Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR(PMA-qPCR)

The detection of viable bacteria in wastewater treatment plants （WWTPs） is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide （PMA） combined with the quantitative polymerase chain reaction （PMA-qPCR） to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.

Microbiological quality of roof tank water in an urban village in southeastern China

Urban villages are unique residential neighborhoods in urban areas in China. Roof tanks are their main form of water supply, and water quality deterioration might occur in this system because of poor hygienic conditions and maintenance. In this study, water samples were seasonally collected from an urban village to investigate the influence of roof tanks as an additional water storage device on the variation in the microbial community structure and pathogenic gene markers. Water stagnation in the roof tank induced significant decreases in chlorine(p < 0.05), residual chlorine was as low as 0.02 mg/L in spring. Propidium monoazide(PMA)-qPCR revealed a one-magnitude higher level of total viable bacterial concentration in roof tank water samples(2.14 ± 1.81 × 105gene copies/mL) than that in input water samples(3.57 ± 2.90 × 104gene copies/mL, p < 0.05), especially in spring and summer. In addition,pathogenic fungi, Mycobacterium spp., and Legionella spp. were frequently detected in the roof tanks. Terminal users might be exposed to higher microbial risk induced by high abundance of Legionella gene marker. Spearman’s rank correlation and redundancy analysis showed that residual chlorine was the driving force that promoted bacterial colonization and shaped the microbial community. It is worth noted that the sediment in the pipe will be agitated when the water supply is restored after the water outages, which can trigger an increase in turbidity and bacterial biomass. Overall, the findings provide practical suggestions for controlling microbiological health risks in roof tanks in urban villages.