Spore-Forming Clostridium (Clostridioides) difficile in Wastewater Treatment Plants in Western Australia

There is growing evidence that shows Clostridium (Clostridioides) difficile is a pathogen of One Health importance with a complex dissemination pathway involving animals, humans, and the environment. Thus, environmental discharge and agricultural recycling of human and animal waste have been suspected as factors behind the dissemination of Clostridium difficile in the community. Here, the presence of C. difficile in 12 wastewater treatment plants (WWTPs) in Western Australia was investigated. Overall, C. difficile was found in 90.5% (114/126) of raw sewage influent, 48.1% (50/104) of treated effluent, 40% (2/5) of reclaimed irrigation water, 100% (38/38) of untreated biosolids, 95.2% (20/21) of anaerobically digested biosolids, and 72.7% (8/11) of lime-amended biosolids. Over half of the isolates (55.3% [157/284]) were toxigenic, and 97 C. difficile ribotypes (RTs) were identified, with RT014/020 the most common (14.8% [42/284]). Thirteen C. difficile isolates with the toxin gene profile A⁺ B⁺ CDT⁺ (positive for genes coding for toxins A and B and the binary C. difficile transferase toxin [CDT]) were found, including the hypervirulent RT078 strain. Resistance to the antimicrobials fidaxomicin, vancomycin, metronidazole, rifaximin, amoxicillin-clavulanate, meropenem, and moxifloxacin was uncommon; however, resistance to clindamycin, erythromycin, and tetracycline was relatively frequent at 56.7% (161/284), 14.4% (41/284), and 13.7% (39/284), respectively. This study revealed that toxigenic C. difficile was commonly encountered in WWTPs and being released into the environment. This raises concern about the possible spillover of C. difficile into animal and/or human populations via land receiving the treated waste. In Western Australia, stringent measures are in place to mitigate the health and environmental risk of recycling human waste; however, further studies are needed to elucidate the public health significance of C. difficile surviving the treatment processes at WWTPs. IMPORTANCE Clostridium difficile infection (CDI) is a leading cause of antimicrobial-associated diarrhea in health care facilities. Extended hospital stays and recurrences increase the cost of treatment and morbidity and mortality. Community-associated CDI (CA-CDI) cases, with no history of antimicrobial use or exposure to health care settings, are increasing. The isolation of clinically important C. difficile strains from animals, rivers, soil, meat, vegetables, compost, treated wastewater, and biosolids has been reported. The objective of this study was to characterize C. difficile in wastewater treatment plants (WWTPs) in Australia. We found that C. difficile can survive the treatment processes of WWTPs, and toxigenic C. difficile was being released into the environment, becoming a potential source/reservoir for CA-CDI.