WO2018115361A1 - Souches humaines de lactobacilli - Google Patents

Souches humaines de lactobacilli Download PDF

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Publication number
WO2018115361A1
WO2018115361A1 PCT/EP2017/084236 EP2017084236W WO2018115361A1 WO 2018115361 A1 WO2018115361 A1 WO 2018115361A1 EP 2017084236 W EP2017084236 W EP 2017084236W WO 2018115361 A1 WO2018115361 A1 WO 2018115361A1
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WIPO (PCT)
Prior art keywords
strain
formulation
difficile
gasseri
lactobacillus
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PCT/EP2017/084236
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English (en)
Inventor
Colin Hill
Paul Ross
Mary Clare Rea
Debebe Alemayehu
Eileen Frances Murphy
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Agriculture And Food Development Authority (Teagasc)
University College Cork - National University Of Ireland, Cork
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Application filed by Agriculture And Food Development Authority (Teagasc), University College Cork - National University Of Ireland, Cork filed Critical Agriculture And Food Development Authority (Teagasc)
Publication of WO2018115361A1 publication Critical patent/WO2018115361A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C12R2001/225—Lactobacillus

Definitions

  • Clostridium difficile is a Gram positive, cytotoxin-producing anaerobic intestinal pathogen with an asymptomatic carriage rate of up to 30 % of people in long-term care facilities (Ziakas et al, 2015).
  • C. difficile may flourish and cause illness varying from mild diarrhoea (usually self- limiting) to pseudomembraneous colitis, fulminant colitis, toxic megacolon and even death (Kachrimanidou and Malisiovas, 2011).
  • the incidences of C. difficile infection (CD I) have rapidly increased since the 1990s, and the mortality rate has also grown markedly (Wiegand et al., 2012).
  • the European Society of Clinical Microbiology and Infection (ESCMID) guidelines for treatment of CDI include antibiotics, toxin-binding resins and polymers, immunotherapy, probiotics and faecal or bacterial intestinal transplantation (Debast et ah, 2014).
  • Antibiotic treatment is typically advised, including the use of metronidazole, vancomycin and fidaxomicin (Debast et ah, 2014).
  • standard therapies for CDI frequently have limited efficacy, the search for alternative therapies such as live therapeutics and bacteriocins that may help to reduce incidences and recurring infections are gaining credence (Evans and Johnson, 2015; Goldstein et ah, 2015; Rea et ah, 2013).
  • bacteriocins and H2O2 and bioactive metabolites e.g. CLA
  • CLA bioactive metabolites
  • GAB A Gamma- aminobutyric acid
  • the effect of bacteria frequently associated with the human gut, such as C. difficle is being increasingly understood within the whole context of the human gut microbiome.
  • the human gut microbiota consists of greater than 1000 bacterial species, with every individual hosting at least 160 different species (Eckburg et al., 2005; Qin et al., 2010; Tap et al., 2009; Walker et al., 2011).
  • the gut microbiota of a healthy adult is, in general, stable with Firmicutes and Bacteroidetes typically accounting for over 90% of bacteria with smaller fractions of Actinobacteria and Proteobacteria (Eckburg et al., 2005; Claesson et al., 2011; Ley et al., 2006; Human Microbiome Project Consortium, 2012).
  • Esckburg et al., 2005; Claesson et al., 2011; Ley et al., 2006; Human Microbiome Project Consortium, 2012 Unfortunately, considerable inter-individual variability exists at species and strain level (Lozupone et al., 2012).
  • Noncommunicable diseases also known as chronic diseases, are diseases that are not caused by infectious agents and are not passed from person to person. They are generally of long duration and slow progression (WHO, 2016) and include diseases such as autoimmune disease, cardiovascular disease, stroke, cancer, osteoporosis, depression, anxiety, autism, Alzheimer's disease, chronic kidney disease, diabetes and obesity. NCDs are the leading cause of death globally (WHO, 2016).
  • Reduced microbial diversity is centrally implicated in many NCDs. Studies have demonstrated associations between reduced microbial diversity and eczema (Bisgaard et al., 2011; Abrahamsson et al., 2012; Ismail et al., 2012; van Nimdorf et al., 2011), asthma (Ege et al., 2011; Abrahamsson et al., 2014) autoimmune disease (Kostic et al., 2015; Knip & Siljander, 2016), cardiovascular disease (Kelly et al., 2016) and obesity and type 2 diabetes (Le Chatelier et al., 2013; Remely et al., 2014; Turnbaugh et al., 2009).
  • Reduced diversity has been observed in patients with Crohn's disease (Sokol et al., 2008), type 1 diabetes, coeliac disease, allergy, autism and cystic fibrosis (Spor et al., 2011), all non-communicable diseases. Reduced diversity has also been associated with an increased risk of adiposity, insulin resistance, high blood lipid levels and inflammation (Cotillard et al, 2013; Le Chatelier et al, 2013) - features that can lead to Non- alcoholic liver disease (NAFLD) and its more serious follow-on Non-alcoholic steatohepatitis (NASH) as well as contributing to many of the disease highlighted above.
  • NAFLD Non- alcoholic liver disease
  • NASH Non-alcoholic steatohepatitis
  • Gut microbiota from obese mice had a lower bacterial diversity than that from lean mice (Turnbaugh et al, 2008).
  • 16S rRNA gene surveys revealed a reduced diversity of gut microbiota in human obese twins compared to their lean twin counterparts (Turnbaugh et al, 2009).
  • gut microbiota diversity may be a contributing factor to disorders of brain function, such as depression, anxiety and autism, through bidirectional signalling via the gut-brain axis (Collins et al, 2012; Cryan and Dinan, 2012; Mayer et al, 2014; Mayer et al, 2015; Stilling et al, 2014).
  • compositional diversity also decreases as we age (Claesson et al, 2011), which has been associated with a less diverse diet and correlates with poor health, increased frailty and markers of inflammation (Claesson et al, 2012).
  • C dijficile Clostridium dijficile associated diarrhoea
  • C dijjicile infection is normally the result of perturbation of the gut microbiota as a result of broad- spectrum antibiotic treatment which results in a decrease in diversity of the gut microbiota (Rea et al, 2012a). Introducing a probiotic strain in a disease state could increase diversity, thus reducing the ability of C.
  • FMT faecal microbiota transplantation
  • Lactobacillus gasseri strain APC678 having NCIMB accession number 42658.
  • the strain is probiotic.
  • the strain may be in the form of a biologically pure culture.
  • the strain may be in the form of viable cells.
  • the strain may be in the form of non-viable cells.
  • the strain may be isolated from human faeces.
  • the strain is in the form of a bacterial broth. In another case the strain is in the form of a freeze-dried powder. Also provided is a formulation comprising a strain of the invention.
  • the formulation may comprise an ingestible carrier.
  • the ingestible carrier may be a pharmaceutically acceptable carrier.
  • the ingestible carrier in some cases is a food product.
  • the food product may, for example, be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, ice cream, milk powder, milk concentrate, cheese spread, dressing and beverage.
  • the formulation may be in the form of a fermented food product.
  • the formulation may be in the form of a fermented milk product. In some cases the carrier does not occur in nature.
  • the formulation in some embodiments, is in the form of a capsule, a tablet, a pellet, or a powder.
  • the strain is present in the formulation at more than 10 6 cfu per gram of ingestible carrier.
  • Also provided is a vaccine comprising a strain of the invention.
  • the invention further provides a strain or a formulation of the invention for use as a probiotic.
  • the strain or the formulation in some cases is used for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection.
  • the strain or the formulation in some cases is used for generating a protective immune response against a pathogenic organism.
  • the invention provides a method of generating a protective immune response in a subject against a pathogenic organism comprising administering to the subject a strain or a formulation of the invention.
  • the invention also provides a method of increasing the bacterial biodiversity in the gastrointestinal tract comprising the step of administering to a subject a strain or a formulation of the invention.
  • the strain or formulation is administered in association with antibiotic treatment.
  • the subject in some embodiments, has a compromised immune system. In some cases the subject is more than 65 years of age.
  • the strain may be used in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract.
  • the formulation may be used in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract.
  • a strain selected from one or more of:-
  • Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;
  • Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659;
  • invention also provides a formulation comprising one or more strains selected from:
  • Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;
  • the formulation may comprise an ingestible carrier.
  • the ingestible may be a pharmaceutically acceptable carrier.
  • the ingestible carrier is a food product.
  • the food product may be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spread, dressing and beverage.
  • the formulation is in the form of a fermented food product.
  • the formulation is in the form of a fermented milk product.
  • the carrier does not occur in nature.
  • the formulation is in the form of a capsule, a tablet, a pellet, or a powder.
  • the strain is present in the formulation at more than 10 6 cfu per gram of ingestible carrier.
  • the invention also provides Lactobacillus gasseri strain APC678 having NCIMB accession number 42658 or mutants or variants thereof.
  • the invention also provides Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661 or mutants or variants thereof.
  • the invention also provides Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659 or mutants or variants thereof.
  • the invention also provides Lactobacillus paracasei strain APC1483 having NCIMB accession number 42660 or mutants or variants thereof.
  • the mutant is a genetically modified mutant.
  • the variant is a naturally occurring variant.
  • the strain(s) may be probiotic.
  • the strain may be in the form of a biologically pure culture.
  • the invention also provides an isolated strain of Lactobacillus gasseri APC678 (NCIMB 42658).
  • the invention also provides an isolated strain of Lactobacillus rhamnosus DPC6111 (NCIMB 42661).
  • the invention also provides an isolated strain of Lactobacillus gasseri DPC6112 (NCIMB 42659).
  • the invention also provides an isolated strain of Lactobacillus paracasei APC1483 (NCIMB 42660).
  • the strain may be in the form of viable cells.
  • the strain may be in the form of non-viable cells.
  • the strain is isolated from human faeces.
  • the strain is in the form of a bacterial broth. In some cases the strain is in the form of a freeze-dried powder.
  • the invention also provides a formulation comprising at least one isolated strain of the invention.
  • the formulation may comprise an ingestible carrier.
  • the ingestible may be a pharmaceutically acceptable carrier.
  • the ingestible carrier is a food product.
  • the food product may be selected from the group comprising acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spread, dressing and beverage.
  • the formulation is in the form of a fermented food product.
  • the formulation is in the form of a fermented milk product.
  • the carrier does not occur in nature.
  • the formulation is in the form of a capsule, a tablet, a pellet, or a powder.
  • the strain is present in the formulation at more than 10 6 cfu per gram of ingestible carrier.
  • the invention also provides a vaccine comprising one or more of the strains of the invention.
  • the strain or a formulation may be for use as a probiotic.
  • the strain or a formulation may be for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection.
  • the invention also provides a method for the prophylaxis and/or treatment of a Clostridium difficile colonisation or infection comprising administering a strain or a formulation of one or more of :
  • Lactobacillus rhamnosus DPC6111 having NCIMB accession number 42661;
  • Lactobacillus gasseri strain DPC6112 having NCIMB accession number 42659;
  • the invention further provides a method for screening a bacterial strain for activity against C difficile comprising: co-culturing a strain of interest with C. difficile in a co-culture medium comprising sugars (such as glucose) in a concentration of from 0.01 g/1 to 2 g/1, the co-culture medium having a pH of from 6.7 to 6.9.
  • the invention also provides a method for screening a bacterial strain for activity against C. difficile comprising:
  • co-culturing a strain of interest with C. difficile in a co-culture medium comprising sugars (such as glucose) in a concentration of from 0.01 g/1 to 01.0 g/1, the co-culture medium having a pH of 6.8
  • the co-culture medium comprises sugars (such as glucose) in a concentration of from 0.05 g/1 to 0.5 g/1.
  • the co-culture medium in some cases comprises sugars (such as glucose) in a concentration of from 0.09 g/1 to 0.11 g/1.
  • the strain of interest may, for example, be a Lactobacillus.
  • the invention also provides a co-culture medium for use in screening a bacterial strain for activity against C. difficile wherein the medium comprises sugars (such as glucose) in a concentration of from 0.01 g/1 to 2.0 g/1 and wherein the co-culture medium has a pH of from 6.7 to 6.9.
  • sugars such as glucose
  • co-culture medium for use in screening a bacterial strain for activity against C. difficile wherein the medium comprises sugars (such as glucose) in a concentration of from 0.01 g/1 to 1.0 g/1 and wherein the co-culture medium has a pH of 6.8.
  • sugars such as glucose
  • the co-culture medium comprises sugars (such as glucose) in a concentration of from 0.05 g/1 to 0.5 g/1.
  • the co-culture medium in some cases comprises sugars (such as glucose) in a concentration of from 0.09 g/1 to 0.1 lg/1.
  • sugars such as glucose
  • the invention also provides a method of increasing the bacterial biodiversity in the gastrointestinal tract comprising the step of administering to a subject a strain or a formulation of the invention. In some embodiments the strain or formulation is administered in association with antibiotic treatment.
  • the subject has a compromised immune system.
  • the subject may be an elderly patient who may be more than 65 years of age.
  • the strain or formulation may be for use in the prophylaxis or treatment of low bacterial biodiversity of the gastrointestinal tract.
  • the isolated strains may be in the form of viable cells.
  • the isolated strains may be in the form of non- viable cells.
  • Formulations comprising one or more of the strains may also comprise an ingestible carrier.
  • the ingestible carrier may be a pharmaceutically acceptable carrier such as a capsule, tablet or powder.
  • the ingestible carrier may be a food product such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages.
  • the strains of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
  • a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
  • Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives.
  • the amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect.
  • a formulation comprising one or more of the strains may also include a bacterial component, a drug entity or a biological compound.
  • a vaccine comprising at least one strain of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.
  • mutants and variants of the deposited strains include mutants and variants of the deposited strains.
  • mutant, variant and genetically modified mutant include a strain whose genetic and/or phenotypic properties are altered compared to the parent strain.
  • Naturally occurring variant includes the spontaneous alterations of targeted properties selectively isolated.
  • Deliberate alteration of parent strain properties is accomplished by conventional (in vitro) genetic manipulation technologies, such as gene disruption, conjugative transfer, etc.
  • Genetic modification includes introduction of exogenous and/or endogenous DNA sequences into the genome of a strain, for example by insertion into the genome of the bacterial strain by vectors, including plasmid DNA, or bacteriophages.
  • Natural or induced mutations include at least single base alterations such as deletion, insertion, transversion or other DNA modifications which may result in alteration of the amino acid sequence encoded by the DNA sequence.
  • mutant, variant and genetically modified mutant also include a strain that has undergone genetic alterations that accumulate in a genome at a rate which is consistent in nature for all micro-organisms and/or genetic alterations which occur through spontaneous mutation and/or acquisition of genes and/or loss of genes which is not achieved by deliberate (in vitro) manipulation of the genome but is achieved through the natural selection of variants and/or mutants that provide a selective advantage to support the survival of the bacterium when exposed to environmental pressures such as antibiotics.
  • a mutant can be created by the deliberate (in vitro) insertion of specific genes into the genome which do not fundamentally alter the biochemical functionality of the organism but whose products can be used for identification or selection of the bacterium, for example antibiotic resistance.
  • mutant or variant strains can be identified by DNA sequence homology analysis with the parent strain. Strains having a close sequence identity with the parent strain without demonstrable phenotypic or measurable functional differences are considered to be mutant or variant strains. A strain with a sequence identity (homology) of 99.5% or more with the parent DNA sequence may be considered to be a mutant or variant. Sequence homology may be determined using on-line homology algorithm "BLAST" program, publicly available at http://www.ncbi.nlm.nih,gov/BLAST/.
  • Mutants of the parent strain also include derived strains having at least 95.5% sequence homology to the 16S rRNA polynucleotide sequence of the parent strain. These mutants may further comprise DNA mutations in other DNA sequences in the bacterial genome.
  • Fig. 1 is Effect of lactobacilli on the survival of Clostridium difficile in co-culture.
  • Black bars ( ⁇ ) show the cell numbers of C difficile at TO and T 24 in the absence of Lactobacillus strains.
  • Bars with dashed lines (Ea) show the cell numbers of C difficile following 24 h co-culture with: L. gasseri APC 678, L. rhamnosus DPC 6111, L. gasseri
  • Fig. 2 is Clostridium difficile detected during faecal shedding.
  • C difficile detected in mouse faeces CFU g "1 faeces) following (a) 24 h, (b) 4 days, (c) 7 days administration of lactobacillus strains and (d) C. difficile levels in mouse colon (CFU colon "1 ) following 7 days injestion of the lactobacillus strains (or control).
  • Control 10 % RSM only; Lactobacillus gasseri APC 678; Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323.
  • Lactobacillus gasseri strain APC678 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB42658.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • Lactobacillus rhamnosus strain DPC6111 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42661.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • Lactobacillus gasseri strain DPC6112 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42659.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • Lactobacillus paracasei strain APC1483 was made at the National Collections of Industrial and Marine Bacteria Limited (NCIMB) Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, UK on September 20, 2016 and accorded the accession number NCIMB 42660.
  • NCIMB National Collections of Industrial and Marine Bacteria Limited
  • Lactobacilli when they grow in the normal growth medium used- MRS (Man Rogasa Sharpe) produce significant amounts of lactic acid from the metabolism of glucose and a fully grown lactobacillus culture can drop the pH over night to -4.4-4.2.
  • MRS Man Rogasa Sharpe
  • Clostridium difficile strains are quite sensitive to acidic conditions and these conditions would not be encountered in the colon (normal pH -6.8) which is where C. difficile would normally be found.
  • a novel co-culture medium containing minimized nutritional composition was developed.
  • the new medium support limited growth of both lactobacilli and C. difficile cells when grown separately.
  • the medium was rationally designed to simulate the lower gut environment in its composition so that cell growth was limited mainly by the amount of energy supply available.
  • the medium was designed to have high buffering capacity to prevent a drop in pH during cell growth.
  • Table 1 Comparison of de Man, Rogosa and Sharpe (MRS), Reinforced Clostridium Medium (RCM) and developed co-culture medium.
  • Lactobacillus strains were maintained at -80°C in 40 % (v/v) glycerol and routinely cultured anaerobically at 37°C on de Man Rogosa Sharpe (MRS) agar (Difco, Beckton Dickinson & Co., New Jersey, USA) for 48 h or overnight in MRS broth.
  • MRS de Man Rogosa Sharpe
  • C difficile strains EM304 (ribotype 027) and VPI 10463 were maintained at -80°C on micro-bank beads (Pro-Lab Diagnostics, Merseyside, UK) and cultured on Fastidious Anaerobic Agar (Lab M, Heywood, Lancashire, UK) supplemented with 7 % defibrinated horse blood (Cruinn Diagnostics, Dublin, Ireland) at 37 °C for 3 days. Fresh cultures were grown overnight at 37 °C in Reinforced Clostridium Medium (RCM) (Merck, Darmstadt, Germany) pre -boiled and cooled under anaerobic conditions.
  • RCM Reinforced Clostridium Medium
  • lactobacilli and Clostridia were grown in an anaerobic chamber (Don Whitley, West Yorkshire, UK) under an anoxic atmosphere (10 % H 2 , 0 % 0 2 , 0 % N 2 ), unless otherwise stated.
  • Table 3 Target strains, growth medium and incubation conditions for well diffusion assays to detect bacteriocin production by strains of interest.
  • This media allowed growth of both Lactobacillus and C. difficile strains by ⁇ 1 to 2 logs over a 24h period. Due to the buffering capacity of the medium, the pH was maintained at near neutral ( ⁇ pH 6.5) following growth for 24 h, eliminating concerns relating to the reduction of C. difficile merely as a result of acid production.
  • Lactobacillus strains (Table 4) from human and animal origin were selected for further screening using a co-culture method, with C. difficile EM 304 (ribotype 027) (Rea et ah, 2012b) as the target strain.
  • the media composition (g L "1 ) was: meat extract, 2 g; peptone, 2 g; yeast extract, 1 g; NaCl, 5 g; sodium acetate, 0.5 g; L-cysteine hydrochloride, 0.5 g; glucose, 0.1 g; NaH 2 PO 4 .H 2 0, 3.7 g; Na 2 HP0 4 .7H 2 0, 6.2 g; pH 6.8 (+ 0.2). Following overnight growth, 1 ml of C. difficile EM304 and each Lactobacillus strain were centrifuged at 14,000 x g. Pelleted cells were washed once in phosphate buffered saline (PBS) under anaerobic conditions and resuspended in fresh PBS.
  • PBS phosphate buffered saline
  • the MGM was inoculated with a test strain of Lactobacillus and C. difficile EM304 at 10 6 CFU ml "1 .
  • the tubes were incubated at 37°C for 24 h.
  • Survival of C. difficile was determined by plating onto Brazier's cefoxitin cycloserine and egg yolk agar (CCEY) (Lab M), and Lactobacillus counts were determined by plating onto MRS agar. Plates were incubated anaerobically at 37°C for 2-3 days and the anti-bacterial ability was determined as a reduction in C. difficile counts compared to the control in the absence of Lactobacillus.
  • Table 4 Lactobacilli screened for anti-Clostridium difficile activity.
  • Lactobacillus 2 1 pig caecum; 1 milk
  • Method 16s rRNA gene sequencing (16S) was performed to identify L. gasseri APC678. Briefly, total DNA was isolated from the strains using 100 ⁇ of Extraction Solution and 25 ⁇ of Tissue Preparation solution (Sigma-Aldrich, XNAT2 Kit). The samples were incubated for 5 minutes at room temperature followed by 2 h at 95 °C. 100 ⁇ of Neutralization Solution (Sigma-Aldrich, XNAT2 kit) was then added. DNA solution was quantified using a Nanodrop spectrophotometer and stored at 4°C. PCR was performed using the 16S primers.
  • the primer pairs used for identification of the both strain were 16S Forward 5'- CTG ATC TCG AGG GCG GTG TGT ACA AGG -3' and 16S Reverse 5'- CTG ATG AAT TCG AGA CAC GGT CCA GAC TCC-3'.
  • the cycling conditions were 94°C for 4 min (1 cycle), 94°C for 45 sec, 56°C for 45 sec, 72°C for 45 sec (30 cycles).
  • the PCR reaction contained 2 ⁇ (100 ng) of DNA, PCR mix (Sigma-Aldrich, Red Taq), 0.025 nM 16S L and R primer (MWG Biotech, Germany). The PCR reactions were performed on an Eppendorf thermocycler.
  • PCR products were run alongside a molecular weight marker (100 bp Ladder, Roche) on a 2 % agarose EtBr stained gel in TAE, to determine the 16S profile.
  • PCR products were purified using the Promega Wizard PCR purification kit.
  • the purified PCR products were sequenced at Beckman Coulter Genomics (UK) using the primer sequences (above) for the 16S rRNA gene region. Sequence data was then searched against the NCBI nucleotide database to determine the identity of the strain by nucleotide homology.
  • the resultant DNA sequence data was subjected to the NCBI standard nucleotide-to-nucleotide homology BLAST search engine (http://www.ncbi.nlm.nih.gov/BLAST/) to identify the nearest match to the sequence.
  • L. gasseri APC678 KU710510.1 Lactobacillus gasseri strain LG202 1024/1028 99% 1026 100% 0%
  • Table 6 Sequence of the 16S rRNA gene region of L. gasseri APC678.
  • Example 3 Screening for survival through gastrointestinal transit
  • GIT gastrointestinal tract
  • a suspension of 10 8 CFU ml - " 1 Lactobacillus was suspended in artificial gastric juice with the following composition: NaCl, 125 mmol L “1 ; KC1, 7 mmol L “1 ; NaHC0 3 , 45 mmol L “1 and pepsin, 3 g L “1 .
  • the final pH was adjusted with HC1 to pH 2 and pH 3 and with NaOH to pH 7.
  • the bacterial suspensions were incubated at 37 °C with agitation (200 rev min " ) to stimulate peristalsis. Aliquots were taken for enumeration of viability after 0, 90 and 180 min.
  • the cells were suspended in simulated intestinal fluid, which was prepared with 0.1 % (w/v) pancreatin (Sigma) and 0.15 % oxgall bile salts (Difco), in water adjusted to pH 8.0 with NaOH, for a further 180 min.
  • the suspensions were incubated at 37°C and samples taken for total viability after 90 and 180 min. Viability was assessed by plating serial dilutions on MRS agar and incubating at 37°C for 48 h. Survival was expressed as log reduction from 0 h.
  • Table 7 Survival of lactobacilli during simulated gastrointestinal tract transit.
  • Example 4 In vivo assessment of strains in C. difficile carriage mouse model
  • L. gasseri APC 678 and L. rhamnosus DPC 6111 were investigated.
  • This model serves two purposes. It acts as a model of diversity reduction post-antibiotic use in addition to being a model of c. difficile colonisation and disease.
  • the well characterised strain L. gasseri ATCC 33323 (Azcarate-Peril et ah, 2008), which did not show any inhibition of C. difficile using the afore-mentioned in vitro assays, was selected as a negative control for the animal study.
  • Mouse model serves two purposes. It acts as a model of diversity reduction post-antibiotic use in addition to being a model of c. difficile colonisation and disease.
  • the well characterised strain L. gasseri ATCC 33323 (Azcarate-Peril et ah, 2008), which did not show any inhibition of C. difficile using the afore-mentioned in vitro assays, was selected as a negative control for the animal study.
  • mice All procedures involving animals were approved by the UCC Animal Experimentation Ethics Committee (#2011/17).
  • C. difficile model 40 female C57BL/6 mice (7 weeks old) were obtained from Harlan Laboratories UK (Bicester, Oxfordshire, UK). All mice used in the experiment were housed in groups of 5 animals per cage under the same conditions. Food, water, bedding and cages were autoclaved before use.
  • mice were made susceptible to C. difficile infection by altering the gut microbiota using a previously described protocol (Chen et al, 2008). Briefly, an antibiotic mixture comprising of kanamycin (0.4 mg mL “1 ), gentamicin (0.035 mg mL “1 ), colistin (850 U mL “1 ), metronidazole (0.215 mg L “1 ) and vancomycin (0.045 mg mL “1 ) was prepared in water (all antibiotics were purchased from Sigma Aldrich, Ireland).
  • C. difficile strain VPI 10463 Adhering to strict anaerobic conditions, C. difficile strain VPI 10463 was grown overnight in RCM. Bacterial cells were collected by centrifugation at 4,050 x g for 5 minutes, washed once in PBS and resuspended in PBS to achieve a preparation of 5 x 10 5 CFU per mouse. Lactobacillus strains for each group - L. gasseri APC 678, L. rhamnosus DPC 6111 and L. gasseri ATCC 33323 (see Table 4 for details) were prepared by growing the strains overnight in MRS broth under anaerobic conditions.
  • CFU g "1 faeces viable C. difficile
  • CFU colon "1 ) total numbers of C. difficile per colon were counted.
  • C. difficile survival was determined by culturing anaerobically at 37°C on CCEY agar for 48 h. C. difficile colonies were confirmed using the Oxoid C. difficile test kit (Oxoid, Basingstoke, UK). At the time of culling caecal contents were collected for compositional sequencing from each individual mouse, snap frozen and stored at -80°C until required.
  • Total metagenomic DNA was extracted for each mouse caecum, following thawing at 4°C, with the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with an additional bead beating step (Murphy et aL , 2010). DNA was quantified using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Ireland). Initially the template DNA was amplified using primers specific to the V3-V4 region of the 16s rRNA gene which also allowed for the Illumina overhang adaptor, where the forward
  • Each PCR reaction contained 2.5 ⁇ DNA template (5 ng), 5 ⁇ forward primer (1 ⁇ ), 5 ⁇ reverse primer (1 ⁇ ) (Sigma) and 12.5 ⁇ Kapa HiFi Hotstart Readymix (2X) (Anachem, Dublin, Ireland).
  • the template DNA was amplified under the following PCR conditions for a total of 25 cycles: 95°C for 3 minutes and 30 seconds respectively (initialization and denaturation), 55°C for 30 seconds (annealing) and 72°C for 30 seconds (elongation), followed by a final elongation step of 5 minutes.
  • PCR products were visualised using gel electrophoresis (IX TAE buffer, 1.5 % agarose gel, 100 V) post PCR reaction.
  • PCR products then underwent the same electrophoresis and cleaning protocols as described above.
  • Samples were quantified using the Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) in conjunction with the broad range DNA quantification assay kit (Biosciences, Dublin, Ireland). All samples were pooled to an eqimolar concentration. Quality of the pool was determined by running on the Agilent Bioanalyser prior to sequencing. The sample pool was then denatured with 0.2 M NaOH, diluted to 4 pM and combined with 10 % (v/v) denatured 4 pM PhiX. Samples were sequenced on the MiSeq sequencing platform (Teagasc Sequencing Centre, Moorepark, Fermoy, Co. Cork, Ireland) using a 2300 cycle V3 Kit following protocols outlined by Illumina. Bioinformatic analysis
  • Raw niumina 300 base pair paired-end sequence reads were merged using Flash (Magoc and Salzberg, 2011) and quality checked using the split libraries script from the Qiime package (Caporaso et ah, 2010). Reads were then clustered into operational taxonomical units (OTUs) and chimeras removed with the 64-bit version of USEARCH (Edgar, 2010). Subsequently OTUs were aligned and a phylogenetic tree generated within Qiime. Taxonomical assignments were reached using the SILVA 16S specific database (version 111) (Quast et al, 2013).
  • PICRUSt phylogenetic investigation of communities by reconstruction of unobserved states analysis was performed on the OTU tables to infer function (Langille et ah, 2013). Alpha and beta diversity analysis was also implemented within Qiime. Principal coordinate analysis (PCoA) plots were then visualised using EMPeror v0.9.3-dev (Vazquez-Baeza et ah, 2013).
  • Non-parametric Mann- Whitney statistical analysis was applied on MiniTab (Version 15; faecal and colon culture data) and SPSS (PASW Statistics version 18; caecal microbiota compositional data) statistical packages, to assess whether differences in C. difficile shedding, microbiota composition and diversity between the control and probiotic-fed groups were significant. Statistical significance was accepted at p ⁇ 0.05, adjusted for ties, where the null hypothesis was rejected.
  • V3-V4 16S rRNA gene amplicons were generated and sequenced using the Illumina MiSeq. Following quality filtering, 4,985,283 sequence reads remained. Diversity, richness and coverage estimations were calculated for each data set (Table 6), all of which indicated good sample richness throughout and the presence of a diverse microbiota. Interestingly, the Simpson and Shannon diversity metrics were significantly higher in the L. gasseri APC 678-fed mice compared to the control mice, and all alpha diversity indices tested were significantly higher in the L. gasseri APC 678- fed mice compared to the those fed L. gasseri ATCC33323 or L.
  • Beta-diversity was estimated using distance matrices built from unweighted Unifrac distances and, subsequently, principal co-ordinate analysis (PCoA) was performed on the distance matrices. The different groups cluster on the basis of strain administrated.
  • the relative abundance of the Phylum Proteobacteria significantly decreased in the mice fed APC 678 or DPC 6111 relative to the control mice or the animals fed ATCC 33323 which in fact showed an increase in Proteobacteria relative to the control. This change was mirrored in the significant reduction of the relative abundance of the genera Escherichia/Shigella in the groups fed L. gasseri APC 678 and L. rhamnosus DPC6111.
  • Elevated Proteobacteria are normally associated with antibiotic use and are elevated in patients with CDI (Cotter et al and Milani et al 2016) indicating that the abundance profiles seen post feeding APC678 or with L. rhamnosus DPC6111 are more consistent with a healthy diversity profile at the phylum level. Differences in effect on abundance of specific bacterial families and genera within phyla seen on administration of different bacterial strains.
  • the relative abundance of the genera Escherichia/Shigella was significantly reduced in the groups fed L. gasseri APC 678 and L. rhamnosus DPC 6111.
  • the decrease in the L. gasseri ATCC 33233-fed group was not significant.
  • the relative abundance of Peptostreptococcaceae Incertae Sedis were significantly reduced in all Lactobacillus-fed groups with both L. gasseri strains showing the largest decrease.
  • Roseburia significantly increased in all Lactobacillus-fed groups but the largest increase was seen in the L. gasseri APC 678-fed group.
  • Roseburia are associated with the production of short chain fatty acid production in the gut.
  • SCFA are known to have antiinflammatory, anti-tumorigenic, and antimicrobial activity and can be metabolised by host epithelial cells in the colon (Rios-Covian et ah, 2016).
  • the relative abundance of Oscillibacter significantly increased in the groups fed both L. gasseri APC 678 or L. rhamnosus DPC 6111 but not in the L. gasseri ATCC 33323-fed group.
  • Oscillibacter Some species of Oscillibacter are associated with the production of SCFA producing predominantly valerate (lino et ah, 2007).
  • Table 8 Alpha diversity indices for sequencing coverage and diversity of microbiota of caecum samples at Day 7 from control and the mice fed the test strains.
  • Table 9 Relative abundance (%) of bacterial phyla in the caecum at Day 7 of the control and probiotic-fed mice (Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323).
  • Table 10 Relative abundance (%) at bacterial phylum, family and genus level in the caecum at Day 7 of the control and test mice (Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323). Only phyla, families and genera with significant differences compared to the control mice are represented.
  • Table 11 Relative abundance (%) of bacterial families in the caecum at Day 7 of the control and test mice (fed Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and
  • Lactobacillus gasseri ATCC 33323 Lactobacillus gasseri ATCC 33323.
  • Cyanobacteria 4C0d-2 uncultured 0.001 0.000 0.000 0.000 0.000
  • Methylobacteriaceae 0.000 0.000 0.000 0.001
  • Table 12 Relative abundance (%) of bacterial genera in the caecum at Day 7 of the control and test mice (fed Lactobacillus gasseri APC 678, Lactobacillus rhamnosus DPC 6111 and Lactobacillus gasseri ATCC 33323).
  • Subdoligranulum 0.000 0.000 0.003 0.000
  • Methylobacterium 0.000 0.000 0.000 0.001
  • Stenotrophomonas 0.000 0.057* 0.001 0.000
  • L. gasseri ATCC 33323 which has been shown to have a number of traits encoded on its genome which are important for its survival and retention in the gastrointestinal tract (Azcarate-Peril et ah, 2008).
  • L. gasseri APC 678 was the lead candidate from this screening and, interestingly while 10 strains of L. gasseri were screened in vitro only 2 L. gasseri strains, L. gasseri APC 678 and L. gasseri DPC 6112, inhibited C. difficile lending credence to the theory that not all strains of the same species have the same effect.
  • the two lead candidates from the in vitro work namely L. gasseri APC 678 and L. rhamnosus DPC 6111, were selected for in vivo analysis. These strains had the added advantage of being capable of survival in significant numbers during simulated gastric transit, at low pH and in the presence of bile and the digestive enzymes encountered in the stomach and upper GIT.
  • the increased survival rate in these environments in the presence of milk is a further bonus as fermented milk products such as yoghurts and cheese are often used as vehicles for oral delivery of live bacteria (Gardiner et ah, 1998; Hickson et ah, 2007).
  • the strains (L. gasseri APC 678, L. rhamnosus DPC 6111 and the aforementioned well- characterised strain L. gasseri ATCC 33323) were tested for their ability to reduce faecal shedding of C. difficile in a murine model of CDI over 7 days.
  • the ability to reduce faecal shedding was significant, when compared to the control group fed RSM, in those animals fed L. gasseri APC 678 four days post infection and this reduction was maintained up to 7 days at which time the animals were euthanized. No significant effect was seen in terms of faecal shedding of C.
  • CDI is normally the result of perturbation of the gut microbiota as a result of broad-spectrum antibiotic treatment which results in a decrease in microbial diversity (Rea et ah, 2012b).
  • One function of a live therapeutic in a disease state would be to increase diversity, thus reducing the ability of C. difficile to survive and multiply due to competition for nutrients.
  • Compositional sequencing showed that in the control fed mice and the test groups there was a diverse microbiota despite the prior administration of antibiotics to make the animals more susceptible to infection.
  • L. gasseri APC 678 increased diversity for all the indices tested, including the number of observed species compared to the other strains studied.
  • FMT in experimental animals has shown that immunologic, behavioural and metabolic phenotypes can be transferred from donor to recipient which may not always be beneficial to the recipient in the long term. (Collins et al., 2013; Di Luccia et al., 2015; Pamer, 2014).
  • L. gasseri APC678 was used successfully to control (clear/reduce significantly) C. difficile infection in an in vivo mouse model.
  • L. gasseri APC678 by reducing the C difficile infection will reduce the immune pathogenesis of Clostridium difficile- associated disease through a reduction of the neutrophil-mediated inflammatory response such as bactericidal reactive oxygen intermediates, defensins and pro-inflammatory cytokines and chemokines thereby stopping the tissue damage and persistent clinical disease.
  • the compositions described herein exhibit immunological properties (Protective immunity) following appropriate administration to a subject. The presence of Protective immunity may be demonstrated as described above and/or by showing that infection by a pathogen (e.g., C.
  • test subjects e.g., human or non-human
  • a suitable amount of time such as 1 week in the model
  • protective immunity may be one that is detrimental to the infectious organism corresponding to the C. difficile and beneficial to the host (e.g., by reducing or preventing infection).
  • protective cellular responses may be reactive with the C. difficile strain, especially when administered in an effective amount and/or schedule.
  • compositions described herein may be used to induce an immune response against C. difficile.
  • An immunological composition that, upon administration to a host, results in a therapeutic (e.g., typically administered during an active infection) and/or protective (e.g., typically administered before or after an active infection).
  • the present invention generally relates to compositions and methods for the prevention or treatment of bacterial infection by the Gram-positive organism, Clostridium difficile, in a vertebrate subject.
  • the methods provide administering an agent to the vertebrate subject in need thereof in an amount effective to reduce, eliminate, or prevent Clostridium difficile bacterial infection or bacterial carriage.
  • the term "immunity” refers to the response of immune system cells to external or internal stimuli ⁇ e.g., antigen, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.
  • external or internal stimuli e.g., antigen, cell surface receptors, cytokines, chemokines, and other cells
  • Protective immunity means that the subject mounts an active immune response to a composition, such that upon subsequent exposure to Clostridium difficile bacteria, the subject is able to combat the infection.
  • a protective immune response will generally decrease the incidence of morbidity and mortality from subsequent exposure to Clostridium difficile bacteria among subjects.
  • Protective immunity will also generally decrease colonization by Clostridium difficile bacteria in the subjects.
  • CDAD C. difficile-associated disease
  • the toxins translocate to cytosol of target cells including IECs, mast cells, fibroblasts, smooth muscle cells, and monocytes (Na et al 2008), eventually resulting in a disruption of the actin cytoskeleton (Voth et al 2005), epithelial cell rounding and eventually cell death and necrosis.
  • This cellular stress response triggers secretion of inflammatory cytokines and expression of leukocyte adhesion molecules (integrins and selectins (Savidge et al 2003) which in turn facilitate neutrophil infiltration into the colon.
  • the infiltrated neutrophils are activated at the site of infection and produce bactericidal reactive oxygen intermediates (He et al 2000), defensins and pro-inflammatory cytokines and chemokines (Kelly et al 2011), leading to an intense neutrophil-mediated inflammatory response, which is believed to be one of the key determinants of disease severity an over exuberant neutrophil response is clearly associated with tissue damage and persistent clinical disease (Bulusu et al 2000).
  • strains of the invention may be administered to animals (including humans) in an orally ingestible form in a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
  • a conventional preparation such as capsules, microcapsules, tablets, granules, powder, troches, pills, suppositories, suspensions and syrups.
  • Suitable formulations may be prepared by methods commonly employed using conventional organic and inorganic additives.
  • the amount of active ingredient in the medical composition may be at a level that will exercise the desired therapeutic effect.
  • the formulation may also include a bacterial component, a drug entity or a biological compound.
  • a vaccine comprising one or more of the strains of the invention may be prepared using any suitable known method and may include a pharmaceutically acceptable carrier or adjuvant.
  • probiotic organisms The introduction of probiotic organisms is accomplished by the ingestion of the micro-organism in a suitable carrier. It would be advantageous to provide a medium that would promote the growth of these probiotic strains in the large bowel.
  • the addition of one or more oligosaccharides, polysaccharides, or other prebiotics enhances the growth of lactic acid bacteria in the gastrointestinal tract.
  • Prebiotics refers to any non-viable food component that is specifically fermented in the colon by indigenous bacteria thought to be of positive value, e.g. bifidobacteria, lactobacilli. Types of prebiotics may include those that contain fructose, xylose, soya, galactose, glucose and mannose.
  • the combined administration of a probiotic strain with one or more prebiotic compounds may enhance the growth of the administered probiotic in vivo resulting in a more pronounced health benefit, and is termed synbiotic.
  • the probiotic strains may be administered prophylactically or as a method of treatment either on its own or with other probiotic and/or prebiotic materials as described above.
  • the bacteria may be used as part of a prophylactic or treatment regime using other active materials such as those used for treating inflammation or other disorders especially those with an immunological involvement.
  • Such combinations may be administered in a single formulation or as separate formulations administered at the same or different times and using the same or different routes of administration.
  • the strains of the invention may be formulated to facilitate controlled release such as a delayed release of the strain.
  • the formulation may be adapted to release the strain at a particular location in the gastrointestinal tract such as the small intestine or in the colon.
  • the strain may be formulated in a capsule which has a coating which is adapted to release the strain at a particular location.
  • a range of coatings are available to facilitate such controlled release.
  • One such family of coatings are those available under the Trade Mark Eudragit.
  • the invention is not limited to the embodiments hereinbefore described, which may be varied in detail.
  • Nebot-Vivinus M., Harkat, C, Bzioueche, Carrier, H. C, Plichon-Dainese, R., Moussa, L., Eutamene, H., Pishvaie, D., Holowacz, S., Seyrig, C, Piche, T. & Theodorou, V. 2014. Multispecies probiotic protects gut barrier function in experimental models. World J. Gastroenterol., 20, 6832-6843.
  • Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine, Gastroenterology 125 (2003) 413e420.
  • Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U SA, 105, 16731-6.
  • PHAST a fast phage search tool. Nucleic Acids Res, 39, W347-52.

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Abstract

La présente invention concerne Lactobacillus gasseri APC678 qui est efficace contre la colonisation ou l'infection par C. difficile. Clostridium difficile est l'une des causes les plus communes de diarrhée acquise à travers des soins de santé, entraînant un spectre de maladie allant d'une diarrhée légère à une maladie potentiellement mortelle.
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