Accepted Manuscript

Occurrence of antibiotics and antibiotic resistance genes in hospital and urban

wastewaters and their impact on the receiving river

Sara Rodríguez-Mozaz , Sara Chamorro , Elisabet Marti , Belinda Huerta , Meritxell

Gros , Alexandre Sànchez-Melsió , Carles M. Borrego , Damià Barceló , Jose Luis
Balcázar
PII: S0043-1354(14)00791-X
DOI: 10.1016/j.watres.2014.11.021
Reference: WR 11004

To appear in: Water Research

Received Date: 14 June 2014

Revised Date: 14 November 2014
Accepted Date: 15 November 2014

Please cite this article as: Rodríguez-Mozaz, S., Chamorro, S., Marti, E., Huerta, B., Gros, M., Sànchez-
Melsió, A., Borrego, C.M., Barceló, D., Balcázar, J.L., Occurrence of antibiotics and antibiotic resistance
genes in hospital and urban wastewaters and their impact on the receiving river, Water Research
(2014), doi: 10.1016/j.watres.2014.11.021.

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1 Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters

2 and their impact on the receiving river

4 Sara Rodríguez-Mozaz a,1, Sara Chamorro a,1, Elisabet Marti a, Belinda Huerta a , Meritxell Gros a,

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5 Alexandre Sànchez-Melsió a, Carles M. Borrego a,b, Damià Barceló a,c, Jose Luis Balcázar a,*

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a
7 Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University

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8 of Girona, Girona, Spain.
b
9 Group of Molecular Microbial Ecology, Institute of Aquatic Ecology, University of Girona,

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10 Girona, Spain.
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11 Water and Soil Quality Research Group, Department of Environmental Chemistry, IDAEA-CSIC,

12 Barcelona, Spain.
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13 Sara Rodríguez-Mozaz and Sara Chamorro contributed equally to this work.
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15 * Corresponding author at: Catalan Institute for Water Research (ICRA), Scientific and

16 Technological Park of the University of Girona, Emili Grahit 101, Girona 17003, Spain. Phone: +34
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17 972 18 33 80; Fax: +34 972 18 32 48.

18 E-mail address: jlbalcazar@icra.cat (J.L. Balcazar).

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26 Abstract

27 Antibiotic resistance has become a major health concern; thus, there is a growing interest in

28 exploring the occurrence of antibiotic resistance genes (ARGs) in the environment as well as the

29 factors that contribute to their emergence. Aquatic ecosystems provide an ideal setting for the

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30 acquisition and spread of ARGs due to the continuous pollution by antimicrobial compounds

31 derived from anthropogenic activities. We investigated, therefore, the pollution level of a broad

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32 range of antibiotics and ARGs released from hospital and urban wastewaters, their removal through

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33 a wastewater treatment plant (WWTP) and their presence in the receiving river. Several

34 antimicrobial compounds were detected in all water samples collected. Among antibiotic families,

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35 fluoroquinolones were detected at the highest concentration, especially in hospital effluent samples.
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36 Although good removal efficiency by treatment processes was observed for several antimicrobial

37 compounds, most antibiotics were still present in WWTP effluents. The results also revealed that
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38 copy numbers of ARGs, such as blaTEM (resistance to β-lactams), qnrS (reduced susceptibility to
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39 fluoroquinolones), ermB (resistance to macrolides), sulI (resistance to sulfonamides) and tetW

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40 (resistance to tetracyclines), were detected at the highest concentrations in hospital effluent and

41 WWTP influent samples. Although there was a significant reduction in copy numbers of these
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42 ARGs in WWTP effluent samples, this reduction was not uniform across analyzed ARGs. Relative

43 concentration of ermB and tetW genes decreased as a result of wastewater treatment, whereas
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44 increased in the case of blaTEM, sulI and qnrS genes. The incomplete removal of antibiotics and
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45 ARGs in WWTP severely affected the receiving river, where both types of emerging pollutants

46 were found at higher concentration in downstream waters than in samples collected upstream from

47 the discharge point. Taken together, our findings demonstrate a widespread occurrence of

48 antibiotics and ARGs in urban and hospital wastewater and how these effluents, even after

49 treatment, contribute to the spread of these emerging pollutants in the aquatic environment.

50 Keywords: Urban and hospital wastewater; antibiotics; antibiotic resistance; aquatic ecosystem
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51 1. Introduction

52 Antimicrobial agents have been used in large quantities for several decades since sulfonamides

53 were introduced in the 1930s. Antibiotics are one of the most important drugs for treating infectious

54 diseases, and large amounts of these compounds are released into municipal wastewater due to

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55 excessive consumption and disposal of unused antibiotics (Rodriguez-Mozaz et al., 2010).

56 Antibiotics are used not only in human medicine but also in veterinary practices, animal husbandry,

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57 agriculture and aquaculture (Zhang et al., 2009; Kümmerer, 2009). They can therefore reach surface

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58 and groundwater bodies through different routes, such as wastewater treatment plant (WWTP)

59 effluents (as they are not completely removed), surface runoff, or infiltration of water used for

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60 agricultural purposes.
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61 It is well known that antibiotics pose a significant risk to environmental and human health, even at

62 low concentrations (Kümmerer, 2009). In addition, the overuse and misuse of antibiotics has led to
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63 the emergence of antibiotic-resistant bacteria, compromising the effectiveness of antimicrobial

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64 therapy because the infectious organisms are becoming resistant to most antibiotics (Pruneau et al.,
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65 2011; Marti et al., 2014a). In fact, the emergence and spread of antibiotic-resistant bacteria have

66 been classified by the World Health Organization (WHO) as one of the three biggest threats to
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67 public health in the 21th century. As a consequence of antibiotic consumption, the normal human

68 microbiota can be altered and enriched in antibiotic-resistant bacteria. Humans can be therefore
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69 considered as a source of both antibiotics and antibiotic resistance genes (ARGs), which may be
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70 released into the environment through sewage systems. Sewer systems collect wastewater not only

71 from domestic origin but also from industrial and hospital sources. Hospital effluents, in particular,

72 constitute a special category of waste that are highly hazardous because of their infectious and toxic

73 characteristics (Verlicchi et al., 2010; Chagas et al., 2011) and they also represent an important

74 source of multi-resistant bacteria (Chagas et al., 2011; Huang et al., 2012) and antibiotics (Santos et

75 al., 2013; Coutu et al., 2013).

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76 Because of the increasing concern about antibiotic pollution in aquatic ecosystems, several studies

77 have been conducted to assess the presence of these emerging pollutants in WWTP effluent

78 discharges (Hirsch et al. 1999; Costanzo et al., 2005; García-Galán et al., 2011; Leung et al., 2012),

79 whereas other studies have paid attention to the ARGs released into the environment and,

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80 consequently, the emergence and potential spread of antibiotic resistance (Pruden et al., 2006;

81 Zhang et al., 2009; Munir et al., 2011; LaPara et al., 2011; Chen and Zhang, 2013; Marti and

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82 Balcázar, 2013; Sidrach-Cardona et al., 2014). Although some studies have analyzed

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83 simultaneously the occurrence of these emerging pollutants, those studies have been focused on the

84 link between the presence of a limited number of antibiotics and the prevalence of some selected

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85 ARGs in environmental settings (Gao et al., 2012; Li et al., 2012; Huerta et al., 2013; Marti et al.,
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86 2013; Varela et al., 2014; Xu et al., 2014), but none of them have performed a study integrating a

87 hospital and urban wastewater system as well as the impacted river.

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88 The aim of this study was therefore to evaluate the pollution level of antibiotics and ARGs released
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89 from hospital and urban wastewaters, their removal through WWTPs and their presence in the
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90 receiving river. A broad range of antibiotics covering different families (β-lactams, lincosamides,

91 macrolides, quinolones/fluoroquinolones, sulfonamides, tetracyclines, dihydrofolate reductase

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92 inhibitors and nitroimidazoles) were selected and monitored. Five ARGs were also selected

93 according to the results of antibiotic detection and clinical importance, and among them, blaTEM
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94 (resistance to β-lactams), qnrS (reduced susceptibility to fluoroquinolones), ermB (resistance to

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95 macrolides), sulI (resistance to sulfonamides) and tetW (resistance to tetracyclines) genes were

96 monitored in the same samples and a relationship between their occurrence and the presence of

97 antibiotics was explored.

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99 2. Materials and Methods

100 2.1 Sampling sites

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101 Five sampling points were selected to study the antibiotic and ARG pollution of both hospital and

102 WWTP effluents to the Ter River (Figure 1). The first sampling point consisted in the wastewater

103 effluents from the main hospital of Girona, which according to the 2010 annual report has 400 beds,

104 11 operating rooms, 94 offices and outpatient visits and 1.649 employees to provide specialized

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105 assistance to approximately 800,000 inhabitants. Second and third sampling points were influent

106 and effluent collectors from the Girona WWTP, which receives the hospital wastewater without any

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107 previous treatment (1,000 – 1,500 m3/day) along with municipal wastewater from the city of Girona

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108 (45,000 – 55,000 m3/day). The two last sampling points were also located in the Ter River,

109 approximately 250 m upstream and downstream of the WWTP discharge point. The Ter River

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110 represents an important source of drinking water, not only for the population of Girona region
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111 (741,899 inhabitants in 2011) but also for the population of the Barcelona metropolitan area

112 (4,777,042 inhabitants in 2011).

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113 2.2 Sampling procedure

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114 Three sampling campaigns were performed in November (2011), December (2011) and January
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115 (2012). Water samples were collected in each of the 5 sampling points and transported to the

116 laboratory in a portable icebox for immediate processing.

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117 2.3 Quantification of antibiotics

118 Samples were analyzed in triplicate for the determination of sixty-two antibiotics following the
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119 protocol previously described (Gros et al., 2013). Briefly, successive filtration of water samples was
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120 done through 2.7 µm, 1.0 µm and 0.45 µm pore-size membranes (Millipore; Billerica, MA, USA).

121 After filtration, water sample was pH-adjusted to 3 with HCl (1.0 M) and EDTA (4%, v/v) and an

122 appropriate volume of each sample (50 ml hospital and WWTP effluents, 25 ml WWTP influent

123 and 250 ml river water) was loaded into Solid Phase Extraction (SPE)-HLB cartridges (60 mg,

124 3mL) (Waters Corp.; Mildford, MA, USA) for analytes preconcentration. Reconstituted extracts

125 were analyzed by chromatographic separation with an ultra performance liquid chromatography
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126 (UPLC) system (Waters Corp.) equipped with a quaternary pump system using an Acquity BEH T3

127 column (50 mm × 2.1 mm i.d., 1.7 µm particle size) (Waters Corp.). The UPLC system was coupled

128 to a triple quadrupole-linear ion trap mass spectrometer (Applied Biosystems; Foster City, CA,

129 USA) with a Turbo V ion spray source. Analysis was performed in positive ionization mode in a

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130 multiple reaction monitoring (MRM) mode.

131 For an accurate quantification, total recoveries were determined for each water matrix (river, and

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132 influent and effluent wastewater; n = 3) and concentrations were calculated by internal calibration

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133 with isotopically labeled standards, according to Gros et al. (2013).

134 2.4 DNA extraction

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135 Water samples were collected in triplicate at selected sites and filtered under sterile conditions
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136 through low protein-binding 0.22-µm-pore-size membranes (Millipore). The collected bacterial

137 cells were then resuspended in lysis buffer (1.2 % Triton X-100, 1 M Tris-Cl, 0.5 M Na2EDTA),
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138 followed by enzymatic digestion with lysozyme and proteinase K. Genomic DNA was extracted
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139 using the DNeasy Blood & Tissue Kit (Qiagen; Valencia, CA, USA), according to the
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140 manufacturer’s instructions. DNA concentration and purity were determined using a NanoDrop

141 spectrophotometer (NanoDrop Technologies; Wilmington, DE, USA). All DNA samples were
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142 stored at –20 °C until analysis.

143 2.5. Quantification of ARGs

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144 Real-time PCR (qPCR) assays were used to quantify five ARGs, including blaTEM, ermB, qnrS, sulI
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145 and tetW, which confer resistance to the main antibiotic families used in human and veterinary

146 medicine. Copy number of the 16S rRNA gene was also quantified for normalization of the data.

147 All qPCR assays were developed using the Brilliant III Ultra-Fast QPCR Master Mix (Agilent

148 Technologies; Santa Clara, CA, USA), with the exception for the blaTEM gene, which was amplified

149 using the SYBR Green Master Mix (Applied Biosystems) due to non-specific amplification. All

150 qPCR assays were conducted on a MX3005P system (Agilent Technologies), as previously
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151 described (Marti et al., 2013). Each gene was amplified using specific primer sets (Table S1) and

152 the PCR conditions included an initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95

153 °C for 15 s and at the annealing temperature given in Table S1 for 20 s. In the case of the 16S rRNA

154 gene, amplification conditions were 35 cycles at 95 °C for 15 s, followed by an annealing

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155 temperature at 60 °C for 1 min. After each qPCR assay, a dissociation curve was constructed by

156 increasing the temperature from 65 to 95 °C in order to confirm the specificity of the amplified

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157 products.

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158 Standard curves were generated by cloning the amplicon from positive controls into the pCR2.1-

159 TOPO vector (Invitrogen, Carlsbad, CA, USA), and the corresponding copy number was calculated

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160 as follows: copy number µl−1 = (A×6.022×1023) (660×B)−1, where A is the plasmid DNA
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161 concentration (g µl−1), B is the plasmid length (bp) containing the cloned sequence, 6.022×1023 is

162 the Avogadro's number and 660 is the average molecular weight of one base pair (Perini et al.,
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163 2011). A ten-fold serial dilution was then used to construct the standard curve for each ARG, which
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164 was run in parallel with the samples to obtain absolute quantification. The copy number of each
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165 ARG was also normalized to the 16S rRNA gene copy number in order to obtain relative

166 quantification.
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167 2.6 Statistical analysis

168 Comparisons of average antibiotic and ARG concentrations among different sampling points were
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169 carried out using ANOVA or Kruskal Wallis tests, as appropriate. Correlations between antibiotic
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170 and ARG values were made using Pearson’s test (all variables satisfied the normality assumption).

171 Differences were considered significant at p < 0.05. All statistical analyses were performed using

172 SPSS 17.0 software (SPSS; Chicago, IL, USA).

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174 3. Results and discussion

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175 3.1 Quantification of antibiotics

176 Thirty-three out of the sixty-two antimicrobial compounds analyzed were detected in different water

177 samples collected over the three sampling campaigns (Table S2). Despite their high consumption,

178 compounds belonging to penicillin and tetracycline families were detected neither in the analyzed

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179 wastewater nor in the river waters, probably because of their chemical instability (Graham et al.,

180 2011).

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181 Among antibiotic families, fluoroquinolones were detected at the highest concentration, especially

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182 in hospital effluent samples. In fact, ciprofloxacin and ofloxacin were present in hospital effluents,

183 ranging from 13.78 µg/L for ciprofloxacin in the third sampling campaign to 14.38 µg/L for

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184 ofloxacin in the first one (Table 1). In contrast, lower concentrations (at least one order of
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185 magnitude) were found in urban wastewater than in hospital effluents. Such high values in hospitals

186 may be related to their medical consumption, as these fluoroquinolones are frequently used in
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187 hospital practice to treat infections (MacDougall et al., 2005; Ray et al., 2005) and, therefore, they
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188 have also been found in other hospital effluents at such high concentrations (Thomas et al., 2007;
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189 Kovalova et al., 2012; Santos et al., 2013). The levels of metronidazole, sulfamethoxazole and

190 trimethoprim in hospital effluent samples were also high in all sampling campaigns (Table 1). The
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191 levels of sulfamethoxazole and trimethoprim were very similar in all samplings probably due to

192 their combined therapeutic use (Batt et al., 2006). However, the ratio between both compounds was
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193 not consistent with the one found in the WWTP influent samples, where sulfamethoxazole
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194 concentrations were higher than trimethoprim concentrations. In the case of metronidazole, similar

195 concentrations have been previously detected in hospital and urban wastewaters (Verlicchi et al.,

196 2012).

197 In contrast, lower concentrations of antibiotics belonging to the group of cephalosporins, such as

198 cefazolin and cefotaxime, were detected in hospital effluent samples compared to those found in

199 urban WWTP samples (Table 1). Although the hospitals are typically considered the major source
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200 of cephalosporins (Kümmerer, 2009), the concentrations detected in the present study were lower

201 than those found in WWTP influent samples. These results are, nevertheless, in agreement with

202 other studies where cefazolin and cefotaxime were also lower in hospital effluents than in the inlet

203 of a WWTP (Gros et al., 2013). Low levels of macrolides, such as azythromycin and clarithromycin

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204 were detected in hospital effluent samples as compared with those detected in WWTP influents,

205 except in the second sampling campaign, where clarithromycin was detected up to 0.94 µg/L in

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206 hospital effluent samples. Clarithromycin was indeed the antibiotic that showed higher

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207 concentration variability in hospital effluents among samplings. Unlike fluoroquinolones and

208 cephalosporins, macrolide consumption is more widespread in households than in clinical settings

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209 (Kümmerer et al., 2003), and they are rather applied to treat specific diseases. For instance, the high
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210 levels of clarithromycin described in the second campaign could be attributed to pneumonia or

211 bronchitis outbreaks since clarithromycin is often used to treat lung infections (Tanaka et al., 2002).
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212 Although good removal efficiency was observed for several antimicrobial compounds (Table 1 and
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213 Table S2), most antibiotics were still present in WWTP effluents at concentrations that may affect
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214 microbial communities. The presence of such high concentrations of antibiotics in effluents was in

215 fact impacting the receiving river water, where antibiotics such as ofloxacin, azythromycin,
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216 trimethoprim and metronidazole, not measured in samples collected upstream the WWTP

217 discharge, were detected at high concentrations in downstream river samples (up to 131.0 ng/L for
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218 ofloxacin). In line with this, ciprofloxacin and sulfamethoxazole showed approximately tenfold
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219 higher concentrations in downstream than in upstream samples. However, an increase in cefazolin

220 concentrations was not detected in the water samples analyzed probably due to its high removal

221 efficiency by the WWTP. In fact, previous studies have reported removal efficiencies between 75

222 and 100% for this compound (Lin et al., 2009). Finally, it is important to note that around 80% of

223 the detected antibiotics (33 antimicrobial compounds out of the 62 target compounds) were found in
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224 river water samples (Table S2). This observation indicates the widespread occurrence of these

225 compounds in the aquatic ecosystems (Zhang et al. 2012).

226 3.2 Quantification of ARGs

227 Five ARGs blaTEM, qnrS, ermB, sulI and tetW and the 16S rRNA gene were quantified using qPCR

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228 assays in the different water samples (Figure 2). High R2 values (average 0.997) and high

229 efficiencies (from 95.8 to 106.5%) obtained from the standard curves demonstrated the linearity and

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230 sensitivity of each qPCR assay (Figure S1).

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231 The blaTEM gene is one of the most frequently detected plasmid-borne antimicrobial resistance

232 genes, which confers resistance to penicillins and extended-spectrum cephalosporins (Mroczkowska

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233 and Barlow, 2008). The qnrS gene is associated with plasmid-borne fluoroquinolone resistance that
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234 has become increasingly prevalent in anthropogenically-influenced environments (Marti et al.,

235 2014b). The ermB gene encodes resistance to macrolides, lincosamides and streptogramin
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236 antibiotics and is generally found on conjugative genetic elements (Negreanu et al., 2012). The sulI
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237 gene encodes dihydropteroate synthase that confers resistance to sulfonamides and is generally
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238 harbored in class 1 integrons containing other resistance genes (Antunes et al., 2005). The tetW

239 gene encodes a ribosomal protection protein that confers resistance to tetracycline (Aminov et al.,
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240 2001).

241 Higher absolute copy numbers of these ARGs (p < 0.05) were detected in hospital effluent and
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242 WWTP influent samples than those found in other water samples (Figure 2). A significant reduction
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243 of these ARGs (p < 0.05) was also observed in WWTP effluent samples, which decreased more

244 than hundredfold in some instances. However, ARGs were detected in downstream wasters,

245 indicating a moderate removal efficiency of the WWTP and their persistence in natural waters. In

246 fact, ermB, qnrS and sulI genes showed significantly higher values (p < 0.05) in water samples

247 collected downstream of the WWTP discharge than in upstream waters. These results agree with

248 previous studies suggesting that WWTP discharges could contribute to the spread of ARGs into
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249 aquatic environments. Storteboom et al. (2010) observed that the abundance of selected ARGs in

250 the Poudre River was higher in anthropogenically impacted areas than those from upstream of the

251 river. Likewise, Marti et al. (2013) suggested that the WWTP was a significant point source of

252 several ARGs into the receiving river. Czekalski et al. (2014) recently demonstrated that the

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253 abundance of ARGs in close proximity of the WWTP discharge point was up to two hundredfold

254 above levels determined at a remote site of Lake Geneva and decreased exponentially with distance.

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255 Altogether, these observations undoubtedly demonstrate the contribution of WWTP discharges to

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256 the spread of antibiotic resistance.

257 The quantitative analysis also demonstrated that the relative concentration (copy numbers

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258 normalized to the 16S rRNA gene copy number) of ermB and tetW genes decreased (p < 0.05) as a
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259 result of wastewater treatment, whereas the relative concentration of blaTEM, qnrS and sulI genes

260 was higher in effluent samples than those found in WWTP influent samples (Figure S2). A
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261 plausible explanation might be related to the spread of some ARGs among bacterial cells in
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262 activated sludge (Szczepanowski et al., 2009; Rizzo et al., 2013), which convert WWTPs into hot
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263 spots for horizontal gene transfer. In the case of the qnrS gene, this problem may become more

264 severe due to detected fluoroquinolone concentrations (ciprofloxacin with average values of 932.56
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265 and 139.34 ng/L, and ofloxacin with average values of 913.08 and 104.90 ng/L in the WWTP

266 influent and effluent, respectively), since cumulative amounts of these antibiotics might increase the
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267 concentration well above the minimum inhibitory concentrations (MICs) for several bacteria,
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268 causing a selective advantage to resistant bacteria (Liu et al., 2011).

269 3.3 Correlation between concentrations of antibiotics and corresponding ARGs

270 We carried out a correlation analysis between the absolute concentrations of ARG and the

271 antibiotics to which they confer resistance to determine potential links between both variables.

272 Statistical calculations were conducted for all ARG except for the tetW gene since no tetracycline

273 was detected in water samples. Significant positive correlations between the concentrations of
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274 antibiotics and their corresponding ARGs were observed (Figure 3). In fact, there were correlations

275 between ciprofloxacin and the qnrS gene (r = 0.86, p = 0.001), ofloxacin and the qnrS gene (r =

276 0.81, p = 0.001), cefazolin and the blaTEM gene (r = 0.84, p = 0.001), cefotaxime and the blaTEM

277 gene (r = 0.74, p = 0.002), clarithromycin and the ermB gene (r = 0.89, p = 0.001), and

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278 sulfamethoxazole and the sulI gene (r = 0.83, p = 0.001); however, no significant correlation was

279 found between azythromycin and the ermB gene. The correlations also showed that ARGs increase

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280 with the concentration of antibiotics (Figure 3). These results are consistent with previous studies

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281 suggesting that exposure to antibiotics could lead to selective pressure for ARGs (Wu et al., 2010;

282 Li et al., 2012). A previous study demonstrated a significant correlation between the total plasmid-

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283 mediated quinolone resistance genes (qnrD, qnrS, qepA, oqxA and oqxB) and fluoroquinolone
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284 residues in wastewater and soil samples from swine feedlots and their surrounding environment (Li

285 et al., 2012). Similarly, a significant correlation has been demonstrated between total tet gene copies
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286 (tetM, tetO, tetQ and tetW genes) and tetracycline residues in soils adjacent to swine feedlots (Wu et
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287 al., 2010) but not in WWTP environments, where a significant correlation was found only between
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288 sulfonamides and sul genes (Gao et al., 2012). Likewise, a recent study demonstrated that relative

289 tet gene copies (tetB and tetW) were strongly correlated with the concentrations of tetracycline
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290 residues in water samples collected from a WWTP and its surrounding environment, whereas no

291 significant correlations were observed between sul gene copies (sulI, sulII and sulIII) and
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292 sulfonamides (Xu et al., 2014). Although we have demonstrated a significant correlation between
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293 almost all ARGs analyzed and the environmental concentration of the antibiotics to which they

294 confer resistance, further studies should be conducted in environments exposed to different

295 pollution levels in order to provide a better insight into these correlations.

296

297 4. Conclusions
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298 Our study evaluated the presence of a large number of antibiotics and ARGs in an integrated urban

299 wastewater system including the contribution of hospital effluent, as well as river water receiving

300 WWTP discharges. Results revealed a larger occurrence of some antibiotics, such as ciprofloxacin

301 and ofloxacin, in hospital samples than in the WWTP influents whereas no significant differences

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302 were found between both samples in relation to ARGs. The WWTP was unable to totally remove

303 antibiotics and ARGs from urban effluents, releasing them into the environment and contributing to

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304 their persistence and spread in river waters. Because rivers are the main source of water, either

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305 directly or indirectly, for human and animal consumption, antibiotic pollution may pose a serious

306 risk for human and animal health through the spread of antibiotic-resistant bacteria. Further research

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307 is needed to unequivocally demonstrate that WWTP discharges promote horizontal gene transfer
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308 among aquatic bacterial populations.

309
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310 Acknowledgements
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311 This study has been supported by the European Union through the European Regional Development
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312 Fund (FEDER) and the Generalitat de Catalunya (Consolidated Research Group: Catalan Institute

313 for Water Research 2014 SGR 291). We are grateful to the operators and staff at the Girona WWTP
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314 (Trargisa) for their assistance. J.L.B. acknowledges the Ramon y Cajal research fellowship (RYC-

315 2011-08154) from the Spanish Ministry of Economy and Competitiveness.

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316
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317 References

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346 Gros M, Rodríguez-Mozaz S, Barceló D. Rapid analysis of multiclass antibiotic residues and some

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380 hospitals. Clin Infect Dis 2005;41:435–440.

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395 antibiotic resistance in agricultural soils. Environ Sci Technol 2012;46:4800–4808.

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398 environment. PLoS ONE 2011;6:e17699.

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418 Storteboom H, Arabi M, Davis JG, Crimi B, Pruden A. Identification of antibiotic-resistance-gene

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422 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment

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432 residues and bacterial populations in a hospital-urban wastewater treatment plant system. Water Res

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442 Xu J, Xu Y, Wang H, Guo C, Qiu H, He Y, Zhang Y, Li X, Meng W. Occurrence of antibiotics and

443 antibiotic resistance genes in a sewage treatment plant and its effluent-receiving river.

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446 Biotechnol 2009;82:397–414.

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448 the Laizhou Bay, China: Impact of river discharge. Ecotoxicol Environ Saf 2012;80:208–215.

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449

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467 TABLES AND FIGURES

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469 Table 1. Occurrence of the 9 antibiotics detected at the highest concentration in the different water

470 samples.

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471 Figure 1. Geographical location of the different sampling sites (adapted from Google Maps).

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472 Figure 2. Absolute concentration of ARGs in the different water samples. Within the box plot chart,

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473 the crosspieces of each box plot represent (from top to bottom) maximum, upper-quartile, median

474 (black bar), lower-quartile and minimum values.

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475 Figure 3. Correlations between the concentrations of antibiotics and their corresponding ARGs.

476 Sample locations are represented by: black squares (hospital effluents), red squares (WWTP
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477 influents), brown squares (WWTP effluents), white squares (upstream) and green squares

478 (downstream). Light gray lines show 95% confidence intervals.

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480 Table S1. Primer pairs used in qPCR assays for specific detection and quantification of ARGs.
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481 Table S2. Concentrations of 62 antibiotics, expressed in ng/L, detected in the different water
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482 samples analyzed.

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483 Figure S1. Standard curves for each qPCR assay. Each standard curve was generated by plotting

484 mean Ct values versus tenfold serial dilutions of plasmid DNA.

485 Figure S2. Relative concentration of ARGs in the different water samples. Crosspieces of each box

486 plot represent (from top to bottom) maximum, upper-quartile, median (black bar), lower-quartile

487 and minimum values.

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

Concentration (ng/L)

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Ciprofloxacin Ofloxacin Cefazolin Cefotaxime Azythromycin Clarithromycin Sulfamethoxazole Trimethoprim Metronidazole*
First sampling
Hospital

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8,372. 9±67.8 14,377.8±50.9 83.4±3.6 143.7±4.2 20.1±5.7 167.3±10.7 751.7±2.4 594.3±13.5 937.4±111.8
effluent
WWTP
639.1±59.2 592.9±2.5 94.7±0.9 363.5±1.6 ND 460.9±7.4 330.7±8.0 87.8±0.4 ND

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influent
WWTP
108.0±4.2 82.0±6.3 14.6±2.5 229.2±7.7 75.5±9.4 92.3±0.6 64.9±10.1 97.2±26.5 ND
effluent

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Upstream 8.1±0.22 <MQL 8.4±0.1 93.1±11.8 ND 35.4±1.1 <MQL ND ND
Downstream 50.0±0.9 100.7±2.3 8.4±0.9 147.5±3.6 55.9±0.8 61.3±2.1 40.2±3.5 50.7±1.6 28.4±1.9

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Second sampling
Hospital
8,305.1±48.0 5,700.0±33.3 44.6±2.7 240.4±14.3 59.9±7.1 941.1±1.9 190.2±6.1 136.3±5.0 523.9±8.2
effluent

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WWTP
1,307.0±6.6 581.7±10.2 146.6±4.8 252.8±0.4 189.0±3.5 551.3±17.2 417.4±5.0 122.9±4.5 240.1±13.7
influent

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WWTP
135.2±0.4 60.9±2.7 22.1±0.3 207.9±2.2 134.1±6.3 129.0±10.8 73.0±2.8 107.6±7.7 29.6±4.2
effluent

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Upstream 9.4±1.5 <MQL 6.6±0.4 68.5±1.7 ND 35.8±0.4 3.9±0.4 <MQL ND
Downstream 72.4±5.1 137.6±3.2 7.9±1.2 130.8 ± 0.8 70.7±0.5 96.3±0.9 64.8±3.3 70.5±3.4 17.1±2.6
Third sampling
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Hospital
13,779.7±24.0 4,750.0±23.6 <MQL 236.8±4.7 59.9±7.1 ND 4,816.7±212.1 3,8.26±48.6 1,792.9±32.6
effluent
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WWTP
851.6±5.9 1.564.6±9.4 116.2±7.2 340.5±8.9 214.5±16.3 471.3±27.0 401.6±1.1 179.5±4.7 274.5±11.2
influent
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WWTP
174.8±8.1 171.8±12.8 24.8±1.5 223.4±2.6 135.0±2.6 115.1±3.3 56.2±3.8 124.9±13.7 144.0±9.1
effluent
Upstream 4.7±0.1 <MQL 10.5±1.1 67.98±0.5 ND 44.7±3.6 7±0.6 <MQL ND
Downstream 48.2±7.9 131±1.6 3.4±0.1 165.6 ±4.0 115.5±0.2 61.1±17.9 71.8±2.2 92.7±3.8 23.2±7.9

* This value includes the concentration of metronidazole-OH (metabolite). ND, not detected. MQL, method quantification limit.
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 Absolute concentration [Log (ARG copies / ml)]

0
2
4
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tetW
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sulI

Total
ermB
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7 7
r = 0.86 r = 0.81
p = 0.001 p = 0.001
6 6
Log (qnrS copies/ml)

Log (qnrS copies/ml)

5 5

4 4

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3 3

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2 2

1.0 2.0 3.0 4.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

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Log (ciprofloxacin concentrations) Log (ofloxacin concentrations)

6 5
r = 0.84 r = 0.74

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p = 0.001 p = 0.002
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Log (blaTEM copies/ml)

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0.5 1.0 1.5 2.0 2.5 1.8 2.0 2.2 2.4 2.6
Log (cefazolin concentrations) Log (cefotaxime concentrations)
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7 7
r = 0.89 r = 0.83
p = 0.001 p = 0.001
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Log (ermB copies/ml)

Log (sulI copies/ml)

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3

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2

1 2

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 -1.0 0 1.0 2.0 3.0 4.0
Log (clarithromycin concentrations) Log (sulfamethoxazole concentrations)
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Highlights

• Antibiotics and ARGs studied in a hospital-urban wastewater system and receiving river.
• Higher levels of fluoroquinolones were found in hospital than in urban wastewater.
• Similar levels of ARGs were found in hospital and urban wastewaters.

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• Urban treated effluents contribute to antibiotic pollution and resistance in the aquatic
environment.
•

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Correlations between antibiotic concentration and corresponding ARG were observed.

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Table S1. Primer pairs used in qPCR assays for specific detection and quantification of

Target Annealing Product

Primer Sequence (5’–3’) References
gene temp (°C) (bp)

16S F1048 GTGSTGCAYGGYTGTCGTCA

60 146 [1]
rRNA R1194 ACGTCRTCCMCACCTTCCTC
RTblaTEMFX GCKGCCAACTTACTTCTGACAACG
blaTEM 60 247 [2]
RTblaTEMFR CTTTATCCGCCTCCATCCAGTCTA

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qnrSrtF11 GACGTGCTAACTTGCGTGAT
qnrS 62 240 [3]
qnrSrtR11 TGGCATTGTTGGAAACTTG
erm(B)-91f GATACCGTTTACGAAATTGG

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ermB 58 364 [4]
erm(B)-454r GAATCGAGACTTGAGTGTGC
sul(I)-FX CGCACCGGAAACATCGCTGCAC
sulI 65 163 [5]

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sul(I)-RX TGAAGTTCCGCCGCAAGGCTCG
tet(W)-FX GAGAGCCTGCTATATGCCAGC
tetW 60 168 [6]
tet(W)-FV GGGCGTATCCACAATGTTAAC

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ARGs. AN
References
[1] Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, et al. Quantitative real-time
PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans,
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Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunol
Med Microbiol 2003;39:81–86.
[2] Xi C, Xi CW, Zhang YL, Marrs CF, Ye W, Simon C, et al. Prevalence of antibiotic resistance in
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drinking water treatment and distribution systems. Appl Environ Microbiol 2009;75:5714–5718.
[3] Marti E, Balcázar JL. Real-time PCR assays for quantification of qnr genes in environmental
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water samples and chicken feces. Appl Environ Microbiol 2013;79:1743–1745.

[4] Chen J, Yu Z, Michel FC, Wittum T, Morrison M. Development and application of real-time
PCR assays for quantification of erm genes conferring resistance to macrolide-lincosamides-
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streptogramin B in livestock manure and manure management systems. Appl Environ Microbiol
2007;14:4407–4416.
[5] Pei R, Kim SC, Carlson KH, Pruden A. Effect of river landscape on the sediment concentrations
of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res 2006;40:2427–
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2435.
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[6] Aminov RI, Garrigues-Jeanjean N, Mackie RI. Molecular ecology of tetracycline resistance:
Development and validation of primers for detection of tetracycline resistance genes encoding
ribosomal protection proteins. Appl Environ Microbiol 2001;67:22–32.
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Table S2. Concentrations of 62 antibiotics, expressed in ng/L, detected in the different water
samples analyzed.

Sampling Hospital WWTP WWTP River River

Campaign Effluent influent effluent Upstream Downstream
Sulfonamides
Sulfamethoxazole 1st 751.67±2.36 330.74±8.00 64.90±10.07 <mql 40.19±3.51
2nd 190.22±6.06 417.43±4.96 73.02±2.80 3.94±0.40 64.77±3.51

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3rd 4,816.67±212.13 401.65±1.08 56.18±3.81 6.98±0.64 71.85±2.23
Sulfadiazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Sulfamethazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Sulfaquinoxaline 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Sulfathiazole 1st nd nd nd nd nd
2nd nd nd nd nd nd
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3rd nd nd nd nd nd
Sulfadimethoxine 1st 31.52±7.70 12.67±2.01 nd nd nd
2nd 3.18±1.49 4.45±0.43 nd <mql 4.71±0.31
3rd <mql <mql nd nd 3.56±1.24
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Sulfapyridine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd 44.93±1.10 nd nd nd
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Sulfamerazine 1st nd 23.77±0.98 nd nd nd

2nd nd nd nd nd nd
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3rd nd nd nd nd nd
Sulfamethizole 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd 44.93±1.10 nd nd nd
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Sulfamethoxypyridazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Sulfisoxazole 1st nd nd nd nd nd
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2nd nd nd nd nd nd
3rd nd nd nd nd nd
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Sulfanitran 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Sulfabenzamide 1st nd nd nd nd nd
2nd nd nd nd <mql nd
3rd nd nd nd nd <mql
Sulfapyridine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd 44.93±1.10 nd nd nd
N-acetylsulfadiazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
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N-acetylsulfamethazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
N-acetylsulfapyridine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
N-acetylsulfamerazine 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Fluoroquinolones
Cinoxacin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Ciprofloxacin 1st 8,372.88±67.80 639.07±59.20 108.00±4.24 8.12±0.22 49.96±0.91
2nd 8,305.08±47.94 1,306.98±6.58 135.25±0.35 9.35±1.50 72.45±5.12
3rd 13,779.66±23.97 851.63±5.92 174.75±8.13 4.69 ±0.03 48.23±7.90

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Difloxacin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

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Marbofloxacin 1st nd nd nd nd nd
2nd nd nd nd nd nd
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3rd nd nd nd nd nd
Ofloxacin 1st 14,377.78±50.92 592.92±2.50 82.00±6.27 <mql 100.73±2.30
2nd 5,700.00±33.33 581.71±10.22 60.87±2.71 <mql 137.61±3.23
3rd 4,750.00±23.57 1,564.60±9.37 171.83±12.79 <mql 130.98±1.62
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st
Orbifloxacin 1 nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
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Enrofloxacin 1st nd nd nd 17.48±0.03 <mql

2nd nd nd nd 109.18±2.00 3.15±0.19
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3rd nd nd nd <mql <mql

Norfloxacin 1st 880.65±14.78 575.16±25.88 <mql nd 41.23±1.23
2nd <mql 475.52±6.92 <mql nd 76.23 ±2.28
3rd 9,177.42±22.81 338.13±1.51 <mql nd 48.64±24.57
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Danofloxacin 1st <mql nd nd nd nd

2nd <mql nd <mql nd nd
3rd nd nd nd nd nd
st
Enoxacin 1 nd nd nd nd nd
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2nd nd nd nd nd nd
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3rd nd nd nd nd nd
Quinolones
Flumequine 1st nd <mql <mql <mql <mql
2nd 6.06±0.01 <mql nd <mql <mql
3rd 6.41±0.14 <mql nd <mql <mql
Oxolinic acid 1st nd <mql nd nd nd
2nd nd nd nd <mql nd
3rd nd <mql nd nd nd
Nalidixic acid 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Pipemidic acid 1st nd 133.61±10.70 33.87±4.85 nd 18.49±0.20
2nd nd 49.58±1.19 38.41±1.12 nd 19.55±1.77
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3rd nd 56.81±2.38 35.25±2.46 nd 10.59±2.07
Cephalosporins
Cefazolin 1st 83.42±3.58 94.72±0.96 14.58±2.52 8.43±0.13 8.39±0.95
2nd 44.60±2.68 146.58±4.78 22.09±0.30 6.63±0.38 7.94±1.21
3rd <mql 116.17±7.17 24.79±1.51 10.50±1.41 3.38±0.02
Cefalexin 1st nd 40.01±4.61 9.79±1.15 5.97±0.74 7.81±0.09
2nd nd 16.99±1.91 22.73 ±4.34 4.49±1.13 4.06±0.92
3rd nd 32.41±2.04 24.06±0.84 8.39 ±0.09 5.77±1.09
Cefotaxime 1st 143.69±4.16 363.53±1.58 229.25±7.71 93.06±11.82 147.47±3.61

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2nd 240.38±14.27 252.83±0.44 207.87±2.16 68.49±1.67 130.82±0.79
3rd 236.75±4.73 340.49±8.89 223.36±2.65 67.98±0.48 165.61±3.97
Cefuroxime 1st nd nd nd nd nd
2nd nd nd nd nd nd

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3rd nd nd nd nd nd
Ceftiofur 1st 1,341.55±1.13 851.48±7.55 451.83±0.74 53.32±0.50 51.61±0.77
2nd 569.04±4.51 815.60±6.41 417.82±1.47 54.04±1.27 51.72±0.19

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3rd 645.57±8.44 917.58±1.41 453.11±4.48 50.73±1.10 47.80±2.49
Cefapirin 1st nd nd nd nd nd
2nd nd nd nd nd nd

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3rd nd nd nd nd nd
Penicillins
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Amoxicillin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Ampicillin 1st nd nd nd nd nd
M

2nd nd nd nd nd nd
3rd nd nd nd nd nd
Penicillin V 1st 97.09±8.58 nd nd <mql 19.56±0.88
D

2nd nd nd nd <mql 10.21±0.29

3rd nd nd nd <mql 67.35±0.47
TE

Penicillin G 1st nd nd 175.71±33.58 nd nd

2nd nd nd nd nd nd
3rd nd 199.38±38.87 nd nd nd
Cloxacillin 1st nd nd nd nd nd
EP

2nd nd nd nd nd nd
3rd nd nd nd nd nd
Oxacillin 1st nd nd nd nd nd
C

2nd nd nd nd nd nd
3rd nd nd nd nd nd
AC

Piperacillin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Macrolides
Erythromycin 1st 29.46±0.17 <mql 14.49±0.29 nd 17.14±0.30
2nd 71.39±2.42 <mql 9.22±0.56 nd 13.96±0.89
3rd nd 4.08±0.17 4.75±0.27 nd 5.74±1.42
Erythromycin – H2O 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Azithromycin 1st 20.14±5.68 nd 75.46±9.36 nd 55.89±0.76
2nd 59.94±7.06 189.00±3.54 134.07±6.29 nd 70.71±0.51
3rd 59.94±7.06 214.50±16.26 135.00±2.62 nd 115.54±0.25
 ACCEPTED MANUSCRIPT
Clarithromycin 1st 167.26±10.73 460.87±7.38 92.26±0.64 35.37±1.10 61.31±2.09
2nd 941.10±1.94 551.30±17.22 129.02±10.85 35.79±0.45 96.28±0.89
3rd nd 471.30±27.05 115.11±3.26 44.66±3.58 61.10±17.88
Roxithromycin 1st <mql 55.77±9.02 <mql 2.86±0.07 22.74±1.89
2nd nd 54.41±2.72 <mql 2.98±0.16 23.03±0.70
3rd nd 61.86±5.86 <mql 2.98±0.48 25.09±0.42
Spiramycin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

PT
Tilmicosin 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Tylosin 1st nd nd nd nd nd

RI
2nd nd nd nd nd nd
3rd nd nd nd nd nd
Tetracyclines

SC
Tetracycline 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd

U
Doxycycline 1st nd nd nd nd nd
2nd nd nd nd nd nd
AN
3rd nd nd nd nd nd
Chlortetracycline 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
M

Oxytetracycline 1st nd nd nd nd nd
2nd nd nd nd nd nd
3rd nd nd nd nd nd
D

Lincosamides
Lincomycin 1st nd nd nd nd 5.89±0.44
TE

2nd nd nd nd nd nd
3rd nd nd nd nd nd
Clindamycin 1st 1,638.64±3.21 nd nd nd nd
2nd nd nd nd nd nd
EP

3rd 3,795.45±32.14 nd nd nd nd
Nitroimidazoles
Metronidazole 1st 519.23±42.74 nd nd nd 28.40±1.89
C

2nd 92.49±6.37 164.62±7.74 nd nd 17.11±2.39

3rd 1,399.73±21.37 228.89±7.19 109.58±5.40 nd 23.20±7.92
AC

Metronidazole – OH 1st 418.21±69.03 nd nd nd nd

2nd 431.40±1.87 75.47±5.97 29.55±4.16 nd nd
3rd 393.14±11.19 45.63±3.98 34.38±3.66 nd nd
Dihydrofolate reductase inhibitors
Trimethoprim 1st 594.23±13.51 87.84±0.38 97.22±26.46 nd 50.69±1.59
2nd 136.31±4.97 122.88±4.50 107.62±7.74 <mql 70.47±3.35
3rd 3,787.26±48.64 179.46±4.71 124.92±13.74 <mql 92.68±3.77

Values expressed as mean ± SD.

nd, not detected.
<mql, below method quantification limit.
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tetW 16S rRNA gene

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 Relative concentration [Log (ARG copies / 16S rRNA copies)]

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