Quantitative 16S rRNA Gene Amplicon Sequencing for Comprehensive Pathogenic Bacterial Tracking in a Municipal Wastewater Treatment Plant

Quantification of bacterial pathogens in the environment is crucial for determining their potential risk of pathogenicity and infection. Here, we applied quantitative sequencing (qSeq) based on the 16S rRNA gene with spike-in internal standards for comprehensive pathogen quantification in a municipal wastewater treatment plant (WWTP). A novel bacterial pathogen database based on biosafety levels (BSLs) was constructed for rapid pathogen identification. Pathogen taxonomies were obtained from the constructed database using a basic local alignment search tool, and amounts of pathogens were quantified by regression analysis of spike-in internal standards. The total pathogen concentrations were gradually decreased from 6.8 ± 4.9 × 107 copies/mL in the influent to 1.6 ± 1.4 × 105 copies/mL in the effluent. BSL2 pathogens (e.g., Aeromonas spp., Mycobacterium spp., Klebsiella spp., and pathogenic Escherichia spp.) were detected more than 4.0 × 103 copies/mL at the effluent. The genus Arcobacter (BSL1) showed the highest abundance throughout the WWTP (8.8 ± 8.1 × 104 to 2.5 ± 3.5 × 107 copies/mL). BSL2 Mycobacterium spp. noticeably increased at the anaerobic treatment (2.3 × 106 copies/mL) and showed least removal at the effluent. The qSeq analysis with the pathogen database provided a holistic view of the microbial pathogen dynamics and ecology in the WWTP.