Article pubs.acs.org/est
Fluoroquinolones and qnr Genes in Sediment, Water, Soil, and Human Fecal Flora in an Environment Polluted by Manufacturing Discharges Carolin Rutgersson,† Jerker Fick,‡ Nachiket Marathe,†,§ Erik Kristiansson,∥ Anders Janzon,† Martin Angelin,⊥ Anders Johansson,# Yogesh Shouche,§ Carl-Fredrik Flach,† and D. G. Joakim Larsson*,† †
Institute of Biomedicine, Department of Infectious Diseases, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden ‡ Department of Chemistry, Umeå University, Umeå, Sweden § Microbial Culture Collection, National Centre for Cell Science, Pune, India ∥ Department of Mathematical Statistics, Chalmers University of Technology, Gothenburg, Sweden ⊥ Infectious Diseases, Department of Clinical Microbiology, Umeå University, Umeå, Sweden # The Laboratory for Molecular Infection Medicine Sweden (MIMS), Department of Clinical Microbiology, Umeå University, Umeå, Sweden S Supporting Information *
ABSTRACT: There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.
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INTRODUCTION
We have previously found exceptionally high concentrations of fluoroquinolone antibiotics (FQs) in the final effluent from a treatment plant, PETL, (Patancheru Enviro Tech Limited), receiving process water from bulk drug production near Hyderabad, India. Highly multiresistant bacteria are very common within the treatment plant.15,16 The FQs has also contaminated river sediment, surface, ground, and well water in the area.1,3,17 Shotgun metagenome pyrosequencing accordingly showed high levels of quinolone resistance genes (qnr), in the heavily FQ-polluted river sediments.17 The qnr-encoded proteins typically confer resistance up to low mg/L levels of FQ.18,19 They can also facilitate the emergence of additional
Environmental pollution with active pharmaceutical ingredients from drug manufacturing has been documented in recent years in different parts of the world.1−4 The overall consequences of environmental discharge of pharmaceutical waste are not yet completely understood, but it can undoubtedly have effects on local wildlife.1,5−8 In the case of antibiotics, a growing body of evidence supports the hypothesis that not only an increased use of antibiotics, but also an increased exposure of microbial communities outside of our bodies, contributes to the recruitment and spread of antibiotic resistance genes among human pathogens.9,10 If the concentrations of antibiotics in the environment reach sufficiently high levels, environmental bacteria carrying resistance genes are expected to increase in abundance, leading to more opportunities for transfer of their genes to bacteria that are able to colonize the human body and possibly cause disease.11−14 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 7825
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participants were women. Institutional ethical clearance (IEC, National Centre for Cell Science, Pune) was obtained for the collection of fecal samples from Indian volunteers while sampling of fecal matter from the Swedish study subjects was approved by the regional ethical review board in Umeå (2011357-32M). DNA Extraction. DNA from composite soil samples and river sediments was extracted using the MOBIO PowerSoil DNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA) as previously described.17 The alternative lysis method was performed following the manufacturer’s instructions; samples and solution C1 were incubated at 70 °C for 10 min, with brief vortexing after 5 min, to ensure complete homogenization and cell lysis. The well water remaining after the sample sent for chemical analysis had been removed (approximately four hundred milliliters) was filtered through 0.22 μm Millipore filters (Millipore Corporation, Billerica, MA) and DNA was extracted from the filters using PowerWater DNA Isolation Kit (Mo Bio Laboratories Inc.). DNA from fecal samples was extracted with the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. All DNA concentrations were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the samples were kept at −20 °C until analysis. Polymerase Chain Reaction. Primer sequences for 16S rDNA and qnrA were adopted from previous studies.24,25 For the remaining qnr genes, primers were designed with Primer3Plus software26 and compared to the Genbank database using a nucleotide BLAST search to ensure specificity. All primers were synthesized at Eurofins MWG Operon (Ebersberg, Germany) and PCR followed by gel electrophoresis confirmed that amplicons of the anticipated length were generated for each respective primer pair. Primer sequences are presented as SI Table 2. Serial dilutions of recombinant pCR2.1 TOPO vectors with the respective target gene inserted (Eurofins MWG Operon) were used as standards. Since cloning of qnrB was unsuccessful, serial dilutions of a PCR product purified from agarose gel served as standard curve for this gene. Five or 20 nanograms of DNA, from environmental samples and feces respectively, was mixed with primers (0.3 μM), Power SYBR Green Mastermix (Applied Biosystems, Warrington, UK) in a final reaction volume of 10 μL. Each sample was run in triplicate in a 384-well plate format using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Singapore). On each 384-well plate, standards ranging over at least 5 orders of magnitude were included as well as template controls containing only Mastermix and water to check for potential contamination. The PCR program contained an initial 10 min denaturation step at 95 °C, followed by 40 amplification cycles consisting of an initial denaturation at 95 °C for 30 s followed by 30 s at 60 °C and 30 s at 72 °C. Analysis of melting curves ranging from 60 °C-95 °C was performed for all samples. The SDS 2.4 software was used for analysis using default settings. The median threshold cycle (Ct) output values for the technical replicates were normalized with the median Ct value from the 16S rDNA gene of the same sample to enable a relative quantification of each gene. A subset of amplicons (including representatives for each qnr gene) was Sanger sequenced and the resulting sequences were examined and confirmed by nucleotide BLAST (http://blast.ncbi.nlm.nih. gov/Blast.cgi) searches.
resistance mechanisms by allowing the bacteria to survive under a moderately elevated FQ-selection pressure.20−22 In this study, we address the hypothesis that environmental FQ-contamination has caused an enrichment of qnr in well water and soil as well as in human fecal flora of local villagers. We have thus expanded the sampling area into presumably unaffected areas. We have used quantitative PCR (qPCR) as a targeted and considerably more sensitive method than shotgun pyrosequencing to analyze qnr. As well water is frequently used for irrigation we also investigated whether FQ antibiotics have contaminated farmland. To put results in broader context, qnr prevalence in fecal samples was compared between Indian residents and students from Sweden using the same analytical protocol. In an international perspective, Swedes have low quinolone consumption and correspondingly modest quinolone resistance levels.23
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MATERIALS AND METHODS Environmental Sampling. Information about both Indian and Swedish river sediment sampling points and their coordinates has previously been published.17 In short, Indian sediments from six sites (4−6 subsamples from each site), two upstream from PETL, one just downstream from the PETL effluent discharge site and three sites further downstream (up to 17 km downstream) were sampled on the 28th of March, 2008. Sediments up- and downstream from a municipal Swedish sewage treatment plant with no input from pharmaceutical manufacturers were sampled on the third of May 2009. Five samples were taken 5−100 m upstream and six samples 25−250 m downstream from the plant. All sediments were sampled right at the water’s edge, from surface down to approximately 10 cm depth. Indian well water and soil samples were collected on the 12th of January and on the 13th and 14th of June 2011, respectively, from 15 villages, located within the Patancheru industrial area as well as villages farther away (Supporting Information (SI) Figure 1). For each well water and soil sample, the closest distance to waterways with previously documented FQ contamination3 was determined using Google Earth. Distances, sampling dates and coordinates for soil and well water samples are shown in SI Table 1. Approximately 500 mL of well water and a composite soil sample, made up of six spoonfuls (roughly 100 mL in total) of soil taken approximately 1 m apart and a couple of centimeters below surface, were sampled at each village and kept in separate sterile plastic bottles. Upon arrival to the laboratory, to improve sample homogeneity, all soil and well water samples were individually blended and about 15 mL of soil and 100 mL of well water from each village were put into individual sterile tubes and frozen until chemical analysis. Fecal Sampling. All participants were asked to fill in a form including questions on medicine use during the previous six months prior to sampling and anyone reporting on taking antibiotics was excluded from the study. Indian fecal samples were collected from a total of 11 villages. Samples from villages I−VIII (n = 86) were collected on the 16th and 18th of July 2010, and samples from villages IX-XIII (n = 68) were collected on the 18th of April 2011. Two of the villages (Gandigudem and Rai Bollaram Thanda) were sampled at both time points, but no individual included in this study was sampled twice. The age of the participants ranged from 4 to 75 years, and 53% of the participants were female. The Swedish fecal samples were collected between April 2010 and May 2012 (n = 37). The age range of the participants was 22−34 years, and 78% of 7826
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Analysis of Fluoroquinolones in Well Water and Soil. The methodology used for the chemical analysis, including the setting of key parameters, SRM transitions, absolute recoveries etc., has been described previously.27 In short, samples were extracted off-line and analyzed using a triple stage quadrupole MS/MS TSQ Quantum Ultra EMR (Thermo Fisher Scientific, San Jose, CA) coupled with an Accela LC pump (Thermo Fisher Scientific, San Jose, CA) and a PAL HTC autosampler (CTC Analytics AG, Zwingen, Switzerland). Detailed information on chemicals, sample preparation and instrumental analysis for the determination of fluoroquinolone concentration in well water and soil is presented in the online SI. Statistics. The well water samples were classified as polluted or nonpolluted based on whether FQs were detected or below the detection limit, respectively. Differences in qnr prevalence between FQ-polluted and nonpolluted well water were assessed using Fisher’s exact test. All nondetects were given the value corresponding to the lowest qnr/16S rDNA ratio divided by two. No formal statistical analysis on qnr in soil was performed due to few hits. Regression analyses of qnr levels, taking into account the absolute level of FQ detected in the samples did not provide more clear results (data not shown). For analysis of fecal samples, Indian villages were separated into two groups depending on whether FQs were detected in the corresponding well water or soil at any time point. The differences in the abundance of qnrB and qnrS genes in fecal samples from people living in FQ-polluted or FQ-free villages were analyzed using a hierarchical linear model with an effect parameter for each village. The number of fecal samples with detectable levels of the qnrD gene was markedly lower than for the qnrS and qnrB genes, and the data for this gene was therefore stratified into discrete groups of “detected” and “nondetected” and analyzed using a logistic regression model. For both regression models, sex and age were included as covariates. An increase in qnr abundance in polluted villages was tested using a one-sided F-test for the linear model and the one-sided Wald test for the logistic regression model. Indian fecal samples from 2010 and 2011 were analyzed both individually and together. The difference in qnr prevalence between individuals from India and Sweden was assessed using Fisher’s exact test. Tests resulting in a p-value less than 0.05 were considered statistically significant.
Figure 1. Total fluoroquinolone (FQ) concentrations in well water and soil (average from January and June samplings) related to the distance to waterways with previously documented FQ contamination. Nondetects were set at half the detection limit for the corresponding FQ.
six well water sites in January and three in June. In addition, qnrD and qnrB were found in three and one village, respectively, in January water samples. Neither qnrA nor qnrC were detected in any samples. No difference in qnr prevalence was seen in well water from FQ-contaminated villages compared to villages where no FQs were detected; p = 1.00 and p = 0.23 for January and June sampling, respectively, (Figure 2a). For soil, only qnrVC was found, which was detected in two of the June samples (Figure 2b). Fluoroquinolones and qnr in River Sediment. The concentration of FQs in the river sediments have been published previously,17 and ranged between 5.2 and 915.3 μg/g organic matter (0.45−54 μg/g dry weight) for Indian sites, whereas no FQs were detected in Swedish river sediments. Data from the qPCR analysis showed that qnr genes were highly prevalent and abundant in Indian sediments compared with the Swedish samples (Figure 2c). The qnrB, qnrS, and qnrVC genes were detected in >94% of Indian sediment samples and qnrVC was also the most abundant gene at all sites. The qnrD and qnrA genes were somewhat less prevalent and found in >73% of Indian samples but in similar abundances as qnrB and qnrS. Although the primers for qnrC were shown to be functional using recombinant bacteria, this gene was not detected in any sediment sample. In the Swedish sediments, qnrS and qnrVC were detected in one sample, taken a few meters downstream from the effluent discharge site. The abundances of these genes were more than 103 and 105 times lower in the Swedish sediment samples than in the Indian river sediment (Figure 2c). qnr in Fecal Samples. The abundance of qnr in fecal samples was also generally much lower in the Swedish fecal samples compared to Indian fecal samples (Figure 3). There was no significant enrichment of qnr in the fecal samples from Indian inhabitants living in villages with local environmental pollution with FQs compared with nonpolluted villages (p = 1.00, 1.00, and 0.96 for qnrB, qnrS and qnrD, respectively). There was a general trend toward lower qnr levels in samples from 2011, but even when the 2010 and 2011 data were analyzed separately, there was no indication that feces from people living in polluted villages had higher levels of qnr than samples from people living in nonpolluted villages (p = 0.93,
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RESULTS Fluoroquinolones and qnr in Well Water and Soil. FQs were detected in well water or soil samples in all villages located up to three kilometers from previously documented quinolonepolluted waterways.3 No FQs were found in sites farther away from the contaminated rivers and lakes (Figure 1). In January water samples, the total FQ concentration was up to 915 ng/L, which decreased to 399 ng/L in June samples. For soil samples, the January total FQ concentration was up to 179 ng/g organic matter (44 ng/g dry weight). In June, FQs were found in only two soils as opposed to seven in January, but the total FQ concentration in these samples were more than 30 times higher than the levels detected in January. Concentrations of the individual FQ antibiotics detected in well water and soil samples are shown in SI Table 1. The proportion of villages where one or more qnr genes were detected in well water varied from 73% (8 of 11 villages; 4 samples were lost) in the January samples, to 20% (3 of 15 villages) in June. The most commonly detected and most abundant qnr gene in well water was qnrVC, which was found in 7827
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Figure 3. Village average of the total copy number of qnr/16S rDNA in human fecal samples. Error bars represent SEM and the number of fecal samples analyzed is given in the parentheses. The total FQ concentration in well water and soil (average from January and June samplings) are given for the corresponding Indian villages. Nondetects were substituted with values corresponding to half the detection limit for the FQs.
with Sweden. The remaining qnr genes were only occasionally detected in Indian fecal samples (three hits for qnrA and two hits for qnrVC), while none of these genes were found in Swedish fecal samples. The qnrC gene was not detected in any of the samples. Two or more qnr genes were detected in 117 of 154 (76%) of Indian fecal samples. Most commonly, qnrS and qnrB coexisted; in 89% of samples with qnrS, qnrB was also detected, and in 27% of the samples with qnrS, both qnrB and qnrD were found (Figure 4c). A single Swedish fecal sample contained more than one qnr gene (qnrB, qnrD and qnrS). Complete qnr data for individual sediment and fecal samples are available in SI Tables 3 and 4, respectively.
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Figure 2. Individual copy number of qnr/16S rDNA in relation to the concentration of FQs in the same samples. The different qnr alleles are denoted with respective capital letter. In samples without detected FQ, values are set to half the detection limit for each corresponding substance. Graphs represent samples from (a) well water, (b) farmland soil, (c) river sediments sampled up- and downstream from wastewater treatment plants. Data for Swedish sediment samples are shown on the y-axis because no FQs were detected in these samples.
DISCUSSION To our best knowledge, this is the first study exploring the link between local environmental antibiotic contamination and the gut flora of human residents, with the underlying hypothesis that environmental pollution plays a role in the spread of antibiotic resistance genes to human pathogens. No such link could be established in this study. However, it needs to be stressed that we found a widespread occurrence of the investigated resistance genes in residents from all investigated Indian villages, something that would easily mask a small flow of resistance genes from the environment. Furthermore, we believe this is the first study documenting antibiotic contamination of farmland as a consequence of industrial discharges. The high levels of FQs found in contaminated river sediment correlated with high abundances of qnr, whereas the lower levels found in well water and soil did not. Such field observations are valuable in the quest for establishing selective concentration of antibiotics in different environments, with the ultimate intent to motivate and direct management of antibiotic pollution. The total FQ concentration detected in well water in the present study (up to 915 ng/L) is approximately 10-fold higher than what is generally found in treated sewage effluents in Sweden28 and markedly exceeds the very low ng/L levels occasionally found in drinking water.29 Well water from six of the villages included in the present study has been analyzed
0.49, 0.67 and p = 0.89, 1.00, 0.64 for qnrB, qnrS, and qnrD in 2010 and 2011, respectively). In Indian fecal samples (n=154), qnrS and qnrB were the most abundant and prevalent qnr genes followed by qnrD, found in 85, 83 and 26% of samples respectively (Figure 4a-b). The same genes were found in the Swedish fecal samples (n = 37) but were considerably less common (qnrS 11%; qnrB 8%; qnrD 11%). The proportion of individuals with at least one detected qnr was significantly higher in Indian (91.6%) compared to Swedish individuals (24.3%, p < 10−15). The differences in qnrS and qnrB abundance in fecal samples between India and Sweden were approximately 2 orders of magnitude, although a few samples from Swedish study subjects had similar levels of qnr as observed in Indian samples. For qnrD, the variation between countries was less pronounced. In some Indian villages the gene was not found in any study subjects, however, when qnrD was detected it was generally approximately 10-fold more abundant in India as compared 7828
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of FQs in 2011 in villages upstream from the plant, (Sultanpur, Gandigudem, and Kazipally), where the groundwater contamination is more likely to have been caused by discharges from isolated industries or illegal dumping.3,31 In soil samples, ciprofloxacin was found in concentrations up to 1900 ng/g dry weight (7200 ng/g organic matter), which is much less than the levels detected in Indian river sediments (up to 54 μg/g dry weight) but only slightly higher than the concentrations previously found in Swiss soils, where up to 400 ng ciprofloxacin/g dry matter was detected 21 months after sludge application.32 However, in general the FQ concentration in soils was considerably lower; with two exceptions all samples contained less than 20 ng/g dry weight for individual FQs, which is similar to the levels previously detected in vegetable farmland and agricultural soil irrigated with wastewater.33−35 It is known that plants may take up drugs, including FQs, from soil36,37 suggesting a possible direct human exposure route of FQs via ingestion. However, the most recognized human health risks coupled with environmental antibiotic exposure are not direct effects but rather the risks for promoting the development and dissemination of resistance in bacterial pathogens.38 The FQ concentrations in well water and soil showed considerable variability between the two sampling occasions. The yearly monsoon period had just begun a week prior to the second sampling occasion, an event which has a vast impact on the area’s topography and land-fresh water distribution and may have thus affected the FQ concentrations in the environmental samples. Nevertheless, for both water and soil, the same two villages had the highest concentrations of FQs in both the January and June samples, which indicate that the (composite) grab sampling methodology applied is valid. In Indian river sediments, qnr were abundant compared to Swedish samples which is in concordance with previous studies.17 This is likely the result of a selection pressure exerted by the high concentrations of FQs, also in sediments sampled upstream from PETL where local authorities have reported on illegal dumping of industrial waste.31 In contrast, we did not observe an enrichment of qnr genes in soil or well water contaminated with, in general, relatively low levels of FQs. This suggests that the FQs, at the levels detected, do not exert a major selection pressure to select for bacteria expressing the investigated qnr. This may partly be explained by the FQs tendency to readily adsorb to particles and form complexes with cations in, for example, soil39,40 which could cause limited bioavailability of these substances.33 The FQ levels detected in well water samples (up to 915 ng/L) is also well below the nonspecies-related clinical resistance breakpoint for most FQs at 1 mg/L (www.eucast.org). However, competition experiments have shown an enrichment of certain resistant mutants in as little as 100 ng/L ciprofloxacin, which is 230 times below the minimum inhibitory concentration of the wild type strain.41 Additionally, quinolones are able to provoke the SOS response in bacteria at low μg/L concentrations which in turn may promote horizontal gene transfer.42 The lack of observed enrichment of qnr does therefore not exclude a selective effect on other resistance factors. The composition of different qnr alleles varied somewhat between different sample matrices. In environmental samples, qnrVC was most prevalent and for Indian sediment, qnrVC was the most abundant qnr gene at all sites. In our previous metagenome sequencing study of the same river sediments qnrD was the most commonly detected qnr.17 This gene was detected on a novel plasmid which subsequently has been
Figure 4. (a) Average abundance of qnr gene copy number per 16S rDNA in fecal samples. Error bars represent SEM. (b) Average prevalence of qnr genes in fecal samples. Error bars represent the standard error of the estimated prevalence and were calculated under the assumption of Bernoulli-distributed data. (c) Graphical representation of the co-occurrence of qnr genes detected in individual fecal samples from Indian villagers.
previously.3 In comparison with the previously detected levels in well water samples from March 2008, up to 14 000 ng/L of ciprofloxacin alone, the concentrations of FQs were lower in the water samples from 2011. Although there might be seasonal influences on groundwater status that could have affected the concentrations in the wells, the findings may also be explained by decreased local discharges of industrial waste. The Andhra Pradesh Contamination Control Board reports that an 18 km pipeline was connected to the PETL outlet in July 2009, and since then a gradually increasing portion of treated effluent from the plant has been transported to another treatment plant in Amberpet, reaching 100% of PETL discharges in March 2010. 30 While this is consistent with the lower FQ concentrations in 2011 in the three villages downstream from the PETL discharge site (Baithole, Pocharam and Ganapahigudem), it is probably not the direct cause for the lower levels 7829
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identified in human pathogens.43,44 However, it should be noted that, due to the requirement of larger amounts of high quality DNA for metagenome sequencing, sediment DNA was amplified by Repli-G in our previous study.17 It is known that whole genome amplification approaches, including Repli-G, can lead to a biased amplification of DNA45,46 indicating an overestimation of the relative number of qnrD copies in river sediments in our previous study compared with the results of the qPCR analyses of unamplified DNA. We found no support for the hypothesis of an enrichment of qnr in Indian fecal samples from villagers from FQ-polluted sites compared to people from villages where no FQs were detected. However, we would like to stress that the lack of correlation does not prove that transfer of qnr from environmental bacteria to human gut has not occurred at an earlier time point. As the transfer of a resistance gene from the environment to the human microflora may be a rare or even one-time event, any spread beyond the first human recipient may be mainly governed by person-to-person transmission rather than additional transfer of the same gene from environmental bacteria.11 In such a case, it would be exceedingly difficult to identify the origin of a resistance factor without obtaining a detailed time series from multiple regions, preferably also covering the time before and just after the emergence and spread of the resistance gene in the human flora. It is important to acknowledge that we have only studied one snapshot in time, while resistant bacteria can rapidly spread with individuals over long distances47,48 why the generally high prevalence of qnr in Indian fecal sample may mask an earlier difference between villages. A possible explanation behind the significant difference in fecal qnr prevalence between India and Sweden is that the broad usage and availability of FQs has provided a selective advantage for microbes carrying quinolone resistance determinants in the Indian community compared with Sweden, where the use of FQs is more restricted.49 The same three qnr alleles (qnrB, qnrS and qnrD) were most commonly detected in both Indian and Swedish fecal samples. Data from a recent study, investigating qnrB and qnrS prevalence in fecal samples from healthy Dutch people before and after international travel to e.g. the Indian subcontinent, is well in line with the results presented in this study for Swedish and Indian individuals, respectively.50 The finding of qnrD in both well water and fecal samples indicates that this allele is worth further examination in future studies, for example, to pursue its genetic contexts and likely origin, also in relation to the previously described qnrD plasmid.17 Such information could provide clues to questions relating to the recruitment of mobile resistance genes to human pathogens. As raw human sewage including fecal matter is added at PETL with the intent to maintain biological treatment efficiency1 it is possible that residual fecal bacteria escaping from the treatment plant could be a major source of qnr genes found in sediment downstream from the treatment plant. However, qnrVC was detected in only two Indian fecal samples but was highly prevalent in sediment. Thus, human fecal contamination is unlikely to be the source of qnrVC in this environment, but it is rather is the result of environmental selection. We acknowledge that to firmly establish causality between antibiotic exposure and enrichment of resistance genes in the environment, controlled laboratory studies are also required to define concentration−response relationships.
Nevertheless, to avoid pitfalls associated with oversimplified experimental systems, it is also important to study this course of events in the more complex reality, as we have done here.
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ASSOCIATED CONTENT
S Supporting Information *
This article has additional Supporting Information on analytical procedures, location of sampling sites as well as detailed information on FQ concentration and qnr data for individual samples. This material is available free of charge via the Internet at http://pubs.acs.org/.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +46-31-342 46 25; e-mail: joakim.larsson@fysiologi. gu.se. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by FORMAS, the Swedish Research Council VR, MISTRA, the Research School for Environment and Health in Gothenburg, the Adlerbertska Research Foundation and the Swedish International Development Cooperation Agency (SIDA). N.M. is grateful to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for fellowship. We thank Gamana and Mr. Anil Dayakar at for the sampling of soil and assistance with fecal sampling in India; Helena Palmgren, Birgitta Evengård, Margareta Granlund, Joakim Forsell, Maria Casserdahl, and Helén Edebro in their work with organizing the fecal sample collection in Sweden; Lina Gunnarsson for appreciated help with the qPCR analyses and the Genomics Core Facility platform at the Sahlgrenska Academy, University of Gothenburg, for providing qPCR and sequencing instruments and Professor Edward RB Moore for valuable discussions.
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REFERENCES
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