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

Abstract

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.

Quantitative detection and reduction of potentially pathogenic bacterial groups of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex, and Mycobacterium in wastewater treatment facilities

“…The relative abundance of the genus Arcobacter (the sum of the relative abundances of the five above-mentioned Arcobacteraceae genera in the reference database) was markedly high (≥ 5.0%) in seven out of the nine analyzed samples, indicating the presence of Arcobacter as a dominant prokaryotic genus in most of the analyzed wastewater influent samples. The relatively high abundance of the genus Arcobacter in the influent wastewater prokaryotic communities is in good agreement with the previously reported 16S rRNA gene-targeted amplicon sequencing data [ 8 – 10 ]. Although the reason for the particularly high abundance of the genus Arcobacter in various influent wastewater samples is still under debate, a compelling explanation is their ability to grow in hydrogen sulfide-rich and oxygen-limited sewer pipe environments [ 65 ].…”

“…Recent advances in high-throughput DNA sequencing approaches (e.g., 16S ribosomal RNA [rRNA] gene amplicon sequencing and metagenomic sequencing) have enabled researchers to reveal the phylogenetic diversity, relative abundance, and putative pathogenic traits of potentially pathogenic bacteria in wastewater environments [ 7 – 10 ]. Representative and frequently observed potentially pathogenic bacteria in wastewater, identified by conventional cultivation and high-throughput DNA sequencing approaches, include those from the genera Aeromonas , Arcobacter , Klebsiella , and Mycobacterium [ 7 – 11 ].…”

“…Representative and frequently observed potentially pathogenic bacteria in wastewater, identified by conventional cultivation and high-throughput DNA sequencing approaches, include those from the genera Aeromonas , Arcobacter , Klebsiella , and Mycobacterium [ 7 – 11 ]. Galagoda et al recently investigated the dynamics of potentially pathogenic bacteria in a Japanese municipal WTF using quantitative 16S rRNA gene sequencing and a novel pathogen database [ 10 ]. While 69 potentially pathogenic bacterial genera were detected in the influent wastewater, only 13 potentially pathogenic bacterial genera, including abundant levels of Aeromonas , Arcobacter , Klebsiella , and Mycobacterium , were quantitatively detected in the chlorine-disinfected, treated effluent.…”

“…These data indicate the potential importance of better understanding the abundance and persistence of these potentially pathogenic bacteria in WTFs to reduce health risks associated with treated wastewater. However, to the best of our knowledge, the group-level abundance and persistence of Aeromonas , Arcobacter , Klebsiella , and Mycobacterium in the existing WTFs in East Asian regions remain largely unknown owing to limited investigation [ 10 , 12 ].…”

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes

“…In recent years, quantitative sequencing methods have been developed to spike microbial DNA (Lin et al, 2018; Smets et al, 2016) or viable bacteria absent in the sample (Stämmler et al, 2016), or to add synthetic 16S rRNA (Tourlousse et al, 2017) or 16S‐18S‐internal transcribed spacer regions (Tkacz et al, 2018; Wang et al, 2021) as standards for high‐throughput quantitative microbial community analysis. These quantitative sequencing methods can be divided into two quantitative patterns: (i) the total number of a target gene in a sample is calculated from the internal standard gene (ISG) read ratio and the total number of ISG molecules added to the sample (Galagoda et al, 2023; Lin et al, 2018; Smets et al, 2016; Stämmler et al, 2016; Tkacz et al, 2018) or (ii) the target gene read numbers are converted to copy numbers using a dose–response curve as an internal standard curve, which is estimated based on the ISG read numbers with known gene copy numbers (Tourlousse et al, 2017; Wang et al, 2021). However, neither method is widespread enough to replace qPCR.…”