Strategy to Evaluate Changes in Bacterial Community Profiles and Bacterial Pathogen Load Reduction After Sewage Disinfection

Abstract

Sewage effluent discharge is a major source of pathogenic contamination to the environment. The disinfection process is critical for the elimination of pathogens in sewage. In this study, we examined the impact of chlorine disinfection on the total, viable, and culturable populations of indicator bacteria, pathogens, and bacterial communities in two contrasting types of effluents (primarily treated saline and secondarily treated freshwater). Effluents collected bimonthly over 1 year were examined using cultivation, quantitative PCR (qPCR), and 16S rRNA gene amplicon sequencing coupled with or without propidium monoazide (PMA) treatment. The results showed that each type of effluent was characterized by a specific set of representative genera before disinfection. Salinity appeared to be the major abiotic factor associated with the differences in bacterial community compositions. The pathogen analysis pipeline revealed over 20 viable clinically important pathogenic species in the effluents. Although the bacterial communities differed markedly between the two types of effluents before disinfection, the species of pathogens persisting after disinfection were similar, many of them were members of Enterobacter and Vibrio. The relative abundances of all pathogens identified in the amplicon sequences were multiplied by the 16S rRNA gene copy numbers of total bacteria detected by PMA-qPCR to estimate their concentrations. Pathogens remained viable after disinfection reached 8 log10 16S rRNA copies ml−1 effluent. Meanwhile, around 80 % of the populations of three indicator bacteria including Escherichia coli, Enterococcus, and Bacteroidales were viable after disinfection, but over 99 % of the viable E. coli and Enterococcus were in the non-culturable state. We estimated the total pathogen load by adding the concentrations of all viable pathogens and examined their correlations with indicator bacteria of different types, physiological states, and effluents. The results showed that the PMA-qPCR measurement of E. coli is a reliable proxy of bacterial pathogen loads in both types of effluents. The utility of viable indicator bacteria as a biological index to assess the overall bacteriological hazards in effluents is discussed.

Progress in methods for the detection of viable Escherichia coli

“…144 Tang et al found that PMA-qPCR was effective in detecting viable bacteria in chlorine-disinfected wastewater. 145…”

Effects of chlorination on the survival of sewage bacteria in seawater microcosms

“…Differences of less than 3.2 (equivalent to 91.9% efficiency) were regarded as amplification inhibition, and a higher level of dilution would be needed to alleviate the inhibition. The final gene copy numbers were corrected by the DNA extraction and PMA treatment efficiencies using the recovered gene copies of the extraction and PMA controls (Tang & Lau, 2022), calculated as below: $$$$ \mathrm{Extraction}\ \mathrm{efficiency}\left(\%\right)=\frac{\mathrm{Detected}\ \mathrm{TaqMan}\ \mathrm{control}\ \mathrm{DNA}\ \mathrm{copy}}{\mathrm{Spiked}\ \mathrm{TaqMan}\ \mathrm{control}\ \mathrm{DNA}\ \mathrm{copy}}\times 100\% $$$$ $$$$ \mathrm{PMA}\ \mathrm{efficiency}\left(\%\right)={\displaystyle \begin{array}{l}\left(1-\frac{\mathrm{Detected}\ \mathrm{salmon}\ \mathrm{DNA}\ \mathrm{copy}}{\mathrm{Spiked}\ \mathrm{salmon}\ \mathrm{DNA}\ \mathrm{copy}}\right)\\ {}\times 100\%\end{array}} $$$$ …”

“…To quantify the viable populations of total bacteria, Enterococcus and E. coli , the samples were treated with PMA before DNA extraction, following the procedures in our previous study (Tang & Lau, 2022 ). Briefly, triplicates of 500‐mL aliquots from each sample were centrifuged and resuspended in autoclaved PBS, then added with a final concentration of 100 μM of PMA.…”

“…Differences of less than 3.2 (equivalent to 91.9% efficiency) were regarded as amplification inhibition, and a higher level of dilution would be needed to alleviate the inhibition. The final gene copy numbers were corrected by the DNA extraction and PMA treatment efficiencies using the recovered gene copies of the extraction and PMA controls (Tang & Lau, 2022 ), calculated as below: …”

“…The incomplete removal of bacteria after disinfection may arise from practical considerations, such as the cost of disinfection processes, formation of disinfection byproducts and types of effluent reuse. Additionally, unintended consequences of microbial disinfectant resistance, biofilm formation on distribution pipes, outdated discharge standards and monitoring schemes may also contribute to the persistence of bacteria (Chandy & Angles, 2001 ; Naidoo & Olaniran, 2013 ; Tang & Lau, 2022 ; Xie et al, 2022 ). Therefore, designing an effective disinfection scheme requires careful consideration of the intended outcomes and in‐depth understanding of microbial responses.…”