Targeted Restoration of the Intestinal Microbiota with a Simple, Defined Bacteriotherapy Resolves Relapsing Clostridium difficile Disease in Mice

Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.

[1]  G. Macfarlane,et al.  Regulation of short-chain fatty acid production , 2003, Proceedings of the Nutrition Society.

[2]  N. Salzman,et al.  Prolonged Impact of Antibiotics on Intestinal Microbial Ecology and Susceptibility to Enteric Salmonella Infection , 2009, Infection and Immunity.

[3]  Stuart Johnson,et al.  An epidemic, toxin gene-variant strain of Clostridium difficile. , 2005, The New England journal of medicine.

[4]  C. Pothoulakis,et al.  Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins. , 2001, American journal of physiology. Gastrointestinal and liver physiology.

[5]  A. Richardson,et al.  Simultaneous determination of volatile and non‐volatile acidic fermentation products of anaerobes by capillary gas chromatography , 1989 .

[6]  Jeffrey D Goldsmith,et al.  A mouse model of Clostridium difficile-associated disease. , 2008, Gastroenterology.

[7]  M. Woolhouse,et al.  Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157 , 2008, Nature Reviews Microbiology.

[8]  O. Dekkers,et al.  Time interval of increased risk for Clostridium difficile infection after exposure to antibiotics. , 2012, The Journal of antimicrobial chemotherapy.

[9]  Patrick D Schloss,et al.  The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection , 2011, Gut microbes.

[10]  Andries J. van Tonder,et al.  Evolutionary dynamics of Clostridium difficile over short and long time scales , 2010, Proceedings of the National Academy of Sciences.

[11]  Ken Dewar,et al.  A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. , 2005, The New England journal of medicine.

[12]  L. Mcfarland Evidence-based review of probiotics for antibiotic-associated diarrhea and Clostridium difficile infections. , 2009, Anaerobe.

[13]  Harald Meier,et al.  46. ARB: A Software Environment for Sequence Data , 2011 .

[14]  E. Kuijper,et al.  Clostridium difficile infection in Europe: a hospital-based survey , 2011, The Lancet.

[15]  J. Parkhill,et al.  Dominant and diet-responsive groups of bacteria within the human colonic microbiota , 2011, The ISME Journal.

[16]  D. Gerding Clostridium difficile 30 years on: what has, or has not, changed and why? , 2009, International journal of antimicrobial agents.

[17]  D. Gerding,et al.  Decreased cure and increased recurrence rates for Clostridium difficile infection caused by the epidemic C. difficile BI strain. , 2012, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[18]  N. Fairweather,et al.  The Clostridium difficile spo0A Gene Is a Persistence and Transmission Factor , 2012, Infection and Immunity.

[19]  Paramvir S. Dehal,et al.  FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments , 2010, PloS one.

[20]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[21]  M. Sakamoto,et al.  Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. , 2006, International journal of systematic and evolutionary microbiology.

[22]  C. Donskey,et al.  Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[23]  Mathias J Friedrich,et al.  CD25+/Foxp3+ T cells regulate gastric inflammation and Helicobacter pylori colonization in vivo. , 2006, Gastroenterology.

[24]  T. Borody,et al.  Fecal microbiota transplantation and emerging applications , 2012, Nature Reviews Gastroenterology &Hepatology.

[25]  M. Tvede,et al.  Bacteriotherapy for chronic relapsing Clostridium difficile in six patients , 1989 .

[26]  J. Bartlett,et al.  Clinical practice. Antibiotic-associated diarrhea. , 2002, New England Journal of Medicine.

[27]  Y. Benjamini,et al.  More powerful procedures for multiple significance testing. , 1990, Statistics in medicine.

[28]  A. Tatem,et al.  Clostridium difficile PCR ribotype 027: assessing the risks of further worldwide spread , 2010, The Lancet Infectious Diseases.

[29]  N. Fairweather,et al.  Antibiotic Treatment of Clostridium difficile Carrier Mice Triggers a Supershedder State, Spore-Mediated Transmission, and Severe Disease in Immunocompromised Hosts , 2009, Infection and Immunity.

[30]  M. Quail,et al.  Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium , 2009, Genome Biology.

[31]  J. Wehland,et al.  Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria , 2009, PLoS pathogens.

[32]  J. Bakken Fecal bacteriotherapy for recurrent Clostridium difficile infection. , 2009, Anaerobe.

[33]  Julian Parkhill,et al.  The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome , 2006, Nature Genetics.

[34]  M. Wilcox,et al.  The potential for airborne dispersal of Clostridium difficile from symptomatic patients. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[35]  S. Tzipori,et al.  Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models. , 2012, The Journal of infectious diseases.

[36]  S. Roos,et al.  Lactobacillus reuteri prevents colitis by reducing P-selectin-associated leukocyte- and platelet-endothelial cell interactions. , 2009, American journal of physiology. Gastrointestinal and liver physiology.

[37]  T. Borody,et al.  Bacteriotherapy Using Fecal Flora: Toying With Human Motions , 2004, Journal of clinical gastroenterology.

[38]  J. Bakken,et al.  Treating Clostridium difficile infection with fecal microbiota transplantation. , 2011, Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association.

[39]  D. Relman,et al.  Host Transmission of Salmonella enterica Serovar Typhimurium Is Controlled by Virulence Factors and Indigenous Intestinal Microbiota , 2007, Infection and Immunity.

[40]  Martin Hartmann,et al.  Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities , 2009, Applied and Environmental Microbiology.

[41]  R. D. de Souza,et al.  Colonic Health: Fermentation and Short Chain Fatty Acids , 2006, Journal of clinical gastroenterology.

[42]  D. Gerding,et al.  Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain. , 2009, Gastroenterology.

[43]  J. Rask-Madsen,et al.  BACTERIOTHERAPY FOR CHRONIC RELAPSING CLOSTRIDIUM DIFFICILE DIARRHOEA IN SIX PATIENTS , 1989, The Lancet.

[44]  B. Finlay,et al.  Antibiotic-Induced Perturbations of the Intestinal Microbiota Alter Host Susceptibility to Enteric Infection , 2008, Infection and Immunity.

[45]  James R. Cole,et al.  The Ribosomal Database Project: improved alignments and new tools for rRNA analysis , 2008, Nucleic Acids Res..

[46]  A. Sarah Walker,et al.  Characterisation of Clostridium difficile Hospital Ward–Based Transmission Using Extensive Epidemiological Data and Molecular Typing , 2012, PLoS medicine.

[47]  E. van Nood,et al.  Struggling with recurrent Clostridium difficile infections: is donor faeces the solution? , 2009, Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin.

[48]  D. Gerding,et al.  Distribution of Clostridium difficile strains from a North American, European and Australian trial of treatment for C. difficile infections: 2005-2007. , 2009, Anaerobe.

[49]  Peer Bork,et al.  Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation , 2007, Bioinform..

[50]  T. Louie,et al.  A new macrocyclic antibiotic, fidaxomicin (OPT-80), causes less alteration to the bowel microbiota of Clostridium difficile-infected patients than does vancomycin. , 2010, Microbiology.

[51]  D. Relman,et al.  Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation , 2010, Proceedings of the National Academy of Sciences.

[52]  T. Clark,et al.  Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens , 2010, Applied and Environmental Microbiology.

[53]  J. Sirard,et al.  The Microbiota Mediates Pathogen Clearance from the Gut Lumen after Non-Typhoidal Salmonella Diarrhea , 2010, PLoS pathogens.

[54]  R. Seaton,et al.  Faecal transplant for recurrent Clostridium difficile-associated diarrhoea: a UK case series. , 2009, QJM : monthly journal of the Association of Physicians.

[55]  Hilary G. Morrison,et al.  Reproducible Community Dynamics of the Gastrointestinal Microbiota following Antibiotic Perturbation , 2009, Infection and Immunity.

[56]  D. Antonopoulos,et al.  Decreased diversity of the fecal Microbiome in recurrent Clostridium difficile-associated diarrhea. , 2008, The Journal of infectious diseases.

[57]  A. Viale,et al.  Profound Alterations of Intestinal Microbiota following a Single Dose of Clindamycin Results in Sustained Susceptibility to Clostridium difficile-Induced Colitis , 2011, Infection and Immunity.

[58]  J. Ballard,et al.  Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile , 2010, PLoS pathogens.

[59]  J. Brazier Clostridium difficile: from obscurity to superbug , 2008, British journal of biomedical science.

[60]  S. Mazmanian,et al.  A microbial symbiosis factor prevents intestinal inflammatory disease , 2008, Nature.

[61]  G. Dougan,et al.  Salmonella enterica Serovar Typhimurium Exploits Inflammation to Compete with the Intestinal Microbiota , 2007, PLoS biology.

[62]  S. Clare,et al.  Proteomic and Genomic Characterization of Highly Infectious Clostridium difficile 630 Spores , 2009, Journal of bacteriology.

[63]  K. Schleifer,et al.  ARB: a software environment for sequence data. , 2004, Nucleic acids research.

[64]  Barak A. Cohen,et al.  Phylogeny based discovery of regulatory elements , 2006, BMC Bioinformatics.

[65]  Pamela Sears,et al.  Fidaxomicin versus vancomycin for Clostridium difficile infection. , 2011, The New England journal of medicine.

[66]  Steven Salzberg,et al.  Identifying bacterial genes and endosymbiont DNA with Glimmer , 2007, Bioinform..

[67]  Courtney J. Robinson,et al.  From Structure to Function: the Ecology of Host-Associated Microbial Communities , 2010, Microbiology and Molecular Biology Reviews.

[68]  Jon Brazier,et al.  Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe , 2005, The Lancet.

[69]  J. Handelsman,et al.  Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness , 2005, Applied and Environmental Microbiology.

[70]  D. Gerding Clindamycin, cephalosporins, fluoroquinolones, and Clostridium difficile-associated diarrhea: this is an antimicrobial resistance problem. , 2004, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.