ABSTRACT
BACKGROUND: Alterations in the intestinal microbiota are thought to be involved in the pathogenesis of inflammatory bowel diseases (IBD). Klebsiella oxytoca is an intestinal pathobiont that can produce a cytotoxin (tillivaline). AIM: We aimed to elucidate the pathogenetic relevance of toxin-producing K. oxytoca in patients with IBD flares and investigated the clonal relationship of K. oxytoca isolates from IBD patients using multilocus sequence typing (MLST). METHODS: Fecal samples of 235 adult IBD patients were collected from January 2008 to May 2009 and were tested for K. oxytoca, C. difficile toxin, and other pathogens by standard microbiological methods. Clinical data and disease activity scores were collected. K. oxytoca isolates were tested for toxin production using cell culture assays. A total of 45 K. oxytoca isolates from IBD patients, healthy, asymptomatic carriers and from patients with antibiotic-associated hemorrhagic colitis in part from our strain collection were tested for their clonal relationship using MLST. RESULTS: The prevalence of K. oxytoca in IBD overall was 4.7%. Eleven K. oxytoca isolates were detected. Two of 11 isolates were tested positive for toxin production. There was no significant difference in the distribution of K. oxytoca isolates between the groups (active vs. remission in UC and CD). MLST yielded 33 sequence types. K. oxytoca isolates from IBD did not cluster separately from isolates from asymptomatic carriers. CONCLUSIONS: Our data demonstrate that toxin (tilivalline)-producing K. oxytoca is not associated with IBD flares.
Subject(s)
Colitis, Ulcerative/microbiology , Crohn Disease/microbiology , Intestines/microbiology , Klebsiella Infections/microbiology , Klebsiella oxytoca/isolation & purification , Adult , Bacterial Typing Techniques , Benzodiazepinones/isolation & purification , Case-Control Studies , Colitis, Ulcerative/diagnosis , Crohn Disease/diagnosis , DNA, Bacterial/genetics , Disease Progression , Feces/microbiology , Female , Humans , Intestines/pathology , Klebsiella Infections/diagnosis , Klebsiella oxytoca/classification , Klebsiella oxytoca/genetics , Male , Middle Aged , Multilocus Sequence Typing , Prospective Studies , Risk Factors , Young AdultABSTRACT
Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.
Subject(s)
Anti-Bacterial Agents/adverse effects , Benzodiazepinones/toxicity , Colitis/chemically induced , Enterotoxins/toxicity , Peptides/toxicity , Actinobacteria/metabolism , Animals , Apoptosis/drug effects , Caspase 3/metabolism , Colitis/pathology , Cytotoxins/toxicity , Epithelial Cells/drug effects , Epithelial Cells/microbiology , Epithelial Cells/pathology , Humans , Klebsiella oxytoca/genetics , Mice, Inbred C57BL , Molecular Sequence Data , Multigene Family , Penicillins/pharmacology , Peptide Synthases/metabolism , RibosomesABSTRACT
Klebsiella oxytoca acts as a pathobiont in the dysbiotic human intestinal microbiota, causing antibiotic-associated hemorrhagic colitis (AAHC), but it also infects other organs, resulting in pneumonia and urinary tract and skin infections. The virulence of K. oxytoca is still poorly understood. The production of a specific cytotoxin has been linked to AAHC pathogenesis. To investigate the clonal relationships of K. oxytoca with regard to clinical origin and virulence attributes, we established a multilocus sequence typing (MLST) method and analyzed 74 clinical K. oxytoca isolates from asymptomatic carriers and patients with AAHC, respiratory infections, and other infections. The isolates were phenotypically characterized, typed, and compared phylogenetically based on the sequences of seven housekeeping genes. MLST analysis yielded 60 sequence types, 12 of which were represented by more than one isolate. The phylogenetic tree distinguished clusters of K. oxytoca isolates between patients with AAHC and those with respiratory infections. Toxin-positive and -negative strains were observed within one sequence type. Our findings indicate that AAHC isolates share a genetic background. Interestingly, K. oxytoca isolates from nosocomial pneumonia showed a different genetic clustering, suggesting that these strains do not originate from the intestines or that they are specialized for respiratory tract colonization. Our results further indicate a polyphyletic origin and possible horizontal transfer of the genes involved in K. oxytoca cytotoxin production. This work provides evidence that K. oxytoca isolates colonizing the two main clinically relevant habitats (lower gastrointestinal [GI] tract and respiratory tract) of the human host are genetically distinct. Applications of this MLST analysis should help clarify the sources of nosocomial infections.
