Microbial source tracking using molecular and cultivable methods in a tropical mixed-use drinking water source to support water safety plans.

Microbial contamination deteriorates source water quality, posing a severe problem for drinking water suppliers worldwide and addressed by the Water Safety Plan framework to ensure high-quality and reliable drinking water. Microbial source tracking (MST) is used to examine different microbial pollution sources via host-specific intestinal markers for humans and different types of animals. However, the application of MST in tropical surface water catchments that provide raw water for drinking water supplies is limited. We analyzed a set of MST markers, namely, three cultivable bacteriophages and four molecular PCR and qPCR assays, together with 17 microbial and physicochemical parameters, to identify fecal pollution from general, human-, swine-, and cattle-specific sources. Seventy-two river water samples at six sampling sites were collected over 12 sampling events during wet and dry seasons. We found persistent fecal contamination via the general fecal marker GenBac3 (100 % detection; 2.10-5.42 log copies/100 mL), with humans (crAssphage; 74 % detection; 1.62-3.81 log copies/100 mL) and swine (Pig-2-Bac; 25 % detection; 1.92-2.91 log copies/100 mL). Higher contamination levels were observed during the wet season (p < 0.05). The conventional PCR screening used for the general and human markers showed 94.4 % and 69.8 % agreement with the respective qPCR results. Specifically, in the studied watershed, coliphage could be a screening parameter for the crAssphage marker (90.6 % and 73.7 % positive and negative predictive values; Spearman's rank correlation coefficient = 0.66; p < 0.001). The likelihood of detecting the crAssphage marker significantly increased when total and fecal coliforms exceeded 20,000 and 4000 MPN/100 mL, respectively, as Thailand Surface Water Quality Standards, with odds ratios and 95 % confidence intervals of 15.75 (4.43-55.98) and 5.65 (1.39-23.05). Our study confirms the potential benefits of incorporating MST monitoring into water safety plans, supporting the use of this approach to ensure high-quality drinking water supplies worldwide.