Metagenomic insights into chlorination effects on microbial antibiotic resistance in drinking water
Graphical abstract
Highlights
► Metagenomic analysis reveals prevalence of antibiotic resistance genes in drinking water. ► Chlorination helps to concentrate antibiotic resistance genes in drinking water. ► Pipeline transportation can alleviate antibiotic resistance in the chlorinated water.
Introduction
The prevalence of antibiotic resistant bacteria (ARB) and resistance genes (ARGs) has conferred enormous and complicated impacts on environmental safety (Taylor et al., 2011) and human health (Walsh et al., 2011). Growing evidence suggests that drinking water is a reservoir for ARB and ARGs (Armstrong et al., 1981, Schwartz et al., 2003), thus the induced public health problems may receive great concerns (Ram et al., 2008). Notably, previous studies have indicated that chlorination, a commonly used disinfection technology in drinking water or wastewater treatment, can contribute to the enrichment of ARB (Armstrong et al., 1982) and spread of ARGs (Xi et al., 2009). Cross- or co-resistance of disinfectants and ARGs might be the underlying mechanisms responsible for the antibiotic resistance promotion (Dukan and Touati, 1996, Greenberg et al., 1990, Nakajima et al., 1995). Mobile genetic elements (MGEs) including plasmids, integrons and insertion sequences act as facilitators in the prevalence of ARB and ARGs in environment (Wright et al., 2008, Ciric et al., 2011), since bacteria can capture different ARGs housed on MGEs through horizontal transfer (Martinez, 2008). Furthermore, the mobile resistome can easily spread among species including human pathogens, which deserves more public health concerns (Canton, 2009, Nikaido, 2009).
Currently, efforts are focused on the isolation of ARB from drinking water, while few studies have been conducted based on the metagenome DNA extraction due to the extremely low biomass and high microbial diversity (Lebkowska, 2009). However, comprehensive characterization of ARB and ARGs in natural environment or artificial compartments using culture-dependent methods remains challenging since most of environmental bacteria are unculturable (Schmieder and Edwards, 2012). Metagenomic analysis combined with high-throughput sequencing is considered as a promising tool for analysis of ARGs diversity (Zhang et al., 2011) and abundance (Uyaguari et al., 2011), as well as discoveries of novel genes (Torres-Cortes et al., 2011), since this method has been applied to detect ARGs in various environmental compartments including soils (Monier et al., 2011), activated sludge (Parsley et al., 2010), sediments (Kristiansson et al., 2011) and feces (Durso et al., 2011).
In this study, culture-independent methods including high-throughput sequencing and quantitative real-time PCR (q-PCR) were used for comprehensive assessment of chlorination effects on microbial resistance patterns in drinking water. This is the first study investigating the abundance and diversity of ARGs and MGEs in drinking water using metagenomic methods based on Illumina high-throughput sequencing. PCRs and q-PCRs were also performed to determine the effects of chlorination on occurrence and abundance of various ARGs. Meanwhile, we also characterized the antibiotic resistance phenotypes and genotypes of the bacterial isolates from drinking water after microbial community structure analysis. The results of this study may help to extend our knowledge about the antibiotic resistance in drinking water treatment system which might be practically useful for detecting the ARGs in water environments.
Section snippets
Water sampling and DNA extraction
As illustrated in Figure S1, filtered water (FW) and chlorine-disinfected water (DW) were simultaneously sampled from chlorination tank and clear water tank of Beihekou Tap Water Plant (Nanjing, China), and tap water (TW) was also sampled at Environmental Biotechnology Laboratory of Nanjing University (Nanjing, China) at the same time. To avoid the temporal variation, the water sampling was repeated for four times separately in August, September, October and November of 2011. The information
Chlorination effect on antibiotic resistance pattern of intestinal bacteria in drinking water
Phylogenic analysis on the bacterial clones showed that Escherichia dominated in FW (63.0%), followed by Enterobacter (18.5%), Klebsiella (16.7%) and Acinetobacter (1.9%) (Figure S2). After chlorine disinfection in the water treatment plant, Pseudomonas was enriched in DW (27.8%). Drinking water pipeline transportation further enhanced the relative abundance of Pseudomonas (63.0%) and Citrobacter (33.3%) in TW. Antibiotic susceptibility analyses demonstrated that FW, DW and TW contained the
Discussion
In this study, intestinal bacteria were isolated from FW, DW and TW to preliminarily investigate the antibiotic resistance patterns in drinking water. Antibiotic susceptibility assay suggested elevated resistance to several tested antibiotics after chlorine disinfection of drinking water. These results are not completely consistent with the PCRs with environmental DNA as template due to unfeasibility for culture of most environmental bacteria, the diversiform genetic determinants for antibiotic
Conclusions
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Results of PCR, high-throughput sequencing, metagenomic analysis and culture-dependent methods reveal prevalence of a variety of ARB and ARGs in drinking water.
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Chlorine disinfection can greatly affect the microbial structure and cause enrichment of ARB, ARGs and MGEs in drinking water. Plasmid over-replication, microbial community shift and multidrug efflux pumps-mediated co-resistance might be responsible for the enrichment of ARGs under chlorine stress.
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Although water pipeline transportation
Acknowledgments
This study was financially supported by National Natural Science Foundation of China (51278240, 50938004 and 51008153) and Program for Changjiang Scholars and Innovative Research Team in University of China.
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