STOTEN-24352; No of Pages 10

Science of the Total Environment xxx (2017) xxx–xxx

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Science of the Total Environment

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Occurrence and diversity of antibiotic resistance in untreated

hospital wastewater
Qiang Wang a,b, Panliang Wang a, Qingxiang Yang a,b,⁎
a
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
b
Key Laboratory for Microorganisms and Functional Molecules (Henan Normal University), University of Henan Province, Xinxiang 453007, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Antibiotic resistance was assessed in

untreated hospital wastewater.
• High-frequency ARGs were detected
with high-capacity qPCR.
• Some clinical pathogenic or opportunis-
tic MARB were detected with high prev-
alence.
• Positive correlations between MGEs and
some ARGs were detected.
• High concentrations of ARGs and MEGs
were detected.

a r t i c l e i n f o a b s t r a c t

Article history: Antibiotics, antibiotic-resistant bacteria (ARB), antibiotic-resistance genes (ARGs), and mobile genetic elements
Received 1 September 2017 (MGEs) have been reported in many environments. However, the investigation of their occurrence and diversity
Received in revised form 13 October 2017 in untreated hospital wastewater is still insufficient. High concentrations of antibiotic residues were found in hos-
Accepted 13 October 2017
pital wastewater using solid-phase extraction and UPLC–MS/MS analysis. The concentrations of six of 14 antibi-
Available online xxxx
otics reached μg/L levels in the hospital wastewater, which is higher than reported in other aquatic environments.
Editor: D. Barcelo Results of high-throughput sequencing analysis indicated that sequences affiliated to genera Escherichia and
Acinetobacter were the predominant in the cultivable multiple-antibiotic-resistant bacteria (CMARB) recovered
Keywords: from the wastewater of three hospitals in China, with compositions of 34%–74%. Notably, several genera contain-
Antibiotic-resistant bacteria ing clinically pathogenic or opportunistic CMARB (e.g., Escherichia, Acinetobacter, Aeromonas, Myroides, Enterococ-
Antibiotic-resistance gene cus, Proteus, Pseudomonas, and Streptococcus) were detected at high relative abundances in the wastewaters of
Mobile genetic element the three hospitals. High-capacity quantitative PCR showed that 131–139 unique ARGs of the 178 targeted
Gene cassette genes were detected in the hospital wastewaters. The high prevalence of five MGEs and 12 ARGs was confirmed
Hospital wastewater
with qPCR, and some positive correlations between ARGs and MGEs were identified, such as between intI1 and
qnrD, intI2 and sul3, intI3 and tetX, Tn916/Tn1545 and sul2, and ISCR1 and sul3. These results suggest that highly
abundant antibiotic-resistant pathogens and highly mobile ARGs already exist in the human body, and that their
release from hospitals without effective treatment poses high risks to environments and human health.
© 2017 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: College of Life Sciences, Henan Normal University, Xinxiang 453007, China.
E-mail address: yangqx@htu.edu.cn (Q. Yang).

https://doi.org/10.1016/j.scitotenv.2017.10.128
0048-9697/© 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
2 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx
1. Introduction
Since their introduction into human medicines in the 1940s, antibi-
otics have been the most important drugs for the treatment of infectious
diseases. However, antibiotic-resistant pathogens have emerged and
spread among human and animal populations worldwide in response
to the selection pressure exerted by the overuse of antibiotics. Some
antibiotic-resistant pathogens, such as methicillin-resistant Staphylo-
coccus aureus (Grundmann et al., 2006) and carbapenem-resistant En-
terobacteriaceae (Patrice et al., 2011), pose significant risks to human
health. Hospital wastewater is highly hazardous because it is not
only infectious and toxic, but is also an important source of antibiotic-
resistant bacteria (ARB) and antibiotic-resistance genes (ARGs).
Without suitable treatment, ARB or ARGs from clinical sources can be
dispersed and even thrive in the environments, accelerating the devel-
opment of multiple-antibiotic-resistant bacteria (MARB).
With the increasing concerns about the antibiotic pollution of
aquatic environments, several studies have assessed the presence of
antibiotics, ARGs, and ARB in hospital effluents (Le et al., 2016;
Rodríguez-Mozaz et al., 2015; Timraz et al., 2017; Varela et al., 2016),
and other studies have examined the correlation between antibiotics
and ARGs (Li et al., 2016) or between antibiotics and ARB (Varela
et al., 2014). However, there have been far fewer studies of hospital
aquatic ecosystems than of other environments, such as urban waste-
water. Most reports focused on the influents or effluents from hospital
treatment plants, in which the distribution and diversity of antibiotics,
ARGs, and ARB were altered in response to the selective pressure im-
posed by long-term antibiotic exposure. Few reports were found to
study the occurrence of antibiotic resistance in the untreated hospital
wastewater, in which the antibiotics unlikely to affect ARB and ARGs.
These wastewaters reflect the integrated discharges from humans, es-
pecially when patient density is high, and therefore reflect the original
ARB and ARGs in human clinical samples. The human microflora, espe-
cially intestinal bacteria, is a reservoir for diverse ARGs. These bacteria
not only exchange resistance genes among themselves, but can also in-
teract with the bacteria that pass through the body, causing those bac-
teria to acquire and transmit ARGs (Sommer et al., 2010; Van den
Braak et al., 1998). Furthermore, the ARB and ARGs that are released
from the human body might then return to their original habitats
(e.g., food, water, soil) and pose an increased environmental risk
(Salyers et al., 2004). Therefore, investigation of antibiotic resistance
in untreated hospital wastewater is helpful for understanding how clin-
ical antibiotic resistance affects the environmental ARB.
Levels of antibiotic resistance in hospital wastewater may be
different from other aquatic environments due to different antibiotic
application patterns. For example, some antibiotics, such as cefotiam, pi-
peracillin, and vancomycin, are mainly used in hospitals (Kümmerer
and Henninger, 2003). Therefore, the aim of this study was to evaluate
the pollution levels of antibiotics, ARB, ARGs, and mobile genetic
elements (MGEs) in untreated hospital wastewater to identify the po-
tential risks of the discharge of clinical ARB and ARGs into the environ-
ment. A broad range of antibiotics from different families, including
sulfonamides, tetracyclines, quinolones, macrolides, cephalexin, linco-
mycin, and trimethoprim, were selected and monitored in the waste-
water from three tertiary public hospitals located in Xinxiang city,
central China.
2. Materials and methods
2.1. Study sites and sampling
Hospital wastewater samples were collected from three tertiary
public hospitals (abbreviated as H1, H2, and H3) located in Xinxiang
city, central China, in January and July 2016. The three hospitals are
comprehensive hospitals with general departments that each admit
800–1300 patients per day and have bed capacities of 1740, 1200, and
Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
700, respectively. The wastewater was collected from the outflow
from daily medical applications in sterile plastic bottles. The collected
samples were transported to the laboratory in a portable icebox for im-
mediate processing.
2.2. Quantitation of antibiotics in hospital wastewater with solid-phase ex-
traction and ultra-performance liquid chromatography–tandem mass
spectrometry (UPLC–MS/MS)
The procedures for the extraction of the antibiotics were according
to Zhang et al. (2011). Briefly, the collected water samples were filtered
through 0.45 μm fiber filters. The pH of the filtrate was adjusted to 3.0
with 6 M HCl, and Na2EDTA was added (to a final concentration of
0.2 g/L) to minimize any interference during the analysis. The ex-
tracted antibiotic compounds were analyzed with a Waters UPLC-
TQD system (Waters, USA) equipped with a Waters ACQUITY FTN
AutoSampler, Waters Xevo TQ MS, and Waters Acquity UPLC BEH
C18 column (2.1 × 50 mm, 1.7 μm). The specific reaction conditions
and method of analysis were according to Gros et al. (2013).
Tetracycline, oxytetracycline, erythromycin, spiramycin, sulfadia-
zine, sulfamethazine, sulfamethoxazole, cefalexin, ciprofloxacin,
ofloxacin, enrofloxacin, norfloxacin, lincomycin, and trimethoprim
were purchased from Dr. Ehrenstorfer (Augsburg, Germany) as stan-
dard compounds. The standard curve for each antibiotic was construct-
ed with at least five concentrations (R2 N 0.99). The recovery and matrix
effects were estimated according to Matuszewski et al. (2003) and are
shown in Table 1.
2.3. Enumeration of THCB and CMARB
Colony-forming units (CFUs) of total heterotrophic cultivable het-
erotrophic bacteria (THCB) and CMARB were measured with a standard
plate dilution technique on Luria–Bertani (LB) agar, Salmonellae Shigel-
la agar (SS), or Mueller–Hinton (MH) agar. Samples of 1 mL of hospital
wastewater were transferred into centrifuge tube containing 9 mL of
stroke-physiological saline solution, and mixed thoroughly. For
CMARB culture, tri-resistant media were prepared by mixing three of
the eight antibiotics tested, as listed in Table S1. Eight different antibi-
otics (tetracycline, cephalexin, erythromycin, gentamicin, chloram-
phenicol, kanamycin, ciprofloxacin, and amoxicillin) were purchased
from Solarbio Technology Co., Ltd. (Beijing, China) and used for the cul-
ture and isolation of CMARB in hospital wastewater. The working con-
centrations of these antibiotics were higher than the minimum
inhibitory concentrations for resistant bacteria according to CLSI guide-
lines (Franklin et al., 2012), as listed in Table S2. Plates of LB, SS, or MH
agar containing all of the different combinations of antibiotic were inoc-
ulated with 0.2 mL of the diluted wastewater sample, and incubated at
25 °C for 48–72 h (Qingxiang et al., 2017). To culture THCB, LB, SS, or MH
plates without antibiotics were inoculated and incubated under the
same conditions. The abundance of CMARB was determined based on
the ratio of CMARB to THCB on each type of agar plate. Each hospital
wastewater sample was diluted in a series of consecutive decimal
steps and each dilution was plated by triplicate.
2.4. Community characterization of total bacteria (TB) and CMARB
Total DNA was extracted from the three hospital wastewater sam-
ples to characterize the communities of TB. Each hospital wastewater
sample (200 mL) was filtered through a polycarbonate membrane
(0.22 μm; Xinya, Shanghai, China), and the filter membranes were
then used for DNA extraction with the E.Z.N.A.® Water DNA Kit
(Omega, Solon, OH, USA), according to the manufacturer's instructions.
To determine the diversity of CMARB cultivated on the tri-resistant
media described above, all colonies were harvested and mixed and the
total genomic DNA was extracted. To obtain template DNA from the ag-
gregate colonies present on tri-resistant media, the surface of the agar
 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx 3

Table 1
Concentrations and validation parameters of antibiotic residues in three hospital wastewater samples.

Antibiotics Concentrations Validation parameters

H1 (ng/L) H2 (ng/L) H3 (ng/L) Mean valuesa Recovery rate (%) LOD (ng/L) LOQ (ng/L)

Sulfadiazine (SD) 119.24 124.94 125.82 123.33 ± 3.57 46.82–65.24 7.18 23.93
Sulfamethazine (SM2) 11.65 BLDb BLD NAc 41.87–63.24 10.32 54.40
Sulfamethoxazole (SMX) 434.3 576.28 587.97 532.85 ± 85.55 41.84–55.23 4.38 14.60
Ofloxacin (OFL) 2596.51 2020.94 2372.09 2329.85 ± 290.10 42.43–61.36 14.90 49.67
Norfloxacin (NOR) 1879.76 1383.85 1624.58 1629.40 ± 247.99 46.72–59.64 31.26 104.20
Ciprofloxacin (CIP) 1236.95 1223.52 1541.82 1334.10 ± 180.02 47.62–76.24 9.46 31.53
Enrofloxacin (ENR) BLD 59.72 BLD NA 57.53–82.67 6.54 21.80
Tetracycline (TC) 1147.83 1487.78 1598.26 1411.29 ± 234.75 53.78–67.42 25.10 83.67
Oxytetracycline (OTC) 1287.42 1727.05 1423.89 1479.45 ± 225.02 46.73–71.36 4.82 16.07
Spiramycin (SPM) BLD BLD 58.73 NA 57.53–82.67 13.72 45.73
Erythromycin (ERY) 599.4 404.28 433.14 478.94 ± 105.31 56.83–78.29 8.28 27.60
Lincomycin (LIN) 118.25 140.08 136.81 131.71 ± 11.77 52.75–67.43 10.99 36.63
Trimethoprim (TMP) 345.63 184.78 273.85 268.09 ± 80.58 41.32–62.31 12.35 41.17
cefalexin (CLX) 2459.25 2669.14 2048.43 2392.27 ± 315.73 38.79–57.81 3.75 12.50
a
Values are means of the three hospital samples ± standard deviation.
b
BLD, below detection limit.
c
NA, not applicable.

media was flooded with 2 mL of stroke-physiological saline solution,
and a sterile spreader was used to suspend as much colony material as
possible (Stevenson et al., 2004). The concentrations of DNA were de-
termined with spectrophotometry (NanoDrop 2000, Thermo Fisher Sci-
entific, Waltham, MA, USA) and the integrity of the DNA was checked
with 0.7% (w/v) agarose gel electrophoresis (60 V, 2 h).
The purified DNA was sent to Novogene (Beijing, China) for Illumina
HiSeq 2500 (Pair-end reads of 250 nt in length) sequencing. The V4 region
of the 16S rRNA genes was amplified with primer pair 515F (5′-GTGCCA
GCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′)
and sequenced, and the diversity of the TB and CMARB was analyzed
(Caporaso et al., 2011). The samples were individually barcoded to
allow multiplex sequencing. Raw sequencing data were in FASTQ format.
Paired-end reads were then preprocessed using Trimmomatic software to
detect and remove ambiguous bases (N). Low quality sequences with av-
erage quality score below 20 using sliding window trimming approach.
After trimming, paired-end reads were assembled using FLASH software.
The parameters of assembly were as follows: minimum 10 bp of overlap,
maximum 200 bp of overlap, and maximum 20% mismatch rate. Se-
quences were performed further denoising and chimera removal by
using QIIME software (version 1.8.0). Primer sequences were removed
and clean reads were clustered to generate operational taxonomic units
(OTUs) using UPARSE software with 97% similarity cutoff (Edgar et al.,
2011). A BLAST analysis of the taxonomic classification to the phylum
level was performed with RDP Classifier (version 2.2, http://sourceforge.
net/projects/rdp-classifier/) (Wang et al., 2007) in the GreenGene data-
base (http://greengenes.lbl.gov/cgi-bin/nphindex.cgi) (DeSantis et al.,
2006). The sequencing dataset for TB and CMARB were deposited in Se-
quence Read Archive with accession numbers SRP118400 and
SRP118450, respectively.
2.5. Detection and quantification of ARGs
The samples collected in January 2016 were used to detect almost all
the major classes of ARGs with high-capacity qPCR (Wcgene, Shanghai,
China). For this, the total DNA was extracted with the E.Z.N.A.® Water
DNA Kit (Omega), according to the manufacturer's instructions.
The 258 qPCR primers designed for the 178 unique ARGs tested, two
MGEs (class 1 integron intI1 and transposon Tn916/Tn1545), and 16S
rRNA genes assessed are listed in Table S3. Amplification was conducted
in 100 nL reactions containing (final concentrations) 1 × LightCycler
480 SYBR Green I Master Mix (Roche Inc., Indianapolis, IN, USA),
1 ng/μL bovine serum albumin, 3 ng/μL DNA template, and 1 μM each
primer. The thermal cycling parameters were initial denaturation at
95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for
30 s and annealing at 60 °C for 30 s, with a melting curve analysis
autogenerated by the program. For each primer set, amplification was
conducted in triplicate and a no-template control was included.
Based on the results of the high-capacity qPCR, 12 frequently detect-
ed genes were selected for further quantification with qPCR. These in-
cluded three tetracycline-resistance genes (TRGs: tetX, tetM, tetO),
three macrolide-resistance genes (MRGs: ereA, ermA, ermB), three
sulfonamide-resistance genes (SRGs: sul1, sul2, sul3), and three
quinolone-resistance genes (QRGs: qnrA, qnrB, oqxB). The 16S rRNA
genes were also quantified in all samples. Plasmids containing the target
gene fragments were constructed with the pMD™19-T vector (Takara,
Japan) and were used as controls for qPCR standard curves. The 20 μL re-
actions contained 10 μL of AceQ SYBR® Green Master Mix (Vazyme,
Nanjing, China), 0.2 mM each primer, and 2 μL of DNA template. Real-
Time PCR was performed with the LightCycler® 96 qPCR System
(Roche, Switzerland) using the following reaction program: initial de-
naturation at 95 °C for 5 min, and 40 cycles consisting of 95 °C for
10 s, annealing temperature (Table S3) for 15 s, and 72 °C for 15 s. The
melting curve was generated by the Roche system. The standard curves
for the qPCR primer sets covered at least five orders of magnitude (R2 ≥
0.99).
2.6. Detection of MGEs and associated gene cassettes
Five MGEs were detected in the hospital wastewaters, including
integrons (intI1, intI2, and intI3), Tn916/Tn1545, and ISCR1 (class 1 in-
sertion sequences common region). The gene cassettes carried by the
integrons or ISCR1 were amplified and sequenced. The primers used
are listed in Table S4. The total DNA in the hospital wastewater was
used as the template. The PCR assays were performed in 25 μL reactions
containing 12.5 μL of 2 × PCR Taq MasterMix (ABM, Richmond, BC,
Canada), 0.8 μL of 10 mM forward and reverse primers, 1 μL of template
DNA, and 9.9 μL of sterile double-distilled H2O. The temperature param-
eters were as follows: initial denaturation at 95 °C for 5 min, followed by
35 cycles of denaturation at 94 °C for 1 min, annealing temperature for
30 s, and extension at 72 °C for 90 s; with a final extension at 72 °C for
10 min.
The PCR products were checked with 1.0% (w/v) agarose gel electro-
phoresis and then sent to Sangon Biotech (Shanghai, China) for se-
quencing. The gene cassette sequences were analyzed online at the
National Center for Biotechnology Information database (https://blast.
ncbi.nlm.nih.gov/Blast.cgi) and the integron database INTEGRALL
(http://integrall.bio.ua.pt/).

Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
4 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx
2.7. Statistical analysis
The results of high-capacity qPCR were analyzed with the SmartChip
qPCR software (v2.7.0.1). Wells with multiple melting peak and wells
with amplification efficiencies beyond 1.8–2.2 were omitted. A cycle
threshold (Ct) of 30 was used as the detection limit. Only samples in
which amplification occurred in all three replicates were considered
positive. Pearson's correlation was used to analyze the relationships
between antibiotics and ARGs. The threshold for significance was p b
0.05. All statistical analyses were performed with SPSS 18.0 (SPSS Inc.,
Chicago, Illinois), and diagrams were drawn with Origin 8.0 (OriginLab
Inc., Hampton, Massachusetts).
3. Results
3.1. Occurrence and abundance of antibiotics
Fourteen antibiotics, including sulfonamides, tetracyclines, quino-
lones, macrolides, cephalexin, lincomycin, and trimethoprim, were de-
tected in the wastewaters of the three hospitals, as shown in Table 1.
Among these, 11 antibiotics were detected in all three hospitals, where-
as sulfamethazine, enrofloxacin, and spiramycin were each present in
only one hospital. The concentrations of six of the 14 antibiotics reached
the μg/L level. The high detection frequencies and concentrations sug-
gest that hospital wastewater is a major source of antibiotic contamina-
tion. Among these 14 antibiotics, the concentrations of quinolones,
tetracyclines, and cephalexin were notably higher than those of
macrolides and sulfonamides in all three hospitals.
3.2. Population and community characteristics of THCB and CMARB in hos-
pital wastewater
The concentrations of THCB and CMARB in the January and July
wastewaters from the three hospitals are shown in Fig. 1A. The concen-
tration of THCB in January wastewater (2.03–3.31 × 109 CFU/L) was not
significantly different from the concentration of THCB in July wastewa-
ter (1.75–2.47 × 109 CFU/L) (p N 0.05). The CMARB that were resistant
to combinations of any three of the eight antibiotics were 1–2
orders of magnitude lower than the THCB in each wastewater sample
(9.93 × 107–2.34 × 108 CFU/L in January and 1.01–2.00 × 108 CFU/L
in July). The percentages of CMARB to THCB (4.89%–8.11%) in the
wastewater samples in these two months are shown in Fig. 1B. The
prevalence of CMARB was higher in the July samples than in the January
samples (p b 0.05).
High-throughput sequencing was used to determine the composi-
tions of the bacterial communities of the collected TB (Fig. 2A) and
CMARB (Fig. 2B). At the genus level, although the composition of
CMARB differed between the three hospital wastewaters, sequences af-
filiated to the genera Escherichia and Acinetobacter were the predomi-
nant in all three wastewaters, and with compositions of 34%–74%. The
effect of season on the CMARB communities was significant (p b 0.05),
whereas there were no significant differences among the three hospital
samples collected in the same month (p N 0.05). The diversity fraction of
CMARB was greater in the samples from July than in samples from Jan-
uary. Sequences affiliated to the genera Aeromonas, Comamonas,
Citrobacter, Pseudochrobactrum, and Morganella were detected in high
abundance in the July samples, whereas they were not detected in
any January sample. The CMARB and TB community compositions dif-
fered significantly (p b 0.01). For example, although Escherichia was
the most prevalent CMARB in almost all samples (21.78%–70.99% of
total CMARB), it constituted only 0.12%–6.11% of the total bacteria.
Arcobacter constituted b1% of CMARB in all samples, but it was the pre-
dominant genus in the TB (6.41%–45.42% of TB). Notably, sequences af-
filiated to several genera associated with opportunistic pathogens
(e.g., Escherichia, Acinetobacter, Aeromonas, Myroides, Enterococcus,
Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
Proteus, Pseudomonas, Streptococcus, etc.) were detected at relatively
high abundances in the three hospital wastewaters.
3.3. Occurrence of ARGs in hospital wastewater
The 178 ARGs assayed in this study confer resistance to seven classes
of antibiotics and include three resistance mechanisms. The counts of
ARGs detected in the three hospital wastewaters are shown in Fig. 3.
Of the 178 targeted ARGs, 131, 160, and 139 unique ARGs were detected
in the wastewaters from H1, H2, and H3, respectively. One hundred
twenty-six ARGs were present in the samples from all three hospitals,
whereas 17 ARGs were not detected in any of the three hospital waste-
waters. The high prevalence of ARGs indicates that ARGs occur widely in
hospital wastewater.
Based on the results of high-capacity quantification, the concentra-
tions of 12 ARGs, including tetracycline-, macrolide-, sulfonamide-,
and quinolone-resistance genes, were confirmed with qPCR. The con-
centrations of the 12 ARGs and 16S rRNA genes are shown in Table 2.
The TRG tetO was the most abundant in wastewater from both H1 and
H2 samples. The SRG sul1 was the most abundant resistance gene in
wastewater from H3. The MRG ermA was found at only several copies
per milliliter in samples from all of the hospitals. However, two other
MRGs, ereA and ermB, were detected at high concentrations. QRGs
were found at the lowest concentrations in the three hospital wastewa-
ters. The number of gene copies was normalized to the 16S rRNA gene
copy number (Fig. 4). The overall trend in the relative abundances of
the four ARG families was TRGs ≈ SRGs N MRGs N QRGs, although
there were some differences in the relative abundances of the ARGs
among the three hospital samples.
3.4. High prevalence of MGEs in hospital wastewater
Five MGEs, including three integrons (intI1, intI2, and intI3), Tn916/
Tn1545, and ISCR1, were quantified in this study (Table 2 and Fig. 5).
The most abundant MGE was ISCR1, with concentrations of (3.02 ±
0.58) × 107 to (4.51 ± 0.29) × 108 copies/mL, which were higher than
the concentrations of the 16S rRNA gene. The concentrations of the
three classes of integrons were also high (especially those of the class
1 integrons). Transposon Tn916/Tn1545 was detected at concentrations
of (8.08 ± 0.22) × 104 to (4.49 ± 0.13) × 105 copies/mL. The integron-
and ISCR1-associated gene cassettes were also investigated. When the
gene cassettes were detected, bright and well-size bands were excised
from the agarose gel after PCR amplification. Many gene cassettes
with low copy numbers in the samples or that did not match the de-
signed primers were ignored. Some bands of a similar size were also ig-
nored. Ultimately, 13 PCR products were sequenced, and six specific
gene cassettes were identified, including five class-1-integron-
associated gene cassettes (aadA–aacC, emrE–aadA–blaVIM-4, catB3–
blaOXA-4–aadA, catB3–aadA–blaVIM-4, and aadA–aacC–aac3-VI) and one
ISCR1-associated gene cassette (orf513–sul1) (Fig. 6). No class 2 or
class 3 integron-associated gene cassettes were detected with our
method. The aadA gene, encoding an aminoglycoside adenylating en-
zyme, which confers resistance to streptomycin and spectinomycin,
was observed in all the class 1 integron-associated gene cassettes
detected.
4. Discussion
The results of this study reflect the intrinsic characteristics of the an-
tibiotics, ARGs, ARB, and MGEs found in the hospital environment. The
major source of the ARB, ARGs, and MGEs detected in this study was
human excreta, and they therefore reflect the environmental risks
posed by human clinical sources, especially those derived from patients.
The occurrence and diversity of the antibiotics detected also reflect their
medical use.
 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx 5

Fig. 1. Number of colony-forming units of total heterotrophic cultivable bacteria (THCB) and cultivable multiple-antibiotic-resistant bacteria (CMARB) in hospital wastewater. (A),
Abundance of THCB and CMARB, (B), Percentage of CMARB relative to THCB. H1-1–H3-1, samples collected from three hospital wastewaters in January; H1-7–H3-7, samples collected
from three hospital wastewaters in July.

Among the antibiotics detected in this study, cephalexin and
ofloxacin were clearly most abundant, which may be attributed to
their high medical consumption. Higher concentrations of most antibi-
otics were found in the hospital wastewaters than in urban river
water or urban wastewater, especially the cephalexin, quinolones, and
tetracyclines (Luo et al., 2011; Varela et al., 2014; Xu et al., 2016b). Sim-
ilar results have been reported in other hospital wastewaters (Le et al.,
2016; Rodríguez-Mozaz et al., 2015). However, lower concentrations
of sulfonamides were detected in hospital wastewater samples than
have been reported in studies of urban wastewater samples (Michael
et al., 2013; Xu et al., 2015). One possible reason for this is that sulfon-
amides are primarily used for animals (Malintan and Mohd, 2006),
and only a small percentage are used as human medicines. Similar to
sulfonamides, macrolides are used more in households than in hospitals
(Kümmerer and Henninger, 2003) which may explain why they were
detected at very low concentrations. It is notable that β-lactams are
also widely used in hospitals, and their presence in wastewaters should
be assessed in future studies.
Our THCB and CMARB counts suggested that the influence of season
on the bacterial population is insignificant (p N 0.05). This result is con-
trary to most previous reports (Banerjee et al., 2016; Jung and
Matthews, 2016). However, our result is just that the CMARB was
from human intrinsic ARB, rather than from horizontal gene transfer
under the pressure of environmental factors. The intrinsic ARB were
likely derived from human intestinal excreta because Escherichia was
the most prevalent CMARB in almost all samples.
Notably, some sequences affiliated to clinical and opportunistic
pathogens were prevalent in the hospital wastewater samples. For

Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
6 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx

Fig. 2. Relative abundance of sequences affiliated to different bacterial genera in samples of (A) TB and (B) CMARB. H1-1–H3-1, samples collected from three hospital wastewaters in
January; H1-7–H3-7, samples collected from three hospital wastewaters in July.

example, E. coli, which, although most strains are innocuous residents of
the gastrointestinal tract can cause diarrheal and extraintestinal dis-
eases (Croxen et al., 2013), was highly abundant among the ARB. In ad-
dition, antibiotic-resistant E. coli strains have many opportunities to
transfer their ARGs to other enteropathogens. The genus Acinetobacter
is frequently isolated in hospital-acquired infections, especially in inten-
sive care units. In this study, this genus was highly abundant in both TB
and CMARB, especially the species Acinetobacter baumannii.
A. baumannii is a frequent cause of nosocomial pneumonia and other in-
fections, such as skin and wound infections, infective endocarditis, bac-
teremia, urinary tract infections, and meningitis (Dent et al., 2010).
Several studies have demonstrated that A. baumannii is resistant to
many of the antibiotics used in the hospital context (Fournier and
Richet, 2006; Hu et al., 2011). Opportunistic pathogens Aeromonas
spp. have been implicated in a number of infections in humans,
including bacteremia and gastroenteritis, and members of the genus
Aeromonas can carry genes encoding β-lactam and plasmid-mediated
quinolone resistance that can be spread by horizontal gene transfer.
Other human pathogens or opportunistic pathogens, such as members
of the genera Myroides, Enterococcus, Proteus, Pseudomonas, and Strepto-
coccus, were also prevalent in the MARB communities in the hospital
wastewaters. All of these bacteria were resistant to more than three
classes of clinical antibiotics, in addition to their intrinsic resistance. Al-
though the number of ARB in hospital wastewaters is reduced after
treatment with sewage processing techniques (Huang et al., 2012),
wastewaters still contain higher proportions of various resistance fac-
tors than surface waters (Goñi-Urriza et al., 2000; Schwartz et al.,
2003). Several reports have even indicated that wastewater treatment
plants (WWTPs) provide conditions conducive to the establishment
and propagation of ARB (Kim et al., 2007; Marcinek et al., 1998).

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(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx 7

Table 2 Using PCR or qPCR to determine the presence of ARGs is an accurate

Concentrations of ARGs, MGEs, and 16S rRNA genes in three hospital wastewater but inefficient method that is not applicable to the broad-spectrum
samples.a
scanning of ARGs in environmental samples. High-capacity qPCR is a
Concentrations of ARGs and 16S rRNA genes (copies/mL)a useful complementary approach. In this study, the results of high-
Hospital 1 Hospital 2 Hospital 3 capacity qPCR revealed the high abundance and diversity of antibiotic
8 7 resistance genes in hospital wastewater. Of the 178 targeted ARGs,
16S rRNA (2.26 ± 0.30) × 10 (8.81 ± 4.29) × 10 (1.11 ± 0.34) × 108
tetX (4.14 ± 0.14) × 106 (1.30 ± 0.17) × 106 (3.78 ± 0.74) × 106 126 were detected in all the samples from the three hospitals, and
tetM (4.15 ± 0.45) × 105 (8.59 ± 1.45) × 104 (4.10 ± 0.75) × 104 therefore pose a risk of their dissemination. Environments other than
tetO (8.34 ± 1.23) × 107 (2.45 ± 1.99) × 107 (1.38 ± 0.08) × 106 hospital wastewater, such as raw municipal sewage (Karkman et al.,
ereA (1.06 ± 0.03) × 105 (6.81 ± 1.11) × 104 (3.50 ± 1.03) × 105 2016), soil fertilized with sewage sludge and chicken manure (Chen
ermA 1.88 ± 0.00 1.88 ± 0.00 1.88 ± 0.00
ermB (3.20 ± 0.38) × 106 (1.99 ± 0.73) × 106 (8.28 ± 1.38) × 105
et al., 2016), and even drinking-water treatment system (Xu et al.,
sul1 (7.24 ± 1.49) × 107 (1.89 ± 0.25) × 107 (1.30 ± 0.68) × 107 2016a), have also been shown to contain high levels of ARGs and
sul2 (5.14 ± 0.24) × 106 (7.83 ± 0.64) × 106 (1.29 ± 0.09) × 106 MGEs. Although wastewater treatment processes are reported to effi-
sul3 (4.84 ± 0.17) × 104 (6.40 ± 0.47) × 102 (3.84 ± 0.17) × 104 ciently remove several ARGs, most ARGs are still detectable at high con-
qnrA (1.58 ± 0.35) × 104 (1.01 ± 0.18) × 104 (4.79 ± 1.35) × 104
centrations in treated hospital effluents and WWTP effluents (Laht et al.,
qnrD (3.72 ± 1.34) × 103 (1.72 ± 0.23) × 103 (2.58 ± 1.34) × 103
oqxB (1.60 ± 0.48) × 104 (3.34 ± 0.28) × 103 (1.85 ± 0.28) × 104 2014; Li et al., 2016; Quoc et al., 2016). Rodríguez-Mozaz and co-
intI1 (2.88 ± 0.75) × 107 (4.57 ± 1.58) × 107 (1.51 ± 0.24) × 107 workers (Rodríguez-Mozaz et al., 2015) reported that relative concen-
intI2 (1.52 ± 0.29) × 104 (1.52 ± 0.58) × 102 (2.87 ± 0.29) × 103 trations of the ermB and tetW genes decreased after wastewater treat-
intI3 (1.62 ± 0.44) × 103 (1.26 ± 0.18) × 102 (4.07 ± 0.52) × 103 ment, whereas the concentrations of the blaTEM, sulI, and qnrS genes
Tn916/Tn1545 (4.49 ± 0.13) × 105 (8.08 ± 0.22) × 104 (8.70 ± 1.33) × 104
ISCR1 (4.51 ± 0.29) × 108 (3.02 ± 0.58) × 107 (1.26 ± 0.32) × 108
increased. Similarly, Timraz et al. (2017) evaluated the removal efficien-
TRGs (8.80 ± 1.25) × 107 (2.59 ± 1.05) × 107 (5.20 ± 0.80) × 106 cies of ARGs by WWTPs operated on-site at hospitals. They found that
MRGs (3.30 ± 0.38) × 106 (2.06 ± 0.74) × 106 (1.18 ± 0.31) × 106 the sul1 and intl1 genes remained in the treated effluents at up to 105
SRGs (7.76 ± 3.52) × 107 (1.97 ± 2.52) × 107 (1.43 ± 0.08) × 107 copies per mL or at 0.8 copies per 16S rRNA gene. Therefore, although
QRGs (3.56 ± 0.96) × 104 (3.14 ± 0.44) × 104 (6.90 ± 0.34) × 104
hospital wastewaters are treated on-site or at WWTPs, effluents with
MGEs (4.80 ± 2.96) × 108 (7.60 ± 2.44) × 107 (1.41 ± 0.54) × 108
large amounts of detectable ARGs are still a potential source for horizon-
TRGs, tetracycline-resistance genes (tetX, tetM, tetO); MRGs, macrolide-resistance genes tal gene transfer after their discharge into aquatic environments.
(ereA, ermA, ermB); SRGs, sulfonamide-resistance genes (sul1, sul2, sul3); QRGs, quino-
lone-resistance genes (qnrA, qnrB, oqxB), MGEs, mobile genetic elements (intI1, intI2,
Based on the results of high-capacity qPCR, 12 ARGs detected at high
intI3, Tn916/1545, ISCR1). frequencies were assayed with qPCR. Unexpectedly, a negative correla-
a
Values are means of triplicate ± standard deviation. tion was observed between the concentrations of antibiotics and ARGs
in the hospital wastewaters (Fig. S1). This negative correlation may in-
dicate that antibiotics to which there is less microbial drug resistance
Therefore, hospital wastewater, or even treated wastewater, presents a are used more extensively in hospitals. Those antibiotics with high mi-
high risk to human health, with the release of ARB into aquatic environ- crobial drug resistance have reduced therapeutic effects and are used
ments. Compared with other pollution sources, hospital wastewater less frequently. Therefore, they appear at low concentrations in waste-
presents a greater environmental risk not only because they contain water, whereas the corresponding ARGs are common. Considering the
high concentrations of antibiotics but also due to antibiotic-resistant short retention time of collected wastewater, the ARGs detected were
pathogen and/or opportunistic pathogen release. likely pre-existing in patients or others and were not subject to selection

Fig. 3. Prevalence of antibiotic resistance genes in hospital wastewater samples based on high-capacity quantification. FCA = fluoroquinolone, quinolone, florfenicol, chloramphenicol, and
amphenicol resistance genes; MLSB = macrolide-mincosamide-streptogramin B resistance.

Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
8 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx

Fig. 4. Relative abundance of antibiotic resistance genes grouped by antibiotics which they confer resistance to. TRGs, tetracycline-resistance genes (tetX, tetM, tetO); MRGs, macrolide-
resistance genes (ereA, ermA, ermB); SRGs, sulfonamide-resistance genes (sul1, sul2, sul3); QRGs, quinolone-resistance genes (qnrA, qnrB, oqxB).

pressure by the residual antibiotics in the wastewater. In contrast, pos-
itive correlations between antibiotic and ARG concentrations have gen-
erally been found in most other antibiotic-polluted environments, such
as hospital or urban WWTP systems (Li et al., 2016; Varela et al., 2014).
Some antibiotics may play a role in shaping bacterial communities and
promoting the horizontal transfer of ARGs under conditions of long-
term exposure (Corno et al., 2014; Jutkina et al., 2016).
A high positive correlation between ARGs and MGEs has been dem-
onstrated in various environments (Tian et al., 2016; Zhu et al., 2013). In
the present study, five MGEs were detected at high concentrations in all
samples. Some positive correlations between ARGs and MGEs were also
found in the hospital wastewaters, such as between intI1 and qnrD, intI2
and sul3, intI3 and tetX, Tn916/Tn1545 and sul2, and ISCR1 and sul3
(Table S5). This indicates that the ARGs present in the human body
are highly mobile, and the transfer of ARGs among pathogenic and non-
pathogenic bacteria poses a serious risk. Furthermore, if highly mobile
ARGs are dispersed in the environment, they will be further enriched
and widely disseminated. It is noteworthy that the concentrations of
class 1 integrons and ISCR1 elements were higher than the concentra-
tions of 16S rRNA genes. This high occurrence of integrons and insertion
sequences implies their co-presence in single cells and their high mobil-
ity potential among different genera and species.
ARGs normally cluster in genomic or plasmid DNA as ARG cassettes.
Exogenous ARGs carried in gene cassettes are usually accumulate and
are expressed by integrons, especially class 1 integrons. Integrons em-
bedded in gene cassettes provide a genetic platform that accelerates

Fig. 5. Relative abundance of MGEs in the wastewater of the three studied Hospitals.

Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx
Fig. 6. Types of antibiotic resistance gene cassettes present in hospital wastewater.
horizontal gene transfer among bacteria. Among the gene cassettes de-
tected in this study, the aadA gene was found in all the class 1 integron-
associated gene cassettes. According to the literature, gene cassettes
carrying aadA are frequently detected in both clinical and environmen-
tal isolates (Clark et al., 1999; Ma et al., 2013; Michael et al., 2005), sug-
gesting that the aadA gene readily clusters with other ARGs in gene
cassettes. The gene cassette aadA–aacC, which was detected in this
study, has also been found in different pathogens, including Salmonella
enterica (Doublet et al., 2003) and Vibrio fluvialis (Ahmed et al., 2004).
These class 1 integrons embedded in gene cassettes can not only trans-
fer their ARGs to other genera by horizontal gene transfer, but can also
recombine the ARGs they carry to generate new gene cassettes. There-
fore, hospital wastewaters containing large gene cassettes may intensify
the transmission of ARGs and the development of MARB. In contrast, no
class 2 or class 3 integron-associated gene cassettes were detected in
this study. Class 2 and class 3 integrons may be less diverse than class
1 integrons because the gene encoding the class 2 integrase contains a
nonsense mutation in codon 179, leading to a truncated, nonfunctional
protein (Mazel, 2006).
5. Conclusion
In this study, antibiotics, bacterial communities, ARGs, and MGEs
were evaluated in hospital wastewaters. Our results demonstrate a
higher occurrence of some antibiotics, especially cephalexin, quino-
lones, and tetracyclines, in hospital wastewaters than has been reported
in other aquatic environments. However, unlike reports of other envi-
ronments, these antibiotics did not correlate positively with the abun-
dance of various ARB, ARGs, and MGEs, indicating that these ARB,
ARGs, and MGEs were already present in human excreta, especially
the intestinal excreta. In contrast, the positive correlations between
MGEs and some ARGs strongly suggest that mobile ARGs are released
into the environment in human clinical wastewater. Several clinical
pathogenic or opportunistic MARB, such as Escherichia, Acinetobacter,
Aeromonas, Myroides, Enterococcus, Proteus, Pseudomonas, and Strepto-
coccus, were highly prevalent in the hospital wastewaters and some
ARG cassettes were present in the samples. These data highlight the
risks that hospital wastewaters present to the environment and
human health unless suitably treated.
Acknowledgments
This study was supported by the National Natural Science Foundation
of China [NSFC 21477035 and NSFC 21277041], the Training Program of
the National Natural Science Foundation of China [5101049170806], the
Key Project of Natural Science of the Education Department of Henan
Province of China [16A180029], and the PhD research startup foundation
of Henan Normal University [qd15177].
Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
9
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2017.10.128.
References
Ahmed, A.M., Nakagawa, T., Arakawa, E., Ramamurthy, T., Shinoda, S., Shimamoto, T.,
2004. New aminoglycoside acetyltransferase gene, aac(3)-Id, in a class 1 integron
from a multiresistant strain of Vibrio fluvialis isolated from an infant aged 6 months.
J. Antimicrob. Chemother. 53 (6):947–951. https://doi.org/10.1093/jac/dkh221.
Banerjee, G., Ray, A.K., Kumar, R., 2016. Effect of temperature on lateral gene transfer ef-
ficiency of multi-antibiotics resistant bacterium, Alcaligenes faecalis. Sains Malaysiana
45 (6), 909–914.
Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh, P.J.,
et al., 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences
per sample. Proc. Natl. Acad. Sci. 108 (Supplement 1):4516–4522. https://doi.org/
10.1073/pnas.1000080107.
Chen, Q., An, X., Li, H., Su, J., Ma, Y., Zhu, Y.-G., 2016. Long-term field application of sewage
sludge increases the abundance of antibiotic resistance genes in soil. Environ. Int. 92,
1–10 (doi:10.1016/j.envint.2016.03.026).
Clark, N.C., Olsvik, O., Swenson, J.M., Spiegel, C.A., Tenover, F.C., 1999. Detection of a strep-
tomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.
Antimicrob. Agents Chemother. 43 (1), 157–160.
Corno, G., Coci, M., Giardina, M., Plechuk, S., Campanile, F., Stefani, S., 2014. Antibiotics
promote aggregation within aquatic bacterial communities. Front. Microbiol. 5:1–9.
https://doi.org/10.3389/fmicb.2014.00297.
Croxen, M.A., Law, R.J., Scholz, R., Keeney, K.M., Wlodarska, M., Finlay, B.B., 2013. Recent
advances in understanding enteric pathogenic Escherichia coli. Clin. Microbiol. Rev.
26 (4):822–880. https://doi.org/10.1128/cmr.00022-13.
Dent, L.L., Marshall, D.R., Pratap, S., Hulette, R.B., 2010. Multidrug resistant Acinetobacter
baumannii: a descriptive study in a city hospital. BMC Infect. Dis. 10 (1), 1–7 (doi:
10.1186/1471-2334-10-196).
DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., et al., 2006.
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible
with ARB. Appl. Environ. Microbiol. 72 (7):5069–5072. https://doi.org/10.1128/
aem.03006-05.
Doublet, B., Lailler, R., Meunier, D., Brisabois, A., Boyd, D., Mulvey, M.R., et al., 2003. Variant Sal-
monella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar
Albany. Emerg. Infect. Dis. 9 (5):585. https://doi.org/10.3201/eid0905.020609.
Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensi-
tivity and speed of chimera detection. Bioinformatics 27 (16):2194–2200. https://
doi.org/10.1093/bioinformatics/btr381.
Fournier, P.E., Richet, H., 2006. The epidemiology and control of Acinetobacter baumannii
in health care facilities. Clin. Infect. Dis. 42 (5):692. https://doi.org/10.1086/500202.
Franklin, R., Matthew, A., Jeff, A., Michael, N., George, M., 2012. Performance Standards for
Antimicrobial Susceptibility Testing; Twenty-Second Informational Supplement. CLSI
Document M100-S22. Clinical and Laboratory Standards Institute, Wayne, PA https://
doi.org/10.1302/2046-3758.13.2000032.
Goñi-Urriza, M., Capdepuy, M., Arpin, C., Raymond, N., Caumette, P., Quentin, C., 2000. Im-
pact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and
Aeromonas spp. Appl. Environ. Microbiol. 66 (1):125. https://doi.org/10.1128/
aem.66.1.125-132.2000.
Gros, M., Rodríguez-Mozaz, S., Barceló, D., 2013. Rapid analysis of multiclass antibiotic
residues and some of their metabolites in hospital, urban wastewater and river
water by ultra-high-performance liquid chromatography coupled to quadrupole-
linear ion trap tandem mass spectrometry. J. Chromatogr. A 1292 (16):173. https://
doi.org/10.1016/j.chroma.2012.12.072.
Grundmann, H., Aires-De-Sousa, M., Boyce, J., Tiemersma, E., 2006. Emergence and resur-
gence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet
368 (9538):874. https://doi.org/10.1016/s0140-6736(06)68853-3.
10 Q. Wang et al. / Science of the Total Environment xxx (2017) xxx–xxx
Hu, Q., Hu, Z., Li, J., Tian, B., Xu, H., Li, J., 2011. Detection of OXA-type carbapenemases and
integrons among carbapenem-resistant Acinetobactor baumannii in a teaching hospi-
tal in China. J. Basic Microbiol. 51 (5):467–472. https://doi.org/10.1002/
jobm.201000402.
Huang, J.J., Hu, H.Y., Lu, S.Q., Li, Y., Tang, F., Lu, Y., et al., 2012. Monitoring and evaluation of
antibiotic-resistant bacteria at a municipal wastewater treatment plant in China. En-
viron. Int. 42 (1):31–36. https://doi.org/10.1016/j.envint.2011.03.001.
Jung, Y., Matthews, K.R., 2016. Potential transfer of extended spectrum β-lactamase
encoding gene, bla shv18 gene, between Klebsiella pneumoniae in raw foods. Food
Microbiol. 60:39–48. https://doi.org/10.1016/j.fm.2016.06.002.
Jutkina, J., Rutgersson, C., Flach, C.-F., Larsson, D.J., 2016. An assay for determining minimal
concentrations of antibiotics that drive horizontal transfer of resistance. Sci. Total En-
viron. 548, 131–138 (doi:10.1016/j.scitotenv.2016.01.044).
Karkman, A., Johnson, T.A., Lyra, C., Stedtfeld, R.D., Tamminen, M., Tiedje, J.M., et al., 2016.
High-throughput quantification of antibiotic resistance genes from an urban waste-
water treatment plant. FEMS Microbiol. Ecol. 92 (3). https://doi.org/10.1093/
femsec/fiw014.
Kim, S., Jensen, J.N., Aga, D.S., Weber, A.S., 2007. Tetracycline as a selector for resistant bac-
teria in activated sludge. Chemosphere 66 (9):1643–1651. https://doi.org/10.1016/
j.chemosphere.2006.07.066.
Kümmerer, K., Henninger, A., 2003. Promoting resistance by the emission of antibiotics
from hospitals and households into effluent. Clin. Microbiol. Infect. 9 (12):
1203–1214. https://doi.org/10.1111/j.1469-0691.2003.00739.x.
Laht, M., Karkman, A., Voolaid, V., Ritz, C., Tenson, T., Virta, M., et al., 2014. Abundances of
tetracycline, sulphonamide and beta-lactam antibiotic resistance genes in conven-
tional wastewater treatment plants (WWTPs) with different waste load. PLoS One
9 (8), e103705. https://doi.org/10.1371/journal.pone.0103705.
Le, T.-H., Ng, C., Chen, H., Yi, X.Z., Koh, T.H., Barkham, T.M.S., et al., 2016. Occurrences and
characterization of antibiotic-resistant bacteria and genetic determinants of hospital
wastewater in a tropical country. Antimicrob. Agents Chemother. 60 (12),
7449–7456 (doi:10.1128/aac.01556-16).
Li, C., Lu, J., Liu, J., Zhang, G., Tong, Y., Ma, N., 2016. Exploring the correlations between an-
tibiotics and antibiotic resistance genes in the wastewater treatment plants of hospi-
tals in Xinjiang, China. Environ. Sci. Pollut. Res. 23 (15):15111–15121. https://doi.org/
10.1007/s11356-016-6688-z.
Luo, Y., Xu, L., Rysz, M., Wang, Y., Zhang, H., Alvarez, P.J.J., 2011. Occurrence and transport
of tetracycline, sulfonamide, quinolone, and macrolide antibiotics in the Haihe River
Basin, China. Environ. Sci. Technol. 45 (5):1827–1833. https://doi.org/10.1021/
es104009s.
Ma, L., Zhang, X.X., Zhao, F., Wu, B., Cheng, S., Yang, L., 2013. Sewage treatment plant
serves as a hot-spot reservoir of integrons and gene cassettes. J. Environ. Biol. 34
(2), 391–399.
Malintan, N.T., Mohd, M.A., 2006. Determination of sulfonamides in selected Malaysian
swine wastewater by high-performance liquid chromatography. J. Chromatogr. A
1127 (1):154–160. https://doi.org/10.1016/j.chroma.2006.06.005.
Marcinek, H., Wirth, R., Muschollsilberhorn, A., Gauer, M., 1998. Enterococcus faecalis gene
transfer under natural conditions in municipal sewage water treatment plants. Appl.
Environ. Microbiol. 64 (2), 626–632.
Matuszewski, B.K., Constanzer, M.L., Chavezeng, C.M., 2003. Strategies for the assessment
of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal.
Chem. 75 (13):3019–3030. https://doi.org/10.1021/ac020361s.
Mazel, D., 2006. Integrons: agents of bacterial evolution. Nat. Rev. Microbiol. 4 (8):
608–620. https://doi.org/10.1038/nrmicro1462.
Michael, G.B., Cardoso, M., Schwarz, S., 2005. Class 1 integron-associated gene cassettes in
Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in
Brazil. J. Antimicrob. Chemother. 55 (5):776–779. https://doi.org/10.1093/jac/dki081.
Michael, I., Rizzo, L., McArdell, C., Manaia, C., Merlin, C., Schwartz, T., et al., 2013. Urban waste-
water treatment plants as hotspots for the release of antibiotics in the environment: a re-
view. Water Res. 47 (3):957–995. https://doi.org/10.1016/j.watres.2012.11.027.
Patrice, N., Thierry, N., Laurent, P., 2011. Global spread of carbapenemase-producing En-
terobacteriaceae. Emerg. Infect. Dis. 17 (10), 1791 (doi:10.1128/microbe.3.516.1).
Please cite this article as: Wang, Q., et al., Occurrence and diversity of antibiotic resistance in untreated hospital wastewater, Sci Total Environ
(2017), https://doi.org/10.1016/j.scitotenv.2017.10.128
Qingxiang, Y., Tian, T., Tianqi, N., Panliang, W., 2017. Molecular characterization of antibi-
otic resistance in cultivable multidrug-resistant bacteria from livestock manure. Envi-
ron. Pollut. 229:188–198. https://doi.org/10.1016/j.envpol.2017.05.073.
Quoc, T.D., Elodie, M.G., Pierre, L., Fabrice, A., Marie-Jeanne, T., Martine, B., et al., 2016. Fate
of antibiotics from hospital and domestic sources in a sewage network. Sci. Total En-
viron. 575, 758–766 (doi:10.1016/j.scitotenv.2016.09.118).
Rodríguez-Mozaz, S., Chamorro, S., Marti, E., Huerta, B., Gros, M., Sànchez-Melsió, A., et al.,
2015. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban
wastewaters and their impact on the receiving river. Water Res. 69:234–242. https://
doi.org/10.1016/j.watres.2014.11.021.
Salyers, A.A., Gupta, A., Wang, Y., 2004. Human intestinal bacteria as reservoirs for antibi-
otic resistance genes. Trends Microbiol. 12 (9), 412–416 (DOI: 10.1016/
j.tim.2004.07.004).
Schwartz, T., Kohnen, W., Jansen, B., Obst, U., 2003. Detection of antibiotic-resistant bacte-
ria and their resistance genes in wastewater, surface water, and drinking water
biofilms. FEMS Microbiol. Ecol. 43 (3):325–335. https://doi.org/10.1016/s0168-
6496(02)00444-0.
Sommer, M.O., Church, G.M., Dantas, G., 2010. The human microbiome harbors a diverse
reservoir of antibiotic resistance genes. Virulence 1 (4), 299–303 (doi:10.1016/
j.tim.2004.07.004).
Stevenson, B.S., Eichorst, S.A., Wertz, J.T., Schmidt, T.M., Breznak, J.A., 2004. New strategies
for cultivation and detection of previously uncultured microbes. Appl. Environ.
Microbiol. 70 (8):4748–4755. https://doi.org/10.1128/aem.70.8.4748-4755.2004.
Tian, Z., Zhang, Y., Yu, B., Yang, M., 2016. Changes of resistome, mobilome and potential
hosts of antibiotic resistance genes during the transformation of anaerobic digestion
from mesophilic to thermophilic. Water Res. 98:261–269. https://doi.org/10.1016/
j.watres.2016.04.031.
Timraz, K., Xiong, Y., Al Qarni, H., Hong, P.-Y., 2017. Removal of bacterial cells, antibiotic
resistance genes and integrase genes by on-site hospital wastewater treatment
plants: surveillance of treated hospital effluent quality. Environ. Sci.: Water Res.
Technol. 3 (2):293–303. https://doi.org/10.1039/c6ew00322b.
Van den Braak, N., van Belkum, A., van Keulen, M., Vliegenthart, J., Verbrugh, H.A., Endtz,
H.P., 1998. Molecular characterization of vancomycin-resistant enterococci from hos-
pitalized patients and poultry products in The Netherlands. J. Clin. Microbiol. 36 (7),
1927–1932.
Varela, A.R., André, S., Nunes, O.C., Manaia, C.M., 2014. Insights into the relationship be-
tween antimicrobial residues and bacterial populations in a hospital-urban wastewa-
ter treatment plant system. Water Res. 54:327–336. https://doi.org/10.1016/
j.watres.2014.02.003.
Varela, A.R., Nunes, O.C., Manaia, C.M., 2016. Quinolone resistant Aeromonas spp. as carriers
and potential tracers of acquired antibiotic resistance in hospital and municipal wastewa-
ter. Sci. Total Environ. 542:665–671. https://doi.org/10.1016/j.scitotenv.2015.10.124.
Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classifier for rapid as-
signment of rRNA sequences into the new bacterial taxonomy. Appl. Environ.
Microbiol. 73 (16):5261–5267. https://doi.org/10.1128/aem.00062-07.
Xu, J., Xu, Y., Wang, H., Guo, C., Qiu, H., He, Y., et al., 2015. Occurrence of antibiotics and antibi-
otic resistance genes in a sewage treatment plant and its effluent-receiving river.
Chemosphere 119:1379–1385. https://doi.org/10.1016/j.chemosphere.2014.02.040.
Xu, L., Ouyang, W., Qian, Y., Su, C., Su, J., Chen, H., 2016a. High-throughput profiling of an-
tibiotic resistance genes in drinking water treatment plants and distribution systems.
Environ. Pollut. 213:119–126. https://doi.org/10.1016/j.envpol.2016.02.013.
Xu, Y., Guo, C., Luo, Y., Lv, J., Zhang, Y., Lin, H., et al., 2016b. Occurrence and distribution of
antibiotics, antibiotic resistance genes in the urban rivers in Beijing, China. Environ.
Pollut. 213:833–840. https://doi.org/10.1016/j.envpol.2016.03.054.
Zhang, D., Lin, L., Luo, Z., Yan, C., Zhang, X., 2011. Occurrence of selected antibiotics in
Jiulongjiang River in various seasons, South China. J. Environ. Monit. 13 (7):
1953–1960. https://doi.org/10.1039/c0em00765j.
Zhu, Y.G., Johnson, T.A., Su, J.Q., Qiao, M., Guo, G.X., Stedtfeld, R.D., et al., 2013. Diverse and
abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. 110
(9):3435–3440. https://doi.org/10.1073/pnas.1222743110.