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Occurrence of antibiotics and antibiotic resistance


genes in hospital and urban wastewaters and their
impact on the receiving river

Sara Rodriguez-Mozaz a,1, Sara Chamorro a,1, Elisabet Marti a,


Belinda Huerta a, Meritxell Gros a, Alexandre Sa  nchez-Melsio  a,
Carles M. Borrego a,b, Damia Barcelo
 a,c, Jose Luis Balca
 zar a,*
a
Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona,
Spain
b
Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona, Girona, Spain
c
Water and Soil Quality Research Group, Department of Environmental Chemistry, IDAEA-CSIC, Barcelona, Spain

article info abstract

Article history: Antibiotic resistance has become a major health concern; thus, there is a growing interest
Received 14 June 2014 in exploring the occurrence of antibiotic resistance genes (ARGs) in the environment as
Received in revised form well as the factors that contribute to their emergence. Aquatic ecosystems provide an ideal
14 November 2014 setting for the acquisition and spread of ARGs due to the continuous pollution by anti-
Accepted 15 November 2014 microbial compounds derived from anthropogenic activities. We investigated, therefore,
Available online 24 November 2014 the pollution level of a broad range of antibiotics and ARGs released from hospital and
urban wastewaters, their removal through a wastewater treatment plant (WWTP) and their
Keywords: presence in the receiving river. Several antimicrobial compounds were detected in all
Urban and hospital wastewater water samples collected. Among antibiotic families, fluoroquinolones were detected at the
Antibiotics highest concentration, especially in hospital effluent samples. Although good removal
Antibiotic resistance efficiency by treatment processes was observed for several antimicrobial compounds, most
Aquatic ecosystem antibiotics were still present in WWTP effluents. The results also revealed that copy
numbers of ARGs, such as blaTEM (resistance to b-lactams), qnrS (reduced susceptibility to
fluoroquinolones), ermB (resistance to macrolides), sulI (resistance to sulfonamides) and
tetW (resistance to tetracyclines), were detected at the highest concentrations in hospital
effluent and WWTP influent samples. Although there was a significant reduction in copy
numbers of these ARGs in WWTP effluent samples, this reduction was not uniform across
analyzed ARGs. Relative concentration of ermB and tetW genes decreased as a result of
wastewater treatment, whereas increased in the case of blaTEM, sulI and qnrS genes. The
incomplete removal of antibiotics and ARGs in WWTP severely affected the receiving river,
where both types of emerging pollutants were found at higher concentration in

* Corresponding author. Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Emili
Grahit 101, Girona 17003, Spain. Tel.: þ34 972 18 33 80; fax: þ34 972 18 32 48.
 zar).
E-mail address: jlbalcazar@icra.cat (J.L. Balca
1
Contributed equally to this work.
http://dx.doi.org/10.1016/j.watres.2014.11.021
0043-1354/© 2014 Elsevier Ltd. All rights reserved.
w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2 235

downstream waters than in samples collected upstream from the discharge point. Taken
together, our findings demonstrate a widespread occurrence of antibiotics and ARGs in
urban and hospital wastewater and how these effluents, even after treatment, contribute
to the spread of these emerging pollutants in the aquatic environment.
© 2014 Elsevier Ltd. All rights reserved.

Zhang, 2013; Marti and Balca  zar, 2013; Sidrach-Cardona


1. Introduction et al., 2014). Although some studies have analyzed simulta-
neously the occurrence of these emerging pollutants, those
Antimicrobial agents have been used in large quantities for studies have been focused on the link between the presence of
several decades since sulfonamides were introduced in the a limited number of antibiotics and the prevalence of some
1930s. Antibiotics are one of the most important drugs for selected ARGs in environmental settings (Gao et al., 2012; Li
treating infectious diseases, and large amounts of these et al., 2012; Huerta et al., 2013; Marti et al., 2013; Varela et al.,
compounds are released into municipal wastewater due to 2014; Xu et al., 2014), but none of them have performed a
excessive consumption and disposal of unused antibiotics study integrating a hospital and urban wastewater system as
(Rodriguez-Mozaz and Weinberg, 2010). Antibiotics are used well as the impacted river.
not only in human medicine but also in veterinary practices, The aim of this study was therefore to evaluate the pollu-
animal husbandry, agriculture and aquaculture (Zhang et al., tion level of antibiotics and ARGs released from hospital and
2009; Kümmerer, 2009). They can therefore reach surface urban wastewaters, their removal through WWTPs and their
and groundwater bodies through different routes, such as presence in the receiving river. A broad range of antibiotics
wastewater treatment plant (WWTP) effluents (as they are not covering different families (b-lactams, lincosamides, macro-
completely removed), surface runoff, or infiltration of water lides, quinolones/fluoroquinolones, sulfonamides, tetracy-
used for agricultural purposes. clines, dihydrofolate reductase inhibitors and
It is well known that antibiotics pose a significant risk to nitroimidazoles) were selected and monitored. Five ARGs
environmental and human health, even at low concentrations were also selected according to the results of antibiotic
(Kümmerer, 2009). In addition, the overuse and misuse of detection and clinical importance, and among them, blaTEM
antibiotics has led to the emergence of antibiotic-resistant (resistance to b-lactams), qnrS (reduced susceptibility to fluo-
bacteria, compromising the effectiveness of antimicrobial roquinolones), ermB (resistance to macrolides), sulI (resistance
therapy because the infectious organisms are becoming to sulfonamides) and tetW (resistance to tetracyclines) genes
resistant to most antibiotics (Pruneau et al., 2011; Marti et al., were monitored in the same samples and a relationship be-
2014a). In fact, the emergence and spread of antibiotic- tween their occurrence and the presence of antibiotics was
resistant bacteria have been classified by the World Health explored.
Organization (WHO) as one of the three biggest threats to
public health in the 21st century. As a consequence of anti-
biotic consumption, the normal human microbiota can be
2. Materials and methods
altered and enriched in antibiotic-resistant bacteria. Humans
can be therefore considered as a source of both antibiotics and
2.1. Sampling sites
antibiotic resistance genes (ARGs), which may be released into
the environment through sewage systems. Sewer systems
Five sampling points were selected to study the antibiotic and
collect wastewater not only from domestic origin but also
ARG pollution of both hospital and WWTP effluents to the Ter
from industrial and hospital sources. Hospital effluents, in
River (Fig. 1). The first sampling point consisted in the
particular, constitute a special category of waste that are
wastewater effluents from the main hospital of Girona, which
highly hazardous because of their infectious and toxic char-
according to the 2010 annual report has 400 beds, 11 operating
acteristics (Verlicchi et al., 2010; Chagas et al., 2011) and they
rooms, 94 offices and outpatient visits and 1.649 employees to
also represent an important source of multi-resistant bacteria
provide specialized assistance to approximately 800,000 in-
(Chagas et al., 2011; Huang et al., 2012) and antibiotics (Santos
habitants. Second and third sampling points were influent
et al., 2013; Coutu et al., 2013).
and effluent collectors from the Girona WWTP, which receives
Because of the increasing concern about antibiotic pollu-
the hospital wastewater without any previous treatment
tion in aquatic ecosystems, several studies have been con-
(1000e1500 m3/day) along with municipal wastewater from
ducted to assess the presence of these emerging pollutants in
the city of Girona (45,000e55,000 m3/day). The two last sam-
WWTP effluent discharges (Hirsch et al., 1999; Costanzo et al.,
pling points were also located in the Ter River, approximately
2005; Garcı́a-Gala n et al., 2011; Leung et al., 2012), whereas
250 m upstream and downstream of the WWTP discharge
other studies have paid attention to the ARGs released into the
point. The Ter River represents an important source of
environment and, consequently, the emergence and potential
drinking water, not only for the population of Girona region
spread of antibiotic resistance (Pruden et al., 2006; Zhang
(741,899 inhabitants in 2011) but also for the population of the
et al., 2009; Munir et al., 2011; LaPara et al., 2011; Chen and
Barcelona metropolitan area (4,777,042 inhabitants in 2011).
236 w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2

Fig. 1 e Geographical location of the different sampling sites (Adapted from Google Maps).

2.2. Sampling procedure binding 0.22-mm-pore-size membranes (Millipore). The


collected bacterial cells were then resuspended in lysis buffer
Three sampling campaigns were performed in November (1.2% Triton X-100, 1 M Tris-Cl, 0.5 M Na2EDTA), followed by
(2011), December (2011) and January (2012). Water samples enzymatic digestion with lysozyme and proteinase K.
were collected in each of the 5 sampling points and trans- Genomic DNA was extracted using the DNeasy Blood & Tissue
ported to the laboratory in a portable icebox for immediate Kit (Qiagen; Valencia, CA, USA), according to the manufac-
processing. turer's instructions. DNA concentration and purity were
determined using a NanoDrop spectrophotometer (NanoDrop
2.3. Quantification of antibiotics Technologies; Wilmington, DE, USA). All DNA samples were
stored at 20  C until analysis.
Samples were analyzed in triplicate for the determination of
sixty-two antibiotics following the protocol previously 2.5. Quantification of ARGs
described (Gros et al., 2013). Briefly, successive filtration of
water samples was done through 2.7 mm, 1.0 mm and 0.45 mm Real-time PCR (qPCR) assays were used to quantify five ARGs,
pore-size membranes (Millipore; Billerica, MA, USA). After including blaTEM, ermB, qnrS, sulI and tetW, which confer
filtration, water sample was pH-adjusted to 3 with HCl (1.0 M) resistance to the main antibiotic families used in human and
and EDTA (4%, v/v) and an appropriate volume of each sample veterinary medicine. Copy number of the 16S rRNA gene was
(50 ml hospital and WWTP effluents, 25 ml WWTP influent also quantified for normalization of the data. All qPCR assays
and 250 ml river water) was loaded into Solid Phase Extraction were developed using the Brilliant III Ultra-Fast QPCR Master
(SPE)-HLB cartridges (60 mg, 3 mL) (Waters Corp.; Mildford, Mix (Agilent Technologies; Santa Clara, CA, USA), with the
MA, USA) for analytes preconcentration. Reconstituted ex- exception for the blaTEM gene, which was amplified using the
tracts were analyzed by chromatographic separation with an SYBR Green Master Mix (Applied Biosystems) due to non-
ultra performance liquid chromatography (UPLC) system specific amplification. All qPCR assays were conducted on a
(Waters Corp.) equipped with a quaternary pump system MX3005P system (Agilent Technologies), as previously
using an Acquity BEH T3 column (50 mm  2.1 mm i.d., 1.7 mm described (Marti et al., 2013). Each gene was amplified using
particle size) (Waters Corp.). The UPLC system was coupled to specific primer sets (Table S1) and the PCR conditions included
a triple quadrupole-linear ion trap mass spectrometer an initial denaturation at 95  C for 3 min, followed by 40 cycles
(Applied Biosystems; Foster City, CA, USA) with a Turbo V ion at 95  C for 15 s and at the annealing temperature given in
spray source. Analysis was performed in positive ionization Table S1 for 20 s. In the case of the 16S rRNA gene, amplifi-
mode in a multiple reaction monitoring (MRM) mode. cation conditions were 35 cycles at 95  C for 15 s, followed by
For an accurate quantification, total recoveries were an annealing temperature at 60  C for 1 min. After each qPCR
determined for each water matrix (river, and influent and assay, a dissociation curve was constructed by increasing the
effluent wastewater; n ¼ 3) and concentrations were calcu- temperature from 65 to 95  C in order to confirm the specificity
lated by internal calibration with isotopically labeled stan- of the amplified products.
dards, according to Gros et al. (2013). Standard curves were generated by cloning the amplicon
from positive controls into the pCR2.1-TOPO vector (Invi-
2.4. DNA extraction trogen, Carlsbad, CA, USA), and the corresponding copy
number was calculated as follows: copy number
Water samples were collected in triplicate at selected sites ml1 ¼ (A  6.022  1023) (660  B)1, where A is the plasmid
and filtered under sterile conditions through low protein- DNA concentration (g ml1), B is the plasmid length (bp)
w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2 237

containing the cloned sequence, 6.022  1023 is the Avogadro's

Metronidazolea
number and 660 is the average molecular weight of one base

937.4 ± 111.8

1792.9 ± 32.6
240.1 ± 13.7

274.5 ± 11.2
523.9 ± 8.2

144.0 ± 9.1
pair (Perini et al., 2011). A ten-fold serial dilution was then

28.4 ± 1.9

29.6 ± 4.2

17.1 ± 2.6

23.2 ± 7.9
used to construct the standard curve for each ARG, which was
run in parallel with the samples to obtain absolute quantifi-

ND
ND
ND

ND

ND
cation. The copy number of each ARG was also normalized to
the 16S rRNA gene copy number in order to obtain relative
quantification.

Trimethoprim

3,8.26 ± 48.6
594.3 ± 13.5

124.9 ± 13.7
97.2 ± 26.5

136.3 ± 5.0
122.9 ± 4.5
107.6 ± 7.7

179.5 ± 4.7
87.8 ± 0.4

50.7 ± 1.6

70.5 ± 3.4

92.7 ± 3.8
2.6. Statistical analysis

<MQL

<MQL
ND
Comparisons of average antibiotic and ARG concentrations
among different sampling points were carried out using
ANOVA or Kruskal Wallis tests, as appropriate. Correlations

Sulfamethoxazole
between antibiotic and ARG values were made using Pearson's

4816.7 ± 212.1
test (all variables satisfied the normality assumption). Differ-

751.7 ± 2.4
330.7 ± 8.0
64.9 ± 10.1

190.2 ± 6.1
417.4 ± 5.0

401.6 ± 1.1
40.2 ± 3.5

73.0 ± 2.8

64.8 ± 3.3

56.2 ± 3.8

71.8 ± 2.2
3.9 ± 0.4
ences were considered significant at p < 0.05. All statistical

7 ± 0.6
<MQL
analyses were performed using SPSS 17.0 software (SPSS;
Chicago, IL, USA).

Table 1 e Occurrence of the 9 antibiotics detected at the highest concentrations in the different water samples.

Clarithromycin

This value includes the concentration of metronidazole-OH (metabolite). ND, not detected. MQL, method quantification limit.
3. Results and discussion

167.3 ± 10.7

551.3 ± 17.2
129.0 ± 10.8

471.3 ± 27.0
460.9 ± 7.4

941.1 ± 1.9

115.1 ± 3.3

61.1 ± 17.9
92.3 ± 0.6
35.4 ± 1.1
61.3 ± 2.1

35.8 ± 0.4
96.3 ± 0.9

44.7 ± 3.6
3.1. Quantification of antibiotics
Concentration (ng/L)

ND
Thirty-three out of the sixty-two antimicrobial compounds
analyzed were detected in different water samples collected
Azythromycin

over the three sampling campaigns (Table S2). Despite their

214.5 ± 16.3
189.0 ± 3.5
134.1 ± 6.3

135.0 ± 2.6

115.5 ± 0.2
high consumption, compounds belonging to penicillin and
20.1 ± 5.7

75.5 ± 9.4

55.9 ± 0.8

59.9 ± 7.1

70.7 ± 0.5

59.9 ± 7.1
tetracycline families were detected neither in the analyzed
wastewater nor in the river waters, probably because of their
ND

ND

ND

ND
chemical instability (Graham et al., 2011).
Among antibiotic families, fluoroquinolones were detected
at the highest concentration, especially in hospital effluent
Cefotaxime

11.8

14.3
4.2
1.6
7.7

3.6

0.4
2.2
1.7
0.8

4.7
8.9
2.6
0.5
4.0
samples. In fact, ciprofloxacin and ofloxacin were present in
143.7 ±
363.5 ±
229.2 ±
93.1 ±
147.5 ±

240.4 ±
252.8 ±
207.9 ±
68.5 ±
130.8 ±

236.8 ±
340.5 ±
223.4 ±
67.98 ±
165.6 ±
hospital effluents, ranging from 13.78 mg/L for ciprofloxacin in
the third sampling campaign to 14.38 mg/L for ofloxacin in the
first one (Table 1). In contrast, lower concentrations (at least
one order of magnitude) were found in urban wastewater than
Cefazolin

146.6 ± 4.8

116.2 ± 7.2
83.4 ± 3.6
94.7 ± 0.9
14.6 ± 2.5

44.6 ± 2.7

22.1 ± 0.3

24.8 ± 1.5
10.5 ± 1.1
8.4 ± 0.1
8.4 ± 0.9

6.6 ± 0.4
7.9 ± 1.2

3.4 ± 0.1
in hospital effluents. Such high values in hospitals may be
<MQL

related to their medical consumption, as these fluo-


roquinolones are frequently used in hospital practice to treat
infections (MacDougall et al., 2005; Ray et al., 2005) and,
14,377.8 ± 50.9

therefore, they have also been found in other hospital efflu-


5700.0 ± 33.3

4750.0 ± 23.6
Ofloxacin

1.564.6 ± 9.4
581.7 ± 10.2

171.8 ± 12.8
592.9 ± 2.5

100.7 ± 2.3

137.6 ± 3.2

ents at such high concentrations (Thomas et al., 2007;


82.0 ± 6.3

60.9 ± 2.7

131 ± 1.6

Kovalova et al., 2012; Santos et al., 2013). The levels of


<MQL

<MQL

<MQL

metronidazole, sulfamethoxazole and trimethoprim in hos-


pital effluent samples were also high in all sampling cam-
paigns (Table 1). The levels of sulfamethoxazole and
Ciprofloxacin

13,779.7 ± 24.0
8372.9 ± 67.8

8305.1 ± 48.0

trimethoprim were very similar in all samplings probably due


639.1 ± 59.2

1307.0 ± 6.6
108.0 ± 4.2

135.2 ± 0.4

851.6 ± 5.9
174.8 ± 8.1
8.1 ± 0.22
50.0 ± 0.9

72.4 ± 5.1

48.2 ± 7.9

to their combined therapeutic use (Batt et al., 2006). However,


9.4 ± 1.5

4.7 ± 0.1

the ratio between both compounds was not consistent with


the one found in the WWTP influent samples, where sulfa-
methoxazole concentrations were higher than trimethoprim
concentrations. In the case of metronidazole, similar con-
Hospital effluent

Hospital effluent

Hospital effluent
WWTP influent

WWTP influent

WWTP influent
WWTP effluent

WWTP effluent

WWTP effluent
Second sampling

Third sampling

centrations have been previously detected in hospital and


First sampling

Downstream

Downstream

Downstream

urban wastewaters (Verlicchi et al., 2012).


Upstream

Upstream

Upstream

In contrast, lower concentrations of antibiotics belonging


to the group of cephalosporins, such as cefazolin and cefo-
a

taxime, were detected in hospital effluent samples compared


238 w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2

Fig. 2 e Absolute concentration of ARGs in the different water samples. Within the box plot chart, the crosspieces of each
box plot represent (from top to bottom) maximum, upper-quartile, median (black bar), lower-quartile and minimum values.

to those found in urban WWTP samples (Table 1). Although clarithromycin was detected up to 0.94 mg/L in hospital
the hospitals are typically considered the major source of effluent samples. Clarithromycin was indeed the antibiotic
cephalosporins (Kümmerer, 2009), the concentrations detec- that showed higher concentration variability in hospital ef-
ted in the present study were lower than those found in fluents among samplings. Unlike fluoroquinolones and
WWTP influent samples. These results are, nevertheless, in cephalosporins, macrolide consumption is more widespread
agreement with other studies where cefazolin and cefotaxime in households than in clinical settings (Kümmerer and
were also lower in hospital effluents than in the inlet of a Henninger, 2003), and they are rather applied to treat spe-
WWTP (Gros et al., 2013). Low levels of macrolides, such as cific diseases. For instance, the high levels of clarithromycin
azythromycin and clarithromycin were detected in hospital described in the second campaign could be attributed to
effluent samples as compared with those detected in WWTP pneumonia or bronchitis outbreaks since clarithromycin is
influents, except in the second sampling campaign, where often used to treat lung infections (Tanaka et al., 2002).
w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2 239

Fig. 3 e Correlations between the concentrations of antibiotics and their corresponding ARGs. Sample locations are
represented by: black squares (hospital effluents), red squares (WWTP influents), brown squares (WWTP effluents), white
squares (upstream) and green squares (downstream). Light gray lines show 95% confidence intervals. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article.)

Although good removal efficiency was observed for several discharge, were detected at high concentrations in down-
antimicrobial compounds (Table 1 and Table S2), most anti- stream river samples (up to 131.0 ng/L for ofloxacin). In line
biotics were still present in WWTP effluents at concentrations with this, ciprofloxacin and sulfamethoxazole showed
that may affect microbial communities. The presence of such approximately tenfold higher concentrations in downstream
high concentrations of antibiotics in effluents was in fact than in upstream samples. However, an increase in cefazolin
impacting the receiving river water, where antibiotics such as concentrations was not detected in the water samples
ofloxacin, azythromycin, trimethoprim and metronidazole, analyzed probably due to its high removal efficiency by the
not measured in samples collected upstream the WWTP WWTP. In fact, previous studies have reported removal
240 w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2

efficiencies between 75 and 100% for this compound (Lin et al., relative concentration of blaTEM, qnrS and sulI genes was
2009). Finally, it is important to note that around 80% of the higher in effluent samples than those found in WWTP influent
detected antibiotics (33 antimicrobial compounds out of the 62 samples (Fig. S2). A plausible explanation might be related to
target compounds) were found in river water samples (Table the spread of some ARGs among bacterial cells in activated
S2). This observation indicates the widespread occurrence of sludge (Szczepanowski et al., 2009; Rizzo et al., 2013), which
these compounds in the aquatic ecosystems (Zhang et al., convert WWTPs into hot spots for horizontal gene transfer. In
2012). the case of the qnrS gene, this problem may become more
severe due to detected fluoroquinolone concentrations (cip-
3.2. Quantification of ARGs rofloxacin with average values of 932.56 and 139.34 ng/L, and
ofloxacin with average values of 913.08 and 104.90 ng/L in the
Five ARGs blaTEM, qnrS, ermB, sulI and tetW and the 16S rRNA WWTP influent and effluent, respectively), since cumulative
gene were quantified using qPCR assays in the different water amounts of these antibiotics might increase the concentration
samples (Fig. 2). High R2 values (average 0.997) and high effi- well above the minimum inhibitory concentrations (MICs) for
ciencies (from 95.8 to 106.5%) obtained from the standard several bacteria, causing a selective advantage to resistant
curves demonstrated the linearity and sensitivity of each bacteria (Liu et al., 2011).
qPCR assay (Fig. S1).
The blaTEM gene is one of the most frequently detected 3.3. Correlation between concentrations of antibiotics
plasmid-borne antimicrobial resistance genes, which confers and corresponding ARGs
resistance to penicillins and extended-spectrum cephalospo-
rins (Mroczkowska and Barlow, 2008). The qnrS gene is asso- We carried out a correlation analysis between the absolute
ciated with plasmid-borne fluoroquinolone resistance that concentrations of ARG and the antibiotics to which they
has become increasingly prevalent in anthropogenically- confer resistance to determine potential links between both
influenced environments (Marti et al., 2014b). The ermB gene variables. Statistical calculations were conducted for all ARG
encodes resistance to macrolides, lincosamides and strep- except for the tetW gene since no tetracycline was detected in
togramin antibiotics and is generally found on conjugative water samples. Significant positive correlations between the
genetic elements (Negreanu et al., 2012). The sulI gene encodes concentrations of antibiotics and their corresponding ARGs
dihydropteroate synthase that confers resistance to sulfon- were observed (Fig. 3). In fact, there were correlations between
amides and is generally harbored in class 1 integrons con- ciprofloxacin and the qnrS gene (r ¼ 0.86, p ¼ 0.001), ofloxacin
taining other resistance genes (Antunes et al., 2005). The tetW and the qnrS gene (r ¼ 0.81, p ¼ 0.001), cefazolin and the blaTEM
gene encodes a ribosomal protection protein that confers gene (r ¼ 0.84, p ¼ 0.001), cefotaxime and the blaTEM gene
resistance to tetracycline (Aminov et al., 2001). (r ¼ 0.74, p ¼ 0.002), clarithromycin and the ermB gene (r ¼ 0.89,
Higher absolute copy numbers of these ARGs (p < 0.05) were p ¼ 0.001), and sulfamethoxazole and the sulI gene (r ¼ 0.83,
detected in hospital effluent and WWTP influent samples than p ¼ 0.001); however, no significant correlation was found be-
those found in other water samples (Fig. 2). A significant tween azythromycin and the ermB gene. The correlations also
reduction of these ARGs (p < 0.05) was also observed in WWTP showed that ARGs increase with the concentration of antibi-
effluent samples, which decreased more than hundredfold in otics (Fig. 3). These results are consistent with previous
some instances. However, ARGs were detected in downstream studies suggesting that exposure to antibiotics could lead to
wasters, indicating a moderate removal efficiency of the selective pressure for ARGs (Wu et al., 2010; Li et al., 2012). A
WWTP and their persistence in natural waters. In fact, ermB, previous study demonstrated a significant correlation be-
qnrS and sulI genes showed significantly higher values tween the total plasmid-mediated quinolone resistance genes
(p < 0.05) in water samples collected downstream of the (qnrD, qnrS, qepA, oqxA and oqxB) and fluoroquinolone residues
WWTP discharge than in upstream waters. These results in wastewater and soil samples from swine feedlots and their
agree with previous studies suggesting that WWTP discharges surrounding environment (Li et al., 2012). Similarly, a signifi-
could contribute to the spread of ARGs into aquatic environ- cant correlation has been demonstrated between total tet gene
ments. Storteboom et al. (2010) observed that the abundance copies (tetM, tetO, tetQ and tetW genes) and tetracycline resi-
of selected ARGs in the Poudre River was higher in anthro- dues in soils adjacent to swine feedlots (Wu et al., 2010) but
pogenically impacted areas than those from upstream of the not in WWTP environments, where a significant correlation
river. Likewise, Marti and Balca  zar (2013) suggested that the was found only between sulfonamides and sul genes (Gao
WWTP was a significant point source of several ARGs into the et al., 2012). Likewise, a recent study demonstrated that rela-
receiving river. Czekalski et al. (2014) recently demonstrated tive tet gene copies (tetB and tetW) were strongly correlated
that the abundance of ARGs in close proximity of the WWTP with the concentrations of tetracycline residues in water
discharge point was up to two hundredfold above levels samples collected from a WWTP and its surrounding envi-
determined at a remote site of Lake Geneva and decreased ronment, whereas no significant correlations were observed
exponentially with distance. Altogether, these observations between sul gene copies (sulI, sulII and sulIII) and sulfonamides
undoubtedly demonstrate the contribution of WWTP dis- (Xu et al., 2014). Although we have demonstrated a significant
charges to the spread of antibiotic resistance. correlation between almost all ARGs analyzed and the envi-
The quantitative analysis also demonstrated that the ronmental concentration of the antibiotics to which they
relative concentration (copy numbers normalized to the 16S confer resistance, further studies should be conducted in en-
rRNA gene copy number) of ermB and tetW genes decreased vironments exposed to different pollution levels in order to
(p < 0.05) as a result of wastewater treatment, whereas the provide a better insight into these correlations.
w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2 241

Chen, H., Zhang, M., 2013. Occurrence and removal of antibiotic


4. Conclusions resistance genes in municipal wastewater and rural domestic
sewage treatment systems in eastern China. Environ. Int. 55,
Our study evaluated the presence of a large number of an- 9e14.
tibiotics and ARGs in an integrated urban wastewater system Costanzo, S.D., Murby, J., Bates, J., 2005. Ecosystem response to
antibiotics entering the aquatic environment. Mar. Pollut. Bull.
including the contribution of hospital effluent, as well as
51, 218e223.
river water receiving WWTP discharges. Results revealed a
Coutu, S., Rossi, L., Barry, D.A., Rudaz, S., Vernaz, N., 2013.
larger occurrence of some antibiotics, such as ciprofloxacin Temporal variability of antibiotics fluxes in wastewater and
and ofloxacin, in hospital samples than in the WWTP in- contribution from hospitals. PLoS One 8, e53592.
fluents whereas no significant differences were found be- Czekalski, N., Dı́ez, E.G., Bürgmann, H., 2014. Wastewater as a
tween both samples in relation to ARGs. The WWTP was point source of antibiotic-resistance genes in the sediment of
unable to totally remove antibiotics and ARGs from urban a freshwater lake. ISME J. 8, 1381e1390.
Garcı́a-Gala  n, M.J., Dı́az-Cruz, M.S., Barcelo  , D., 2011. Occurrence
effluents, releasing them into the environment and contrib-
of sulfonamide residues along the Ebro river basin: removal in
uting to their persistence and spread in river waters. Because wastewater treatment plants and environmental impact
rivers are the main source of water, either directly or indi- assessment. Environ. Int. 37, 462e473.
rectly, for human and animal consumption, antibiotic Gao, P., Munir, M., Xagoraraki, I., 2012. Correlation of tetracycline
pollution may pose a serious risk for human and animal and sulfonamide antibiotics with corresponding resistance
health through the spread of antibiotic-resistant bacteria. genes and resistant bacteria in a conventional municipal
Further research is needed to unequivocally demonstrate wastewater treatment plant. Sci. Total Environ. 421e422,
173e183.
that WWTP discharges promote horizontal gene transfer
Graham, D.W., Olivares-Rieumont, S., Knapp, C.W., Lima, L.,
among aquatic bacterial populations. Werner, D., Bowen, E., 2011. Antibiotic resistance gene
abundances associated with waste discharges to the
Almendares River near Havana, Cuba. Environ. Sci. Technol. 45,
418e424.
Acknowledgments Gros, M., Rodrı́guez-Mozaz, S., Barcelo  , D., 2013. Rapid analysis of
multiclass antibiotic residues and some of their metabolites in
This study has been supported by the European Union hospital, urban wastewater and river water by ultra-high-
performance liquid chromatography coupled to quadrupole-
through the European Regional Development Fund (ERDF
linear ion trap tandem mass spectrometry. J. Chromatogr. A
operational program for Catalonia 2007e2013) and the Gen- 1292, 173e188.
eralitat de Catalunya (Consolidated Research Group: Catalan Hirsch, R., Ternes, T., Haberer, K., Kratz, K.-L., 1999. Occurrence of
Institute for Water Research 2014 SGR 291). We are grateful to antibiotics in the aquatic environment. Sci. Total Environ. 225,
the operators and staff at the Girona WWTP (Trargisa) for their 109e118.
assistance. J.L.B. acknowledges the Ramon y Cajal research 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
fellowship (RYC-2011-08154) from the Spanish Ministry of
municipal wastewater treatment plant in China. Environ. Int.
Economy and Competitiveness.
42, 31e36.
Huerta, B., Marti, E., Gros, M., Lo  pez, P., Pompe ^o, M., Armengol, J.,
et al., 2013. Exploring the links between antibiotic occurrence,
antibiotic resistance, and bacterial communities in water
Appendix A. Supplementary data
supply reservoirs. Sci. Total Environ. 456e457, 161e170.
Kovalova, L., Siegrist, H., Singer, H., Wittmer, A., McArdell, C., 2012.
Supplementary data related to this article can be found at Hospital wastewater treatment by membrane bioreactor:
http://dx.doi.org/10.1016/j.watres.2014.11.021. performance and efficiency for organic micropollutant
elimination. Environ. Sci. Technol. 46, 1536e1545.
Kümmerer, K., 2009. Antibiotics in the aquatic environment e a
review e Part I. Chemosphere 75, 417e434.
references
Kümmerer, K., Henninger, A., 2003. Promoting resistance by the
emission of antibiotics from hospitals and households into
effluents. Clin. Microbiol. Infec 9, 1203e1214.
Aminov, R.I., Garrigues-Jeanjean, N., Mackie, R.I., 2001. Molecular LaPara, T.M., Burch, T.R., McNamara, P.J., Tan, D.T., Yan, M.,
ecology of tetracycline resistance: development and validation Eichmiller, J.J., 2011. Tertiary-treated municipal wastewater is
of primers for detection of tetracycline resistance genes a significant point source of antibiotic resistance genes into
encoding ribosomal protection proteins. Appl. Environ. Duluth-Superior Harbor. Environ. Sci. Technol. 45, 9543e9549.
Microbiol. 67, 22e32. Leung, H.W., Minh, T.B., Murphy, M.B., Lam, J.C.W., So, M.K.,
Antunes, P., Machado, J., Sousa, J.C., Peixe, L., 2005. Dissemination Martin, M., et al., 2012. Distribution, fate and risk assessment
of sulfonamide resistance genes (sul1, sul2, and sul3) in of antibiotics in sewage treatment plants in Hong Kong, South
Portuguese Salmonella enterica strains and relation with China. Environ. Int. 42, 1e9.
integrons. Antimicrob. Agents Chemother. 49, 836e839. Li, J., Wang, T., Shao, B., Shen, J., Wang, S., Wu, Y., 2012. Plasmid-
Batt, A.L., Bruce, I.B., Aga, D.S., 2006. Evaluating the vulnerability mediated quinolone resistance genes and antibiotic residues in
of surface waters to antibiotic contamination from varying wastewater and soil adjacent to swine feedlots: potential transfer
wastewater treatment plant discharges. Environ. Pollut. 142, to agricultural lands. Environ. Health Perspect. 120, 1144e1149.
295e302. Lin, A.Y.-C., Yu, T.-H., Lateef, S.K., 2009. Removal of
Chagas, T.P.G., Seki, L.M., da Silva, D.M., Asensi, M.D., 2011. pharmaceuticals in secondary wastewater treatment
Occurrence of KPC-2-producing Klebsiella pneumoniae strains in processes in Taiwan. J. Hazard Mater. 167, 1163e1169.
hospital wastewater. J. Hosp. Infect. 77, 281.
242 w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 3 4 e2 4 2

Liu, A., Fong, A., Becket, E., Yuan, J., Tamae, C., Medrano, L., et al., Santos, L.H.M.L.M., Gros, M., Rodriguez-Mozaz, S., Delerue-
2011. Selective advantage of resistant strains at trace levels of Matos, C., Pena, A., Barcelo  , D., et al., 2013. Contribution of
antibiotics: a simple and ultrasensitive color test for detection hospital effluents to the load of pharmaceuticals in urban
of antibiotics and genotoxic agents. Antimicrob. Agents wastewaters: identification of ecologically relevant
Chemother. 55, 1204e1210. pharmaceuticals. Sci. Total Environ. 461e462, 302e316.
MacDougall, C., Powell, J.P., Johnson, C.K., Edmond, M.B., Sidrach-Cardona, R., Hijosa-Valsero, M., Marti, E., Balca  zar, J.L.,
Polk, R.E., 2005. Hospital and community fluoroquinolone use Becares, E., 2014. Prevalence of antibiotic-resistant fecal
and resistance in Staphylococcus aureus and Escherichia coli in 17 bacteria in a river impacted by both an antibiotic production
US hospitals. Clin. Infect. Dis. 41, 435e440. plant and urban treated discharges. Sci. Total Environ.
Marti, E., Variatza, E., Balca  zar, J.L., 2014a. The role of aquatic 488e489, 220e227.
ecosystems as reservoirs of antibiotic resistance. Trends Storteboom, H., Arabi, M., Davis, J.G., Crimi, B., Pruden, A., 2010.
Microbiol. 22, 36e41. Identification of antibiotic-resistance-gene molecular
Marti, E., Variatza, E., Balca  zar, J.L., 2014b. Bacteriophages as a signatures suitable as tracers of pristine river, urban, and
reservoir of extended-spectrum b-lactamase and agricultural sources. Environ. Sci. Technol. 44, 1947e1953.
fluoroquinolone resistance genes in the environment. Clin. Szczepanowski, R., Linke, B., Krahn, I., Gartemann, K.H.,
Microbiol. Infect. 20, O456eO459. Gützkow, T., Eichler, W., et al., 2009. Detection of 140 clinically
Marti, E., Balca zar, J.L., 2013. Real-time PCR assays for relevant antibiotic-resistance genes in the plasmid
quantification of qnr genes in environmental water samples metagenome of wastewater treatment plant bacteria showing
and chicken feces. Appl. Environ. Microbiol. 79, 1743e1745. reduced susceptibility to selected antibiotics. Microbiology
 zar, J.L., 2013. Prevalence of antibiotic
Marti, E., Jofre, J., Balca 155, 2306e2319.
resistance genes and bacterial community composition in a river Tanaka, E., Kimoto, T., Tsuyuguchi, K., Suzuki, K., Amitani, R.,
influenced by a wastewater treatment plant. PLoS ONE 8, e78906. 2002. Successful treatment with faropenem and
Mroczkowska, J.E., Barlow, M., 2008. Fitness trade-offs in blaTEM clarithromycin of pulmonary Mycobacterium abscessus
evolution. Antimicrob. Agents Chemother. 52, 2340e2345. infection. J. Infect. Chemother. 8, 252e255.
Munir, M., Wong, K., Xagoraraki, I., 2011. Release of antibiotic Thomas, K.V., Dye, C., Schlabach, M., Langford, K.H., 2007. Source
resistant bacteria and genes in the effluent and biosolids of to sink tracking of selected human pharmaceuticals from two
five wastewater utilities in Michigan. Water Res. 45, 681e693. Oslo city hospitals and a wastewater treatment works. J.
Negreanu, Y., Pasternak, Z., Jurkevitch, E., Cytryn, E., 2012. Impact Environ. Monit. 9, 1410e1418.
of treated wastewater irrigation on antibiotic resistance in Varela, A., Andre , S., Nunes, O.C., Manaia, C.E., 2014. Insights into
agricultural soils. Environ. Sci. Technol. 46, 4800e4808. the relationship between antimicrobial residues and bacterial
Perini, F., Casabianca, A., Battocchi, C., Accoroni, S., Totti, C., populations in a hospital-urban wastewater treatment plant
Penna, A., 2011. New approach using the real-time PCR system. Water Res. 54, 327e336.
method for estimation of the toxic marine dinoflagellate Verlicchi, P., Galletti, A., Masotti, L., 2010. Management of hospital
Ostreopsis cf. ovata in marine environment. PLoS One 6, e17699. wastewater: the case of the effluent of a large hospital situated
Pruden, A., Pei, R., Storteboom, H., Carlson, K.H., 2006. Antibiotic in a small town. Water Sci. Technol. 61, 2507e2519.
resistance genes as emerging contaminants: studies in Verlicchi, P., Al Aukidy, M., Galletti, A., Petrovic, M., Barcelo  , D.,
Northern Colorado. Environ. Sci. Technol. 40, 7445e7450. 2012. Hospital effluent: investigation of the concentrations
Pruneau, M., Mitchell, G., Moisan, H., Dumont-Blanchette, E., and distribution of pharmaceuticals and environmental risk
Jacob, C.L., Malouin, F., 2011. Transcriptional analysis of assessment. Sci. Total Environ. 430, 109e118.
antibiotic resistance and virulence genes in multiresistant Wu, N., Qiao, M., Zhang, B., Cheng, W.-D., Zhu, Y.-G., 2010.
hospital-acquired MRSA. FEMS Immunol. Med. Microbiol. 63, Abundance and diversity of tetracycline resistance genes in
54e64. soils adjacent to representative swine feedlots in China.
Ray, G.T., Baxter, R., DeLorenze, G.N., 2005. Hospital-level rates of Environ. Sci. Technol. 44, 6933e6939.
fluoroquinolone use and the risk of hospital-acquired Xu J, Xu Y, Wang H, Guo C, Qiu H, He Y, Zhang Y, Li X, Meng W.
infection with ciprofloxacin-nonsusceptible Pseudomonas Occurrence of antibiotics and antibiotic resistance genes in a
aeruginosa. Clin. Infect. Dis. 41, 441e449. sewage treatment plant and its effluent-receiving river.
Rizzo, L., Manaia, C., Merlin, C., Schwartz, T., Dagot, C., Ploy, M.C., Chemosphere. DOI: 10.1016/j.chemosphere.2014.02.040.
et al., 2013. Urban wastewater treatment plants as hotspots Zhang, X.-X., Zhang, T., Fang, H.H.P., 2009. Antibiotic resistance
for antibiotic resistant bacteria and genes spread into the genes in water environment. Appl. Microbiol. Biotechnol. 82,
environment: a review. Sci. Total Environ. 447, 345e360. 397e414.
Rodriguez-Mozaz, S., Weinberg, H.S., 2010. Meeting report: Zhang, R., Zhang, G., Zheng, Q., Tang, J., Chen, Y., Xu, W., et al.,
pharmaceuticals in watereAn interdisciplinary approach to a 2012. Occurrence and risks of antibiotics in the Laizhou Bay,
public health challenge. Environ. Health Perspect. 118, China: impact of river discharge. Ecotoxicol. Environ. Saf. 80,
1016e1020. 208e215.