Digital PCR-based assessment of pathogens in wastewater and antibiotic resistance genes in drinking water of the Kathmandu Valley, Nepal.

Abstract

Digital polymerase chain reaction (dPCR) is a significant advancement in health-related water microbiology, enabling absolute quantification without standard curves. By partitioning samples into thousands of individual reactions, dPCR allows for precise quantification even in the presence of inhibitory substances common in environmental samples. This study evaluated the applicability of dPCR to detect gastroenteritis-causing enteropathogens (Salmonella spp., Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, JC and BK polyomaviruses, and human adenovirus), crAssphage, and four antibiotic resistance genes (ARGs) (sul1, bla_NDM-1, bla_CTX-M, and intI1) in wastewater and drinking water source samples. Wastewater samples were collected in 2018 from two municipal wastewater treatment plants (WWTPs) that used an oxidation ditch system (n = 12) and a stabilization pond system (n = 10) in the Kathmandu Valley, Nepal, whereas the drinking water source samples were collected from shallow wells between 2015 and 2016 (n = 22). The enteropathogens and ARGs were analyzed using the QIAcuity dPCR System. The highest detection ratio was observed by crAssphage in wastewater (100 %, 22/22) and by sul1 and intl1 in drinking water sources (91 %, 20/22). The log₁₀ reduction values evaluated in both WWTPs were <1 using dPCR, consistent with those obtained in previous quantitative PCR studies, and may be used to cross-validate between methods. However, this study also observed low detection ratios and concentrations of enteropathogens, likely due to factors such as low sample volumes, dead volume, thermal conditions, and the dPCR platform used. Thus, optimizing these variables is imperative to enhance the applicability of dPCR in environmental assessments.