Accepted Manuscript New Insights into Antibiotic Resistome in Drinking Water and Management Perspectives: A Metagenomic based Study of Small-sized Microbes Liping Ma, Bing Li, Tong Zhang PII:
S0043-1354(19)30030-2
DOI:
https://doi.org/10.1016/j.watres.2018.12.069
Reference:
WR 14368
To appear in:
Water Research
Received Date: 7 June 2018 Revised Date:
18 December 2018
Accepted Date: 22 December 2018
Please cite this article as: Ma, L., Li, B., Zhang, T., New Insights into Antibiotic Resistome in Drinking Water and Management Perspectives: A Metagenomic based Study of Small-sized Microbes, Water Research, https://doi.org/10.1016/j.watres.2018.12.069. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphic Abstract
Drinking Water
ARG Risks
Antimicrobial Resistome Profiling
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Pathogenic Bacteria Tracking
Pathogenic Host Identification Two-step Filtration
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Pore Size = 0.45 μm Pore Size = 0.2 μm
Uncultivated Small-sized Microbes
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(0.2–0.45 μm)
Public Health
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New Insights into Antibiotic Resistome in Drinking Water and
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Management Perspectives: A Metagenomic based Study of
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Small-sized Microbes
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Authors: Liping Ma1,2, Bing Li3 and Tong Zhang1,4,5*
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Author affiliation:
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Environmental Biotechnology Laboratory, The University of Hong Kong, Hong Kong
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Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, School
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of Ecological and Environmental Sciences, East China Normal University, Shanghai,
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China
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Environmental Safety, Graduate School at Shenzhen, Tsinghua University, China
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School of Public Health, The University of Hong Kong, Hong Kong
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International Center for Antibiotic Resistance in the Environment, School of
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Environmental Science and Engineering, Southern University of Science and Technology,
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Shenzhen, China
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Corresponding author:
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Prof. Tong Zhang
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Environmental Biotechnology Lab, The University of Hong Kong, Pokfulam Road, Hong
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Kong
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Email:
[email protected];
[email protected]
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Tel: +852-28578551
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Fax: +852-28598987
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Guangdong Provincial Engineering Research Center for Urban Water Recycling and
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Abstract
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The proliferation of antibiotic resistance genes (ARGs) in drinking water and their
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potential horizontal transfer to pathogenic microbes may cause failure of antibiotics.
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However, antimicrobial resistome monitoring in drinking water is not currently routine.
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The bacterial hosts of ARGs, especially small-sized microbes in drinking water, may not
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be effectively removed by membrane filtration disinfection and thus pose threats to
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human health. In the present study, using metagenomic based approach, we investigated
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antibiotic resistome of small-sized microbes (0.2–0.45 µm) in 20 household drinking
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water samples from 12 cities in Mainland China, Hong Kong and Singapore. A total of
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265 ARG subtypes belonging to 17 ARG types were detected at abundances ranging
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from 4.0×10-2 to 1.0×100 copies/cell. Multidrug, bacitracin and aminoglycoside resistance
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genes are dominant, and 43 ARG subtypes were specifically carried by small-size
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microbes. Metagenomic assembly strategy revealed fragments of three opportunistic
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pathogen, i.e. Pseudomonas alcaligenes, Pseudomonas aeruginosa and Mycobacterium
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gordonae, carried mexW, aph(3')-I and aac(2')-I, respectively. Drinking water samples
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were classified into three groups based on the presence of ARG, pathogen and ARG-
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carrying pathogen. These new insights into the antibiotic resistome of small microbes in
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drinking water over a broad scale indicate the need for more comprehensive ARGs
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monitoring and surveillance of drinking water supplies. These findings, together with the
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perspectives and strategies proposed in this study, could support initiatives to improve
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drinking water safety.
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Keywords: drinking water, public health, antibiotic resistome, pathogenic host,
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metagenomic sequencing
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1. Introduction
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Increases in the selective pressure to develop resistance to antibiotics used in human
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medicine and livestock breeding have led to the wide distribution and exacerbation of
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antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) throughout
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both natural and man-made environments (Nesme et al. 2014, Rodriguez-Mozaz et al.
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2015). Antimicrobial resistance has thus become a global issue and is considered an
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emerging environmental pollutant (UNEP 2017) that may pose risks to public health
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(WHO 2018). Nevertheless, most relevant research efforts have focused on the sources of
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ARGs, such as hospital environments and sewage (Ashbolt et al. 2013, Le Page et al.
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2017), or management options (e.g., nutrient management, runoff control and
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infrastructure upgrades) with the aim of reducing the emission of ARGs into the
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environment (Pruden et al. 2013). To date, however, inadequate strategies have been
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proposed to monitor and manage ARGs in household drinking water, which potentially
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pose direct threats to human health (Su et al. 2018). Remarkably, although the presence
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of ARGs is a health threat, a more critical issue related to ARGs is the potential
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horizontal transfer of ARGs to pathogenic microbes as the statement implies (Ashbolt
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2015). Accordingly, these implied potential risks make it imperative to better integrate
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monitoring efforts and risk management strategies into public health decision-making to
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enhance drinking water safety.
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Previous studies of ARGs in drinking water have mainly used conventional methods,
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such as bacterial isolation (Vaz-Moreira et al. 2017), PCR (Figueira et al. 2011) and
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qPCR (Su et al. 2018). In the past decade, advances in genomic technologies have
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yielded novel approaches and new visions for environmental health monitoring and ARG
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risk assessment (Cote et al. 2016). The sequencing of whole microbial communities has
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revealed genomic information from the vast majority of unculturable bacteria (Port et al.
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2014). Given the potential health risks associated with drinking water, researchers have
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recently attempted to clarify the antibiotic resistomes (collection of all the ARGs) of
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drinking water microbes using metagenomic sequencing technique (Chao et al. 2013, Shi
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et al. 2013). However, current studies on this resistome have only focused on microbes
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larger than 0.45 µm (Chao et al. 2013, Ma et al. 2017), and no report has addressed the
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resistome of small microbes (50% of the ORFs on a contig were annotated
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as the same taxon, this contig was then assigned to that taxon (Ishii et al. 2013). The
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average coverage, indicating abundance (×/Gb), of each ARG-carrying contig was
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quantified by mapping metagenomic sequencing reads to the gene cassette using CLC
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Genomics Workbench with a minimum similarity of 95% and over 95% of the read
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length. The species-level taxonomic list of ACCs was then further compared with the
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summarized pathogen list proposed in a previous study to identify ARG-carrying
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pathogen (Li et al. 2015a). Furthermore, virulence factors carried by ARG-carrying
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pathogen was screened by using VFDB (Virulence Factors of Pathogenic Bacteria) based
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online annotation pipeline VFanalyzer (Chen et al. 2016).
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2.5. Pathogen tracking from small-sized microbes
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To identify pathogen from small-sized microbes in drinking water, MetaPhlAn2
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(Metagenomic Phylogenetic Analysis) (Truong et al. 2015) was used to conduct
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taxonomic profiling of metagenomic sequences. The species taxa and corresponding
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abundances were subsequently extracted from the obtained taxonomic profiles. The
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species-level taxonomic list was then further compared with a previously summarized
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pathogen list (Li et al. 2015a), to identify small-sized pathogen in the drinking water
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samples.
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2.6. qPCR
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Abundance of important ARGs with prevalence in all samples were further quantified by
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using quantitative real time PCR (qPCR) assay. The unit of ARGs were transformed into
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relative abundance by normalizing to 16S rRNA gene, as copy of ARG per 16S rRNA
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gene. Primers of ARGs and 16S rRNA gene were listed in supplementary materials Table
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S9. Each 20 µL qPCR reaction consisted of 10 µL 2× SYBR® Green Master Mixes (Bio-
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Rad, Hercules, California), 0.5 µM each primer, 5 ng DNA as template and 7 µL
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nuclease-free PCR-grade water. The thermal cycle consisted of a 3-min initial enzyme
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activation at 95 °C, followed by 45 cycles of denaturation at 95 °C for 30 s, annealing at
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60 °C for 30 s and extension 72 °C for 60 s. Reactions were performed on a Bio-Rad
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CFX96 Connect™ Real-Time PCR Detection System and quantified with Bio-Rad CFX
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manager software (Bio-Rad, Hercules, California). All qPCRs were performed in
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technical triplicates with negative controls. Specific amplification of qPCR was
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confirmed by checking the melting curve of the target gene in each sample.
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3. Results and discussion
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3.1. Antibiotic resistome of small-sized microbes in drinking water
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First, a metagenomic technique based on the structured SARG database was used to
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profile the antibiotic resistome of small-sized microbes in drinking water and present a
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comprehensive spectrum of the ARGs carried by these microbes that had
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opportunistically passed through membrane disinfection and thus represent a direct risk
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to human health via drinking water. A total of 265 ARG subtypes classified into 17 ARG
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types were detected in small-sized microbes from the 20 drinking water samples collected
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in 12 cities (Fig. 1, Table 1), with total abundances ranging from 4.0×10-2 to 1.0×100
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copies/cell. Until now, very limited studies had been employed to show the abundance of
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ARGs in household drinking water/tap water, because of its low biomass and limitation
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of sample collection. Among the limited previous studies, Xu et al., 2016 used high-
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throughput qPCR to quantify diverse ARGs in tap water, and the relative abundance of
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ARGs normalizing to cell number ranged from 5.75×10-1 to 8.25×10-1 copies/cell. This is
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in the range of our detection with 4.0×10-2 to 1.0×100 copies/cell.
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Genes encoding multidrug- (1.6×10-2 to 5.0×10-1 copies/cell), bacitracin- (1.4×10-2 to
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2.6×10-1 copy/cell) and beta-lactam-resistance (2.6×10-4 to 1.2×10-1 copies/cell) were
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detected at the highest levels of abundance in these microbes. Furthermore, these top 3
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ARG types, together with genes conferring resistance to macrolide-lincosamide-
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streptogramin (MLS), aminoglycoside, sulfonamide and fosmidomycin, were detected as
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general ARG types in small-sized microbes from all samples. Among the 265 ARG
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subtypes, bacA (bacitracin) and mexF (multidrug) were dominant, with respective
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abundances of 1.4×10-2 to 2.4×10-1 and 1.5×10-3 to 6.5×10-2 copies/cell in the drinking
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water samples.
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Fig. 2 presents the profiles of 56 ARG subtypes carried by small-sized microbes in
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drinking water samples with average abundances of >1.0×10-3 copies/cell. Notably, 27
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ARG subtypes were prevalent in the drinking water samples from all cities. These
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encoded resistance to bacitracin (bacA and bcrA), fosmidomycin (rosA and rosB), MLS
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(macB and macA), multidrug (acrB, acrF, adeB, amrB, bpeF, ceoB, emrB, mdtB, mdtC,
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mdtF, mexB, mexF, mexT, mexW, multidrug ABC transporter, multidrug transporter,
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ompR, oprC), polymyxin (arnA), sulfonamide (sul1) and vancomycin (vanR). Among
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them, 17 ARG subtypes were defined as generalist ARG subtypes present in all samples
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(Fig. 2).
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Moreover, primers of 4 generalist ARGs (bacA, acrB, mexF and sul1) were available in
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previous studies by literature search, and seven samples were randomly selected to
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further verify their prevalence by using qPCR method. All the examined generalist ARGs
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were detected in these samples (supplementary materials Table S10). mexF and sul1 had
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similar abundances comparing with using metagenomic based approach, while bacA and
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acrB were averagely ~100 times less. The metagenomic based ARG detection mainly
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relies on ARG database, and therefore the most complete ARG database SARG v2.0
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(containing 23 ARG types, 1227 ARG subtypes and 12307 reference sequences) was
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used in the present study. In SARG v2.0 database, there are totally 241 non-redundant
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bacA sequences and 117 acrB sequences. Thus, it covers more sequence diversity, not
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only to annotate/amplify for specific sequence or region of target genes.The generalist
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ARG types, namely, bacitracin, multidrug, sulfonamide, beta-lactam and aminoglycoside,
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have previously been reported to be relatively highly abundant in drinking water
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metagenomes (Li et al. 2015b, Xu et al. 2016). Bacterial strains isolated from drinking
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water were also frequently reported to be resistant to these antibiotics (Figueira et al.
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2011, Xi et al. 2009). At the ARG subtype level, bacA, macB, acrB and sul1 have
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previously been detected in drinking water samples (Ma et al. 2017), whereas some of the
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detected ARG subtypes, such as blaLCR-1, blaLRA-17 and blaOXA-142, were
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identified in drinking water for the first time. As this is the first large-scale study to
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determine the dominant and prevalent antibiotic resistome among small-sized microbes in
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drinking water samples, we aimed to further clarify whether some of the detected ARGs
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were specifically carried by specific small-sized microbes in drinking water, and how
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these might pose risks to human health.
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3.2. Specific ARGs in small-sized microbes in drinking water
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To reveal specific ARG patterns in small-sized microbes collected from drinking water,
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we compared the ARG profile with that of large-sized microbes (>0.45 µm) in the same
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samples. Notably, the abundance of ARGs carried by small-sized microbes (4.0×10-2 to
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1.0×100 copies/cell) was, on average, 7.4% less than that carried by large-sized microbes
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(4.9×10-2 to 1.2×100 copies/cell) (P
