Investigating human pathogens in wastewater is crucial for identifying and predicting potential occupational health risks faced by wastewater treatment plant (WWTP) workers. This study aimed to determine the occurrence and levels of Legionella pneumophila, Mycobacterium spp., Arcobacter butzleri, and Aeromonas hydrophila in untreated municipal wastewater. Grab influent, activated sludge, and secondary settling tank (SST) effluent samples were collected bi-weekly over 6 months from 5 WWTPs in Tshwane, South Africa. Mycobacterium spp., A. butzleri, and A. hydrophila were detected using quantitative PCR (qPCR), while Legionella was detected using both a culture method and qPCR. The four pathogens were identified in most samples at varying levels. Legionella pneumophila had a positivity rate of 92%, ranging from 2 to 5.4 log10 MPN/100 mL. Detection rates of Legionella spp., L. pneumophila, and L. pneumophila serogroup 1 were 97%, 75%, and 69%, respectively, with up to 5.3 log10 gene copies (GC)/mL. Importantly, this study demonstrates molecular typing of L. pneumophila serogroup 1 in wastewater, a topic that has been rarely documented. Mycobacterium spp. were detected in all samples at varying levels (log10 GC/mL) in influent (2.8–7.6), activated sludge (4.8–8.9), and SST effluent (3.8–8.9) samples. Arcobacter butzleri and A. hydrophila were detected in 96% and 82% of the samples, respectively, with GC levels in influent, activated sludge, and SST effluent ranging from 0.8 to 6.6, 1.5 to 6.5, and 0.7 to 6.6 log10 GC/mL for A. butzleri, and similar levels for A. hydrophila. These findings underscore the presence of respiratory and enteric pathogens at various treatment points, suggesting potential occupational exposure for WWTP workers. This emphasises the need for microbiological risk assessments (RAs) or reviewing existing RAs and implementing necessary control measures to protect WWTP workers.
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