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

PLOS ONE RESEARCH ARTICLE Quantitative detection and reduction of potentially pathogenic bacterial groups of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex, and Mycobacterium in wastewater treatment facilities Masataka Aoki ID1, Yasuyuki Takemura1¤, Shuji Kawakami2, Wilasinee Yoochatchaval3, Thao Tran P. ID1, Noriko Tomioka1, Yoshitaka Ebie ID4, Kazuaki Syutsubo ID1,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Regional Environment Conservation Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan, 2 Department of Civil Engineering, National Institute of Technology (KOSEN), Nagaoka College, Nagaoka, Niigata, Japan, 3 Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, Bangkok, Thailand, 4 Material Cycles Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan, 5 Research Center of Water Environment Technology, School of Engineering, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ¤ Current address: Department of Civil Engineering, National Institute of Technology (KOSEN), Wakayama College, Gobo, Wakayama, Japan * OPEN ACCESS Citation: Aoki M, Takemura Y, Kawakami S, Yoochatchaval W, Tran P. T, Tomioka N, et al. (2023) Quantitative detection and reduction of potentially pathogenic bacterial groups of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex, and Mycobacterium in wastewater treatment facilities. PLoS ONE 18(9): e0291742. https://doi.org/10.1371/journal. pone.0291742 Editor: Zhi Zhou, Purdue University, UNITED STATES Received: May 29, 2023 Accepted: September 5, 2023 Published: September 28, 2023 Copyright: © 2023 Aoki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The obtained raw 16S rRNA gene amplicon sequence data are available in the DNA Data Bank of Japan (DDBJ) Sequence Read Archive (DRA) (https://ddbj.nig.ac. jp) under accession numbers DRR457499– DRR457516. All other relevant data are within the paper and its Supporting information files. Abstract Water quality parameters influence the abundance of pathogenic bacteria. The genera Aeromonas, Arcobacter, Klebsiella, and Mycobacterium are among the representative pathogenic bacteria identified in wastewater. However, information on the correlations between water quality and the abundance of these bacteria, as well as their reduction rate in existing wastewater treatment facilities (WTFs), is lacking. Hence, this study aimed to determine the abundance and reduction rates of these bacterial groups in WTFs. Sixty-eight samples (34 influent and 34 non-disinfected, treated, effluent samples) were collected from nine WTFs in Japan and Thailand. 16S rRNA gene amplicon sequencing analysis revealed the presence of Aeromonas, Arcobacter, and Mycobacterium in all influent wastewater and treated effluent samples. Quantitative real-time polymerase chain reaction (qPCR) was used to quantify the abundance of Aeromonas, Arcobacter, Klebsiella pneumoniae species complex (KpSC), and Mycobacterium. The geometric mean abundances of Aeromonas, Arcobacter, KpSC, and Mycobacterium in the influent wastewater were 1.2 × 104–2.4 × 105, 1.0 × 105– 4.5 × 106, 3.6 × 102–4.3 × 104, and 6.9 × 103–5.5 × 104 cells mL−1, respectively, and their average log reduction values were 0.77–2.57, 1.00–3.06, 1.35–3.11, and −0.67–1.57, respectively. Spearman’s rank correlation coefficients indicated significant positive or negative correlations between the abundances of the potentially pathogenic bacterial groups and Escherichia coli as well as water quality parameters, namely, chemical/biochemical oxygen demand, total nitrogen, nitrate-nitrogen, nitrite-nitrogen, ammonium-nitrogen, suspended solids, volatile suspended solids, and oxidation-reduction potential. This study provides PLOS ONE | https://doi.org/10.1371/journal.pone.0291742 September 28, 2023 1 / 25 PLOS ONE Funding: This study was financially supported by Internal Competitive Research Funding (Type A) of the National Institute for Environmental Studies (to MA, YT, NT, YE, and KS) (https://www.nies.go.jp/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Quantification and reduction of potentially pathogenic bacterial groups in wastewater treatment facilities valuable information on the development and appropriate management of WTFs to produce safe, hygienic water. Introduction Wastewater treatment facilities (WTFs) are vital components in reducing pathogens in untreated wastewater [1]. The use of reclaimed water derived from treated wastewater for agricultural irrigation; environmental enhancement; groundwater recharge; portable reuse; and industrial, recreational, and urban use is gaining attention owing to rapid urbanization, especially in developing countries, and the increasing global water scarcity [2]. Jones et al. estimated that 40.7 × 109 m3 year−1 of treated wastewater is intentionally reused for human purposes [3]. A major concern regarding the discharge of wastewater into the environment and the use of treated wastewater is the inadequate removal of pathogenic microorganisms. Active pathogenic microorganisms in biologically treated wastewater can be effectively removed using ultraviolet light, ozonation, or chlorination disinfection [4]. Although chlorination disinfection is the most common and cost-effective option, it produces mutagenic/carcinogenic disinfection by-products, such as trihalomethanes and haloacetic acids [5]. As high pathogen reduction rates lead to low final disinfectant demand [6], the development and appropriate management of WTFs with high pathogen reduction rates are crucial to reduce not only disinfection costs but also the formation of disinfection by-products. 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]. 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 pote (...truncated)