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Characterization of Natural and Affected Environments

Metagenomic characterization of antibiotic resistance genes in full-scale reclaimed water distribution systems and corresponding potable systems Emily Garner, Chaoqi Chen, Kang Xia, Jolene Bowers, David Engelthaler, Jean McLain, Marc A. Edwards, and Amy Pruden Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05419 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 9, 2018

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Environmental Science & Technology

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Title: Metagenomic characterization of antibiotic resistance genes in full-scale reclaimed water

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distribution systems and corresponding potable systems

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Authors: Emily Garner1, Chaoqi Chen2, Kang Xia2, Jolene Bowers3, David M. Engelthaler3, Jean

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McLain4, Marc A. Edwards1, Amy Pruden1*

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1

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24061, United States

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2

Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, Virginia

Department of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, Virginia

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24061, United States

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Translational Genomics Research Institute, Flagstaff, Arizona 86005, United States

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Water Resources Research Center, University of Arizona, Tucson, Arizona 85719, United

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States

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*Corresponding Author (e-mail: [email protected])

1 ACS Paragon Plus Environment


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Abstract

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Water reclamation provides a valuable resource for meeting non-potable water demands.

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However, little is known about the potential for wastewater reuse to disseminate antibiotic

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resistance genes (ARGs). Here, samples were collected seasonally in 2014-2015 from four U.S.

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utilities’ reclaimed and potable water distribution systems before treatment, after treatment, and

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at five points of use (POU). Shotgun metagenomic sequencing was used to profile the resistome

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(i.e., full contingent of ARGs) of a subset (n=38) of samples. Four ARGs (qnrA, blaTEM, vanA,

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sul1) were quantified by quantitative polymerase chain reaction. Bacterial community

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composition (via 16S rRNA gene amplicon sequencing), horizontal gene transfer (via

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quantification of intI1 integrase and plasmid genes), and selection pressure (via detection of

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metals and antibiotics) were investigated as potential factors governing the presence of ARGs.

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Certain ARGs were elevated in all (sul1; p≤0.0011) or some (blaTEM, qnrA; p≤0.0145) reclaimed

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POU samples compared to corresponding potable samples. Bacterial community composition

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was weakly correlated with ARGs (Adonis, R2=0.1424-0.1734) and associations were noted

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between 193 ARGs and plasmid-associated genes. This study establishes that reclaimed water

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could convey greater abundances of certain ARGs than potable waters and provides observations

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regarding factors that likely control ARG occurrence in reclaimed water systems.

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Table of Contents Art

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1 Introduction

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Reclamation and reuse of municipal wastewater effluent is increasingly relied on to offset

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demand on traditional potable water sources. Water reuse can help address challenges such as

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water shortages, groundwater depletion, surface water contamination, increasing demand due to

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population growth, and exacerbated water stress due to climate change.1 However, even as its

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application expands worldwide, there are technical challenges and public health concerns that

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must be assessed, such as trace contaminants, including antibiotics and personal care products,1

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and microbial constituents, such as viruses2,3 and antibiotic resistance elements.4

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The potential of water reuse to contribute to the spread of antibiotic resistance has drawn

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attention.4,5 Antibiotic resistance is a critical public health challenge, with over two million

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antibiotic resistant bacterial infections documented in the U.S. each year6 and even more

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globally.7 Strategies to mitigate the spread of antibiotic resistance have primarily focused on

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optimizing clinical use, limiting application in agriculture, and improving hygiene in hospitals.7–

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10

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implementation and because they do not take into consideration broader sources and routes of

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resistance dissemination associated with natural and built environments.8 Correspondingly, there

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is now growing movement towards identifying comprehensive mitigation strategies to prevent

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the evolution and spread of antibiotic resistance as an environmental “contaminant”.8,11,12 In this

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context, water reuse and the water cycle in general have the potential to contribute to the

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proliferation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs).

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Numerous studies have now documented the abundance of ARB and ARGs in wastewater, which

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like many microorganisms, are not always removed completely by traditional wastewater

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treatment.13–18 Previous studies have indicated that a diverse range of ARB and ARGs are

While such efforts are vital, they are limited in effectiveness due to difficulty of



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present in reclaimed (i.e., non-potable) water,19–21 but, given that antibiotic resistance is a natural

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phenomenon occurring in many aquatic and soil bacteria, it is important to move towards

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advancing understanding of which ARB and ARGs actually pose risk to human health. For

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example, samples collected from ancient permafrost and unexplored caves contain a surprisingly

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diverse array of ARGs.22–24 To address the presence of ARGs in even pristine environments,

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benchmarking the presence of ARGs in water reuse treatment and distribution systems to that of

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corresponding potable water systems can help provide a frame of reference for assessing

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potential risks associated with water reuse compared to water derived from surface and

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groundwater. Discriminating amongst various classes of ARGs and their location in the genome

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is also important, with ARGs that encode resistance to clinically-important antibiotics and that

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are capable of disseminating resistance via horizontal gene transfer being of greatest concern.25,26

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Municipal sewage represents a composite reservoir of excreted bacteria and associated

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DNA, where its collection and treatment will likely select for certain strains and, in some cases,

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could create conditions ideal for horizontal transfer of clinically-important ARGs.27,28

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Wastewater treatment plants (WWTP) have been proposed as potential “hot spots” for ARB

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proliferation.15 Correspondingly, poor efficiency of ARB and ARG removal has been noted with

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some conventional WWTPs.5,15,29 In particular, antibiotics, and other potential selective agents;

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such as heavy metals, herbicides, and disinfectants, have been associated with the loading of

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ARGs in water and soil environments30–33 and their presence in wastewater is expected to play a

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similar role. Further, shotgun metagenomic approaches and tracking of plasmids and other gene

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transfer elements have revealed evidence of high rates of horizontal transfer of ARGs among

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densely populated activated sludge bacteria core to the WWTP biological treatment process.34–37


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While considerable research has been devoted to understanding these phenomena in

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WWTPs and receiving environments, few studies have explored the potential for dissemination

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of ARB and ARGs via subsequent water reuse. While some studies have examined the

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implications of irrigation38–40 or groundwater recharge41,42 with reclaimed water for ARG

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dissemination, only recently has the potential for bacterial regrowth of ARG-carrying bacteria

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during reclaimed water distribution been reported in the peer-reviewed literature.19 Given that

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indicator organisms43 and opportunistic pathogens44 have both exhibited patterns of regrowth

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during distribution in reclaimed water systems, the potential for regrowth of ARG-carrying

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bacteria warrants consideration. Distribution system biofilms are also worthy of investigation

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given that biofilms have been identified as reservoirs for pathogenic bacteria in drinking water

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systems45 and supportive environments for horizontal gene transfer.46

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Here we used quantitative polymerase chain reaction (qPCR) to survey four ARGs;

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blaTEM, qnrA, vanA, and sul1, and the intI1 integrase gene, known to facilitate horizontal gene

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transfer, in full-scale reclaimed water distribution systems located in four U.S. cities that

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implement non-potable reuse. Shotgun metagenomic sequencing was applied towards profiling

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the broader “resistome” (i.e., full contingent of ARGs)47 in a cross-section of samples and the

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bacterial community composition was tracked using 16S rRNA amplicon sequencing to explore

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potential microbial ecological drivers of ARG occurrence. The specific objectives of this work

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were to: 1) characterize the resistome of reclaimed water distribution systems relative to

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corresponding potable systems; 2) quantify abundances of ARGs inhabiting the water versus the

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biofilm; 3) measure removal of ARGs during treatment and any subsequent increases during

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transport of reclaimed water to the point of use (POU); 4) explore associations between ARG

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occurrence and the bacterial community composition; 5) investigate potential for ARG



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dissemination via horizontal gene transfer; and 6) examine associations between water

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chemistry, particularly potential selective agents, and the abundance of ARGs. Realization of the

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objectives of this work will provide context for understanding the potential for water reuse to

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disseminate ARB and ARGs and inform development of management strategies for limiting

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dissemination of ARB and ARGs via distribution system operation.

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2 Methods

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2.1 Site description, sample collection, and preservation

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Four water utilities utilizing tertiary wastewater treatment to produce reclaimed water

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participated in sampling and are described in Table 1. Details about the four seasonal collection

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dates conducted for each utility are provided in Table S1. For each reclaimed system, samples

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were collected of raw wastewater influent, following treatment at the point of entry (POE) to the

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distribution system, and at five POUs. For each potable system, samples were collected of source

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water, at the POE, and at five POUs. After flushing for 30 seconds, water samples for molecular

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analysis were collected via distribution system sampling ports in sterile one liter polypropylene

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containers. Samples for organic carbon analysis were collected in acid-washed, baked 250

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milliliter amber glass bottles. All bottles were prepared with 48 milligrams sodium thiosulfate

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per liter to quench chlorine, and bottles for molecular analysis were also prepared with 292

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milligrams ethylenediaminetetraacetic acid per liter to chelate metals. Water was collected in

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acid-washed 250 milliliter bottles for chemical analyses. For Utilities A and B, after collecting

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water samples, biofilm samples were collected at each POU by inserting a sterile cotton-tipped

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applicator (Fisher Scientific, Waltham, MA) into the distribution system pipe and swabbing the

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upper 180º of the circumference of the pipe in a single pass. The sample end of the swab was

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transferred directly to a sterile DNA extraction lysing tube. 6 ACS Paragon Plus Environment

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Samples were shipped overnight on ice and processed immediately upon arrival, within

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approximately 24 hours of sample collection. Samples for molecular analysis were concentrated

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onto 0.22 micron mixed cellulose esters membrane filters (Millipore, Billerica, MA) until the

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entire one liter sample passed or until the filter became clogged. Filters were folded into quarters,

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torn into 1 cm2 pieces using sterile forceps, transferred to lysing tubes, and preserved at -20ºC.

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DNA was extracted from filters and biofilm swabs using a FastDNA SPIN Kit (MP Biomedicals,

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Solon, OH).

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2.2 Water chemistry

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Free chlorine (4500Cl G), total chlorine (4500Cl G), temperature (2550 B), dissolved

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oxygen (4500-O G), pH (4500-H+ B), turbidity (2130 B), and electrical conductivity (2510 B)

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were measured on-site according to standard methods.48 Upon return to the lab, one 30 milliliter

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aliquot was taken for total organic carbon (TOC) and a second aliquot was filtered through pre-

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rinsed 0.22 micron pore size mixed cellulose esters membrane filters (Millipore, Billerica, MA)

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for dissolved organic carbon (DOC). Biodegradable dissolved organic carbon (BDOC) was

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measured as previously described by Servais et al.49 with an incubation time extended to 45 days.

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Samples were analyzed on a Sievers 5310C portable TOC analyzer (GE, Boulder, CO) according

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to Standard Method 5310C.48 A host of 28 metals, including sodium, magnesium, phosphorus,

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chloride, calcium, iron, copper, zinc, and lead were measured using an Electron X-Series

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inductively coupled plasma mass spectrometer (ThermoFisher, Waltham, MA) according to

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Standard Method 3125B.48 Nitrate, nitrite, phosphate, and sulfate were quantified via a Dionex-

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500 ion chromatography system (ThermoFisher, Waltham, MA) according to Standard Method

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4110B.48 Antibiotics were extracted from samples using solid phase extraction according to Sui

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et al.50 with minor modifications described in the supporting information. The following



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antibiotics were analyzed using an ultra-performance liquid chromatography-tandem mass

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spectrometer (UPLC-MS/MS; Agilent 1290 UPLC/Agilent 6490 Triple Quad tandem MS,

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Agilent Technologies Inc., Santa Clara, CA): cefotaxime, chlorotetracycline, erythromycin,

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flumequine, nalidixic acid, ormetoprim, ornidazole (anti-protozoan), oxolinic acid,

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sulfamethazine, sulfamethoxazole, tetracycline, and tylosin.

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2.3 Quantification of antibiotic resistance genes

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Gene copies were quantified on a CFX96 Real Time System (BioRad, Hercules, CA)

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from DNA extracts in triplicate reactions using qPCR with previously published protocols for

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16S rRNA,51 blaTEM,52 qnrA,53 vanA,54 sul1,55 and intI156 genes. These genes were selected

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based on relevance to human health and environmental transmission of ARGs. The genes

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represent a spectrum of typical documented prevalence in the environment. For example, sul1

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has been widely documented in wastewater impacted environments19,57 and the gene corresponds

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to a class of antibiotics (sulfonamides) for which resistance of human pathogens has become

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commonplace.58 In contrast, vanA encodes resistance to a “last resort” drug and is less common

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in wastewater impacted environments.4,19 A subset of samples was initially analyzed at 5, 10, 20,

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and 50 fold dilutions to determine the optimal dilution effective for minimizing inhibition. A ten-

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fold dilution was selected and applied to all extracts. A triplicate negative control and triplicate

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standard curves of ten-fold serially diluted standards, constructed as described in the supporting

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information, of each target gene ranging from 101 to 107 gene copies/µl were included on each

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96-well plate. The limit of quantification was 10 gene copies per milliliter of sampled water and

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103 gene copies per biofilm swab.


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2.4 Shotgun metagenomics and 16S rRNA amplicon sequencing To profile the resistome, shotgun metagenomic sequencing was conducted on DNA

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extracted from the POE and greatest water age POU samples from each reclaimed system on

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each collection date as previously described,59 with sequencing conducted on an Illumina HiSeq

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with 2x100-cycle paired end reads at the Biocomplexity Institute of Virginia Tech Genomics

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Sequencing Center (BI; Blacksburg, VA). One source water sample, the POE, and the greatest

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water age POU sample from the potable system of each utility (all collected during the summer

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collection from each utility) were also submitted for sequencing. All potable samples from

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Utilities C and D and a subset of samples from Utility A yielded insufficient DNA to conduct

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metagenomic sequencing.

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Functional genes were annotated via the MetaStorm platform60 according to default

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parameters (amino acid identity≥80%; e-value cutoff=1e-10; minimum alignment length=25

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amino acids) using annotation to the Comprehensive Antibiotic Resistance Database (CARD,

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version 1.0.6) for ARGs,61 the Silva ribosomal RNA database for 16S rRNA genes (version

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123),62 the BacMet database (version 1.1) for metal resistance genes,63 and the ACLAME

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database (version 0.4) for plasmid-associated genes.64 Functional genes were normalized to 16S

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rRNA genes as previously described by Li et al.65 Absolute abundances were calculated by

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multiplying relative abundance of target functional genes by total abundance of 16S rRNA genes

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quantified by qPCR (Figure S1). All metagenomes generated in this study are publicly available

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via MG-RAST66 under project number 12943 (see Table S2 for sample IDs and read and

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assembly statistics). Reads were assembled de novo in MetaStorm according to default

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parameters and scaffolds were annotated as described above for reads.



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Bacterial communities were profiled using gene amplicon sequencing targeting the V4

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region of the 16S rRNA gene with barcoded primers (515F/806R).67 Some archaea are also

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detected by these primers. Triplicate PCR products were combined and 240 ng of each

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composite was pooled and purified using a QIAquick PCR Purification Kit (Qiagen, Valencia,

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CA). Sequencing was conducted at BI on an Illumina MiSeq using a 2x250-cycle paired-end

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protocol. Processing of reads was conducted using the QIIME pipeline68 with phylogenetic

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inference based on alignment against the Greengenes database (May 2013 release).69 Samples

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were rarefied to 10,000 randomly selected reads (Table S3). Field, filtration, DNA extraction

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blanks, and a least one PCR blank per lane were included in the analysis.

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2.5 Statistical Analysis

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A Wilcoxon rank sum test for multiple comparisons was applied in JMP (version 13,

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SAS, Cary, NC) to determine differences between abundances of ARGs across groups of

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samples. Spearman’s rank sum correlation coefficients were calculated in JMP to assess

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correlations between ARGs, phyla, Gram-stain type (identified from the literature for each phyla

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to the extent possible), and water quality parameters. Canonical correspondence analysis was

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conducted in R (version 3.4.1) using the Adonis function from the vegan package70 to identify

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ARGs that are significantly correlated with the bacterial community structure. Abundance of

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ARGs based on metagenomic data were imported into PRIMER-E (version 6.1.13) for Bray-

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Curtis resemblance matrix construction and one-way analysis of similarities (ANOSIM) to

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compare profile differences across groups of samples. A significance cutoff of α=0.05 was used

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for all analyses. Co-occurrences of annotated genes on scaffolds were characterized via network

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analysis visualization using Gephi (version 0.8.2).


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3 Results and discussion

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3.1 Metagenomic characterization of the resistome in reclaimed versus potable water

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Shotgun metagenomic sequencing was used to investigate the abundance of known

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ARGs by annotating reads against the CARD database. Seventeen classes of antibiotic

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resistance, as well as multidrug resistance, were identified across all samples (n=38, Figure 1).

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Across the dataset, 590 different ARGs were annotated, with between 16 and 372 different

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ARGs detected in each sample (Table S4). The top 25 most abundant ARGs overall included

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four aminoglycoside (aadA23, ant(2”)-la, aadA, aadA17), one sulfonamide (sul1), one

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trimethoprim (dfrE), one polymyxin (pmrE), one rifampin (rbpA), two beta-lactam (blaOXA-

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256, blaOXA-129), two tetracycline (tetC, tetQ), one fluoroquinolone (qnrS6), one macrolide

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(ereA2), and eight multidrug (msrE, mtrA, mexF, mexK, CRP, adeG, mexW, acrB) ARGs. The

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average number of sequencing reads aligning to different ARGs per sample was 26.8±9.2 per ten

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million reads in the potable water and 69.0±42.7 per ten million reads in the reclaimed water,

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though differences were not significant (Wilcoxon; p=0.0891).

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Among the potable samples that were successfully sequenced, multidrug ARGs were

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common (8.4-33.4% of total ARGs) and the most abundant classes of ARGs were trimethoprim

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(10.6-49.7%), aminocoumarin (0-18.5%), beta lactam (1.3-38.5%), polymyxin (0.4-11.1%), and

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aminoglycoside (0.9-9.8%) resistance. The abundance of total ARGs in potable water ranged

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from 3.93–6.83 log gene copies per milliliter and 4.33–5.32 log gene copies per swab in the

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biofilm.

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Reclaimed water ARG profiles were distinct from that of potable samples (ANOSIM;

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R=0.705, p=0.001; Figure 1; Figure S2). In the reclaimed water, Utility A’s ARG profile stood

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out from that of other utilities (R=0.695-0.932, p=0.001), dominated by aminoglycoside (34.511 ACS Paragon Plus Environment


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66.8% of total ARGs) and sulfonamide (29.2-51.9%) ARGs, whereas Utilities B, C, and D were

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generally dominated by multidrug (10.0-40.8%), trimethoprim (8.4-50.5%), sulfonamide (0-

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38.3), tetracycline (0.97-13.2%), and beta-lactam (1.07-16.9%) ARGs. The biofilms of Utilities

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A and B exhibited patterns that were more similar to the respective water samples of those

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utilities than to each other, dominated by aminoglycoside (44.3%), sulfonamide (22.3%), and

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trimethoprim ARGs (13.8%) for Utility A and multidrug (44.6%), rifampin (24.0%), and

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trimethoprim (17.9%) for Utility B. The abundance of total ARGs in the reclaimed water ranged

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from 5.24–6.53 log gene copies per milliliter and 4.82–6.14 log gene copies per biofilm swab.

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The resistance profile of the treated water at the POE was markedly different from that of

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the influent wastewater for each utility (ANOSIM; R=0.971, p=0.002), consistent with the

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expectation that the treatment process shifts the types and numbers of ARGs relative to raw

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sewage. As expected, the greatest abundances of total ARGs were generally found in the raw

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wastewater influent samples (6.81–7.88 log gene copies per milliliter). Influent wastewater

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tended to be dominated by multidrug (16.1-35.4% of total ARGs), aminoglycoside (12.7-30.8%),

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beta-lactam (3.1-21%), tetracycline (6.3-16.7%), macrolide (2.4-10.4%), and sulfonamide

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resistance (1.2-19.5%), though there was variation across utilities (Figure 1).

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While metagenomic analysis provides tremendous potential for characterizing the

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resistome and allowing broad detection of all known ARGs, the approach is cost-prohibitive,

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limiting the number of samples that can be analyzed, as well as being only semi-quantitative. In

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addition, the lack of sufficient DNA from many samples in this study was a critical limitation to

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being able to fully compare reclaimed water samples to potable samples, which were much lower

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in biomass. This consequentially limited sample size for many of the categories (Figure 1) and

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correspondingly limited the ability to draw statistically significant conclusions about the data.


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The metagenomic analysis completed herein should be viewed as an exploratory

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characterization, particularly with respect to the potable water resistome.

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3.2 Abundance of target ARGs in water and biofilms

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To precisely quantify a selection of ARGs corresponding to a range of critically and

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highly important classes, as defined by the World Health Organization,71 across the full dataset,

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qPCR was utilized. Important to note is that qPCR, as applied in this study, does not directly

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distinguish live and dead organisms or intracellular versus extracellular DNA. However, tracking

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numbers of ARGs through the water systems represents an indicator of net amplification and

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decay of the target genes, via horizontal transfer and/or growth or death and degradation,

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respectively.57

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Several significant differences were noted in target gene numbers when comparing the

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paired reclaimed and potable distribution systems (Table 2). A consistent difference noted across

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all four utilities was that both 16S rRNA (a proxy for total bacterial cells) and sul1 gene copies

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per milliliter were more abundant in the reclaimed than potable distribution system samples

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(Wilcoxon; p≤0.0011). Further, blaTEM was more abundant in the reclaimed than the potable

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distribution system of Utility D (p

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