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

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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
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.
1. Introduction
Antimicrobial agents have been used in large quantities for
several decades since sulfonamides were introduced in the
1930s. Antibiotics are one of the most important drugs for
treating infectious diseases, and large amounts of these
compounds are released into municipal wastewater due to
excessive consumption and disposal of unused antibiotics
(Rodriguez-Mozaz and Weinberg, 2010). Antibiotics are used
not only in human medicine but also in veterinary practices,
animal husbandry, agriculture and aquaculture (Zhang et al.,
2009; Kümmerer, 2009). They can therefore reach surface
and groundwater bodies through different routes, such as
wastewater treatment plant (WWTP) effluents (as they are not
completely removed), surface runoff, or infiltration of water
used for agricultural purposes.
It is well known that antibiotics pose a significant risk to
environmental and human health, even at low concentrations
(Kümmerer, 2009). In addition, the overuse and misuse of
antibiotics has led to the emergence of antibiotic-resistant
bacteria, compromising the effectiveness of antimicrobial
therapy because the infectious organisms are becoming
resistant to most antibiotics (Pruneau et al., 2011; Marti et al.,
2014a). In fact, the emergence and spread of antibiotic-
resistant bacteria have been classified by the World Health
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
altered and enriched in antibiotic-resistant bacteria. Humans
can be therefore considered as a source of both antibiotics and
antibiotic resistance genes (ARGs), which may be released into
the environment through sewage systems. Sewer systems
collect wastewater not only from domestic origin but also
from industrial and hospital sources. Hospital effluents, in
particular, constitute a special category of waste that are
highly hazardous because of their infectious and toxic char-
acteristics (Verlicchi et al., 2010; Chagas et al., 2011) and they
also represent an important source of multi-resistant bacteria
(Chagas et al., 2011; Huang et al., 2012) and antibiotics (Santos
et al., 2013; Coutu et al., 2013).
Because of the increasing concern about antibiotic pollu-
tion in aquatic ecosystems, several studies have been con-
ducted to assess the presence of these emerging pollutants in
WWTP effluent discharges (Hirsch et al., 1999; Costanzo et al.,
2005; Garcı́a-Gala n et al., 2011; Leung et al., 2012), whereas
other studies have paid attention to the ARGs released into the
environment and, consequently, the emergence and potential
spread of antibiotic resistance (Pruden et al., 2006; Zhang
et al., 2009; Munir et al., 2011; LaPara et al., 2011; Chen and
235
© 2014 Elsevier Ltd. All rights reserved.
Zhang, 2013; Marti and Balca  zar, 2013; Sidrach-Cardona
et al., 2014). Although some studies have analyzed simulta-
neously the occurrence of these emerging pollutants, those
studies have been focused on the link between the presence of
a limited number of antibiotics and the prevalence of some
selected ARGs in environmental settings (Gao et al., 2012; Li
et al., 2012; Huerta et al., 2013; Marti et al., 2013; Varela et al.,
2014; Xu et al., 2014), but none of them have performed a
study integrating a hospital and urban wastewater system as
well as the impacted river.
The aim of this study was therefore to evaluate the pollu-
tion level of antibiotics and ARGs released from hospital and
urban wastewaters, their removal through WWTPs and their
presence in the receiving river. A broad range of antibiotics
covering different families (b-lactams, lincosamides, macro-
lides, quinolones/fluoroquinolones, sulfonamides, tetracy-
clines, dihydrofolate reductase inhibitors and
nitroimidazoles) were selected and monitored. Five ARGs
were also selected according to the results of antibiotic
detection and clinical importance, and among them, blaTEM
(resistance to b-lactams), qnrS (reduced susceptibility to fluo-
roquinolones), ermB (resistance to macrolides), sulI (resistance
to sulfonamides) and tetW (resistance to tetracyclines) genes
were monitored in the same samples and a relationship be-
tween their occurrence and the presence of antibiotics was
explored.
2. Materials and methods
2.1. Sampling sites
Five sampling points were selected to study the antibiotic and
ARG pollution of both hospital and WWTP effluents to the Ter
River (Fig. 1). The first sampling point consisted in the
wastewater effluents from the main hospital of Girona, which
according to the 2010 annual report has 400 beds, 11 operating
rooms, 94 offices and outpatient visits and 1.649 employees to
provide specialized assistance to approximately 800,000 in-
habitants. Second and third sampling points were influent
and effluent collectors from the Girona WWTP, which receives
the hospital wastewater without any previous treatment
(1000e1500 m3/day) along with municipal wastewater from
the city of Girona (45,000e55,000 m3/day). The two last sam-
pling points were also located in the Ter River, approximately
250 m upstream and downstream of the WWTP discharge
point. The Ter River represents an important source of
drinking water, not only for the population of Girona region
(741,899 inhabitants in 2011) but also for the population of the
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
Three sampling campaigns were performed in November
(2011), December (2011) and January (2012). Water samples
were collected in each of the 5 sampling points and trans-
ported to the laboratory in a portable icebox for immediate
processing.
2.3. Quantification of antibiotics
Samples were analyzed in triplicate for the determination of
sixty-two antibiotics following the protocol previously
described (Gros et al., 2013). Briefly, successive filtration of
water samples was done through 2.7 mm, 1.0 mm and 0.45 mm
pore-size membranes (Millipore; Billerica, MA, USA). After
filtration, water sample was pH-adjusted to 3 with HCl (1.0 M)
and EDTA (4%, v/v) and an appropriate volume of each sample
(50 ml hospital and WWTP effluents, 25 ml WWTP influent
and 250 ml river water) was loaded into Solid Phase Extraction
(SPE)-HLB cartridges (60 mg, 3 mL) (Waters Corp.; Mildford,
MA, USA) for analytes preconcentration. Reconstituted ex-
tracts were analyzed by chromatographic separation with an
ultra performance liquid chromatography (UPLC) system
(Waters Corp.) equipped with a quaternary pump system
using an Acquity BEH T3 column (50 mm  2.1 mm i.d., 1.7 mm
particle size) (Waters Corp.). The UPLC system was coupled to
a triple quadrupole-linear ion trap mass spectrometer
(Applied Biosystems; Foster City, CA, USA) with a Turbo V ion
spray source. Analysis was performed in positive ionization
mode in a multiple reaction monitoring (MRM) mode.
For an accurate quantification, total recoveries were
determined for each water matrix (river, and influent and
effluent wastewater; n ¼ 3) and concentrations were calcu-
lated by internal calibration with isotopically labeled stan-
dards, according to Gros et al. (2013).
2.4. DNA extraction
Water samples were collected in triplicate at selected sites
and filtered under sterile conditions through low protein-
binding 0.22-mm-pore-size membranes (Millipore). The
collected bacterial cells were then resuspended in lysis buffer
(1.2% Triton X-100, 1 M Tris-Cl, 0.5 M Na2EDTA), followed by
enzymatic digestion with lysozyme and proteinase K.
Genomic DNA was extracted using the DNeasy Blood & Tissue
Kit (Qiagen; Valencia, CA, USA), according to the manufac-
turer's instructions. DNA concentration and purity were
determined using a NanoDrop spectrophotometer (NanoDrop
Technologies; Wilmington, DE, USA). All DNA samples were
stored at 20  C until analysis.
2.5. Quantification of ARGs
Real-time PCR (qPCR) assays were used to quantify five ARGs,
including blaTEM, ermB, qnrS, sulI and tetW, which confer
resistance to the main antibiotic families used in human and
veterinary medicine. Copy number of the 16S rRNA gene was
also quantified for normalization of the data. All qPCR assays
were developed using the Brilliant III Ultra-Fast QPCR Master
Mix (Agilent Technologies; Santa Clara, CA, USA), with the
exception for the blaTEM gene, which was amplified using the
SYBR Green Master Mix (Applied Biosystems) due to non-
specific amplification. All qPCR assays were conducted on a
MX3005P system (Agilent Technologies), as previously
described (Marti et al., 2013). Each gene was amplified using
specific primer sets (Table S1) and the PCR conditions included
an initial denaturation at 95  C for 3 min, followed by 40 cycles
at 95  C for 15 s and at the annealing temperature given in
Table S1 for 20 s. In the case of the 16S rRNA gene, amplifi-
cation conditions were 35 cycles at 95  C for 15 s, followed by
an annealing temperature at 60  C for 1 min. After each qPCR
assay, a dissociation curve was constructed by increasing the
temperature from 65 to 95  C in order to confirm the specificity
of the amplified products.
Standard curves were generated by cloning the amplicon
from positive controls into the pCR2.1-TOPO vector (Invi-
trogen, Carlsbad, CA, USA), and the corresponding copy
number was calculated as follows: copy number
ml1 ¼ (A  6.022  1023) (660  B)1, where A is the plasmid
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
the hospitals are typically considered the major source of
cephalosporins (Kümmerer, 2009), the concentrations detec-
ted in the present study were lower than those found in
WWTP influent samples. These results are, nevertheless, in
agreement with other studies where cefazolin and cefotaxime
were also lower in hospital effluents than in the inlet of a
WWTP (Gros et al., 2013). Low levels of macrolides, such as
azythromycin and clarithromycin were detected in hospital
effluent samples as compared with those detected in WWTP
influents, except in the second sampling campaign, where
clarithromycin was detected up to 0.94 mg/L in hospital
effluent samples. Clarithromycin was indeed the antibiotic
that showed higher concentration variability in hospital ef-
fluents among samplings. Unlike fluoroquinolones and
cephalosporins, macrolide consumption is more widespread
in households than in clinical settings (Kümmerer and
Henninger, 2003), and they are rather applied to treat spe-
cific diseases. For instance, the high levels of clarithromycin
described in the second campaign could be attributed to
pneumonia or bronchitis outbreaks since clarithromycin is
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
antimicrobial compounds (Table 1 and Table S2), most anti-
biotics were still present in WWTP effluents at concentrations
that may affect microbial communities. The presence of such
high concentrations of antibiotics in effluents was in fact
impacting the receiving river water, where antibiotics such as
ofloxacin, azythromycin, trimethoprim and metronidazole,
not measured in samples collected upstream the WWTP
discharge, were detected at high concentrations in down-
stream river samples (up to 131.0 ng/L for ofloxacin). In line
with this, ciprofloxacin and sulfamethoxazole showed
approximately tenfold higher concentrations in downstream
than in upstream samples. However, an increase in cefazolin
concentrations was not detected in the water samples
analyzed probably due to its high removal efficiency by the
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.,
2009). Finally, it is important to note that around 80% of the
detected antibiotics (33 antimicrobial compounds out of the 62
target compounds) were found in river water samples (Table
S2). This observation indicates the widespread occurrence of
these compounds in the aquatic ecosystems (Zhang et al.,
2012).
3.2. Quantification of ARGs
Five ARGs blaTEM, qnrS, ermB, sulI and tetW and the 16S rRNA
gene were quantified using qPCR assays in the different water
samples (Fig. 2). High R2 values (average 0.997) and high effi-
ciencies (from 95.8 to 106.5%) obtained from the standard
curves demonstrated the linearity and sensitivity of each
qPCR assay (Fig. S1).
The blaTEM gene is one of the most frequently detected
plasmid-borne antimicrobial resistance genes, which confers
resistance to penicillins and extended-spectrum cephalospo-
rins (Mroczkowska and Barlow, 2008). The qnrS gene is asso-
ciated with plasmid-borne fluoroquinolone resistance that
has become increasingly prevalent in anthropogenically-
influenced environments (Marti et al., 2014b). The ermB gene
encodes resistance to macrolides, lincosamides and strep-
togramin antibiotics and is generally found on conjugative
genetic elements (Negreanu et al., 2012). The sulI gene encodes
dihydropteroate synthase that confers resistance to sulfon-
amides and is generally harbored in class 1 integrons con-
taining other resistance genes (Antunes et al., 2005). The tetW
gene encodes a ribosomal protection protein that confers
resistance to tetracycline (Aminov et al., 2001).
Higher absolute copy numbers of these ARGs (p < 0.05) were
detected in hospital effluent and WWTP influent samples than
those found in other water samples (Fig. 2). A significant
reduction of these ARGs (p < 0.05) was also observed in WWTP
effluent samples, which decreased more than hundredfold in
some instances. However, ARGs were detected in downstream
wasters, indicating a moderate removal efficiency of the
WWTP and their persistence in natural waters. In fact, ermB,
qnrS and sulI genes showed significantly higher values
(p < 0.05) in water samples collected downstream of the
WWTP discharge than in upstream waters. These results
agree with previous studies suggesting that WWTP discharges
could contribute to the spread of ARGs into aquatic environ-
ments. Storteboom et al. (2010) observed that the abundance
of selected ARGs in the Poudre River was higher in anthro-
pogenically impacted areas than those from upstream of the
river. Likewise, Marti and Balca  zar (2013) suggested that the
WWTP was a significant point source of several ARGs into the
receiving river. Czekalski et al. (2014) recently demonstrated
that the abundance of ARGs in close proximity of the WWTP
discharge point was up to two hundredfold above levels
determined at a remote site of Lake Geneva and decreased
exponentially with distance. Altogether, these observations
undoubtedly demonstrate the contribution of WWTP dis-
charges to the spread of antibiotic resistance.
The quantitative analysis also demonstrated that the
relative concentration (copy numbers normalized to the 16S
rRNA gene copy number) of ermB and tetW genes decreased
(p < 0.05) as a result of wastewater treatment, whereas the
relative concentration of blaTEM, qnrS and sulI genes was
higher in effluent samples than those found in WWTP influent
samples (Fig. S2). A plausible explanation might be related to
the spread of some ARGs among bacterial cells in activated
sludge (Szczepanowski et al., 2009; Rizzo et al., 2013), which
convert WWTPs into hot spots for horizontal gene transfer. In
the case of the qnrS gene, this problem may become more
severe due to detected fluoroquinolone concentrations (cip-
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
WWTP influent and effluent, respectively), since cumulative
amounts of these antibiotics might increase the concentration
well above the minimum inhibitory concentrations (MICs) for
several bacteria, causing a selective advantage to resistant
bacteria (Liu et al., 2011).
3.3. Correlation between concentrations of antibiotics
and corresponding ARGs
We carried out a correlation analysis between the absolute
concentrations of ARG and the antibiotics to which they
confer resistance to determine potential links between both
variables. Statistical calculations were conducted for all ARG
except for the tetW gene since no tetracycline was detected in
water samples. Significant positive correlations between the
concentrations of antibiotics and their corresponding ARGs
were observed (Fig. 3). In fact, there were correlations between
ciprofloxacin and the qnrS gene (r ¼ 0.86, p ¼ 0.001), ofloxacin
and the qnrS gene (r ¼ 0.81, p ¼ 0.001), cefazolin and the blaTEM
gene (r ¼ 0.84, p ¼ 0.001), cefotaxime and the blaTEM gene
(r ¼ 0.74, p ¼ 0.002), clarithromycin and the ermB gene (r ¼ 0.89,
p ¼ 0.001), and sulfamethoxazole and the sulI gene (r ¼ 0.83,
p ¼ 0.001); however, no significant correlation was found be-
tween azythromycin and the ermB gene. The correlations also
showed that ARGs increase with the concentration of antibi-
otics (Fig. 3). These results are consistent with previous
studies suggesting that exposure to antibiotics could lead to
selective pressure for ARGs (Wu et al., 2010; Li et al., 2012). A
previous study demonstrated a significant correlation be-
tween the total plasmid-mediated quinolone resistance genes
(qnrD, qnrS, qepA, oqxA and oqxB) and fluoroquinolone residues
in wastewater and soil samples from swine feedlots and their
surrounding environment (Li et al., 2012). Similarly, a signifi-
cant correlation has been demonstrated between total tet gene
copies (tetM, tetO, tetQ and tetW genes) and tetracycline resi-
dues in soils adjacent to swine feedlots (Wu et al., 2010) but
not in WWTP environments, where a significant correlation
was found only between sulfonamides and sul genes (Gao
et al., 2012). Likewise, a recent study demonstrated that rela-
tive tet gene copies (tetB and tetW) were strongly correlated
with the concentrations of tetracycline residues in water
samples collected from a WWTP and its surrounding envi-
ronment, whereas no significant correlations were observed
between sul gene copies (sulI, sulII and sulIII) and sulfonamides
(Xu et al., 2014). Although we have demonstrated a significant
correlation between almost all ARGs analyzed and the envi-
ronmental concentration of the antibiotics to which they
confer resistance, further studies should be conducted in en-
vironments exposed to different pollution levels in order to
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
4. Conclusions
Our study evaluated the presence of a large number of an-
tibiotics and ARGs in an integrated urban wastewater system
including the contribution of hospital effluent, as well as
river water receiving WWTP discharges. Results revealed a
larger occurrence of some antibiotics, such as ciprofloxacin
and ofloxacin, in hospital samples than in the WWTP in-
fluents whereas no significant differences were found be-
tween both samples in relation to ARGs. The WWTP was
unable to totally remove antibiotics and ARGs from urban
effluents, releasing them into the environment and contrib-
uting to their persistence and spread in river waters. Because
rivers are the main source of water, either directly or indi-
rectly, for human and animal consumption, antibiotic
pollution may pose a serious risk for human and animal
health through the spread of antibiotic-resistant bacteria.
Further research is needed to unequivocally demonstrate
that WWTP discharges promote horizontal gene transfer
among aquatic bacterial populations.
Acknowledgments
This study has been supported by the European Union
through the European Regional Development Fund (ERDF
operational program for Catalonia 2007e2013) and the Gen-
eralitat de Catalunya (Consolidated Research Group: Catalan
Institute for Water Research 2014 SGR 291). We are grateful to
the operators and staff at the Girona WWTP (Trargisa) for their
assistance. J.L.B. acknowledges the Ramon y Cajal research
fellowship (RYC-2011-08154) from the Spanish Ministry of
Economy and Competitiveness.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.11.021.
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