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Antimicrobial
Resistance
in Environmental
Waters
Edited by
Karina Yew-Hoong Gin and Charmaine Ng
Printed Edition of the Special Issue Published in Water

www.mdpi.com/journal/water
Antimicrobial Resistance
in Environmental Waters
Antimicrobial Resistance
in Environmental Waters

Special Issue Editors

Karina Yew-Hoong Gin
Charmaine Ng

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Special Issue Editors
Karina Yew-Hoong Gin Charmaine Ng
National University of Singapore National University of Singapore
Singapore Singapore

Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal
Water (ISSN 2073-4441) from 2017 to 2019 (available at: https://www.mdpi.com/si/water/
Antimicrobial-Resistance-Environmental-Waters).

For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year, Article Number,
Page Range.

ISBN 978-3-03897-608-0 (Pbk)

ISBN 978-3-03897-609-7 (PDF)

Cover image courtesy of Charmaine Ng.


c 2019 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license, which allows users to download, copy and build upon
published articles, as long as the author and publisher are properly credited, which ensures maximum
dissemination and a wider impact of our publications.
The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons
license CC BY-NC-ND.
Contents

About the Special Issue Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Antimicrobial Resistance in Environmental Waters” . . . . . . . . . . . . . . . . . . ix

Charmaine Ng and Karina Yew-Hoong Gin

Monitoring Antimicrobial Resistance Dissemination in Aquatic Systems
Reprinted from: Water 2019, 11, 71, doi:10.3390/w11010071 . . . . . . . . . . . . . . . . . . . . . . 1

Kevin Lambirth, Matthew Tsilimigras, Anju Lulla, James Johnson, Abrar Al-Shaer,
Orion Wynblatt, Shannon Sypolt, Cory Brouwer, Sandra Clinton, Olya Keen,
Molly Redmond, Anthony Fodor and Cynthia Gibas
Microbial Community Composition and Antibiotic Resistance Genes within a North Carolina
Urban Water System
Reprinted from: Water 2018, 10, 1539, doi:10.3390/w10111539 . . . . . . . . . . . . . . . . . . . . . 8

Hsin-Chi Tsai, Ming-Yuan Chou, Yi-Jia Shih, Tung-Yi Huang, Pei-Yu Yang, Yi-Chou Chiu,
Jung-Sheng Chen and Bing-Mu Hsu
Distribution and Genotyping of Aquatic Acinetobacter baumannii Strains Isolated from the Puzi
River and Its Tributaries Near Areas of Livestock Farming
Reprinted from: Water 2018, 10, 1374, doi:10.3390/w10101374 . . . . . . . . . . . . . . . . . . . . . 29

Asli Aslan, Zachariah Cole, Anunay Bhattacharya and Oghenekpaobor Oyibo

Presence of Antibiotic-Resistant Escherichia coli in Wastewater Treatment Plant Effluents Utilized
as Water Reuse for Irrigation
Reprinted from: Water 2018, 10, 805, doi:10.3390/w10060805 . . . . . . . . . . . . . . . . . . . . . 41

Pierangeli G. Vital, Enrico S. Zara, Cielo Emar M. Paraoan, Ma. Angela Z. Dimasupil,
Joseth Jermaine M. Abello, I ñigo Teodoro G. Santos and Windell L. Rivera
Antibiotic Resistance and Extended-Spectrum Beta-Lactamase Production of Escherichia coli
Isolated from Irrigation Waters in Selected Urban Farms in Metro Manila, Philippines
Reprinted from: Water 2018, 10, 548, doi:10.3390/w10050548 . . . . . . . . . . . . . . . . . . . . . 52

Muhammad Raihan Jumat, Muhammad Fauzi Haroon, Nada Al-Jassim, Hong Cheng and
Pei-Ying Hong
An Increase of Abundance and Transcriptional Activity for Acinetobacter junii Post
Wastewater Treatment
Reprinted from: Water 2018, 10, 436, doi:10.3390/w10040436 . . . . . . . . . . . . . . . . . . . . . 63

Junying Lu, Zhe Tian, Jianwei Yu, Min Yang and Yu Zhang
Distribution and Abundance of Antibiotic Resistance Genes in Sand Settling Reservoirs and
Drinking Water Treatment Plants across the Yellow River, China
Reprinted from: Water 2018, 10, 246, doi:10.3390/w10030246 . . . . . . . . . . . . . . . . . . . . . 87

Zheng Xu, Yue Jiang, Shu Harn Te, Yiliang He and Karina Yew-Hoong Gin
The Effects of Antibiotics on Microbial Community Composition in an Estuary Reservoir during
Spring and Summer Seasons
Reprinted from: Water 2018, 10, 154, doi:10.3390/w10020154 . . . . . . . . . . . . . . . . . . . . . 99

Yue Jiang, Cong Xu, Xiaoyu Wu, Yihan Chen, Wei Han, Karina Yew-Hoong Gin and
Yiliang He
Occurrence, Seasonal Variation and Risk Assessment of Antibiotics in Qingcaosha Reservoir
Reprinted from: Water 2018, 10, 115, doi:10.3390/w10020115 . . . . . . . . . . . . . . . . . . . . . 118

v
Yihan Chen, Hongjie Chen, Li Zhang, Yue Jiang, Karina Yew-Hoong Gin and Yiliang He
Occurrence, Distribution, and Risk Assessment of Antibiotics in a Subtropical
River-Reservoir System
Reprinted from: Water 2018, 10, 104, doi:10.3390/w10020104 . . . . . . . . . . . . . . . . . . . . . 133

Jennipher Quach-Cu, Bellanira Herrera-Lynch, Christine Marciniak, Scott Adams,

April Simmerman and Ryan A. Reinke
The Effect of Primary, Secondary, and Tertiary Wastewater Treatment Processes on Antibiotic
Resistance Gene (ARG) Concentrations in Solid and Dissolved Wastewater Fractions
Reprinted from: Water 2018, 10, 37, doi:10.3390/w10010037 . . . . . . . . . . . . . . . . . . . . . . 149

Joyce Seow Fong Chin, Sheetal Sinha, Anjaiah Nalaparaju, Joey Kuok Hoong Yam, Zhiqiang
Qin, Luyan Ma, Zhao-Xun Liang, Lanyuan Lu, Surajit Bhattacharjya and Liang Yang
Pseudomonas aeruginosa Psl Exopolysaccharide Interacts with the Antimicrobial Peptide LG21
Reprinted from: Water 2017, 9, 681, doi:10.3390/w9090681 . . . . . . . . . . . . . . . . . . . . . . . 167

vi
About the Special Issue Editors
Karina Yew-Hoong Gin is an Associate Professor with the Department of Civil and Environmental
Engineering, National University of Singapore. She received her Bachelor degree in Civil Engineering
from the University of Melbourne in 1988 and her M.Eng Degree from the National University of
Singapore in 1991. She obtained her Doctor of Science (ScD) Degree jointly from the Massachusetts
Institute of Technology and the Woods Hole Oceanographic Institution in 1996. Her research
specialization is in the area of water quality, fate and transport of emerging contaminants, and
ecosystem processes.

Charmaine Ng is a Research Fellow with the Department of Surgery, National University of

Singapore. She graduated with a PhD in microbiology at the University of New South Wales,
Australia in 2011. Her research interests include marine and freshwater microbial ecology,
antimicrobial resistance, and the study of the human microbiome in relation to different disease states.

vii
Preface to ”Antimicrobial Resistance in
Environmental Waters”
In recent years, the emergence of antimicrobial resistance has drawn heightened global concern
because of its severe ramifications on the treatment of microbial infections. In particular, the issue
of antibiotic resistance arises due to the overuse and misuse of antibiotics in both developed and
developing countries. Bacteria develop antibiotic resistance in the presence of residual levels of
antibiotics, and these antibiotic-resistant bacteria are in turn able to spread their resistance to other
bacteria through mechanisms such as horizontal gene transfer, mediated by mobile genetic elements
(e.g., plasmids, integrons) or co-selecting agents such as biocides and toxic metals. There is a worrying
trend that pathogens are developing antibiotic resistance to a degree where last-resort antibiotics are
no longer effective. This, in turn, has severe implications for public health and healthcare costs.
In an effort to better understand the rising levels of antimicrobial resistance, surveillance
studies have been undertaken across countries in a common effort to explore the occurrence of
antimicrobial resistance in both clinical and natural environments. Implementing such initiatives
by assessing the types of antibiotics used, antibiotic-resistant bacteria (ARB) present, and associated
antibiotic resistant genes (ARGs) in microbiomes enables a better understanding of the impact of
antibiotics in the medicine, agriculture, and aquaculture industries. Aquatic environments harbor
diverse freshwater bacterial communities which may be subjected to anthropogenic pressures,
while domestic wastewaters receive direct loads of antibiotics and pathogenic bacteria from human
excretion. The nature of these environments allows them to function as hotspots for resistance
through the selection of ARB and the circulation of ARGs through the stimulation of horizontal gene
transfer between members of the microbiome.
The aims of this Special Issue are to present current trends in antimicrobial/antibiotic
resistance in diverse environmental waters, ranging from the detection and occurrence of
antimicrobial factors (e.g., antimicrobials, antibiotics, ARB, ARGs) to their fate and transformations
in different environments such as surface waters, groundwaters, biofilms, and water and wastewater
treatment processes. This knowledge is needed to assist in the management and control of
antimicrobial/antibiotic resistance and, ultimately, the protection of public health.

Karina Yew-Hoong Gin, Charmaine Ng

Special Issue Editors

ix
 water
Editorial
Monitoring Antimicrobial Resistance Dissemination
in Aquatic Systems
Charmaine Ng 1 and Karina Yew-Hoong Gin 2,3, *
1 Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore,
Singapore 117411, Singapore; [email protected]
2 Department of Civil and Environmental Engineering, National University of Singapore,
Singapore 138602, Singapore
3 NUS Environmental Research Institute (NERI), Singapore 138602, Singapore
* Correspondence: [email protected]; Tel.: +65-65168104

Received: 13 December 2018; Accepted: 28 December 2018; Published: 3 January 2019 

Abstract: This special issue on Antimicrobial Resistance in Environmental Waters features 11

articles on monitoring and surveillance of antimicrobial resistance (AMR) in natural aquatic systems
(reservoirs, rivers), and effluent discharge from water treatment plants to assess the effectiveness of
AMR removal and resulting loads in treated waters. The occurrence and distribution of antimicrobials,
antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs) and mobile genetic elements
(MGEs) was determined by utilizing a variety of techniques including liquid chromatography—mass
spectrometry in tandem (LC-MS/MS), traditional culturing, antibiotic susceptibility testing (AST),
molecular and OMIC approaches. Some of the key elements of AMR studies presented in this
special issue highlight the underlying drivers of AMR contamination in the environment and
evaluation of the hazard imposed on aquatic organisms in receiving environments through ecological
risk assessments. As described in this issue, screening antimicrobial peptide (AMP) libraries for
biofilm disruption and antimicrobial candidates are promising avenues for the development of new
treatment options to eradicate resistance. This editorial puts into perspective the current AMR
problem in the environment and potential new methods which could be applied to surveillance and
monitoring efforts.

Keywords: Antimicrobial Resistance; Environmental Waters; water treatment plants; water reuse;
ecological risk assessment

1. Introduction
The release of antimicrobials, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes
(ARGs) originating from human and animal waste to the environment is a global problem which
has serious ramifications on public health. In response to this growing health threat, the World
Health Organization (WHO) launched a global action plan on Antimicrobial Resistance (AMR)
in 2015 with 5 strategic objectives, one of which was to strengthen knowledge of the spread of
AMR through surveillance and research [1]. As a guide, the WHO has drawn up a priority list
of AMR pathogens based on the threat they pose on human infections, response to antibiotic
treatment, transmissibility between humans and animals, and whether there are antibiotics in current
research and development pipelines to treat infections caused by these pathogens. Those of highest
priority are carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa,
and carbapenem-resistant, extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae [2].
The WHO’s Global Antimicrobial Surveillance System (GLASS) report for 2018 revealed widespread
occurrence of antibiotic resistance among half a million people with suspected bacterial infections

Water 2019, 11, 71; doi:10.3390/w11010071 1 www.mdpi.com/journal/water

Water 2019, 11, 71

across 22 countries [3]. An AMR risk assessment of the South East Asian Region by Chereau et al. [4]
concluded a high likelihood of emergence and dissemination among humans. Low stewardship on
antibiotic prescription in treatment of human infections and the absence of legal frameworks for
antibiotic use in animal husbandry and aquaculture are the main drivers for the selection of ARB in
South East Asia [5]. The direct release or insufficient treatment of wastewater effluents from healthcare,
livestock, aquaculture, and agriculture sites into receiving environments also poses a significant risk.
In 2013, China alone produced 92,700 tonnes of antibiotics, 48% of which were consumed by humans
and 52% by animals [6]. It was reported that almost half of all antibiotics were released in rivers
through wastewater effluents and the practice of manure and sludge land spreading [6].
In South East Asia, AMR risk ranking across humans, animals, and environmental compartments
show that human to human transmission in community and hospital settings represent the highest
risk of the emergence and selection of AMR vectors (ARB, ARGs), followed by food- and waterborne
transmission to humans through ingestion of contaminated sources [4]. Although transmission via
contact with contaminated environments (through soil, water, and air), and livestock/animals is
regarded as low risk in comparison to human to human transmission, it is still considered a valid
route of exposure, particularly in countries that are water-scarce and reliant on water reuse. Hence,
it is crucial to address the impact of antimicrobials, ARB and ARGs on AMR prevalence in receiving
environments, specifically in countries where data availability is scant and guidelines for AMR
stewardship frameworks have yet to be established. It is only through identifying and tracking sources
and sinks of AMR in the environment, where intervention strategies can be devised to prevent and
control the spread of the problem.

2. Measuring Vectors of AMR

2.1. Antimicrobials
Currently, there are various practices and methods of monitoring AMR dissemination and the
fate of ARB and ARGs in aquatic environmental systems. Highly sensitive analytical protocols have
been developed to detect antimicrobials using liquid chromatography-tandem mass spectrometry
in environmental water samples and effluents from wastewater treatment plants (WWTPs) [7,8],
while culture-based techniques are used to enumerate ARB.

2.2. ARB
Most environmental and wastewater treatment AMR surveys use methods applied in clinical
settings, where media is supplemented with antibiotics at concentrations above the recommended
minimum inhibitory concentration (MIC) breakpoints implemented by the Clinical and Laboratory
Standards Institute [9] or the European Committee on antimicrobial Susceptibility Testing [10–17].
For non-clinically relevant bacteria (such as environmental bacteria), epidemiological cut-off values
(ECOFF) are an alternative method used to gauge non-wild-type bacteria that display reduced
susceptibility to certain antimicrobials or biocides [18]. Further testing of multidrug resistance
(MDR) of ARB isolates are performed using broth dilution assays, Kirby-Bauer disk diffusion test on
Muller-Hinton agar, or high-throughput platforms such as the VITEK system by bioMerieux (France).

2.3. ARGs, MGEs

To detect the prevalence of ARGs and vectors such as mobile genetic elements (MGE) that may
facilitate horizontal gene transfer of ARGs, traditional quantitative polymerase chain reaction (qPCR),
and the more recent high-throughput qPCR (HT-qPCR) platform with capabilities of detection of ~200
different ARGs and mobile genetic elements (MGE), has been used to compare relative concentrations of
AMR contamination across a variety of aquatic environments including water treatment plants [19–24].
OMIC approaches such as metagenomics are able to provide a holistic picture of the diversity of
ARGs, MGE and vectors (e.g., integrons, plasmids) that assist horizontal gene transfer, and the overall

2
Water 2019, 11, 71

microbial community structure (bacteria, viruses) in environmental systems and wastewaters [25–29].
ResCap, a targeted capture platform (TCP) designed to analyze ~78,000 ARGs, metal resistance and
plasmid markers is a targeted metagenomics approach for qualitative and quantitative resistome
analysis [30]. Other OMIC approaches, such as metatranscriptomics, enable the identification of active
microbial members within a community and, in the context of AMR, enables the measurement of
transcription activity of ARB through ARG expression [31].

3. Key Outcomes of this Special Issue

This special issue comprises of 11 research articles that fall within the scope of AMR. Broadly,
topics extend from AMR monitoring and surveillance in environmental resources and effluents from
water/wastewater treatment plants [32–39], antimicrobial ecological risk assessments of two river
reservoir systems in China that are sources of drinking water supply [40,41], and exploring novel
strategies of using engineered antimicrobial peptides (AMP) to target specific bacteria to disrupt
biofilms that are major causes of chronic and persistent infections [42].

AMR Monitoring and Surveillance

Knowledge of removal efficiencies of technology employed at WWTPs is essential in AMR
surveillance, whether effluents are intended for discharge into the environment or reuse for
irrigation purposes.
To determine the presence of antibiotic resistant E. coli in a conventional WWTP in Georgia,
Aslan et al. [36] isolated E. coli from post-secondary, post-UV and post-chlorination effluent and
performed antibiotic susceptibility tests on the isolates. They reported that ~5.2 log removal of E. coli
and an additional 1.1 log reduction post chlorination was obtained. However, the MICs of E. coli
isolated in the finished water were higher than those at the other treatment stages. The selection of more
resistant organisms in the finished water underscores the urgent need to evaluate the health risks of
using reclaimed water for downstream irrigation. In a qPCR assessment of ARGs of a full-scale tertiary
water reclamation plant, Quach-Cu et al. [39] showed that tertiary-stage WWTPs with disinfection had
superior removal of ARGs (sul1, blaSHV/TEM ), ~3–4 logs compared to reliance of secondary treatment
alone where the removal was only ~1–3 logs. To assess the impact of treated effluent on receiving
environments, Lambirth et al. [35] measured the removal efficiency of ten antibiotics and assayed
resistomes upstream, downstream and within various treatment steps of two urban WWTPs (secondary
and disinfection treatments). The authors found elevated concentration of all 10 antibiotics surveyed
in downstream receiving waters compared to waters upstream of the WWTP. The relative abundance
of ARG signatures encoding for resistance to carbapenems and ESBL antibiotics were much lower
than those detected upstream and sampling points within the WWTP, which debunks the notion that
the wastewater treatment process selects for ARG resistance. Instead, the authors hypothesized that
antibiotics discharged from treated wastewater effluent into the downstream environment may have
an effect on natural microbial communities.
Jumat et al. [37] conducted an ESBL study of diversity and transcriptional activity of bacteria in
a WWTP in Saudi Arabia using metagenomics, metatranscriptomics and real-time qPCR. They found
an increase in the relative abundance of Acinetobacter junii in MBR- and chlorinated treated effluent.
Survival and predominance of A. junii was explained by metatranscriptomics data that showed
an upregulation of gene associated with active cell repair, resistance, virulence (efflux transporters
involved in metal and antibiotic resistance) and cell signaling. These adaptive cellular mechanisms
enable A. junii to withstand depletion of nutrients and counter the effects of chlorination. However,
the authors indicate that the low concentrations of viable A. junii isolated from MBR effluents may
not present that huge a risk. The varying results from WWTP studies covered in this issue makes it
challenging to establish whether WWTPs are indeed hotspots of AMR dissemination. Rather, it is likely
that differences in global and plant operating process contribute to variation in antibiotic resistance
elements detection.

3
Water 2019, 11, 71

In countries with high agricultural productivity, water reuse is commonly practiced to meet
the high water demands of the industry [43]. On a global scale, there are no clear guidelines
implemented on assessment of water quality for reuse purposes, although a few countries have
drawn up recommended microbiological parameters to monitor the quality of recycled water [44].
A recent review by Hong et al. [44] highlights the urgency of understanding the risks of microbiological
and ARG contamination linked to water reuse. In the Philippines, surface waters contaminated
with fecal coliform are frequently used for irrigating urban farms in densely populated cities [45].
Vital et al. [33] evaluated the antibiotic resistance profiles of 212 E. coli strains isolated from irrigation
water, soil, and vegetables from six urban agricultural farms. Of the total isolates, 36.5% were resistant
to more than three antibiotics tested, with the most multidrug-resistant (MDR) isolates being detected
in irrigation water, followed by soil and vegetables. Of the MDR E. coli isolated from irrigation water,
7 of them were ESBL producers that carried either blaTEM or blaCTX-M genes, which raises public health
concerns in primary production environments such as agricultural soils and fresh produce grown in
these areas.
The use of antimicrobials in food animals is widespread, and runoffs originating from
animal waste may carry unmetabolized antibiotics or ARB and ARGs that have direct impact
on surrounding water bodies [46]. Tsai et al. [32] linked significantly higher concentrations
of A. baumannii along the Puzi River in China to sampling sites of livestock wastewater
channels and tributaries adjacent to livestock farms. Further testing of 20 A. baumannii isolates
against 7 antibiotics (ciprofloxacin, cefepime, gentamicin, imipenem, ampicillin-sulbactam,
sulfamethoxazole/trimethoprim, tetracycline) by the Kirby-Bauer disk diffusion test showed that only
10% had resistance to sulfamethoxazole/trimethoprim and 5% had resistance to tetracycline. Although
the A. baumannii isolated by Tsai et al. [32] did not display MDR patterns which are regarded a serious
AMR threat by WHO, their epidemiological potential warrants further studies on prevalence and AMR
developmental trends in the environment.
In China, the Yellow River in the North serves as an important source of drinking water and is
flanked by cities along its banks and watersheds. Previous studies have reported high turbidity and
concentrations of antimicrobials in the Yellow River Catchment [47]. This prompted Lu et al. [38] to
investigate the distribution and abundance of ARGs in Sand Settling Reservoirs (SSR) and Drinking
Water Treatment Plants (DWTP) along the Yellow River. By targeting 17 ARGs as a proxy for AMR
removal through the treatment process, the total concentrations of ARGs decreased from 104 copies/mL
in influent river waters to 103 copies/mL in SSR effluent to 102 copies/mL in finished water. The 2 MGE
targets decreased by at least an order of magnitude, from 106 copies/mL in influent river waters to
105 copies/mL in finished waters.
Horizontal gene transfer (HGT) of ARGs in the mammalian gut through the ingestion of
contaminated food or water generally poses a low risk due to harsh conditions in the gut. However,
this may present a greater risk for propagation in the environment through transformation and
transduction [44]. In another study by Xu et al. [34], the authors studied the relationship of 16
antibiotics with environmental water quality parameters and the impact of antibiotic concentrations on
the microbial community structure along Qingcaosha Reservoir, the largest estuary reservoir in China.
This reservoir has a similar function to the Yellow River, in that it compensates for drinking water
shortages in Shanghai. From the study, the authors concluded that upstream runoffs and anthropogenic
activity along the river contributed to the concentrations of antibiotics measured within the reservoir,
and that tylosin, penicillin G and erythromycin-H2 O showed significant correlations with variations
in bacterial community structure. Further to this study, Jiang et al. [40] studied seasonal variations
of antibiotics in surface waters of Qingcaosha Reservoir. By using risk quotients (RQs) based on the
European technical guidance documentation (TGD) on risk assessment, they showed that out of the
17 antibiotics monitored, four antibiotics (doxycycline, penicillinV, norfloxacin, ciprofloxacin) posed
a high risk to relevant sensitive aquatic organisms, as well as imposed selective stress on microbial
communities. In another study, of a subtropical river-reservoir system located in the Headwater Region

4
Water 2019, 11, 71

of the Dongjiang River which supplies drinking water to three major cities in China, Chen et al. [41]
conducted an ecological risk assessment which showed that concentrations of ciprofloxacin and
norfloxacin posed a moderate risk, while tetracycline posed a higher risk to the aquatic ecosystem.
Finally, through screening a local antimicrobial peptide (AMP) library, Chin et al. [42] identified
LG21, an AMP that specifically binds to exopolysaccharide PsI of P. aeruginosa that has a functional role
of biofilm formation, which provides a protective environment for tolerance and resistance towards
antibiotic treatment. This strategy of exploring AMP to target specific biofilm matrix components to
disrupt formation and development of biofilms is a promising line of treatment to eradicate antibiotic
resistant biofilms in both environmental and clinical settings.

Acknowledgments: The guest editors are grateful to the authors that contributed their work to this special issue.
We would also like to thank the reviewers and journal editors who dedicated their time and expertise towards
evaluating the articles.
Conflicts of Interest: The authors declare no conflict of interest.

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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

7
 water
Article
Microbial Community Composition and Antibiotic
Resistance Genes within a North Carolina Urban
Water System
Kevin Lambirth 1,2 , Matthew Tsilimigras 1 , Anju Lulla 1 , James Johnson 1 , Abrar Al-Shaer 3 ,
Orion Wynblatt 2 , Shannon Sypolt 4 , Cory Brouwer 1,2 , Sandra Clinton 5 , Olya Keen 6 ,
Molly Redmond 7 , Anthony Fodor 1 and Cynthia Gibas 1, *
1 Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, 9201 University
City Blvd, Charlotte, NC 28223, USA; [email protected] (K.L.); [email protected] (M.T.);
[email protected] (A.L.); [email protected] (J.J.); [email protected] (C.B.); [email protected] (A.F.)
2 Bioinformatics Services Division, The University of North Carolina at Charlotte, 150 N Research Campus Dr,
Kannapolis, NC 28081, USA; [email protected]
3 Department of Nutrition, The University of North Carolina at Chapel Hill, 135 Dauer Drive,
Chapel Hill, NC 27599, USA; [email protected]
4 Charlotte Water, Charlotte-Mecklenburg Utility Department, 5100 Brookshire Blvd,
Charlotte, NC 28216, USA; [email protected]
5 Department of Geography and Earth Sciences, The University of North Carolina at Charlotte,
9201 University City Blvd, Charlotte, NC 28223, USA; [email protected]
6 Department of Civil and Environmental Engineering, The University of North Carolina at Charlotte,
9201 University City Blvd, Charlotte, NC 28223, USA; [email protected]
7 Department of Biological Sciences, The University of North Carolina at Charlotte, 9201 University City Blvd,
Charlotte, NC 28223, USA; [email protected]
* Correspondence: [email protected]; Tel.: +1-704-687-8378

Received: 13 September 2018; Accepted: 24 October 2018; Published: 29 October 2018 

Abstract: Wastewater treatment plants (WWTPs) are thought to be potential incubators of antibiotic
resistance. Persistence of commonly used antibiotics in wastewater may increase the potential for
selection of resistance genes transferred between bacterial populations, some of which might pose a
threat to human health. In this study, we measured the concentrations of ten antibiotics in wastewater
plant influents and effluents, and in surface waters up- and downstream from two Charlotte area
treatment facilities. We performed Illumina shotgun sequencing to assay the microbial community
and resistome compositions at each site across four time points from late winter to mid-summer
of 2016. Antibiotics are present throughout wastewater treatment, and elevated concentrations
of multiple antibiotics are maintained in moving stream water downstream of effluent release.
While some human gut and activated sludge associated taxa are detectable downstream, these seem
to attenuate with distance while the core microbial community of the stream remains fairly consistent.
We observe the slight suppression of functional pathways in the downstream microbial communities,
including amino acid, carbohydrate, and nucleic acid metabolism, as well as nucleotide and amino
acid scavenging. Nearly all antibiotic resistance genes (ARGs) and potentially pathogenic taxa are
removed in the treatment process, though a few ARG markers are elevated downstream of effluent
release. Taken together, these results represent baseline measurements that future studies can utilize
to help to determine which factors control the movement of antibiotics and resistance genes through
aquatic urban ecosystems before, during, and after wastewater treatment.

Keywords: metagenomics; antibiotic resistance; wastewater; environmental ecology

Water 2018, 10, 1539; doi:10.3390/w10111539 8 www.mdpi.com/journal/water

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