Fate of Clostridium difficile in Sewage Waste Water Treatment and Biosolids Amended Soil

Introduction: There have been an increasing number of Community Associated Clostridium difficile infections across developed and developing nations although the primary source of the pathogen has yet to be elucidated. Foodborne and zoonotic transfer has been suggested although considered minor sources by which the population is exposed to C. difficile. The role of environmental exposure (e.g. recreation waters, soil) in disseminating C. difficile has yet to be considered and hence requires to be evaluated.

Purpose:  The following study aimed to determine the incidence of C. difficile in sewage and the fate of the pathogen during sludge digestion and subsequent disposal of the effluent/biosolids into the environment.

Methods:  A total of 59 biosolids samples from waste water treatment plants in Ontario were collected and C. difficile was isolated. Toxigenic profile (toxin A, toxin B, and binary toxin) and genotypes (PCR ribotyping, toxinotyping and pulsed field gel electrophoresis) of each isolates were determined. The survival ability of ribotype 078 (5 log CFU/ml) and ribotype 027 (6 log CFU/ml) in biosolids-amended soils (sandy and loam) in farms was determined, sampling was carried out once a month.

Results:  C. difficile was found in 72.9% (43/59) biosolids samples and 35 (81.4%) of the isolates were toxigenic while all of the toxigenic isolates possessed tcdA and tcdB and 17 (48.6%) also possessed cdtA. Fifteen of the 17 (88.2%) A+B+CDT+ isolates were classified as PCR ribotype 078, toxinotype V, 13 of them were NAP7 and 2 were NAP8. From May to Nov 2013, ribotype 027 in sandy and loam soils stayed stable around 6 log CFU/ml. Ribotype 078 in sandy soil remained steadily around 5 log CFU/ml with a sharp decrease to 4 log CFU/ml in August and back to 5 log CFU/ml in September again. In loam soils, ribotype 078 declined sharply from 5 to 1 log CFU/ml from May to August and rose back to 4 log CFU/ml in September followed with slightly decrease.

Significance: Toxigenic C. difficile is highly prevalent within raw sewage and survives the waste water treatment process. The effluent and biosolids resulting from sewage treatment represents a significant environmental burden of C difficile.