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.

Assessment of Carbon Dioxide, Carbon Dioxide/Oxygen, Isoflurane and Pentobarbital Killing Methods in Adult Female Sprague-Dawley Rats.

BACKGROUND: Exposure to carbon dioxide (CO2) gas as a killing method is aversive and exposure to high concentrations is likely to be painful. Bradycardia during exposure to CO2 is associated with nociception and pain. However, it is unclear if bradycardia occurs before loss of consciousness as definitions of loss of consciousness vary in the literature. The objectives of this study were to explore the relationship between recumbency, loss of righting reflex (LORR) and a quiescent electromyograph as measures of loss of consciousness, and identify the onset of bradycardia in relation to these measures. Our primary hypothesis was that CO2 exposure would result in bradycardia, which would precede LORR. METHODS: Thirty-two adult, female Sprague-Dawley rats were instrumented with a telemetry device and randomly assigned to one of four killing methods (concentrations of 100% CO2, CO2 (70%)/O2 (30%), isoflurane (5%) and intraperitoneal pentobarbital (200 mg/kg). Time to achieve recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph, heart rate and apnea were recorded. RESULTS: The general order of progression was recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph and apnea. Recumbency preceded LORR in the majority of animals (CO2; 7/8, CO2/O2; 8/8, isoflurane; 5/8, pentobarbital; 4/8). Bradycardia occurred before recumbency in the CO2 (p = 0.0002) and CO2/O2 (p = 0.005) groups, with a 50% reduction in heart rate compared to baseline. The slowest (time to apnea) and least consistent killing methods were CO2/O2 (1180 ± 658.1s) and pentobarbital (875 [239 to 4680]s). CONCLUSION: Bradycardia, and consequently nociception and pain, occurs before loss of consciousness during CO2 exposure. Pentobarbital displayed an unexpected lack of consistency, questioning its classification as an acceptable euthanasia method in rats.