1 Title: Metagenomic characterization of antibiotic ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45

Environmental Science & Technology

1

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

2

distribution systems and corresponding potable systems

3 4

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

5

McLain4, Marc A. Edwards1, Amy Pruden1*

6 7

1

8

24061, United States

9

2

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

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

10

24061, United States

11

3

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

12

4

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

13

States

14 15

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

1 ACS Paragon Plus Environment


16

Abstract

17

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

18

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

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

POU samples compared to corresponding potable samples. Bacterial community composition

29

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

30

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

31

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

32

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

33

Table of Contents Art

34 2 ACS Paragon Plus Environment

Page 2 of 45

Page 3 of 45

35


1 Introduction

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

10

49

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

50

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

51

is now growing movement towards identifying comprehensive mitigation strategies to prevent

52

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

53

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

54

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

55

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

56

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

57

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



58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

Municipal sewage represents a composite reservoir of excreted bacteria and associated

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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


Page 4 of 45

Page 5 of 45

80


While considerable research has been devoted to understanding these phenomena in

81

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

82

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

83

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

84

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

85

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

86

indicator organisms43 and opportunistic pathogens44 have both exhibited patterns of regrowth

87

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

88

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

89

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

90

systems45 and supportive environments for horizontal gene transfer.46

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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



103

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

104

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

105

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

106

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

107

dissemination of ARB and ARGs via distribution system operation.

108

2 Methods

109

2.1 Site description, sample collection, and preservation

110

Four water utilities utilizing tertiary wastewater treatment to produce reclaimed water

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

Page 6 of 45

Page 7 of 45

126


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

127

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

128

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

129

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

130

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

131

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

132

Solon, OH).

133

2.2 Water chemistry

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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



149

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

150

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

151

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

152

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

153

sulfamethazine, sulfamethoxazole, tetracycline, and tylosin.

154

2.3 Quantification of antibiotic resistance genes

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

103 gene copies per biofilm swab.


Page 8 of 45

Page 9 of 45

170 171


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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

metagenomic sequencing.

180

Functional genes were annotated via the MetaStorm platform60 according to default

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

parameters and scaffolds were annotated as described above for reads.



192

Bacterial communities were profiled using gene amplicon sequencing targeting the V4

193

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

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

2.5 Statistical Analysis

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

analysis visualization using Gephi (version 0.8.2).


Page 10 of 45

Page 11 of 45


214

3 Results and discussion

215

3.1 Metagenomic characterization of the resistome in reclaimed versus potable water

216

Shotgun metagenomic sequencing was used to investigate the abundance of known

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

biofilm.

234

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

235

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

236

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


237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

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

253

While metagenomic analysis provides tremendous potential for characterizing the

254

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

255

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

256

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

257

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

258

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

259

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


Page 12 of 45

Page 13 of 45


260

The metagenomic analysis completed herein should be viewed as an exploratory

261

characterization, particularly with respect to the potable water resistome.

262

3.2 Abundance of target ARGs in water and biofilms

263

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

264

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

265

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

266

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

267

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

268

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

269

respectively.57

270

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

271

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

272

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

273

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

274

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

275

distribution system of Utility D (p

1 Title: Metagenomic characterization of antibiotic ... - ACS Publications

Recommend Documents