Prevalence and fate of Clostridium difficile during sewage treatment, land application and composting

Toxigenic Clostridium difficile was isolated from primary and digested municipal sewage sludge along with biosolids, effluent and sediments from a local water shed. Genotypes of toxigenic C. difficile were identified by PCR ribotyping, toxinotyping, and pulsed field gel electrophoresis (PFGE). C. difficile was isolated from 92% (108/117) of the primary sludge samples and 96% (106/110) of the digested sludge samples, as well as 73% (43/59) of dewatered biosolids samples and 39% (25/64) of rivers sediment samples where effluent was discharged. Ribotype 078 which is commonly associated with community associated C. difficile infection (CA-CDI) was recovered from 19% of primary sludge (21/108), 8% of digested sludge (8/106), 35% of biosolids (15/43) and 60% of river samples (15/25). The persistence of five isolates of C. difficile ribotype 078 under mesophilic and thermophilic digestion were compared. It was found that spore levels associated with the C. difficile-inoculated sludge remained constant at mesophilic temperatures (36 and 42°C) but decreased when digestion was performed at 55°C. The mode of action by applying thermophilic sludge digestion was through inducing germination of spores followed by inactivation of the subsequent vegetative cells. Acidification of the sludge through addition of acetic acid (6 g/l) or HCl inhibited germination through reducing the pH. The presence of endogenous microflora also reduced the germination rate although this varied amongst the strains tested. The viability of C. difficile spores was also reduced by composting biosolids where the temperature reached was >55°C. The survival of C. difficile ribotype 078 and 027 associated with biosolids applied to different soils (sandy and sandy-loam) was studied. It was found that 078 underwent germination and regrowth when applied under the soil subsurface although 027 levels remained constant throughout the 12 month trial. In conclusion, the study demonstrated that C. difficile associated with primary sewage sludge can survive the wastewater treatment processes and be disseminated through land application of biosolids, in addition to effluent discharged in rivers. However, through thermophilic digestion of sludge or composting of biosolids levels of C. difficile can be reduced thereby reducing the environmental burden of the pathogen.