Development of a sensitive and false-positive free PMA-qPCR viability assay to quantify VBNC Escherichia coli and evaluate disinfection performance in wastewater effluent

- We developed a PMA-qPCR method to assess the VBNC E. coli in wastewater effluent.
- The qPCR protocol employs native Taq polymerase assuring no false positive signals.
- The use of native Taq allows for an improved detection limit.
- The method uses salmon testes DNA to validate PMA effectiveness.
- Using the method, we showed that high numbers of VBNC E. coli survive chlorination.

The detection and quantification of viable Escherichia coli cells in wastewater treatment plant effluent is very important as it is the main disinfection efficacy parameter for assessing its public health risk and environmental impact. The aim of this study was to develop a sensitive and false-positive free propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay to quantify the viable but non-culturable (VBNC) E. coli present in secondary wastewater effluent after chlorine disinfection. The qPCR target was the E. coli uidA gene, and native Taq was used to eliminate false positives caused by the presence of contaminant E. coli DNA in recombinant Taq polymerase reagents. Due to issues with qPCR inhibitors in wastewater, this study explored several pre-DNA extraction treatment methods for qPCR inhibitor removal. PMA-qPCR validation was done using salmon testes DNA (Sketa DNA) as an exogenous control added directly to the wastewater samples and amplified using a separate qPCR assay. After disinfection of secondary effluent with 2 ppm chlorine at the plant, the mean Log10 CFU reduction in E. coli was 2.85 from a mean CFU of 3.48/10 mL compared to 0.21 Log10 CCE mean reduction of the uidA gene from a mean CCE of 3.16/10 mL. The VBNC cell concentrations were calculated as 2.32 Log10/10 mL by subtracting the colony forming units (CFU) obtained from membrane filtration from the calculated CFU equivalent (CCE) values obtained from PMA-qPCR. These results demonstrate the effective use of a PMA-qPCR method for the quantification of the E. coli uidA gene and indicate there are high numbers (2.01 × 103 CCE/100 mL) of VBNC E. coli cells leaving the wastewater treatment plant in the final effluent after chlorine treatment. VBNC bacterial cells are of concern as they have the potential to resuscitate and grow, regain virulence, affect natural microbiome in the discharge sites, and pass on antimicrobial resistant genes to other microorganisms.