Persistence of spore-forming Clostridium (Clostridioides) difficile through 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 C. 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 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 profile A+B+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 spill-over 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 diarrhoea in healthcare 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 healthcare 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 characterise C. difficile in wastewater treatment plants (WWTPs) in Australia. We found that C. difficile can persist through the treatment processes of WWTPs and toxigenic C. difficile was being released into the environment becoming a potential source/reservoir for CA-CDI. ### Competing Interest Statement The authors have declared no competing interest.