Degradation of sulfonamides as a microbial resistance mechanism

Accepted Manuscript Degradation of sulfonamides as a microbial resistance mechanism Maria Vila-Costa, Rosalinda Gioia, Jaume Acena, Sandra Perez, Emilio O. Casamayor, Jordi Dachs PII:

S0043-1354(17)30174-4

DOI:

10.1016/j.watres.2017.03.007

Reference:

WR 12744

To appear in:

Water Research

Received Date: 28 October 2016 Revised Date:

2 February 2017

Accepted Date: 4 March 2017

Please cite this article as: Vila-Costa, M., Gioia, R., Acena, J., Perez, S., Casamayor, E.O., Dachs, J., Degradation of sulfonamides as a microbial resistance mechanism, Water Research (2017), doi: 10.1016/j.watres.2017.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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DEGRADATION OF SULFONAMIDES AS A MICROBIAL RESISTANCE

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MECHANISM

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Maria Vila-Costa1,2*, Rosalinda Gioia1,3*, Jaume Acena1, Sandra Perez1, Emilio O.

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Casamayor2, Jordi Dachs1

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Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-24,

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Barcelona 08034, Catalunya, Spain.

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CSIC, Accés Cala St. Francesc 14, E-17300 Blanes, Spain.

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*Current address: The REACH Centre, Lancaster University, Lancaster, LA1 4YQ

Corresponding author e-mails: [email protected], [email protected]

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Integrative Freshwater Ecology Group, Centre for Advanced Studies of Blanes, CEAB-

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ACCEPTED MANUSCRIPT Abstract

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Two of the main mechanisms of bacterial resistance to sulfonamides in aquatic systems,

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spread of antibiotic resistance genes (ARG) among the microbial community and in-situ

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bacterial sulfonamide degradation, were studied in mesocosms experiments using water

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and cobble biofilms from upstream (pristine waters) and downstream (polluted waters)

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from the Llobregat river, NE Iberian Peninsula. Mesocosms were prepared at two

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different concentrations (5000 ng/L and 1000ng/L) of sulfonamides antibiotics

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(sulfamethazine

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sulfonamides and their degradation products were measured during the time course of

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the experiments. Sulfonamides were efficiently degraded by the biofilms during the first

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four weeks of the experiment. The abundance of ARG in biofilms sharply decreased

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after addition of high concentrations of sulfonamides, but this was not observed in the

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mesocosms treated with low concentrations of sulfonamides. Sulfonamide degradation

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was faster in polluted waters and at high concentrations of sulfonamide (and lower ARG

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abundances), suggesting that both degradation and ARG are two complementary

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resistance strategies employed by the microbial community. This study shows that

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microbial degradation of antibiotics is an efficient resistance mechanism coupled with

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the presence of ARG, and suggests that in situ degradation prevails at high

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concentrations of antibiotics whereas physiological adaptation by ARG spread would be

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more important under relatively lower concentrations of antibiotics.

Concentrations

of

ARG,

nutrients,

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sulfamethoxazole).

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and

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keywords: microbial sulfonamide degradation, sulfonamides, riverine, biofilms,

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antibiotic resistance, degradation products.

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ARG: antibiotic resistance genes

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ARB: antibiotic-resistant bacteria

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SMX: sulfamethoxazole

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SMZ: and sulfamethazine

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TP: transformation products

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chlorophyll a (Chla) and ash free dry mass (AFDM)

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MeOH: methanol

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qPCR: quantitative polymerase chain reaction

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DNA: Deoxyribonucleic acid

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TCA: Trichloroacetic acid

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N-Hydroxy-SMX : Hydroxy-sulfamethoxazole,

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desamino-SMX : desamino-sulfamethoxazole

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N-acetyl-SMX: acetyl-sulfamethoxazole

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4-nitro-SMX: nitro-sulfamethoxazole

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N-acetyl-SMZ: acetyl sulfamethazine

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NH4 : ammonium

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Abbreviations:

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ACCEPTED MANUSCRIPT 1. Introduction

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Antibiotics are widely used, and often abused, in human medicine and stockbreeding

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operations, for both infectious disease therapy and growth promotion. Antibiotics are

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ubiquitously found in rivers and there is a raising concern about the wide occurrence of

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antibiotic resistance bacteria (ARB) in natural waters (Ding and He, 2010; Martinez,

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2008; Stoll et al., 2012)⁠ . Antibiotic resistance in microbial communities can arise

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through: (1) microbial antibiotic degradation, that is, chemical transformation of the

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antibiotic by an existing enzyme of the cell, (2) by spontaneous mutation or acquisition

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of antibiotic resistance genes (ARG) from other cells by lateral gene transfer that yield

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alterated enzymes in a way that the antibiotic target site is not recognized by the

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antibiotic. (3) physical removal of intracellular antibiotic by activating membrane efflux

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pumps (Allen et al., 2010; Dever and Dermody, 2013; Pu et al., 2016a). No all

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microorganisms have all three mechanisms, and in fact, the relative importance of each

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mechanism and factors influencing their occurrence in natural systems is not well

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understood.

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Allen et al. (2010)⁠ states that some organisms and some environments harbour ARGs

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irrespective of human activity which leads to the hypothesis that native roles of

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resistance exist in natural communities. However, little is known about the selection of

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pressures on ARGs in environments where there is little or no human activity, and how

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the ARG mechanism is coupled with microbial degradation of the antibiotics.

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Sulfonamides such as sulfamethoxazole (SMX) and sulfamethazine (SMZ) were widely

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used in the past as antibiotics for human treatments. Nowadays, there are limited uses

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for sulfonamide antibiotics due to increasing bacterial resistance, potential for adverse

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effects, and the availability of more active antibiotics. Nevertheless, sulfonamides are

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ACCEPTED MANUSCRIPT frequently used in pigs and cattle for the treatment of bacterial diseases (Hruska and

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Franek, 2012)⁠ . Sulfonamides have been detected at relatively high concentrations in

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sewage treatment plant effluent, surface waters and ground water in diverse world

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regions (Auerbach et al., 2007; Pei et al., 2006; Pruden et al., 2007; Zhang et al.,

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2009)⁠ . Antimicrobial resistance to sulfonamides by propagation of ARG occurred

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quite rapidly after their first introduction (Davies and Davies, 2010)⁠ . Resistance to

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sulfonamides has been thought to be caused by the acquisition of the genes sul1, sul2,

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and sul3 (Randall, 2004; Sköld, 2000)⁠ . Sul1 and sul2 genes are widespread in the

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environment and they have been detected in lagoons (McKinney et al., 2010; Pei et al.,

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2007)⁠ , rivers and surface waters around the globe (Luo et al., 2010; Pruden et al.,

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2006; Stoll et al., 2012)⁠ . The microbial degradation of sulfonamides as a microbial

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resistance mechanism has received little attention, so far.

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Stream biofilms play an important role in controlling the aquatic biogeochemical cycle

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of nutrients and regulate exchange between the water and the hyporheic zone, and is the

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habitat of important microbial communities. Through mutating or acquiring degradative

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genes, bacteria can adapt and proliferate in the environment as a result of the selection

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pressures created by pollutants (van der Meer, 2006)⁠ . For example, biofilms grown in

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water from different river sites before and after the exposure to high levels of

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antibiotics, have shown that the continuous entrance of antibiotics in running waters

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cause significant structural and functional changes in attached microbial communities

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(Proia et al., 2013)⁠ . Degradation of synthetic chemicals is especially efficient in rivers

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(Gioia and Dachs, 2012)⁠ , and several studies have shown that stream biofilms play a

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primary role in attenuating riverine pollutants (Writer et al., 2011a, Battin et al., 2016;

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Romaní and Sabater, 1999; Writer et al., 2011b)⁠ . Besides abiotic transformation of

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sulfonamides by photooxidation (Rizzo et al., 2012, Batchu et al., 2014), microbial

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ACCEPTED MANUSCRIPT degradation of sulfonamides and some of the transformation products (TP) has been

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described in laboratory microcosms (Cetecioglu et al., 2016; Kassotaki et al., 2016;

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Liao et al., 2016; Su et al., 2016)⁠ and sludge (Yang et al., 2016) . Microbial

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degradation of sulfonamides may be a complementary mechanism to ARG alteration,

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attenuating the effects of antibiotics on bacteria in rivers. However, little is known on

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the coupling of microbial degradation with the propagation of ARG as antibiotic

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resistance mechanisms. A priori, the bacterial potential for sulfonamide degradation do

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not need to be coupled with the presence of ARG, since degradation could be driven by

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bacterial communities different than those containing ARG.

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The purpose of this study was to evaluate the relative importance of sulfonamide

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degradation versus the spread of sulfonamide-resistance genes in natural benthic

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biofilm communities as mechanisms of resistance to sulfonamides. Spread of ARG was

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studied quantifying the presence of sul1 and sul2 genes, whereas sulfonamide

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degradation was tracked quantifying sulfonamides concentrations and transformation

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products, since genes encodying for sulfonamide-degrading enzymes are not described

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in natural communities. Both mechanisms were studied under different concentrations

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of SMX and SMZ in mesocosmos experiments using upstream (pristine waters) and

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downstream (polluted waters) samples from the Llobregat river.

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2. Materials and methods

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2.1 Chemicals and materials

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All solvents used: MeOH, acetonitrile and water were purchased from Merck

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(Darmstadt, Germany). Ammonium acetate and formic acid were obtained from VWR

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(Darmstadt, Germany). SMX and SMZ were purchased from Sigma Aldrich (Steinheim, 6

ACCEPTED MANUSCRIPT Germany). The internal standards of SMZ and SMX were obtained from Toronto

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Research Chemicals (Toronto, Canada). The transformation products of SMX and SMZ

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such as N-acetyl-SMX, desamino-SMX, N-Hydroxy-SMX, 4-nitro-SMX and N-acetyl-

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SMZ (see structural formula in Figure S1) were purchased from Toronto Research

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Chemicals (Toronto, Canada). Formic acid (98%-100%) was ACS grade and purchased

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from Sigma Aldrich (Schnelldorf, Germany).Oasis HLB (60 mg), solid-phase extraction

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cartridges were purchased from Waters (Waters, Milford, MA).

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2. 2. Field Sampling

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The Llobregat river (~165 km long) flows from the Pyrenees mountains to the south of

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Barcelona (NE Iberian Peninsula), and drains a catchment area of 4948 km2 inhabited

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by a dense population of more than 3 million people, mostly living in the mid-lower

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part of the river. This part of the river has the biggest anthropogenic impacts by

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receiving discharges from urban and industrial wastewater treatment plants, sewer

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overflows, and diffuse pollution from agricultural fields. Antibiotic concentrations have

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been reported for the lower section of the Llobregat river, with SMX concentrations

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ranging between 213 ng/L and 907 ng/L. In contrast, the upper part of the river is

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pristine with a low population density and almost absent industrial, urban and

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agricultural inputs (Ginebreda et al., 2010; Masiá et al., 2015)⁠ .

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River water and cobbles were collected at six sites along the Llobregat from upstream

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(pristine waters) to downstream (polluted waters) in order to map out the concentration

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gradient of sulfonamide resistance genes along the river. A map of the sites is shown in

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Figure 1. Sampling was conducted in July 2012 and water temperature varied between

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17 ºC at “Les Fonts de Llobregat” (pristine waters) to 24.4 ºC at “St Boi” (polluted

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ACCEPTED MANUSCRIPT waters). At these two sites, a large number of cobbles and water were collected to be

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used in the mesocosm experiements.

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2. 2. Laboratory Experiment

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River water and cobbles were used to prepare mesocosms (3.5 L) in polypropylene

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bakers at two different concentrations (5 µg/L and 1µg/L) of sulfonamides antibiotics

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(SMZ and SMX) and for the two sites; fonts del Llobregat (pristine) and St Boi

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(polluted). Previously to performing these experiments, preliminary experiments

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performed on glass beakers had shown that glass adsorbs sulfonamides and therefore

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were deemed to be unsuitable for the experiment. The mesocosms were prepared

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ensuring that approximately the same area of cobbles was included in each

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polypropylene baker in order to obtain the same amount of benthic microbial biofilms in

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each treatment. A conceptual diagram of the experiment is shown in Figures 1 and S2.

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Samples from the bakers were collected at time 0 and every 15 days thereafter for

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approximately 8 weeks. Eight ‘Chemical mesocosms’ (2 mesocosms with the high

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concentration addition and 2 with the low concentrations addition for each one of the

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two sites) were used to collect duplicate samples for the analysis of sulfonamides and

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their transformation products (TP) in water. Twenty ‘biological mesocosms’ were

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prepared and used to collect cobbles for the analysis of the ARG and other ancillary

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measurements (5 at high concentration addition and 5 at low concentration addition of

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sulfonamides for each one of the two sites). Four of the bakers containing the biological

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mesocosm were sampled every 15 days, corresponding to the two concentration levels

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for the pristine and polluted sites, respectively. As the cobbles were removed for biofilm

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sampling, these bakers could not be used for the following sampling times (Figure S1).

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Chemical and biological mesocosms were treated in the same manner during the course

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ACCEPTED MANUSCRIPT of the experiment, and thus it is assumed that all the mesocosms with the same initial

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addition concentration of sulfonamides were similar in respect of water concentration of

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sulfonamides and biological activity. Chemical controls to monitor abiotic degradation

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were prepared with MilliQ-water with no cobbles by adding the antibiotics at the high

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concentration, while microbiological controls were prepared with river water and

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cobbles without the addition of antibiotics (Figure S1).

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Mesocosms were incubated in replicates for 60 days in 12L:12D photoperiods under

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white light (120µE/s m2). Samples of river water (100 mL) and river cobbles biofilm

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were collected from the ‘chemical mesocosms’ and analysed for chemical concentration

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of sulfonamides. Samples of cobbles were collected from the ‘biological mesocosms’ at

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each sampling time and analysed for chemical concentrations of sulfonamide resistance

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genes and ancillary parameters. Cobbles were scrapped with clean metal brushes and

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washed with MilliQ-water as described elsewhere (Bartrons et al., 2012). Two stones

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generated a total pooled volume of 100-150 ml and were used for chlorophyll a (Chla)

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and ash free dry mass (AFDM) analysis by filtering triplicate subsamples of 4 ml

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through ashed, pre-weighed Whatmann GF/F glass fiber filters. Filters were stored at -

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20 ºC until processing. Replicate samples for DNA analysis were collected from 5-10

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stones, generating a total volume of approximately 30 ml/stone. Each volume was

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centrifuged at 5000 rpm, the supernatant was discarded and the biofilm was collected

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into sterile 2 ml tubes and stored at -20ºC until DNA extraction. Cobble scraped

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surfaces were estimated using aluminum foil and following a weight-to-area

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relationship.

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2. 3. Analytical methods

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Water samples were extracted using an OASIS SPE cartridge (Waters). Forty µL of

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recovery standard d4-SMX at a concentration of 25 ppm was added to the sample prior

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to the SPE extraction. The water samples were passed through SPE cartridges at a

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relatively low flow rate (one drop every two seconds). Cartridges were eluted with 4 x

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2mL of methanol (MeOH) (Gros et al.,

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gentle stream of N2 to about 0.250 mL. The extract was then transferred to LC white

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vials and solvent exchanged to 50:50 MeOH: H2O. An aqueous and MeOH solution of

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d4-SMZ, used as internal standard for quantification, was spiked at a final concentration

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of 100 ng/L. The final volume of the extracts was 0.5 mL. Biofilm samples were freeze-

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dried- for 3-4 days to eliminate or reduce the undesirable effects of water. Samples

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were then homogenised by mixing and stirring of the biofilm. Forty µL of the recovery

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standard d4-SMX (25 mg/L) was added to 1 g of sample. Samples were sonicated with

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80:20 H2O:MeOH (3 x 10 mL x 15 min). The extract was solvent exchanged to a H2O

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solution containing 5% of MeOH. Seventy-five mL of this solution was extracted

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through a SPE cartridge following the same procedure used for water samples. Samples

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were analysed by liquid chromatography and mass spectrometry as reported elsewhere

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(Gros et al.,2009). The MRM transitions of the transformation products were as follows:

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N-acetyl-SMX: m/z 296/134 and m/z 296/65; desamino-SMX: m/z 237/141 and m/z

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237/77; N-hydroxy-SMX: m/z 270/172 and m/z 270/154, and 4-nitro-SMX: m/z

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282/138 and m/z 282/186. A reversed-phase Purospher Star RP-18 end-capped column

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(125 mm × 2.0 mm, particle size 5 µm) was sourced from Merck (Dramstadt,

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Germany). For MS/MS analyses, Symbiosis™ Pico was connected in series with a

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4000QTRAP hybrid triple quadrupole-linear ion trap mass spectrometer equipped with

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a Turbo Ion Spray source from Applied Biosystems-Sciex (Foster City, California,

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USA), where mass spectrometry detection is carried out. 4000QTrap is controlled by

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2009). The MeOH was evaporated under a

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ACCEPTED MANUSCRIPT means of the Analyst 1.4.2 Software from Applied Biosystems-Sciex (Foster City,

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California, USA) and a companion software appendix for controlling the Symbiosis™

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Pico from Spark Holland (Emmen, The Netherlands). Method quantification limits (MQLs)

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according to a signal-to-noise ratio (S/N) of 10 ranged from 0.9 to 9.5 ng/L and from 78to 95

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ng/L for the targeted compounds which were suitable for water and biofilm sample analysis,

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respectively. Recoveries for d4-SMX ranged from 60% to 85% for biofilm samples, and

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from 81 to 110% for water samples. Concentrations were recovery corrected. The

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concentration of sulfonamides in the chemical control remained constant through the

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duration of the experiment showing that there was no abiotic degradation or adsorption

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of the chemical in the mesocosm in absence of biofilm.

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2. 4. AFDM and Chla.

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Biomass of epilithic biofilms (on cobbles) was measured as ash free dry mass (AFDM).

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AFDM material contains both autotrophic and heterotrophic organisms, live and

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senescent organisms, and abiotic carbon. To estimate AFDM, biofilm samples collected

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on glass fiber filters were dried at 60 ºC until constant weight (c. 0.1 mg, Mettler Toledo

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MS204S/01 analytical balance), further combusted at 500 ºC for 5 h, dried in a

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desiccator and weighted. The AFDM was estimated as the mass difference between dry

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and combusted filters. Chla was determined in acetone extracts by spectrophotometry

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(UV-Visible spectrophotometer Varian Cary 300 Bio) following Steinman and Lamberti

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(1996), and corrected for phaeopigments by further acidification.

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2. 5. DNA extraction and quantification of sul genes by quantitative PCR (qPCR).

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DNA was extracted from 0.3 to 1.4 g of wet biofilm using FastDNA® SPIN Kit for Soil

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(Qbiogene, Carlsbad, CA) following the manufacturer instructions with the only

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modification of the use of the homogenizer. Cells were broken by vortexing the mixture

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ACCEPTED MANUSCRIPT of lysing matrix and sample using a MoBio vortex in 2 periods of 10 minutes instead of

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using the FastPrep instrument. Extracted DNA was quantified fluorometrically using the

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Quant-iT dsDNA Assay kit (Invitrogen, Paisley, UK) and Qubit fluorometer

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(Invitrogen). Subsamples of DNA were amplified by 16S PCR to check DNA quality.

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Quantification of sulfonamide resistance genes was performed by qPCR which

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quantified the gene copy numbers of the previously described primers sul1 and sul2 (Pei

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et al., 2006)⁠ . See Table S1 in Supplemental Information for details on the primers and

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the qPCR conditions used.

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environmentally cloned sul1 and sul2 sequences which were subsequently purified by

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QIAquick (QIAGEN), quantified by fluorescence (Qubit fluorometer, Invitrogen), and

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sequenced to check the identity of the target gene. Overall, average efficiencies of all

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quantification reactions ranged from 90% to 110%. Controls without templates resulted

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in undetectable values in all samples. The appropriate size of qPCR products and

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absence of unspecific PCR products were checked by agarose gel electrophoresis (data

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not shown). Gene abundances were normalized to cobble area (cm2) and to AFDM (i.e.

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organic matter) to allow comparison of results among samples.

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2. 6. Bacterial production. Bacterial secondary production was determined in the water

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column at the final time point after incorporation of 3H-leucine into protein using the

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method of Kirchman et al. (1985)⁠ with the modifications of Smith and Azam

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(1992)⁠ . Briefly, 1.2 ml of quadruplicate live and duplicate killed (5% TCA)

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subsamples were incubated with 3H-leucine (40 nmol l-1 final concentration) for about 3

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h at dark and at the in situ temperature. Incubations were stopped by addition of 120 ml

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of cold 50% TCA and then frozen (–20°C) until further processing by centrifugation

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and TCA rinsing.

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Standards were obtained after serial dilutions of

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3. Results and Discussion

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3. 1. Concentration of sulfonamides and their degradation products during the 60

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days laboratory experiment

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The concentrations of sulfonamides and their degradation products, AFDM, Chla,

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nutrients, the ARG abundances, and bacterial production are reported in Tables S2 and

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S3. The averages and standard deviations are reported in Table S4 and S5. Since the

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concentrations of sulfonamides in cobbles biofilm were always found below detection

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limit, only the concentrations of sulfonamides in water are reported.

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Figure 2 shows the concentration of sulfamethazine (SMZ) and sulfamethoxazhole

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(SMX) in both experiments (pristine and polluted sites) for the high (5000 ng/L) and

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low (1000 ng/L) sulfonamide additions from time zero to the end of the experiment (60

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days). The concentrations of both sulfonamides decreased rapidly during the first 30

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days reaching steady-state concentrations. The decrease in chemical concentrations

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during the first 30 days follows the first-order kinetics:

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  = − 


(1)

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Where [Sulf] time is the sulfonamide concentrations at initial time and time t. The half-life

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is given by:

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⁄ =

 

(2)

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ACCEPTED MANUSCRIPT Half-lives of SMZ and SMX are reported in Table 1. The half-lives showed that

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degradation was generally faster in the polluted waters with high concentration addition

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of SMZ and SMX, in which the half-lives ranged from 2.5 to 3.4 d, averaging 2.9 d.

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Conversely, the half-lives of SMZ and SMX in the same polluted water mesocosms but

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with low antibiotic addition ranged from 11d to 22d, averaging 17d, showing a slower

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degradation rate. The degradation of SMZ in the experiment with pristine waters and

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high concentration addition also showed a relatively short half-life (half-life averaged

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8.7 d with a standard deviation of 3.6d). Half-lives for SMX in the pristine low addition

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mesocosms could not be calculated because the regression of ln [SMX] versus exposure

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time were not statistically significant (p>0.01) due to variability between replicates.

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Therefore, it is concluded that irrespective of the initial status of the river water

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(polluted vs pristine), the degradation of sulfonamides was faster in the mesocosms with

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high concentration of sulfonamides.

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Figure 3 shows the transformation products (TPs) of sulfamethoxazole during the

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experiments

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sulfamethoxazole,

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sulfamethazine). The concentration of hydroxy-sulfametoxazole increased between time

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0 and 30 days and then decreased rapidly. The other TPs showed a variable time trend,

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with desamino-SMX and acetyl-SMZ generally showing increasing concentrations over

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time. The decrease/increase trends of SMX and SMZ and their TPs supports the

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evidence that degradation of SMX and SMZ occurred within the first 30 days of the

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experiments but it also shows that other chemical transformations were occurring.

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However, as not all the TPs were identified and quantified, it was not possible to close

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the mass balance.

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(Hydroxy-sulfamethoxazole,

acetyl-

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(acetyl

sulfamethazine

TPs

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desamino-sulfamethoxazole,

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ACCEPTED MANUSCRIPT 3. 2. ARG abundance in the Llobregat River and during the experiments

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Abundance of genes sul1 and sul2 along the Llobregat River are shown in Figure 1.

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Table S3 shows the mean and the standard deviation for each gene abundance at each

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sampling location; sul1 was always the most abundant gene (4.7*108- 6.9*107copies

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/AFDM) at all sites, followed by sul2 (9.0*107 -7.8*106copies /AFDM). The absolute

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abundance of sul1 and sul2 genes was the highest at St. Boi and, in general, increased

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from upstream to downstream. Therefore, in situ sul1 and sul2 were more abundant at

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the polluted site (St. Boi) and less abundant at the pristine site (Les Fonts de Llogregat).

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These two values were also the abundances of sul1 and sul2 in the mesocosm

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experiments at initial time.

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Figure 4 shows the ARG abundances during the course of the experiments. There is a

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significant difference in ARG abundances between high and low sulfonamide addition

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treatments; for the high addition, there is a decrease after time zero and then an increase

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of ARG abundances, whereas in the low concentration addition experiments there is no

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initial decrease of ARG abundances after time zero. For the mesocosms with low

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sulfonamide addition in pristine waters, there was a decrease at time 30 days for both

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sul1 and sul2, but this could be due to an outlier of the AFDM measurement at this time

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point (see also evolution of Chla concentrations over the experiment in Figure S3).

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Abundance of sul1 and sul2 increased during the last 30 days of the experiments

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irrespective of the concentrations and the origin of the river water.

342

The initial decrease of sul1 and sul2 abundance for the mesocosms with high

343

sulfonamide addition of SMX and SMZ, but not under lower concentrations of SMX

344

and SMZ, is consistent with a toxicity effect of sulfonamides to the bacterial groups

345

harboring sul1 and sul2 genes. The effect of a concentration of a chemical (SMX and

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ACCEPTED MANUSCRIPT SMZ in this work) on a biological response (sul1 or sul2 abundances) can be

347

represented with dose-response curves following a sigmoidal model with horizontal

348

asymptotes at both extremes of the dose range (Ritz et al. 2010). Figure 5 shows that

349

when plotting the abundance of ARG versus the concentration of SMX and SMZ

350

normalized by the abundance of the ARG genes, a typical sigmoid-type curve is

351

obtained. For the experiments with low addition of sulfonamides, there is no decrease in

352

ARG abundance due to the fact that the toxicity thresholds of SMX and SMZ are not

353

achieved. Conversely, if the concentrations of SMZ and SMZ are higher than a certain

354

threshold value, then there is a sharp decrease in the abundance of sul1 and sul2 genes

355

as observed during the experiments with high concentration addition of sulfonamides

356

(Figure 4 and 5). This type of sigmoid curve is obtained only if the concentration of

357

SMX+SMZ is normalized by the ARG abundances, indicating that these toxicological

358

thresholds are related to the levels of the ARG genes, and thus suggesting a coupling of

359

the change of sul1 and sul2 abundances and degradation of sulfonamide as antibiotic

360

resistance mechanisms. The concentration threshold of SMX+SMZ (normalized by sul1

361

and sul2 genes) at which a reduction of sul1 and sul2 genes is observed (Figure 5) was

362

surprisingly lower for the polluted waters than for the pristine waters. This could either

363

indicate a lack of acclimation to high sulfonamide concentrations of the microbial

364

community containing sul1 and sul2 genes from polluted waters, which had been

365

previously exposed to sulfonamides at concentrations 5 fold lower, or that antibiotic

366

resistance is primarily supported by degradation rather than by sul1 and sul2 genes at

367

high concentrations of sulfonamides (see below). The microbial community recovered

368

(increase in the genes abundance) after the first 30 days, which coincided with the end

369

of the biodegradation period. Therefore, in the first half of the experiment the bacterial

370

community was degrading the antibiotics; when the concentration of the antibiotic

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ACCEPTED MANUSCRIPT reached steady-state low concentrations, the abundance of ARG started to increase. The

372

residual of sulfonamides and some of the TP might induce potential antimicrobial

373

activity. In the low sulfonamide addition treatments, the sulfonamide concentrations

374

were below the toxicity threshold, with no decrease in ARG abundance although

375

degradation was still observed but with a slower kinetics than when sulfonamide

376

concentrations where higher (Table 1).

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3. 3. Degradation of antibiotic versus spread of ARG

379

The structure of the bacterial community may change after exposure to sulfonamides

380

(Liao et al., 2016; Proia et al., 2013)⁠ . The spread of sul genes by horizontal gene

381

transfer occurs rapidly in natural communities thanks to their linkage to class 1

382

integrons which are often embedded in DNA mobile elements such as plasmids and

383

transposons (Gillings et al., 2015; Sköld, 2000)⁠ . The two anti biotic resistant

384

mechanisms considered here such as the increase of ARG and sulfonamide degradation

385

pathways probably did not occur in the same bacterial groups. One possibility is that the

386

population of sul-harboring bacteria is in part overlapped by the bacterial community

387

with the capacity to degrade SMZ and SMX. Even though this is possible, it may not be

388

the generalized case, since in the mesocosms with polluted water and high addition of

389

sulfonamide (above the toxicity threshold), the degradation was faster (Table 1) with a

390

concurrent decrease of ARG. Therefore, when there was a decrease of ARG, the

391

bacterial degradation was faster suggesting that in polluted waters there was an

392

acclimated population, not found in pristine waters, able to resist sulfonamides by

393

degradation. This resistance mechanism seemed not only complementary to the spread

394

of ARG, but also more effective when the concentration of sulfonamides was highest. It

395

has been suggested that degradation of SMX is enhanced by co-metabolism of ammonia

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17

ACCEPTED MANUSCRIPT oxidizing bacteria which are not targeted by sul1 and sul2 primers (Kassotaki et al.,

397

2016; Pei et al., 2006)⁠ . Figure S4 shows the nutrient concentrations in the

398

experiments. In polluted waters, there was a quick decrease of ammonia and a steady

399

increase of nitrates and phosphate which would have been consistent with SMZ and

400

SMX degradation due to co-metabolism of nitrification. This was in contrast with the

401

NH4 time course in pristine waters where there is an initial increase in NH4

402

concentrations and then a decrease. For pristine waters, the degradation of sulfonamides

403

was slower than in polluted waters (Table 1).

404

The results from the bacterial production (Figure S5) show that there were differences

405

between waters (pristine vs polluted) but not between treatments which suggests that the

406

bacterial communities were recovered from antibiotics from the point of view of this

407

ecological function. For the last sampled time (60 days), the abundance of ARG in

408

controls were not significantly different than those treated with low or high

409

concentrations of sulfonamides for polluted waters (Figure 4). However, for pristine

410

waters, the ARG abundance after 60 days in the controls was similar to that observed in

411

mesocosms where sulfonamides were added at high concentrations, but were lower than

412

those observed in the mesocosms at low concentrations. These results suggest that the

413

potential ARG altering resistance mechanism may not have been changed in polluted

414

waters, and the community resistance relies on its capacity to degrade the antibiotics.

415

Conversely, in pristine waters, the degradation rates were always slower than in

416

polluted waters. The differences of ARG abundance after 60 days, could indicate that

417

the ARG altering mechanism may have been promoted when the bacterial community

418

was challenged with relatively low concentrations of antibiotics, but this did not occur

419

at high concentrations. This could be related to sulfonamide concentrations above the

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ACCEPTED MANUSCRIPT toxicity threshold value (Figure 5), which may have been triggered by changes in the

421

community favoring biodegradation of sulfonamides; however, this could not be

422

demonstrated in this study.

423

4. Conclusions.

424

The work performed here highlights that two of the main mechanisms of sulfonamide

425

resistance, that is, microbial sulfonamide degradation and spread of sulfonamide-

426

resistant genes

427

importance of each mechanism seems to be determined by in situ concentration of

428

sulfonamides. Whereas sulfonamide degradation might prevail at high concentrations of

429

sulfonamides,

430

concentrations. Further research should focus on the phylogenetic identification of the

431

main players of both mechanisms and the assessment of the relative role of the third

432

mechanisms of resistance not included in this study, the increase of membrane efflux

433

pumps (Pu et al., 2016b)⁠ .

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would

be more important

under moderate/low

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ARG spread

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are complementary used in natural ephilitic communities. Relative

5. Acknowledgements

436

This work has been supported by Marie Curie Actions—Intra-European Fellowships

437

(IEF), project number 275863 and by the Spanish Ministry of Economy and

438

Competitiveness project DARKNESS CGL2012-32747 to E.O.C. M.T. Pinnetta, G.

439

Caballero and M.J. Ojeda are acknowledged for sampling assistance, E.Martí for kindly

440

providing river art and M. Kuzmanovic for support with GIS.

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441 442

6. Authors’s contributions

19

ACCEPTED MANUSCRIPT MVC, RG, JD, EOC designed the experiment.

MVC and RG performed the

444

experiments and analyzed data. JA and SP analyzed the degradation products.

445

MVC, RG, and JD wrote the paper. All authors contributed to the discussion and final

446

version of the manuscript.

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ACCEPTED MANUSCRIPT 447

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ACCEPTED MANUSCRIPT Table 1. Half-lives (ranged, mean and standard deviation) for sulfamethazine (SMZ) and sulfamethoxazole (SMX) for the first 30 days of the experiment. Half lives (days) Pristine

Pristine

HIGH

LOW

HIGH

LOW

2.5-3

17-22

6.1-11

2.8±0.3

20±2.4

8.7±3.6

SMX

Not statistically 11-16

significant

3.1±0.4

14±3.8

(P>0.01)

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8.1-14

11±4.6

Not statistically significant (p>0.01)

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SMZ

Polluted

ACCEPTED MANUSCRIPT Figure 1. Map of the sampling locations that shows the abundance of genes sul1 and sul2 corrected by biofilm ash free dry mass (AFDM) at each site and conceptual

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diagram of the experiment set up.

ACCEPTED MANUSCRIPT Figure 2. Concentrations of sulfamethazine and sulfamethoxazole (ng/L) during the curse of the experiment . LOW, HIGH and CTRL refer to the concentrations for the experiments with high and low addition of sulfonamide and the abiotic controls,

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respectively.

ACCEPTED MANUSCRIPT Figure 3. Concentrations of sulfonamides transformation products (Hydroxysulfamethoxazole (HYDORXY), desamino-sulfamethoxazole (DESAMINO), acetylsulfamethoxazole

(ACETYL),

acetyl

sulfamethazine

(ACETYL-SMZ),

nitro-

sulfamethoxazole (NITRO-SMX)) in ng/L during the duration of the experiment. See

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structural formula in Figure S1.

ACCEPTED MANUSCRIPT

Figure 4. Abundance of the ARG sul1 and sul2 during the curse of the experiment. LOW, HIGH and CTRL refer to the concentrations for the experiments with high and low addition of sulfonamide and the biotic controls without sulfonamide addition, re-

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spectively.

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Figure 5. The logarithm of the sul1 or sul2 genes abundances versus the logarithm of the concentration of total sulfonamides normalized by the genes abundance at any given

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time.

ACCEPTED MANUSCRIPT Highlights

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Microbial degradation dominates under high concentrations of sulfonamides. Spread of resistance genes dominates under moderate concentrations of sulfonamides. Pristine and polluted sites have the capacity to decrease sulfonamide concentrations

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• • •