Environment International 121 (2018) 1155–1161

Contents lists available at ScienceDirect

Environment International
journal homepage: www.elsevier.com/locate/envint

Prevalence and transmission of antibiotic resistance and microbiota between T

humans and water environments
Zhen-Chao Zhoua, Wan-Qiu Fenga, Yue Hana, Ji Zhenga, Tao Chena, Yuan-Yuan Weia,
⁎
Michael Gillingsb, Yong-Guan Zhuc, Hong Chena,
a
Institute of Environmental Technology, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
b
Department of Biological Sciences, Macquarie University, Sydney, NSW 2019, Australia
c
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China

A R T I C LE I N FO A B S T R A C T

Handling Editor: Olga-Ioanna Kalantzi The transmission routes for antibiotic resistance genes (ARGs) and microbiota between humans and water en-
Keywords: vironments is poorly characterized. Here, we used high-throughput qPCR analyses and 16S rRNA gene se-
Peri-urban quencing to examine the occurrence and abundance of antibiotic resistance genes and microbiota in both healthy
Aquatic humans and associated water environments from a Chinese village. Humans carried the most diverse assemblage
Wastewater of ARGs, with 234 different ARGs being detected. The total abundance of ARGs in feces, on skin, and in the
Drinking water effluent from domestic sewage treatment systems were approximately 23, 2, and 7 times higher than their
Human abundance in river samples. In total, 53 ARGs and 28 bacteria genera that were present in human feces could
Resistance
also be found in the influent and effluent of rural sewage treatment systems, and also downstream of the effluent
release point. We identified the bacterial taxa that showed a significant association with ARGs (P < 0.01,
r > 0.8) by network analysis, supporting the idea that these bacteria could carry some ARGs and transfer
between humans and the environment. Analysis of ARGs and microbiota in humans and in water environments
helps to define the transmission routes and dynamics of antibiotic resistance within these environments. This
study highlights human contribution to the load of ARGs into the environment and suggests means to prevent
such dissemination.

1. Introduction
Antibiotic resistance is a serious global threat to human health,
causing hundreds of thousands of fatalities each year (Pal et al., 2016;
Pehrsson et al., 2016). The spread of resistant bacteria and their anti-
biotic resistance genes (ARGs) is exacerbated by exchange of resistance
genes between human and environmental microbiota (Bhutani et al.,
2014; Forsberg et al., 2012). Horizontal gene transfer (HGT) can fa-
cilitate the dissemination of ARGs, via mobile genetic elements (MGEs)
such as integrons which are associated to insertion elements, transpo-
sons and plasmids (Aminov, 2011; Stevenson et al., 2017; von
Wintersdorff et al., 2016; Zhu et al., 2017b). Genes that could poten-
tially confer antibiotic resistance can be found in remote and pristine
settings (D'Costa et al., 2011; Pawlowski et al., 2016; Segawa et al.,
2013), but generally some of these are not clinically relevant. In con-
trast, environmental compartments with increased levels of anthro-
pogenic pressure are often highly contaminated with ARGs of concern
for human and animal health (Berendonk et al., 2015). Identifying the
microbiota and ARGs that pose the greatest threat to public health re-
quires an understanding of the distribution and dissemination of ARGs
between various environments.
Populations in rural areas interact directly with the environment,
and often have impacts on water resources. However, little is known
about the diversity and distribution of ARGs in rural drinking water
treatment plants (DWTP), the residents that rely on this water, and the
ARGs that flow through rural domestic sewage treatment systems
(RDSTS). Previous studies have demonstrated ARGs in urban drinking
water (Bai et al., 2015; Shi et al., 2013; Xu et al., 2016), wastewater
treatment systems (Di Cesare et al., 2016; Chen and Zhang, 2013; Mao
et al., 2015), and receiving rivers (Luo et al., 2010; Rodriguez-Mozaz
et al., 2015; Xu et al., 2015; Zheng et al., 2017), highlighting the effect
of different anthropogenic activities on ARGs distribution. The diversity
of human-associated ARGs (Foster et al., 2017; Ma et al., 2016; Sommer
et al., 2010; Zhu et al., 2017a) in human feces (Li et al., 2015; Pal et al.,
2016; Yatsunenko et al., 2012), and on skin (Junker and Schreiber,
2008; Schloissnig et al., 2013) also revealed the prevalence of ARGs.

⁎
Corresponding author.
E-mail address: chen_hong@zju.edu.cn (H. Chen).

https://doi.org/10.1016/j.envint.2018.10.032
Received 14 July 2018; Received in revised form 12 September 2018; Accepted 2 October 2018
Available online 09 November 2018
0160-4120/ © 2018 Elsevier Ltd. All rights reserved.
Z.-C. Zhou et al.
These findings emphasize the role of human/environment interaction in
the dissemination of clinically important resistance genes and provide
fundamental information on the ARGs in these different systems.
Most studies of ARGs in water focus on municipal water treatment
systems or natural water environments. However, the transmission of
ARGs between environments is poorly understood, and important
knowledge gaps remain. Studying microbial communities and ARGs in
humans can contribute to management options for controlling anti-
biotic resistance.
The objective of this work was to investigate the distribution and
characteristics of ARGs in the water transmission network of a peri-
urban village. We examined water treatment plant influent and ef-
fluent, tap water, human feces and skin, sewage treatment influent and
effluent, and the connecting river, both upstream and downstream,
using high-throughput quantitative PCR (HT-qPCR) (Zhu et al., 2013;
Zhu et al., 2017c). Our results provide insights into the fate of ARGs in
rural water supplies and during sewage treatment, and expand our
understanding of ARG prevalence in humans and water environments
in rural areas.
2. Materials and methods
2.1. Subject recruitment and sampling
Healthy male and female volunteers of 23 to 50 years of age were
recruited from a peri-urban village (29°78′N, 121°38′E) located in
Ningbo, Zhejiang Province, China, in May 2017. Briefly, feces (marked
as F1–F10) and skin (marked as S1–S10) microbiota samples of 6 male
and 4 female adults were sampled. Sample collection was approved by
the ethics committee of Zhejiang University School of Medicine, and all
subjects provided written informed consent before participation.
Further, nine water samples (marked as W1–W9) representing different
types of water from the village were collected. W1, DWTP influent; W2,
DWTP effluent; W3–W5, tap water; W6, RDSTS influent; W7, RDSTS
effluent; W8, upstream; W9, downstream. To exclude temporal het-
erogeneity, water samples were collected three times over three days.
Triplicate water samples were collected in sterile polyethylene bottles
which were washed twice with each water sample in advance. All
samples were transported to the laboratory on ice and stored at 4 °C
until processing. Details of sample collection procedures and DNA ex-
traction from feces, skin and water samples are described in
Supplementary Information (S1).
2.2. High-throughput qPCR
A total of 296 primer sets were used to target the ARGs (285 primer
sets targeted resistance genes for all major classes of antibiotics), MGEs
(9 primer sets targeted transposase genes), and one primer set targeted
the clinical class 1 integron-integrase gene and 16S rRNA genes (Table
S1) (Zhu et al., 2017c). High-throughput qPCR was performed using the
Wafergen SmartChip Real-time PCR system. The thermal cycle ampli-
fication conditions consisted of initial denaturation (10 min at 95 °C),
followed by 40 cycles of denaturation (30 s at 95 °C) and annealing (30 s
at 60 °C). Melting curve analyses were automatically generated by
Wafergen software. A non-template negative control was used for each
primer set and all quantitative PCRs were performed in technical tri-
plicates. Exclusion criteria included wells with efficiencies beyond the
range 1.7–2.3 or an r2 under 0.99 and amplicons with multiple melting
curves. Only samples with more than two replicates within the detec-
tion limit (a threshold cycle (Ct) of 31) were regarded as positive
quantification and used in further data analysis (Zhu et al., 2017c).
Gene copy numbers were calculated using the following formula.
Relative gene copy number = 10((31 − Ct)/(10/3))
The relative gene copy numbers of ARGs and MGEs were
1156
Environment International 121 (2018) 1155–1161
transformed into absolute copy numbers by normalization to the ab-
solute 16S rRNA gene copy numbers (Zhu et al., 2013). Absolute 16S
rRNA copy numbers were determined by the standard curve (SC)
method of quantification using the StepOnePlus™ real-time PCR system
(Applied Biosystems, Foster City, CA, USA) as described in a previous
study (Chen and Zhang, 2013).
2.3. Bacterial 16S rRNA gene sequencing
Microbial community compositions were determined by performing
16S rRNA gene sequencing on an Ion Torrent platform. The V4 to V5
regions of the bacterial 16S rRNA were amplified using the universal
primer set 515F/907R (Turner et al., 1999). In order to identify in-
dividual samples in a mixture within a single sequencing run the primer
set was tagged with unique barcodes (7-nucleotide barcodes) for each
sample (Cui et al., 2016). All sequences of each sample were screened
by filtering adaptor sequences and removing low-quality reads, am-
biguous nucleotides and barcodes. High-quality sequences were ana-
lyzed using Quantitative Insights Into Microbial Ecology (QIIME). The
sequences were clustered into operational taxonomic units (OTUs) at
the 97% similarity level using UCLUST.
2.4. Statistical analysis
Shannon-Weiner and Simpson indices were calculated for each
sample. Pearson and Spearman correlations coefficients were de-
termined by SPSS 20 (IBM, Armonk, NY, USA). Comparisons of ARG
abundances among different samples were carried out using Kruskal
Wallis tests with Bonferroni correction. Bar charts and Ternary plots
were generated by OriginPro 9.0 (Origin Lab Corporation, USA). The
principal coordinate analysis (PCoA) based on Bray-Curtis distance was
evaluated for the ARG and bacterial community profiles between dif-
ferent samples, and redundancy analysis (RDA) was applied to search
for potential links between ARGs and bacterial communities by using
Canoco software (V 5.0). Graphs were generated using R3.4.0 (R
Foundation for Statistical Computing) with the pheatmap and ggplot2
package, respectively. ANOSIM analyses were performed in R (vegan
package) with 999 permutations.
Network visualization was conducted using Frucherman Reingold
algorithms in the Gephi platform (Bastian et al., 2009; Hu et al., 2017).
Only statistically robust correlations with Spearman's correlation coef-
ficient (ρ) > 0.7 and significance level (P) < 0.01 were used to form
the final networks (Junker and Schreiber, 2008).
3. Results and discussion
3.1. The persistent bacteria in humans and water environments
The composition of bacterial communities was different among
feces, skin and water (Anosim statistic R = 0.7579, P < 0.001).
Bacteroidetes and Firmicutes were the dominant phyla in human feces
accounting for 89.5% of total bacterial 16S rRNA gene sequences,
Actinobacteria and Proteobacteria were the dominant phyla (69.3%) on
human skin, and Bacteroidetes and Proteobacteria were the dominant
phyla (70.2% to 86.1%) in water environments (Fig. S1).
To further explore ARG transfer in water environments, we tracked
the circulation of bacteria between feces and various water samples
(Fig. S2). A total of 28 bacteria genera were all found in feces, influent,
effluent and downstream, these bacteria were also persistent, here
called type-I bacteria. A total of 9 bacteria were not found in the river
water, and only persist into the effluent, here called type-II bacteria.
Some 37 bacteria were successfully removed by treatment, since they
only moved as far as the influent samples, here called type-III bacteria.
Details of type-I, II, and III bacteria were shown in Table S2.
Type-I bacteria were hardly removed by treatment, and these bac-
teria showed high abundance in feces, influent, effluent and the river.
Z.-C. Zhou et al. Environment International 121 (2018) 1155–1161

They may be the hosts of the persistent ARGs that subsequently transfer
into aquatic environment. In contrast, type-II bacteria were not found in
downstream water samples. These bacteria were generally anaerobic or
facultative anaerobic bacteria that might not survive in well-oxyge-
nated river water. Type-III bacteria were removed by treatment. These
were often enteric bacteria and may not tolerate sewage treatment.
Some Type-III bacteria require specific environmental condition, for
instance species of the genus Aquabacterium that were commonly iso-
lated from drinking water biofilms (Kalmbach et al., 1999). The change
bacterial species composition could affect the distribution of ARGs (Zhu
et al., 2017c), especially if these ARGs were located on chromosomes
rather than mobile genetic elements.
3.2. The characterization of ARGs in various environments
Using the HT-qPCR method, the total numbers of distinct resistance
genes detected in all the combined samples from feces, skin and water
were 234, 150 and 226, respectively. Individual samples exhibited
lower diversity than these totals, with the highest diversity occurring in
a fecal sample (149 ARGs) and the lowest in a skin sample (21 ARGs). In
general, the highest diversity occurred in fecal samples, river water,
sewage influent and effluent. Lower diversity characterized samples
from the drinking water treatment plant, tap water and skin (Fig. 1a).
Genes encoding resistance to beta-lactams, MLSB, tetracycline, ami-
noglycosides, vancomycin, and genes for efflux pumps were present in
almost every sample, although their relative proportions varied (Fig. 1).
Because the bacterial load in each sample type varied considerably,
we normalized the abundance of ARGs to copies per bacterial cell, using
the abundance of the 16S rRNA gene as a proxy for cell number
(Fig. 1b). Fecal samples exhibited the highest number of ARGs per cell,
and these ARGs were dominated by genes conferring resistance to tet-
racycline, beta-lactams and MLSB. The only other sample to show si-
milarly high normalized abundance score was the influent (W6) to the
sewage treatment system (Fig. 1b), reflecting the abundance in feces.
Some individual ARGs were consistently found at high abundance in
all feces samples. These included the genes aacA_aphD, cfxA, ermB,
ermF, tet(Q), tet(X), and tet(32) (Fig. S3). On skin, the resistance genes
cphA-01, fox5, mepA, strB, and ttgB were consistently detected at high
abundance. This demonstrated the different distribution of ARGs in
human bodies, probably driven by the different ecological niches of the
bacterial species in which they reside (Gibson et al., 2015; Schloissnig
et al., 2013; Smillie et al., 2011). The similarities of ARGs within
sample types was confirmed by PCoA (Fig. 2), which showed strong
clustering of feces samples, well separated from skin samples. Samples
from the drinking water treatment plant clustered together, consistent
with their low abundance and diversity of ARGs (Fig. 1), while the
remaining water samples form a separate cluster. From the PCoA ana-
lysis, drinking water and human skins were clustered together such as
W3 and W4, indicating the relationship between drinking water and
human skins. Previous study used animals to demonstrated the effected
of drinking water on mice gut bacterial community (Dias et al., 2017),
but the impacts that antibiotic resistance genes in drinking water may
have on human health were still unclear (Vaz-Moreira et al., 2014).
The diversity of ARGs detected in most feces and skin samples were

Fig. 1. Diversity and abundance of antibiotic resistance genes in human feces (F1 to F10), on human skin (S1 to S10) and in water (W1 to W9). Number of distinct
ARGs detected (a) and abundance of resistance genes normalized as copies per cell, based on 16S rRNA gene abundance (b). Error bars represent standard deviation.
Resistance genes were classified based on the antibiotics to which they conferred resistance, these being: aminoglycosides, beta-lactams, fluoroquinolone/quinolone/
florfenicol/chlora phenicol/amphenicol (FCA), macrolide-lincosamide-streptogramin B (MLSB), sulfonamide, tetracycline, vancomycin and ‘others’, largely com-
prising efflux pumps.

1157
Z.-C. Zhou et al.
Fig. 2. ARGs distribution among human feces, human skins and water.
Principal coordinate analysis (PCoA) based on Bray-Curtis distance, showing
the overall distribution pattern of ARG assemblages in each of the different
samples. F, fecal samples; S, skin samples; W1, DWTP influent; W2, DWTP ef-
fluent; W3–W5, tap water; W6, RDSTS influent; W7, RDSTS effluent; W8, up-
stream; W9, downstream.
similar (Fig. S4a, b). Inverse Simpson and Shannon indices calculations
consistently indicated that ARG diversity in water generally increased
along the water transmission pathway from the drinking water treat-
ment plant to tap water, and then to the sewage treatment influent (Fig.
S4c). Treatment of sewage did not significantly reduce ARG diversity,
and effluent exhibited much higher diversity than upstream river
samples (W8). Further, downstream samples (W9) exhibited sig-
nificantly higher ARG diversity than upstream (P < 0.05), indicating
that the sewage effluent significantly increased the ARG diversity in the
river (Guo et al., 2017; Pruden et al., 2012; Yang et al., 2014).
The absolute abundance of ARGs ranged from about 5 × 105 to
4.5 × 1012 copies per gram in feces, and about 1.3 × 103 to 1.3 × 106
copies per cm2 on skin (Fig. S4). Abundance of these genes in water
samples was much lower, with ARGs being some 3.9 × 103 to
8.7 × 1010 copies per L and MGEs ranging between 1 × 105 to 3 × 1010
copies per L (Fig. S4). Among water samples, RDSTS effluent contained
1158
Environment International 121 (2018) 1155–1161
a higher absolute abundance of ARGs (Fig. S4f), indicating the removal
of ARGs was poor in RDSTS compared with municipal sewage treat-
ment systems (Chen and Zhang, 2013), and that RDSTS may be a hot-
spot for ARG replication and propagation. The rural domestic sewage
treatment system harbored diverse and abundant ARGs that conferred
resistance to almost all antibiotics, showing that it could be a major
conduit for transferring ARGs into the environment.
3.3. The persistent ARGs in humans and water environments
To understand the transfer of ARGs in water environments, we
tracked the circulation of ARGs between feces and various water sam-
ples (Fig. 3). A total of 53 ARGs were found in feces, sewage influent,
effluent and the river downstream from the effluent point. These ARGs
were persistent, and were not removed by treatment, here called type-I
ARGs. A total of 32 ARGs did not survive in the river water, and only
persist into the effluent, here called type-II ARGs. A total of 43 ARGs
only move as far as the influent samples, and appeared to be success-
fully removed by treatment, here called type-III ARGs. Details and
identities of type-I, II, III ARGs were shown in Table S3.
Persistent ARGs may reflect a high level of risk for public health,
since persistent ARGs were inefficiently removed by treatment systems.
Genes for resistance to beta-lactams were persistent in both humans and
water environments such as blaCTX, blaOXA, blaTEM, blaVEB and
blaVIM, and beta-lactams were among the top three most consumed
antibiotics worldwide (You et al., 2018). Among the persistent ARGs
some of those were undoubtedly widespread in different bacterial
species such as ermB, tetM, sul2 and so on.
Large numbers of ARGs found in feces were also present in treat-
ment plant influent, where the lower absolute abundance was pre-
sumably a result of dilution. A significant proportion of these ARGs was
not removed by treatment, and were also found in effluent. The
abundance of some ARGs does decline during treatment, and this may
correspond to the removal of the organisms they reside in.
Chromosomally located resistance determinants could be prone to re-
moval if the cells they reside in are preferentially removed by treat-
ment, while ARGs that can move between species by lateral gene
transfer could survive by moving between organisms. Some ARGs
present in the effluent were not found in the river water. Perhaps this is
because the organisms they reside in do not survive in the river water
environment. The analysis presented in Fig. 3 demonstrates the differ-
ential survival of ARGs, showing transmission from human, to treat-
ment plant, to effluent, and then back into the receiving river.
Fig. 3. Absolute abundance of ARGs in humans and water
samples from various environmental compartments. Red
points (n = 53) represent the ARGs that appear in feces,
influent, effluent and the downstream river. These ARGs
are persistent, and are not removed by treatment, here
called Type-I ARGs. Some ARGs do not survive in the
river water, and only persist into the effluent - green
points (n = 32), here called Type-II ARGs, while some
ARGs appear to be successfully removed by treatment,
only moving as far as the influent samples - blue points
(n = 43), here called Type-III ARGs. (For interpretation
of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Z.-C. Zhou et al. Environment International 121 (2018) 1155–1161

Fig. 4. The Network analysis depicting the co-occurrence patterns between types of ARGs and bacteria. The nodes were coloured according to types of ARGs and
genus. A connection represents a strong (Spearman's correlation coefficient (ρ) > 0.8) and significant (P < 0.01) correlation. Node size was weighted according to
the number of connections (that is the degree) and edges weighted according to the correlation coefficient. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

3.4. Co-occurrence of ARGs and bacteria between humans and water

environments

To address the question of whether specific bacterial species might

host particular ARGs, the co-occurrence patterns of ARGs and bacterial
types were explored by network analysis (Fig. 4) (Junker and Schreiber,
2008). The visualization of the network consisted of 137 nodes (nodes
represented type-I, II, III ARGs or bacteria) and 256 edges. The mod-
ularity index of network was 0.741 (values > 0.4) suggested that the
network had a significantly modular structure (Zhang et al., 2017).
Genes in the same module may interact more frequently among them-
selves than with genes in other modules. These genes were potentially
carried by some specific MGEs or bacteria, and could then share similar
dynamics and transmission between environmental compartments.
Determining the co-occurrence of ARGs and bacteria would help to
Fig. 5. Correlation of absolute abundance of ARGs with absolute abundance of track transmission between humans and water environments and
MGEs. identify emerging ARG subtypes, these could then be targeted to con-
trolling ARG spread in village communities.
As shown in Fig. 4, Type-I ARGs and Type-I bacteria clustered

1159
Z.-C. Zhou et al.
together and had more interconnections within the network. This
clustering strongly suggested that these ARGs were transferred from
feces through influent, effluent and into the river by their residence
within similarly persistent bacteria. Since the network was based on
correlation, it was not really possible to assign residence of particular
resistance genes to particular Bacterial genera. However, it was likely
that the cloud of Type-1 ARGS associated with Type-1 Bacteria was
collectively housed within these Bacteria, and it was possible that the
ARGs were shared among these genera by lateral gene transfer.
Some Type-1 resistance genes do show network patterns that were
consistent with survival through the treatment process by lateral gene
transfer. For instance, the network cluster containing ARGs of the aadA
family were strongly cross connected, but not significantly connected to
any named bacterial genera. This family of ARGs and the similarly
unconnected blaOXA10 were borne on integron gene cassettes
(Partridge et al., 2009). Gene cassettes are DNA elements which can be
captured by integrons (Gillings, 2014). Consequently, these ARGs might
persist through water treatment by lateral gene transfer between di-
verse hosts, or by survival as extracellular DNA.
For some Type-II and Type-III ARGs, the network analysis demon-
strated few relationships with particular bacterial genera, such as the
module containing the acr-family and yce-family. Among Type-III bac-
teria and Type-III ARGs, Clostridium was correlated with ARG subtypes
of MLSB resistance genes (mefA), as was Enterococcus (ermK-02), which
also showed correlation with tetracycline resistance genes (tetS). These
associations have been reported in previous studies (Li et al., 2015;
Martins et al., 2006; Oravcova et al., 2014).
The network analysis (Fig. 4) allowed predictions to be made about
the fates of ARGs as they flow through various compartments. In some
cases, ARGs might be moving between different cell hosts during the
treatment process, in other cases ARGs were eliminated when their host
cells die. Analyses like these will help identify critical points during
sewage treatment that can be addressed to improve the removal of
ARGs.
3.5. The influence of bacteria and MGEs on ARGs distribution
Redundancy analysis was conducted to examine the relationships
between entire ARG profiles and bacterial communities (Fig. S5). The
two primary axes explained 70.5% of total variance in ARG profiles.
Bacteroidetes, Firmicutes and Fusobacteria were significantly corre-
lated with Axis 1 and genes conferring resistance to aminoglycoside,
beta-lactams, MLSB, other/efflux and tetracycline.
The total absolute abundance of ARGs was significantly correlated
with MGEs (P < 0.01, r = 0.989) (Fig. 5). This strongly suggested co-
occurrence and of ARGs and MGEs in the samples examined. In turn,
this suggested that much of the survival of persistent ARGs could de-
pend on their residence on MGEs, where they had the ability to move
between different bacterial species. Our results and previous studies
(Zheng et al., 2017; Zhou et al., 2017; Zhu et al., 2017c) consistently
indicated the potential effect of MGEs on ARG distribution and trans-
mission.
4. Conclusion
In general, this work identified humans as a major contributor to the
load of ARGs entering the environment. In this scenario, ARGs could be
acquired from water, food, domestic animals, or other village members.
ARGs then take up residence in the gut and skin microbiota, where their
abundance will increase under any antibiotic therapy. Such microbiota,
with their cargo of ARGs, enter waste water via washing or sewage. The
domestic sewage treatment systems examined here do eliminate some
bacteria and ARGs, but significant numbers survive the treatment
process to emerge as effluent for disposal into the river. Survival
through the system might occur via cell types that resist elimination,
but is also likely to be driven by ARGs being able to transfer between
1160
Environment International 121 (2018) 1155–1161
different cells and species during treatment. Pollution of river water
with these bacteria and their resident ARGs closes the transmission
circle, since downstream settlements can acquire these resistance genes
via consumption of river water. Understanding the mechanisms of
movement between different environmental compartments is a first
step towards better control of antibiotic resistance. According to the
results of this study, we suggested possible options for antibiotic re-
sistance control: 1) control antibiotic use in rural and urban clinics
especially for personal antibiotic use and raise the awareness of ARGs
among rural residents; 2) focus on the ARGs elimination in rural DWTP
to provide drinking water with low ARGs abundance; 3) set more
RDSTS in rural area to increase the sewage collection rate and
strengthen the tail water treatment.
Acknowledgements
This work was supported by Natural Science Foundation of China
(21876147, 41571130064 and 21677121).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.envint.2018.10.032.
References
Aminov, R.I., 2011. Horizontal gene exchange in environmental microbiota. Front.
Microbiol. 2, 158.
Bai, X., Ma, X., Xu, F., Li, J., Zhang, H., Xiao, X., 2015. The drinking water treatment
process as a potential source of affecting the bacterial antibiotic resistance. Sci. Total
Environ. 533, 24–31.
Bastian, M., Heymann, S., Jacomy, M., 2009. Gephi: an open source software for ex-
ploring and manipulating networks. In: International Conference on Weblogs and
Social Media. 8. ICWSM, pp. 361–362 2009.
Berendonk, T.U., Manaia, C.M., Merlin, C., Fattakassinos, D., Cytryn, E., Walsh, F.,
Bürgmann, H., Sørum, H., Norström, M., Pons, M.N., 2015. Tackling antibiotic re-
sistance: the environmental framework. Nat. Rev. Microbiol. 13, 310.
Bhutani, N., Muraleedharan, C., Talreja, D., Rana, S.W., Walia, S., Kumar, A., Walia, S.K.,
2014. Occurrence of multidrug resistant extended spectrum beta-lactamase-produ-
cing bacteria on iceberg lettuce retailed for human consumption. Biomed. Res. Int.
2015, 547547.
Chen, H., Zhang, M., 2013. Occurrence and removal of antibiotic resistance genes in
municipal wastewater and rural domestic sewage treatment systems in eastern China.
Environ. Int. 55, 9–14.
Cui, E., Wu, Y., Zuo, Y., Chen, H., 2016. Effect of different biochars on antibiotic re-
sistance genes and bacterial community during chicken manure composting.
Bioresour. Technol. 203, 11–17.
D'Costa, V.M., King, C.E., Kalan, L., Morar, M., Sung, W.W., Schwarz, C., Froese, D.,
Zazula, G., Calmels, F., Debruyne, R., 2011. Antibiotic resistance is ancient. Nature
477, 457.
Di Cesare, A., Eckert, E.M., D'Urso, S., Bertoni, R., Gillan, D.C., Wattiez, R., Corno, G.,
2016. Co-occurrence of integrase 1, antibiotic and heavy metal resistance genes in
municipal wastewater treatment plants. Water Res. 94, 208–214.
Dias, M.F., Reis, M.P., Acurcio, L.B., Carmo, A.O., Diamantino, C.F., Motta, A.M.,
Kalapothakis, M., Nicoli, J.R., Nascimento, A.M.A., 2017. Changes in mouse gut
bacterial community in response to different types of drinking water. Water Res.
132, 79.
Forsberg, K.J., Reyes, A., Wang, B., Selleck, E.M., Sommer, M.O., Dantas, G., 2012. The
shared antibiotic resistome of soil bacteria and human pathogens. Science 337,
1107–1111.
Foster, K.R., Schluter, J., Coyte, K.Z., Rakoffnahoum, S., 2017. The evolution of the host
microbiome as an ecosystem on a leash. Nature 548, 43–51.
Gibson, M.K., Forsberg, K.J., Dantas, G., 2015. Improved annotation of antibiotic re-
sistance determinants reveals microbial resistomes cluster by ecology. ISME J. 9,
207–216.
Gillings, M.R., 2014. Integrons: past, present, and future. Microbiol. Mol. Biol. Rev. 78,
257–277.
Guo, J., Li, J., Chen, H., Bond, P.L., Yuan, Z., 2017. Metagenomic analysis reveals was-
tewater treatment plants as hotspots of antibiotic resistance genes and mobile genetic
elements. Water Res. 123, 468–478.
Hu, H.W., Wang, J.T., Li, J., Shi, X.Z., Ma, Y.B., Chen, D., He, J.Z., 2017. Long-term nickel
contamination increases the occurrence of antibiotic resistance genes in agricultural
soils. Environ. Sci. Technol. 51, 790.
Junker, B.H., Schreiber, F., 2008. Analysis of Biological Networks (Wiley Series in
Bioinformatics). Wiley-Interscience.
Kalmbach, S., Manz, W., Wecke, J., Szewzyk, U., 1999. Aquabacterium gen. nov., with
description of Aquabacterium citratiphilum sp. nov., Aquabacterium parvum sp. nov.
and Aquabacterium commune sp. nov., three in situ dominant bacterial species from
Z.-C. Zhou et al.
the Berlin drinking water system. Int. J. Syst. Bacteriol. 2 (49 Pt), 769–777.
Li, B., Yang, Y., Ma, L., Ju, F., Guo, F., Tiedje, J.M., Zhang, T., 2015. Metagenomic and
network analysis reveal wide distribution and co-occurrence of environmental anti-
biotic resistance genes. ISME J. 9 (11), 2490–2502.
Luo, Y., Mao, D., Rysz, M., Zhou, Q., Zhang, H., Xu, L., P, J.J.A., 2010. Trends in antibiotic
resistance genes occurrence in the Haihe River, China. Environ. Sci. Technol. 44,
7220–7225.
Ma, L., Xia, Y., Li, B., Yang, Y., Li, L.G., Tiedje, J.M., Zhang, T., 2016. Metagenomic
assembly reveals hosts of antibiotic resistance genes and the shared resistome in pig,
chicken and human feces. Environ. Sci. Technol. 50 (1), 420–427.
Mao, D., Yu, S., Rysz, M., Luo, Y., Yang, F., Li, F., Hou, J., Mu, Q., Alvarez, P.J., 2015.
Prevalence and proliferation of antibiotic resistance genes in two municipal waste-
water treatment plants. Water Res. 85, 458–466.
Martins, d.C.P., Vazpires, P., Bernardo, F., 2006. Antimicrobial resistance in Enterococcus
spp. isolated in inflow, effluent and sludge from municipal sewage water treatment
plants. Water Res. 40, 1735–1740.
Oravcova, V., Zurek, L., Townsend, A., Clark, A.B., Ellis, J.C., Cizek, A., Literak, I., 2014.
American crows as carriers of vancomycin-resistant enterococci with vanA gene.
Environ. Microbiol. 16, 939–949.
Pal, C., Bengtsson-Palme, J., Kristiansson, E., Larsson, D.G.J., 2016. The structure and
diversity of human, animal and environmental resistomes. Microbiome 4, 54.
Partridge, S.R., Tsafnat, G., Coiera, E., Iredell, J.R., 2009. Gene cassettes and cassette
arrays in mobile resistance integrons. FEMS Microbiol. Rev. 33, 757–784.
Pawlowski, A.C., Wang, W., Koteva, K., Barton, H.A., McArthur, A.G., Wright, G.D., 2016.
A diverse intrinsic antibiotic resistome from a cave bacterium. Nat. Commun. 7,
13803.
Pehrsson, E.C., Tsukayama, P., Patel, S., Mejíabautista, M., Sosasoto, G., Navarrete, K.M.,
Calderon, M., Cabrera, L., Hoyosarango, W., Bertoli, M.T., 2016. Interconnected
microbiomes and resistomes in low-income human habitats. Nature 533, 212–216.
Pruden, A., Arabi, M., Storteboom, H.N., 2012. Correlation between upstream human
activities and riverine antibiotic resistance genes. Environ. Sci. Technol. 46,
11541–11549.
Rodriguez-Mozaz, S., Chamorro, S., Marti, E., Huerta, B., Gros, M., Sànchez-Melsió, A.,
Borrego, C.M., Barceló, D., Balcázar, J.L., 2015. Occurrence of antibiotics and anti-
biotic resistance genes in hospital and urban wastewaters and their impact on the
receiving river. Water Res. 69, 234–242.
Schloissnig, S., Arumugam, M., Sunagawa, S., Mitreva, M., Tap, J., Zhu, A., Waller, A.,
Mende, D.R., Schommer, N.N., Gallo, R.L., 2013. Structure and function of the human
skin microbiome. Trends Microbiol. 21, 660–668.
Segawa, T., Takeuchi, N., Rivera, A., Yamada, A., Yoshimura, Y., Barcaza, G., Shinbori, K.,
Motoyama, H., Kohshima, S., Ushida, K., 2013. Distribution of antibiotic resistance
genes in glacier environments. Environ. Microbiol. Rep. 5, 127–134.
Shi, P., Jia, S., Zhang, X.X., Zhang, T., Cheng, S., Li, A., 2013. Metagenomic insights into
chlorination effects on microbial antibiotic resistance in drinking water. Water Res.
47, 111.
Smillie, C.S., Smith, M.B., Friedman, J., Cordero, O.X., David, L.A., Alm, E.J., 2011.
Ecology drives a global network of gene exchange connecting the human micro-
biome. Nature 480, 241–244.
Sommer, M.O., Church, G.M., Dantas, G., 2010. The human microbiome harbors a diverse
1161
Environment International 121 (2018) 1155–1161
reservoir of antibiotic resistance genes. Virulence 1, 299–303.
Stevenson, C., Hall, J.P., Harrison, E., Wood, A.J., Brockhurst, M.A., 2017. Gene mobility
promotes the spread of resistance in bacterial populations. ISME J. 11 (8),
1930–1932.
Turner, S., Pryer, K.M., Miao, V.P., Palmer, J.D., 1999. Investigating deep phylogenetic
relationships among Cyanobacteria and plastids by small subunit rRNA sequence
analysis. J. Eukaryot. Microbiol. 46, 327–338.
Vaz-Moreira, I., Nunes, O.C., Manaia, C.M., 2014. Bacterial diversity and antibiotic re-
sistance in water habitats: searching the links with the human microbiome. FEMS
Microbiol. Rev. 38 (4), 761–778.
von Wintersdorff, C.J., Penders, J., van Niekerk, J.M., Mills, N.D., Majumder, S., van
Alphen, L.B., Savelkoul, P.H., Wolffs, P.F., 2016. Dissemination of antimicrobial re-
sistance in microbial ecosystems through horizontal gene transfer. Front. Microbiol.
7, 173.
Xu, J., Xu, Y., Wang, H., Guo, C., Qiu, H., He, Y., Zhang, Y., Li, X., Wei, M., 2015.
Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant
and its effluent-receiving river. Chemosphere 119, 1379–1385.
Xu, L., Ouyang, W., Qian, Y., Su, C., Su, J., Chen, H., 2016. High-throughput profiling of
antibiotic resistance genes in drinking water treatment plants and distribution sys-
tems. Environ. Pollut. 213, 119–126.
Yang, Y., Li, B., Zou, S., Fang, H.H., Zhang, T., 2014. Fate of antibiotic resistance genes in
sewage treatment plant revealed by metagenomic approach. Water Res. 62, 97–106.
Yatsunenko, T., Rey, F.E., Manary, M.J., Trehan, I., Dominguez-Bello, M.G., Contreras,
M., Magris, M., Hidalgo, G., Baldassano, R.N., Anokhin, A.P., 2012. Human gut mi-
crobiome viewed across age and geography. Nature 486, 222.
You, X., Wu, D., Wei, H., Xie, B., Lu, J., 2018. Fluoroquinolones and β-lactam antibiotics
and antibiotic resistance genes in autumn leachates of seven major municipal solid
waste landfills in China. Environ. Int. 113, 162–169.
Zhang, S.Y., Su, J.Q., Sun, G.X., Yang, Y., Zhao, Y., Ding, J., Chen, Y.S., Shen, Y., Zhu, G.,
Rensing, C., 2017. Land scale biogeography of arsenic biotransformation genes in
estuarine wetland. Environ. Microbiol. 19 (6).
Zheng, J., Gao, R., Wei, Y., Chen, T., Fan, J., Zhou, Z., Makimilua, T.B., Jiao, Y., Chen, H.,
2017. High-throughput profiling and analysis of antibiotic resistance genes in East
Tiaoxi River, China. Environ. Pollut. 230, 648–654.
Zhou, Z.C., Zheng, J., Wei, Y.Y., Chen, T., Dahlgren, R.A., Shang, X., Chen, H., 2017.
Antibiotic resistance genes in an urban river as impacted by bacterial community and
physicochemical parameters. Environ. Sci. Pollut. Res. 24, 1–10.
Zhu, Y.G., Johnson, T.A., Su, J.Q., Qiao, M., Guo, G.X., Stedtfeld, R.D., Hashsham, S.A.,
Tiedje, J.M., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine
farms. Proc. Natl. Acad. Sci. U. S. A. 110, 3435–3440.
Zhu, Y.G., Gillings, M., Simonet, P., Stekel, D., Banwart, S., Penuelas, J., 2017a. Human
dissemination of genes and microorganisms in Earth's Critical Zone. Glob. Chang.
Biol. 1–12.
Zhu, Y.G., Gillings, M., Simonet, P., Stekel, D., Banwart, S., Penuelas, J., 2017b. Microbial
mass movements. Science 357, 1099–1100.
Zhu, Y.G., Zhao, Y., Li, B., Huang, C.L., Zhang, S.Y., Yu, S., Chen, Y.S., Zhang, T., Gillings,
M.R., Su, J.Q., 2017c. Continental-scale pollution of estuaries with antibiotic re-
sistance genes. Nat. Microbiol. 2, 16270.