It takes two to tango: Klebsiella oxytoca overgrowth and production of Tilivalline, a cytotoxin, are the key events causing Antibiotic-Associated Hemorrhagic Colitis

Background: Klebsiella oxytoca is a member of the enteric microflora of humans that typically behaves as a commensal. However, β-lactamase expression enables this bacterium to dominate the intestinal flora during antibiotic therapy and cause antibiotic-associated hemorrhagic colitis (AAHC). Here we define the structure and function of a cytotoxin secreted by K. oxytoca and its involvement in AAHC.

Methods and Results: General and site-directed mutagenesis of a toxin-producing K. oxytoca strain, combined with an in vitro screen for epithelial cytotoxicity identified a cluster of non-ribosomal peptide synthesis genes. The toxic peptide was isolated and preparative HPLC followed by HiRes-MS and NMR analyses identified the pentacyclic pyrrolobenzodiazepine (PBD) tilivalline. Contribution of tilivalline to virulence was evaluated in a mouse model for AAHC. Following inoculation per os, AAHC was triggered via intraperitoneal administration of augmentin. Indometacin was given subcutaneously. Histological analysis of colon tissue revealed that the tilivalline knockout strain was strongly attenuated in AAHC-specific virulence compared to the tilivalline-producing wild-type strain. Induced epithelial apoptosis was the major pathological feature of colitis in vivo. Apoptosis in vivo was caspase-dependent as was the effect of tilivalline treatment on cultured epithelial cells.

Conclusions: We conclude that tilivalline is the main virulence factor of K. oxytoca in development of AAHC. Tillivallin, a PBD, represents a novel class of bacterial toxins capable of causing colitis. The induced epithelial apoptosis together with massive K. oxytoca overgrowth in the colon during antibiotic therapy are key steps in AAHC emergence. The K. oxytoca genes encoding a pentacyclic PBD toxin – unique in Gram-negative bacteria – may be transferred horizontally.