6-plex Crystal Digital PCR® for comprehensive surveillance of respiratory and foodborne bacterial pathogens in wastewater.

Bacterial wastewater surveillance (WS) is less explored area compared to viral WS despite high burden of bacterial respiratory and gastrointestinal infections worldwide. This study established a 6-plex Crystal Digital PCR® (cdPCR) system, to comprehensively monitor an acute respiratory pathogen - Group A Streptococcus (GAS) pyogenes, foodborne disease (FBD) pathogens - Clostridium perfringens, Salmonella spp., Campylobacter jejuni, and Campylobacter coli, and an indicator bacterium, Escherichia coli in wastewater. Fifty-two grab influent samples collected weekly from a wastewater treatment plant in Yamanashi Prefecture, Japan, between June 2023 and May 2024 were centrifuged, followed by DNA extraction and cdPCR. cdPCR was performed using the naica® system (Stilla Technologies). The 6-plex cdPCR assays showed strong performance. Among the 52 samples, 100 % of samples were positive for C. perfringens, 98 % for Salmonella spp., 56 % for C. jejuni, 25 % for C. coli, and 63 % for S. pyogenes, with concentrations ranging between 4.2 ± 0.3 to 7.5 ± 0.2 log10 copies/L. The concentration of C. perfringens was significantly higher than that of other pathogens (p < 0.05), indicating its dominance. Salmonella spp. had high detection rate, implying increased Salmonella infection in the population. Seasonal variation was not observed in any of FBD pathogens, except for the detection rate of C. coli. S. pyogenes concentrations were significantly higher in spring than in other seasons, agreeing with the trend of GAS pharyngitis cases in the catchment. In conclusion, the 6-plex cdPCR system is a valuable tool for comprehensive WS, offering significant implications for public health monitoring.