Tracking antibiotic resistome during wastewater treatment using high throughput quantitative PCR
Introduction
Antibiotic resistant pathogens have posed serious threat to human health worldwide owing to the increasing infection and mortality as well as treatment cost (Andersson and Hughes, 2010). Municipal wastewater treatment plants (WWTPs) are significant reservoirs for releasing antibiotic resistant bacteria and resistance determinants into environments (Chen et al., 2016; LaPara et al., 2011; Negreanu et al., 2012). WWTPs receive sewages from various sources (e.g. hospitals, industries and households) (Mao et al., 2015; Pruden et al., 2013), of which human commensal microorganisms are the main bacterial inputs to WWTPs. A proportion of human commensal microorganisms are resistant to antibiotics and thus resistant bacteria and resistance genes might arise in WWTPs. WWTPs are also considered as hotspots for horizontal gene transfer (HGT), enabling the development and dissemination of ARGs between bacteria (Karkman et al., 2016; Rizzo et al., 2013). High density of bacteria from different sources can interact and exchange resistance genes via HGT. In addition, chemical compounds (e.g. antibiotics and heavy metals) with varying concentrations can provide persistent pressure for selecting resistant bacteria, facilitating emergence of novel resistance determinants (LaPara et al., 2011; Novo and Manaia, 2010). Discharge of treated wastewater and sludge would lead to release of ARGs to downstream environments (Szczepanowski et al., 2009; LaPara et al., 2011; Caucci and Berendonk, 2014). In this regard, WWTPs link human activities and the environment, contributing to the occurrence, spread and persistence of resistant bacteria and ARGs in the environments (Chen et al., 2016; LaPara et al., 2011; Negreanu et al., 2012).
Previous efforts in investigating the occurrence and abundance of resistance genes in WWTPs indicated the removal efficiencies varied for different ARGs. Although most of studies have reported wastewater treatment can efficiently reduce the abundance of resistance genes (Diehl and LaPara, 2010; Novo et al., 2013; Zhang et al., 2009), there are reports showing that the effect of wastewater treatment on the relative abundance of ARGs is little and some resistance genes can even be enriched in effluent (Mao et al., 2015; Yang et al., 2014). This discrepancy may be partially due to the limited number of targeted ARGs. Profiling the dynamics of extensive number of ARGs during wastewater treatment process is critical for a comprehensive evaluation on the removal of resistant determinants. Metagenomic analysis and high-throughput qPCR array have been adopted to characterize ARGs in a limited number of WWTP(s), showing that the relative abundance of resistance genes varies with seasons and antibiotics in WWTPs show no direct selection for resistance genes (Bengtsson-Palme et al., 2016; Karkman et al., 2016; Yang et al., 2014). WWTPs receive wastewater from various sources daily with diverse bacterial communities and chemical components (Caucci et al., 2016; Sahoo et al., 2010), and thus the resistant bacteria loads during wastewater treatment might also change accordingly. However, the spatial and temporal distributions of resistome and bacterial community during the complete wastewater treatment process were not yet well addressed, especially at a large scale sampling level.
In this study, we collected 114 samples including influent, activated sludge and effluent from eleven WWTPs in six Chinese coastal cities. Illumina sequencing of bacterial 16S rRNA gene and high-throughput quantitative PCR (HT-qPCR) were applied (An et al., 2018) to provide an overview of the abundance and the composition of antibiotic resistance genes during wastewater treatment process; and (Andersson and Hughes, 2010) to characterize the seasonal and geographical distributions of resistome and bacterial community. By examining eleven Chinese WWTPs, we expect to obtain an in-depth understanding of the resistome during wastewater treatment process, and to provide data for mitigation of ARGs in WWTPs.
Section snippets
Sample collection and DNA extraction
A total of 114 samples including influent (INF), activated sludge (AS, anoxic activated sludge and aerobic activated sludge), and effluent (EFF) were collected from eleven Chinese WWTPs in Hangzhou, Longyan, Xiamen, Nanjing, Shenzhen and Hong Kong in August 2014 (summer) and February 2014 (winter), respectively (Fig. S1 and Table S1). These WWTPs mainly treat municipal wastewater and use A/A/O (anaerobic/anoxic/aerobic) and oxidation ditch processes, in which two Hong Kong WWTPs (HK_ST and
Diversity and abundance of ARGs in WWTPs
In total, 211 ± 43 ARGs and 5 ± 1 MGEs (including integrase genes and transposase genes) were detected in all samples, where influent samples harbored 118 ± 41 ARGs, activated sludge had 95 ± 46 ARGs and effluent contained 105 ± 50 ARGs (Fig. S2a). Aminoglycoside, multidrug, tetracycline and beta-lactam resistance genes were the most abundant ARG types during treatment process, accounting for 68% to 97% of the total ARG concentrations (Fig. S3). Wastewater treatment significantly reduced the
Discussion
By using high throughput qPCR, we quantified antibiotic resistance genes in samples from eleven full-scale Chinese WWTPs using 295 sets of primers. To our best knowledge, this study provided by far the most comprehensive picture for ARG profiles in WWTPs. Seasonal and geographical distribution of ARGs profiles were observed in WWTP samples. We detected high abundance and diversity of ARGs and MGEs, some of which were persistent and enriched after wastewater treatment, potentially posing a risk
Conclusions
This study presented comprehensive evidences that wastewater treatment could significantly reduce the richness and abundance of ARGs. However, a substantial number of ARGs were detected in wastewater effluents, which could be consequently discharged into downstream environments. Aminoglycoside and beta-lactams resistance genes persisted in all WWTP samples and were closely associated with integron 1 integrase, suggesting the need for monitoring and mitigating these genes in WWTPs.
The following
Acknowledgements
We thank all the volunteers for help in collecting samples. This study was financially supported by the Natural Science Foundation of China (31722004), the National Key Research and Development Program of China-International collaborative project from Ministry of Science and Technology (2017YFE0107300), the Knowledge Innovation Program of the Chinese Academy of Sciences (IUEQN201504), K.C·Wong Education Foundation and Youth Innovation Promotion Association, CAS.
Notes
The authors declare no competing financial interest.
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These authors contributed equally to this work.