gave detection efficiencies of between 23% (for the 1.3 X 10 5 input sample) and 42% (for the 1.3 X 10 3 input sample). Therefore, the plating efficiency of the M17 SNAR strain on VRBA + 25 ⁇ g/mL nalidixic acid is estimated at 32.5%. Combining the detection limit of ⁇ 10 CFU/mL from an undiluted fecal sample with the 32.5% plating efficiency of the M17SNAR on VRBA + 25 ⁇ g/ml nalidixic acid, a detection limit of ⁇ 33 CFU/ml of M17SNAR in a fecal sample was determined. The results are summarized in Table 13 below.
• the Contig 127 PCR assay was performed on a total of 84 randomly chosen colonies from the VRBA + nalidixic acid plates derived from dilutions of samples containing M17 SNAR - TO demonstrate the selectivity of the plating, 132 colonies from VRBA plates without antibiotic were also chosen randomly and tested with the Contig 127 PCR assay. Of the colonies chosen from VRBA plates, 48 were picked from plates where no M17 SNAR had been added to the samples and 84 were chosen from the countable plates of dilutions from samples in which the M17 SNAR had been introduced.
• the overall objective of this study was to develop a tool for the quantification of E. coli M17 SNAR in human fecal samples and a method which would measure the degree and duration of shedding of the probiotic E. coli M 17SNAR strain in fecal samples.
• Fecal samples collected from canines fed M17SNAR cultures during the course of a 14-day toxicology study were used to model this method.
• the approach used was to enumerate total coliforms and M17 SNAR using selective plating on Violet Red Bile Agar (VRBA) and VRBA supplemented with 25 ⁇ g/mL nalidixic acid (as shown for spiked human fecal samples in Examples 5 above).
• Confirmation of M17SNAR was conducted on colonies growing on VRBA + 25 ⁇ g/mL nalidixic acid using the M17s NAR -specif ⁇ c Contig 127 PCR assay.
• a 0.5 mL portion of the undiluted sample was removed with a P-1000 micropipettor and dispensed into 4.5 mL of sterile 0.1% peptone.
• the diluted sample was then serially diluted to a final dilution of 10 "6 .
• 0.1 mL portions of the undiluted sample and the 10 * ' through 10 "6 dilutions were plated in duplicate onto VRBA and VRBA + 25 ⁇ g/mL nalidixic acid.
• the plates were incubated for 36 hours at 37 0 C prior to enumerating colonies.
• Presumptive total coliforms were scored as the number of lactose-positive colonies on VRBA X reciprocal of the dilution.
• Presumptive M17 SNAR were scored as the number of lactose-positive colonies on VRBA + 25 ⁇ g/mL nalidixic acid X reciprocal of the dilution.
• PCR confirmation - To confirm presence of M17SNAR, colonies were picked from VRBA or VRBA + 25 ⁇ g/mL nalidixic acid and tested using the Contig 127 PCR assay. Colonies to be tested were picked using sterile toothpicks and inoculated into 200 ⁇ L cultures of Luria Broth in sterile 96-well assay plates. Controls, including M17 SNAR and DH5 ⁇ , were also inoculated into the appropriate wells. The plates were then covered with a sterile plastic lid and incubated for 15 hours at 37 0 C.
• PCR mix consists of IX PCR buffer (Takara), 250 mM dNTPs (Takara), 1.4 units of Taq DNA polymerase, 1 uM of each primer. The plate was then covered with a 96-well lid and placed into the thermocycler.
• Reactions were heated to 95 0 C for 2.5 minutes followed by 40 cycles of 95 0 C for 30 seconds, 63 0 C for 45 seconds, 72 0 C for 45 seconds. A final cycle of 72 0 C for 5 minutes was then conducted and the samples held at 4 0 C until ready for gel electrophoresis.
• Presumptive and confirmed detection of M17 SN ⁇ R in canine fecal samples Presumptive and confirmed detection of M17 SN ⁇ R in canine fecal samples.
• Detectable shedding of M17 S NAR- The total coliform and total M17 SNAR counts for the four animals are plotted in Figures 3a-d.
• the total coliform counts per animal ranged between 10 4 and 10 7 CFU/g of feces. Shedding of M17SNAR was detectable in all four animals through day 13. Two animals continued to shed detectable levels of M17 SNAR until day 16 ( Figures 3c-d). Beyond day 16, M17 S NAR could no longer be detected in the fecal samples. Thus, after dosing was stopped (day 14) the M17 SNAR population was rapidly eliminated to levels below the detection limit of the assay (10 1 48 or 30 CFU/g).
• M17 SNAR can be specifically detected in the feces of canines dosed with M17 SNAR -
• the M17 SNAR does not appear to stably colonize the bowel as a member of the flora of these animals, and it is decreased to undetectable levels within 2-4 days after the dosing period.
• EXAMPLE 7 Genomic sequencing ofMl7 s ⁇ AR A complete genome sequence of Escherichia coli M17 SNAR was effected in order to identify genomic sequences unique to E. coli M17 SNAR - In order to accomplish this, whole genome shotgun sequencing strategy was used in which the the genomic DNA is "peppered" with enough sequence 'reads' such that they overlap, and yield, when assembled, the complete sequence of the genome. An 8-fold coverage of the Escherichia coli M17 SNAR genome was performed to obtain a comprehensive number of sequence 'reads' and a shotgun assembly was generated from the same. To complete the work, the following steps were taken sequentially,
• 1.0.6a uses hydrodynamic shearing forces to fragment DNA strands into designated sizes.
• Library Construction Experience has demonstrated the need for construction of several libraries including both, small and large-sized DNA inserts.
• the small-insert library is used to obtain an appropriate coverage of the genome and the large-insert library is used to obtain a 'scaffold' of the genome, which is used during the closure phase of the sequencing project.
• the "reads" from the small insert library provides the 'bulk' of the sequence information while the "reads" from the large- insert library help in assembling the sequence information in the correct order.
• a fosmid is similar to a plasmid (circular DNA) but is capable of containing much larger sizes of DNA inserts, up to 50 kb, compared to about 10 kb in a plasmid.
• the large insert size (40 kb) of Fosmid libraries make them particularly attractive, since Fosmid clones can close small physical gaps with fewer walking steps, in comparison to small insert libraries and can therefore reduce redundant sequencing.
• Sequencing template preparation The plasmids containing genomic DNA were isolated from library cultures using Solid Phase Reversible Immobilization technology (SPRI®) [Hawkins, TL., KJ. McKernan, L.B. Jacotot, J.B. MacKenzie, P.M. Richardson and E.S Lander. 1997. A magnetic attraction to high-throughput genomics. Science Vol. 276 (5320), 1887-1889].
• High-copy plasmid templates (3- 4kb and I Okb insert plasmids) were purified using SprintPrepTM SPRI protocol while the low-copy (40kb fosmid) templates were purified using a SPRI® protocol (Agencourt Bioscience Corp., Beverly, MA).
• the SPRI® (Solid Phase Reversible Immobilization) technology uses carboxylate-coated, iron-core, paramagnetic particles to capture DNA of a desired fragment length based on tuned buffering conditions. Once the desired DNA is captured on the particles, they can be magnetically concentrated and separated so that contaminants can be washed away. This procedure harvests plasmid DNA directly from lysed bacterial cultures by trapping both plasmid and genomic DNA to the functional ized bead particles and selectively eluting only the plasmid DNA.
• the DNA templates were sequenced in 384- well format using BigDye® Version 3.1 reactions on ABI3730 instruments (Applied Biosystems, Foster City, CA).
• the BigDye® Version 3.1 contains dye terminators labeled with novel high sensitivity dyes.
• This BigDye terminator chemistry involves a fluorescein donor dye linked to a dRhodamine acceptor dye and is 2-3 times brighter than standard dye terminators and also has narrower emission spectra giving less background noise. This provides an overall improvement of 4-5 times in sensitivity of the capillary analytical procedure.
• the DNA fragments are chemically labeled with these fluorescent dyes, which facilitate the detection and identification of the DNA.
• labeled DNA samples are prepared in 96- or 384-well plates and placed on the ABI3730 Genetic Analyzer machine in which capillary electrophoresis is used in separating the mixture of DNA fragments according to their lengths, providing a profile of the separation and determining the order of the four deoxyribonucleotide bases.
• the DNA molecules from the samples are injected into thin, fuse-silica capillaries that have been filled with polymer.
• the DNA fragments migrate towards the other end of the capillaries, with the shorter fragments moving faster than the longer fragments.
• As the fragments enter a detection cell they move through the path of a laser beam which causes the dye on the fragments to fluoresce.
• This fluorescence is captured by a charge-coupled device (CCD) camera.
• CCD charge-coupled device
• the CCD camera coverts the fluorescence information into electronic information, which is then transferred to a computer workstation for processing by the 3700 data collection software to generate electropherograms which plot relative dye concentration against time for each of the dyes used to label the DNA fragments. The positions and shapes of the electropherogram are used to determine the base sequence of the DNA fragment. Thermal cycling for the sequencing reactions was performed using 384- well Thermocyclers (ABI, MJ Research, Hercules, CA).
• Sequencing reactions were purified using CleanSeq® dye-terminator removal kit from Agencourt Bioscience Assembly Once the shotgun sequencing phase is complete, the sequencing 'reads' generated from random subclones are assembled into contigs (contiguous sequences), followed by a directed, or finishing phase in which the assembly is inspected for correctness and for various kinds of data anomalies (such as contaminant 'reads', unremoved vector sequences, and chimeric or deleted 'reads'), additional data are collected to close gaps and resolve low quality regions, and editing is performed to correct assembly or base-calling errors.
• data anomalies such as contaminant 'reads', unremoved vector sequences, and chimeric or deleted 'reads'
• Phred base calling software version 0.020425c
• Phred Q20 Universality of Washington
• Phred is a base calling software developed at the University of Washington Genome Center which reads DNA sequence chromatogram files, analyzes peaks to call bases and assigns quality scores to each nucleotide (2).
• Phred Q20 calls bases with a Phred quality value of 20 or greater. A Phred score of 20 indicates the existence of one error in 100 bases.
• Clusters of overlapping sequences are constructed and consensus sequences are deduced from these clusters.
• Paracel Genome AssemblerTM version 2.6.2 (Paracel, Pasadena, CA), coupled with the LIMS system (Agencourt Bioscience Corp., Beverly, MA) were used to assemble the sequence data for this project.
• Paracel's scaffold viewer and Consed version 2.6.2
• Pulse Field Gel Electrophoresis of E.coli M17 SNAR genomic DNA The whole-genome shotgun strategy involves randomly breaking DNA into segments of various sizes and cloning these fragments into vectors for sequencing. The success of this strategy is highly dependent on the quality and integrity of the genomic DNA used as the starting material. A pulse field gel electrophoresis was run to check the quality of the genomic DNA and to determine if the DNA was high molecular weight.
• DNA above 30-50 kb migrates with the same mobility regardless of size and is seen in a gel as a single large diffuse band.
• the DNA is forced to change direction during electrophoresis and different sized fragments within this diffuse band begin to separate from each other.
• smaller sized DNA With each reorientation of the electric field relative to the gel, smaller sized DNA begins to move in the new direction more quickly than the larger DNA.
• the larger DNA lags behind providing a separation from the smaller DNA. Any low molecular weight or plasmid DNA can thus be identified.
• the high-throughput shotgun sequencing of E.coli M17 SNAR resulted in 57,408 distinct high-quality 'reads' representing " 36,265,538 bases with good quality scores, providing an approximately 8-fold coverage of the genome.
• the distribution of these sequences across the three different libraries constructed is shown in Table 15, below.
• the high copy standard 3-4 kb genome library offered the most cost efficient method to produce paired-end sequence coverage of the genome, while the 10 kb and Fosmid libraries provided larger physical links from paired-end 'reads' which are useful in ordering and orienting contiguous sequence 'blocks' in the assembly process.
• Phred20 bases Bases which receive a score of 20 or more when subjected to Phred analysis, a base- calling program developed at the University of Washington Genome Center.
• sequence data was vector and quality screened based on Phred quality score information. All the sequence 'reads' from the libraries were first compared to each other. Identities between the sequences of different 'reads' were noted, and then used to align the sequences into contiguous stretches of sequence called contigs. The sequences of two different 'reads' of the same segment of DNA may not be identical because of the quality of the sequencing reaction analysis. Thus for each base in the contig it is usual to require that it is independently confirmed from multiple overlapping 'reads' from both directions.
• Contig building software designed to take into account the "quality" of each base in a 'read' (where quality is a measure of the confidence the Phred software has that the base has been called correctly) were used. Any gaps, discrepancies or ambiguities in the sequence were also identified. Contigs were then ordered and linked together into larger supercontigs by using paired 'reads' lying in different contigs. A total of 464 contigs were assembled and the details are listed in Table 16, below. Whole genome assembly was thus performed using the Paracel Genome AssemblerTM, version 2.6:2, coupled with the Agencourt's LIMS system while, Paracel's scaffold viewer and Consed (version 13.0) were used to finish the assembly.
• BLAST analysis was effected to help determine sequences unique to E. coli M17 SNAR - BLAST, which stands for 'Basic Local Alignment Search Tool' (www.ncbi.nlm.nih.gov/BLAST/) is a program that finds regions of local similarity (alignment) between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches.
• BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. BLAST analysis allows for performing five distinct blast comparisons.
• an E value of 1 assigned to a hit can be interpreted as meaning that in a database of the current size one might expect to see 1 match with a similar score simply by chance. This means that the lower the E- value, or the closer it is to "0" the more "significant" the match is.
• BLAST analysis was effected with each of the 464 contigs first split up into 200bp (base pair) fragments and then individually blasted against two specialized databases.
• the first database constructed was an 'E. coW database, in which four strains of E. coli (www.ncbi.nlm.nih.gov/genomes/lproks.cgi) were included to create a database named NCBIrefseq_ecoli.dna (Escherichia coli strains included were CFT073, K12, O157:H7 and O157:H7 EDL933).
• NCBIrefseq_bacteria.dna For the second database, all bacterial genomes present in the NCBI database on August 19, 2005 were consolidated to create a 'Bacterial' database and named NCBIrefseq_bacteria.dna. Next, each of the 200bp fragments was subjected to a blastn analysis against both consolidated NCBI databases of both E. coli and bacteria, described above. Sequences with 'no hits,' were identified to be unique sequences.
• sequence 200 base pairs in length.
• BLAST 2.2.10 results against the E. coli database, NCBIrefseq_ecoli.dna: No hits were registered for any of the nine sequences.
• NCBIrefseq_bacteria.dna The top ten highest scoring hits are tabulated along with the respective 'E values'.
• NCBIrefseq_ecol ⁇ .dna Posted date: Aug 20, 2005 6:10 AM Number of letters in database: 20,994,025 Number of sequences in database: 6
• GenBank Accession numbers mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application or GenBank Accession number was specifically and individually indicated to be incorporated herein by reference.
• citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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