Prevalence of Clostridium difficile in Canadian Rivers and Fate during Waste Water Treatment

Introduction: Clostridium difficile is a Gram-positive, anaerobic, spore-forming bacterium that is an important cause of enteric disease in humans and some animal species. While traditionally considered primarily a hospital- and antimicrobial-associated pathogen, there has been an increase in community-associated C. difficile infection (CA-CDI) in humans internationally, for reasons that are unclear. Source of exposure outside of the hospital has not been adequately investigated, but concern has been raised about foodborne or environmental sources.

Purpose: Determine the prevalence and compare the toxigenic and genotypic profile of C. difficile encountered in rivers and waste water sources within Southern Ontario; investigate the survival of C. difficile during anaerobic sludge digestion.

Methods: Samples from rivers and waste water treatment plants (WWTPs) were collected and enriched, C. difficile was isolated. Molecular characterization of toxin A, toxin B, binary toxin, and sequencing of tcdC were undertaken to determine the toxigenic profile of the isolates, PCR ribotyping, toxinotyping and pulsed field gel electrophoresis (PFGE) were employed to detect the genotypes of the isolates.

Results: C. difficile was found in 39.1% (25/64) river samples and 93.0% (119/128) sludge samples; 23 (92.0%) of river isolates and 95 (79.8%) from sludge isolates were toxigenic; 69.6% (16/23) toxigenic sediment isolates could be classed as ribotype 078 and toxinotype V, and 13 were NAP7 while 3 were NAP 8, 16.8% (16/95) toxigenic sludge isolates were ribotype 078, toxinotype V, and NAP7. The 118 toxigenic C. difficile isolates were grouped into 41 different PCR ribotypes, 12 toxinotypes and 57 PFGE types. Anaerobic sludge digestion did not alter C. difficile levels or ribotypes.

Significance: Toxigenic C. difficile are present in raw sewage but do not proliferate during the digestion process. The effluent released from WWTPs add to the environmental burden of C. difficile although how this contributes to the incidence of CA-CDI remains unclear.