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.

Keywords: PMA treatment; amplicon sequencing; bacterial community; chlorination; effluent; indicator bacteria; pathogen; quantitative PCR.