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Dec 8, 2010 - Department of Biology, Point Loma Nazarene University, 3900. Lomaland Drive, San Diego, California 92106, United States. Received August...
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Environ. Sci. Technol. 2011, 45, 447–454

Broad Dissemination of Plasmid-Mediated Quinolone Resistance Genes in Sediments of Two Urban Coastal Wetlands DAVID E. CUMMINGS,* KARISA F. ARCHER,† DAVID J. ARRIOLA,‡ PIETER A. BAKER, K. GRACE FAUCETT, JONATHAN B. LAROYA, KELLY L. PFEIL, CODY R. RYAN,† KELSEY R. U. RYAN,† AND DOUGLAS E. ZUILL Department of Biology, Point Loma Nazarene University, 3900 Lomaland Drive, San Diego, California 92106, United States

Received August 26, 2010. Revised manuscript received November 10, 2010. Accepted November 23, 2010.

Contamination of soil and water with antibiotic-resistant bacteria may create reservoirs of antibiotic resistance genes that have the potential to negatively impact future public health through horizontal gene transfer. The plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrS, qepA, and aac(6′)Ib-cr were detected by PCR amplification of metagenomic DNA from surface sediments of the Tijuana River Estuary, a sewage-impacted coastal wetland along the U.S.-Mexico border; sediments of Famosa Slough, a nearby urban wetland that is largely unaffected by sewage, contained only qnrB, qnrS, and qepA. The number of PCR-positive sites and replicates increased in both wetlands after rainfall. Real-time quantitative PCR revealed a significant increase (p < 0.0005) in qnrA abundance (copies per gram sediment or per 16S rDNA copy) in Tijuana River Estuary sediments immediately following rainfall, but no significant change was measured at Famosa Slough (p > 0.1). Nucleotide sequences of cloned qnrA amplicons were all affiliated with qnrA genes found on plasmids of clinical isolates with one exception that was most similar to the chromosomal qnrA gene found in Shewanella algae. Our results suggest that urban wetlands may become reservoirs of antibiotic resistance genes, particularly where wastewater is improperly managed.

Introduction Global public health is facing an epidemic of bacterial infections with reduced susceptibility to front-line clinical antibiotics such as β-lactams, tetracyclines, and (fluoro)quinolones (1). Undoubtedly, the simple use of antibiotics to prevent and cure infections in humans plays an important part in this increased resistance (e.g. ref 2). Many also argue, though, that the use of antibiotics in applications other than human bacterial infections (e.g., growth promotion in food animals, human viral illnesses, etc.) may be contributing to the problem (3, 4). There is lately a growing concern that the * Corresponding author phone: (619)849-2642; fax: (619)849-2598; e-mail: [email protected]. † Current address: Loma Linda University, School of Medicine, 11175 Campus St., Loma Linda, CA 92350. ‡ Current address: Duke University, School of Medicine, Durham, NC 27710. 10.1021/es1029206

 2011 American Chemical Society

Published on Web 12/08/2010

release of antibiotics, antibiotic-resistant bacteria, and antibiotic resistance genes into natural environments may be further exacerbating problems in the clinical setting (5, 6). Previous work on the fate of antibiotic-resistant bacteria and their resistance genes (AR pollutants) in the natural environment has focused largely on the impacts of animal production facilities (e.g. refs 7-9), which employ antibiotics for disease prevention and treatment as well as growth promotion. These operations generate large quantities of AR pollutants (10, 11), which, once released into the environment, become vulnerable to relocation by wind and water to agricultural soils, surface and groundwater, and human settlements (6, 12-14). A largely overlooked mechanism of release of these entities into the natural environment is by way of improperly disposed or accidentally spilled human wastewater. Human sewage has been documented as a reservoir of AR genes (15-17), many of which are encoded on transmissible plasmids, and some release of these genes into the natural environment via sewage has been reported (18, 19). Given the relative ease by which prokaryotes exchange genetic information, even in the absence of an obvious selective pressure (20, 21), the release of plasmid-bound AR genes into natural environments may pose a real public and environmental health risk. (Fluoro)quinolones (e.g., ciprofloxacin, levofloxacin), among the most commonly prescribed antimicrobials in the world, are broad-spectrum drugs that appear to act by inhibiting the normal functions of bacterial type II topoisomerases. Resistance to (fluoro)quinolones is readily accomplished with mutations in key regions of the topoisomerase genes, termed quinolone resistance-determining regions (22, 23). However, plasmid-mediated quinolone resistance (PMQR) is on the rise, especially among the Enterobacteriaceae (24, 25), threatening to accelerate the spread of resistance through horizontal transfer. There are currently three known classes of PMQR determinants: (1) Qnr peptides, which bind to and protect topisomerases from inhibition by (fluoro)quinolones (26, 27 www.lahey.org/ qnrStudies), (2) an acetyltransferase, Aac(6′)-Ib-cr, that inactivates the drug (28), and (3) efflux pumps (29-31), including QepA, which is specific for (fluoro)quinolones (32). We hypothesized that sewage-impacted wetlands would contain antibiotic resistance genes in greater quantities than protected wetlands and that they would persist beyond the time of original deposition. Here we report on the widespread distribution of PMQR genes in surface sediments of a sewageimpacted coastal wetland as well as a nearby, sewage-free urban wetland. To our knowledge, this is the first report of PMQR genes in the natural environment.

Experimental Section Study Sites. The Mediterranean climate of the southwestern United States and northwestern Mexico is characterized by warm, dry summers and cool, rainy winters. The Tijuana River Estuary (TRE) (N32° 33′ W117° 07′) (Figure 1 and Figure S1) is a 10-km2 National Estuarine Research Reserve (NERR), National Wildlife Refuge (NWR), and Wetland of International Importance (Ramsar Site #1452) on the US-Mexico border in Imperial Beach, California (San Diego County), USA (33). The 4400-km2 watershed, two-thirds of which lies in northern Baja California, Mexico, includes urban areas (Tijuana, Tecate, and Imperial Beach), agricultural land, and open chaparral and sage scrub. Approximately 25 million gallons per day of raw sewage from Tijuana is disposed directly into the river and routed into the South Bay International Wastewater Treatment Plant (USA) during dry weather. VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Locations of Famosa Slough (FAM) and the Tijuana River Estuary (TRE) (2) relative to the San Diego River and Tijuana River watersheds and the cities of San Diego, California (USA) and Tijuana, Baja California (Mexico). Inset, location of this study on the North American continent (box in lower left of inset represents study location). Created with ArcGIS 9.3.1 (ESRI Inc.) and data from public sources. Specific sampling locations within each wetland can be found in the Supporting Information (Figures S1 and S2). However, during winter rains, which can bring several billion gallons of stormwater per day, the treatment plant is inoperable, and the sewage-polluted stormwater is released directly to the estuary (34, 35). Famosa Slough (FAM) (N32° 45′ W117° 13′) (Figure 1 and Figure S2) is a small wetland (0.15 km2) in San Diego, California, USA within the San Diego River watershed (1,140 km2). While Famosa Slough receives urban runoff from the greater San Diego area, it has been relatively protected from direct sewage contamination. Both wetlands are marine dominated and receive semidiurnal tidal oscillations throughout the year as well as periodic stormwater inundation during the rainy season. Sediment Sampling and DNA Extraction. A brief description of surface sediments of the TRE and FAM wetlands has been published elsewhere (36). Sediments were sampled at 6 locations each (see Figure S1 and Figure S2) during the dry season and again immediately following a winter storm. Quadruplicate 50-mL surface sediments (uppermost 1 cm) were collected aseptically from exposed tidal channels at low tide and transported to the laboratory on ice. Sediment samples were frozen at -80 °C until processed for DNA extraction. Thawed samples were homogenized by hand, and metagenomic DNA was extracted from 0.5-g subsamples using the FastDNA SPIN Kit for Soil (MP Biomedicals) according to the manufacturer’s recommendations. Conventional PCR. All primers used in this study are described in Table S1 (Supporting Information). Conventional (nonquantitative) PCR was used to determine the presence or absence of select PMQR genes in wetland sediments: qnrA, qnrB, qnrS, qepA, and aac(6′)-Ib. Each PCR reaction contained the following components (final conc.): HotStarTaq DNA Polymerase (Qiagen) (0.25 U) and associated buffer (1X, including 15 mM MgCl2), dNTPs (Invitrogen) (10 mM each), forward and reverse oligonucleotide primers (Invitrogen) (500 nM each), molecular biology-grade bovine serum albumin 448

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(Roche) (0.4 mg mL-1), and molecular biology-grade water (to 20 µL). Five µL of each DNA extract was used as template in the following thermocycling protocol: hot start (95 °C, 15 min) followed by multiple cycles (see Table S1) of denaturation (94 °C, 1 min), primer annealing (see Table S1, 1 min), and extension (72 °C, 1 min). All reactions were finished with a final extension step at 72 °C for 10 min. Positive and negative controls were included in each run (see footnotes to Table S1). Only samples producing a distinct band on an agarose gel of the same size as the positive controls for each PMQR gene were scored as positive; aac(6′)-Ib amplicons were purified and digested with BtsC I (New England BioLabs) to differentiate the wild-type allele, which has the BtsC I restriction site, from the cr variant responsible for quinolone acetylation, which lacks this site (37). Real-Time Quantitative PCR. Quantitative PCR (qPCR) targeting the qnrA gene was carried out using SYBR Green I chemistry and a Bio-Rad Chromo4 real-time PCR instrument. Optimal qPCR conditions were determined empirically. Each 25-µL reaction contained QuantiTect SYBR Green PCR Master Mix (Qiagen) (1X), forward and reverse primers (Invitrogen) (500 nM each), and molecular biology-grade water. Five µL of DNA template was used in each reaction, and all DNA extracts were amplified in triplicate. The thermocycling protocol consisted of hot start (95 °C, 10 min) followed by 35 cycles of denaturation (95 °C, 1 min), primer annealing (57 °C, 1.5 min), and extension (72 °C, 0.5 min). All reactions were subjected to melting curve analysis and agarose gel electrophoresis to confirm specificity. Primers compatible with qPCR chemistry targeting the qnrA family of (fluoro)quinolone resistance genes were developed as part of this study with Primer3 freeware (38) using all full-length plasmid-bound qnrA sequences available at the time (May 2009) in the GenBank database (39) hosted by the National Center for Biotechnology Information (NCBI). Primers qnrA32F (5′-AGGATTTCTCACGCCAGGATT-3′) and

TABLE 1. Partial qnrA-like Genes from the Tijuana River Estuary (TRE) and Famosa Slough (FAM) during Both Dry and Wet Seasons wetland

season

clone ID

frequency

closest match (% similarity)a,b

accession no.c

TRE TRE TRE TRE FAM FAM FAM FAM FAM FAM FAM FAM

wet wet wet wet dry dry dry dry wet wet wet wet

TRE-wet-q1d TRE-wet-q3 TRE-wet-q4 TRE-wet-q8 FAM-dry-q3d FAM-dry-q8 FAM-dry-q12 FAM-dry-q13 FAM-wet-q1d FAM-wet-q11 FAM-wet-q21 FAM-wet-q24

27/30 1/30 1/30 1/30 26/29 1/29 1/29 1/29 27/30 1/30 1/30 1/30

qnrA/clinical isolate plasmids (100%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (100%) qnrA4/S. algae chromosome (100%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (100%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (98%) qnrA/clinical isolate plasmids (98%)

HQ008308 HQ008309 HQ008310 HQ008311 HQ008316 HQ008317 HQ008318 HQ008319 HQ008312 HQ008313 HQ008314 HQ008315

a Closest match(es) in the GenBank database followed by percent sequence similarity between the clone and the database match. b All clones, with the exception of FAM-dry-q8, were most similar to the same set of qnrA genes on plasmids from clinical isolates of Enterobacteriaceae, Pseudomonas, Acinetobacter, and Stenotrophomonas (see Results and Discussion). c GenBank/DDBJ/EMBL sequence accession numbers. d Clones TRE-wet-q1, FAM-dry-q3, and FAM-wet-q1 were 100% identical to one another.

qnrA155R (5′-CCGCTTTCAATGAAACTGCA-3′), spanning nucleotides 32 to 155 on the qnrA gene of pMG252, produced a 124-bp amplicon both in silico (compared to pMG252) and in vitro. The primers were found to be specific for qnrA-like genes when aligned in the GenBank database using the BLAST tool (40), including all of the known plasmid-borne qnrA genes and all known chromosomal qnrA genes in Shewanella putrefaciens and Shewanella algae. When tested against 10fold serial dilutions of a cloned and sequenced qnrA-like gene previously amplified from TRE sediments, the new qPCR primers were accurate over a range of 5 orders of magnitude (102-106 copies per reaction), with a typical slope of -3.4 and an R2 value of g0.998. No-template control reactions yielded Ct values e101 copies per reaction, setting our lower limit of detection to approximately 102 copies per reaction. This is not entirely surprising given the high sensitivity of SYBR Green-based qPCR and the tendency for Taq DNA polymerase preparations to contain contaminating DNA (41). Melting curve analysis of the PCR products suggested that no primer-dimers were formed and that the amplicons were uniform in size and sequence; agarose gel electrophoresis confirmed this observation. Final validation of the specificity of this new qPCR primer pair is found in the sequences of cloned PCR products (Table 1). The number of copies of qnrA-like genes was normalized to both sediment mass (qnrA copies per g sediment (wet wt)) as well as to the number of copies of the 16S rRNA gene present (qnrA copies per 16S rRNA gene copy) using primers Bac331F and Bac797R and the same chemistry and thermocycling protocol as for qnrA. To create qPCR quantitation standards, partial qnrA and 16S rRNA genes were amplified with primers qnrA13F/555R and EUB8F and UNIV1492R, respectively, and cloned into the pGEM-T Easy vector (Promega). After confirming the identity of the PCR products by sequence analysis, the inserts along with flanking DNA were reamplified from the cloning vector using primers M13F and M13R, purified, and quantified by UV absorption. Detection Limits and Statistical Analyses. The lower limits of detection for conventional PCR reactions were determined by the number of cycles possible before generating a visible band in negative (no template) control reactions; this maximum cycle number was determined empirically for each primer pair (Table S1). The qPCR detection limit for qnrA was estimated using the following assumptions: (i) g 102 copies per reaction, (ii) 0.5 g sediment per extraction, and (iii) 2 × 108 copies of 16S rDNA per g sediment (based on the average concentration of this target

in both wetlands). Based on these assumptions, the lower limit of detection of qnrA-like genes was estimated to be 4.0 × 103 copies per g sediment or 2.0 × 10-5 copies per 16S rDNA copy. For statistical analyses, these numbers were imputed where qPCR measurements were below the detection limits. Differences by site or season were determined using the chi-square test for presence/absence data and Student’s two-tailed t-test for quantitative data. Cloning and Sequencing qnrA Amplicons. qPCR-positive samples were reamplified without SYBR Green, pooled by site (TRE or FAM) and season (wet or dry), and cloned into the pGEM-T Easy vector (Promega) according to the manufacturer’s instructions. Thirty transformed colonies from each library were screened for inserts by reamplification with primers M13F/R which were subsequently sequenced once in each direction (Eurofins MWG Operon). Cloned inserts were aligned with the GenBank database using BLAST to identify the most similar sequences. Nucleotide Accession Numbers. All cloned partial qnrA sequences reported in this study have been deposited in the GenBank/DDBJ/EMBL databases under accession numbers HQ008308-HQ008319.

Results and Discussion Detection of PMQR Genes in Wetland Sediments. Overall, PMQR genes were detected more often in TRE sediments after a rain storm (Figure 2 panel D) than in the same wetland during the dry season (panel C, p < 0.01) or either season at FAM (panels A and B, p < 0.01). All five PMQR genes were observed at TRE after rain, many of which were present in 4/4 replicate samples. aac(6′)-Ib-cr was the least common PMQR gene, only detected at two locations in each dry and wet seasons. Similarly, qnrA was only seen at two sites and only after rain. The only PMQR genes found at TRE-South A were qepA and qnrB; this site may be hydrologically different from the other sites, receiving more stormwater directly from nearby Tijuana streets than from the river itself (see Figure S1). The wet season at FAM (panel B) saw an increase in both frequency and diversity of PMQR genes in the sediments relative to the dry season (panel A, p < 0.05). Whereas only qepA was detectable in the dry season, qnrB, qnrS, and qepA were all present after rain. These results suggest that stormwater from San Diego does indeed carry PMQR genes despite its well developed sewage collection infrastructure, though it appears to be far less extensive than what is seen at the TRE. VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Presence/absence of PMQR genes in coastal wetland sediments: (A) FAM dry season; (B) FAM wet season; (C) TRE dry season; and (D) TRE wet season. Data are reported as the number of PCR-positive sediment samples out of four replicate samples at each site. White bars with black spots, qnrA; solid white bars, qnrB; solid gray bars, qnrS; solid black bars, qepA; white bars with black hatches, aac(6′)-Ib-cr. An important question is whether the PMQR genes that are broadly disseminated into these two urban wetlands during rain events persist throughout the year. The data in panels A and C of Figure 2 (both wetlands, dry season) suggest that they do and that they remain much more prevalent in the sewage-contaminated TRE sediments than those of FAM (p < 0.001). qepA, which was the most commonly seen PMQR gene in the study, was the only one present in FAM sediments during the dry season. By contrast, at the TRE during the dry season all of the PMQR genes except qnrA were detected. While qnrB, qnrS, and qepA could be seen in both wetlands, qnrA and aac(6′)-Ib-cr were found only at TRE. The distribution of PMQR genes in these two wetlands is intriguing. In a survey of the literature through 2008, Strahilevitz et al. (25) reported that aac(6′)-Ib-cr was the most common PMQR gene among clinical Enterobacteriaceae isolates, particularly E. coli. In these impacted sediments, though, it appears to be the least common. While data are lacking on the prevalence of qepA, it appears to be relatively rare among clinical enterics (37, 42-44). Yet, it was detected most commonly in this study of wetland sediments. It may be that PMQR genes are distributed differently among hospital patients (from whom clinical isolates are obtained) and healthy individuals (whose intestinal flora would likely dominate untreated wastewater). In any case, the situation in a complex ecosystem is likely to be more complicated than what is observed in the clinical setting. Quantitation of qnrA-like Genes in TRE Sediments during Dry and Wet Seasons. To examine the dissemination of PMQR genes at the TRE and FAM wetlands, new primers targeting qnrA for quantitative real-time PCR were developed as part of this study. qnrA was selected for focused study because of its rarity in environmental bacteria and clear sequence distinctions between alleles located on plasmids and those located chromosomally in Shewanella spp. qnrAlike genes were below detection in all TRE sediments during the dry season (Figure 3); the detection limit (horizontal dashed line) is imputed where a sample was below detection (4.0 × 103 copies per g sediment or 2.0 × 10-5 copies per 16S 450

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rDNA copy). However, after a winter rain brought sewagecontaminated stormwater to the wetland, qnrA concentrations jumped to as high as 3.08 × 106 copies per g sediment (or 2.10 × 10-2 copies per 16S rDNA copy) (site TRE-MC, located adjacent to the main river channel). The lowest concentrations were found in the southern arm of the wetland at sites TRE-South A and TRE-South B; in fact, qnrA was never detectable at TRE-South A during either wet or dry seasons. The difference between qnrA concentrations during the wet and dry seasons at TRE was highly significant when normalization to either sediment mass or 16S rDNA copies (p < 0.0005). To confirm that the new qPCR primers were amplifying only qnrA-like genes from the sediment metagenomic DNA, samples that were qnrA-positive by qPCR were reamplified without SYBR Green I and cloned into the pGEM-T Easy vector (Promega) for sequencing. Of a library of 30 clones from the TRE during the wet season, only four unique sequences were discovered (Table 1). The library was dominated by clone TRE-wet-q1 (27/30) which was 100% identical to numerous plasmid-borne qnrA genes in the GenBank database. These matching sequences were all affiliated with plasmids from clinical isolates of various Enterobacteriaceae (Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Shigella flexneri, Citrobacter freundii), Acinetobacter baumanii, Pseudomonas aeruginosa, and Stenotrophomonas maltophila as well as a group of clones from a wastewater treatment plant. While this relatively short fragment of the qnrA gene provided insufficient information to differentiate among the various clinical plasmid-borne qnrA matches in the database, it was able to distinguish the plasmid-borne qnrA genes from those found on the chromosome of the common wetland bacterium Shewanella algae. Shewanella spp. were identified as predominant facultative anaerobes in TRE sediments under anaerobic Fe(III)-reducing conditions in a previous study (36) and represent one of the only known examples of chromosomal qnrA (45). The sequence of clone TRE-wet-q1

FIGURE 3. Quantitative PCR analysis of qnrA-like genes in surface sediments of the TRE during the dry season (white fill) and immediately following a winter rain (gray fill). Dotted line represents the detection limit of the assay (the detection limit was imputed where concentrations were below detection). Symbols represent x¯ ( 1 σ (n ) 4 replicate sediment samples). Solid bars, qnrA-like genes per g sediment; hatched bars, qnrA-like genes per 16S rDNA copy.

FIGURE 4. Quantitative PCR analysis of qnrA-like genes in surface sediments of FAM during the dry season (white bars) and immediately following a winter rain (gray bars). Dotted line represents the detection limit of the assay (the detection limit was imputed where concentrations were below detection). Symbols represent x¯ ( 1 σ (n ) 4 replicate sediment samples). Solid bars, qnrA-like genes per g sediment; hatched bars, qnrA-like genes per 16S rDNA copy. was only 95% similar to any Shewanella qnrA gene (qnrA2 of Shewanella putrefaciens), supporting our contention that the qnrA found in TRE sediments is of Enterobacteriaceae plasmid origin. Quantitation of qnrA-like Genes in FAM Sediments during Dry and Wet Seasons. During the dry season, low levels of qnrA were detected closest to the inlet of Famosa Slough (Figure 4) (site FAM-1); concentrations decreased rapidly to the detection limit moving away from the inlet (toward site FAM-6), suggesting a riverine source rather than surface runoff. The distribution of qnrA shifted after rainfall, but the difference between qnrA concentrations during the wet and dry seasons was not significant regardless of normalization to sediment mass or 16S rDNA copies (p > 0.1). The highest concentration was measured at FAM-1

during the dry season (2.23 × 105 copies per g sediment, or 1.11 × 10-3 copies per 16S rDNA copy), and the difference between qnrA concentrations at FAM during the wet or dry seasons and TRE during the wet season was highly significant (p < 0.0005). Like TRE during the wet season, only four unique sequences were identified in the FAM dry season clone library (Table 1). To our surprise, the dominant sequence (26/29), FAM-dry-q3, was 100% identical to clone TRE-wet-q1 and the numerous plasmid-borne qnrA genes in the GenBank database described above and only 95% similar to any Shewanella qnrA gene. Two other clones, FAM-dry-q12 and FAM-dry-q13, observed only once each (1/29), were 97% similar to one another and 98% similar to FAM-dry-q3 described above. Accordingly, when compared to the GenVOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Bank database, these two clones shared 98% sequence similarity with the same list of clinical plasmid-borne qnrA genes described above and only 93% similarity with qnrA2 of Shewanella putrefaciens. The fourth unique clone from FAM during the dry season, observed only once in the library (1/29), was FAM-dry-q8, which was distinct from the previous three clones (92-93% sequence similarity). When compared to the GenBank database, FAM-dry-q8 was identical to qnrA4 and qnrA5 described on the chromosome of Shewanella algae as well as qnrA6 on a plasmid held by a clinical isolate of Proteus mirabilis. This clone also shared 98% sequence similarity with a mix of other Shewanella chromosomal genes and Enterobacteriaceae plasmid genes. The diversity of qnrA genes at FAM did not change much after a heavy winter rain (Table 1). Again, only four unique sequences were discovered in the wet season library of 30 clones, the most common of which, clone FAM-wet-q1 (27/ 30), was 100% identical to the predominant dry season clone at FAM (FAM-dry-q3) and the most abundant wet season clone at TRE (TRE-wet-q1). The remaining three clones in the wet season library, FAM-wet-q11, FAM-wet-q21, and FAM-wet-q24, observed only once each (1/30), were 97% similar to one another and 98% similar to FAM-wet-q1. They shared 98% sequence similarity with the same set of plasmidborne clinical qnrA genes in the GenBank database as the other clones and e95% similarity with the chromosomal Shewanella qnrA genes. The sequence analyses suggest that qnrA genes in the FAM wetland are also of Enterobacteriaceae plasmid origin. Sewage-Impacted Natural Environments As Reservoirs of Antibiotic Resistance Genes. Urban stormwater in coastal cities is notorious for sewage contamination (35, 46-48) that closes beaches and costs municipalities millions of dollars each year (49). The problems are confounded in developing nations where wastewater infrastructure is lacking. Acute public health threats from bacteria and viruses are usually alleviated within 72 h, likely as a result of dilution, osmotic stress due to marine salinity, and photoinactivation from exposure to direct sunlight (50). The chronic threats caused by repeated inoculation with biological pollutants, however, are largely unknown. We know that human and farm animal feces contain elevated levels of AR pollutants (8, 51) as does the wastewater within treatment systems (15-17, 52). A limited but growing body of data suggests that improper handling of wastewater can release AR pollutants into natural habitats (18, 19). It is reasonable to speculate that AR pollutants may find their way back into the human community via vectors (e.g., shore birds, insects, marine animals) or through direct contact (e.g., swimming, fishing, etc.) (see Figure S5). Limited evidence has been published for the role of shorebirds as vectors of AR pollutants in Europe (53). It is believed that AR genes evolved in the natural environment and subsequently moved into clinically relevant bacteria through horizontal gene transfer (54). Indeed, the sources of many of the qnr genes have been traced to marine organisms (45, 55, 56). Thus, the contamination of natural environments with plasmid-bound AR genes may have significant future public health consequences.

Acknowledgments The authors wish to thank Mike Dorrell (PLNU) for assistance with qnrA qPCR primer design and the critical comments of Mike Lehman (US Department of Agriculture) and three anonymous reviewers. Jeff Crooks (Tijuana River Estuary National Estuarine Research Reserve), Brian Collins (US Fish and Wildlife), and Karolynn Estrada (City of San Diego) facilitated field work. Positive control plasmids were kindly provided by George Jacoby (Lahey Clinic, Burlington, MA), Patrice Nordmann (Hoˆpital Biceˆtre, Paris), and Kunikazu 452

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Yamane (National Institute of Infectious Diseases, Tokyo). Thanks to Steven Di Donna (County of San Diego) for providing watershed GIS data. This work was funded in part under NOAA grant # NA04OAR4170038, through NOAA’s National Sea Grant College Program, U.S. Dept. of Commerce. The statements, findings, conclusions, and recommendations are those of the authors and do not necessarily reflect the views of California Sea Grant or the U.S. Dept. of Commerce. We are also grateful for the support of the Howard Hughes Medical Institute and Point Loma Nazarene University.

Supporting Information Available A table of primer characteristics (Table S1), maps of specific sampling locations within each wetland (Figures S1 and S2), conventional and quantitative PCR showing the flux of qnrA in TRE sediments in the midst of winter storms (Figures S3 and S4), and a conceptual model of the movement of AR genes between the human population and the natural environment (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Levy, S. B.; Marshall, B. Antibacterial resistance worldwide: causes, challenges and responses. Nat. Med. 2004, 10, S122– S129. (2) Hsu, L.-Y.; Tan, T.-Y.; Tam, V. H.; Kwa, A.; Fisher, D. A.; Koh, T.-H. Network for Antimicrobial Resistance Surveillance (Singapore). Surveillance and correlation of antibiotic prescription and resistance of Gram-negative bacteria in Singaporean hospitals. Antimicrob. Agents Chemother. 2010, 54, 1173–1178. (3) Levy, S. B. The antibiotic paradox: how the misuse of antibiotics destroys their curative powers; Perseus Publishing: Cambridge, MA, 2002. (4) Salyers, A. A.; Whitt, D. D. Revenge of the microbes: how bacterial resistance is undermining the antibiotic miracle; ASM Press: Washington, DC, 2005. (5) Knapp, C. W.; Dolfing, J.; Ehlert, P. A. I.; Graham, D. W. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environ. Sci. Technol. 2010, 44, 580–587. (6) Pruden, A.; Pei, R.; Storteboom, H.; Carlson, K. H. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environ. Sci. Technol. 2006, 40, 7445–7450. (7) Aubrey-Damon, H.; Grenet, K.; Sall-Ndiaye, P.; Che, D.; Cordeiro, E.; Bougnoux, M.-E.; Rigaud, E.; Le Strat, Y.; Lemanissiere, V.; Armond-Lefe`vre, L.; Delzescaux, D.; Desenclos, J.-C.; Lie´nard, M.; Andremont, A. Antimicrobial resistance in commensal flora of pig farmers. Emerg. Infect. Dis. 2004, 10, 873–879. (8) Chen, J.; Yu, Z.; Michel, F. C., Jr.; Wittum, T.; Morrison, M. Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolideslincosamides-streptogramin B in livestock manure and manure management systems. Appl. Environ. Microbiol. 2007, 73, 4407– 4416. (9) van den Bogaard, A. E.; Willems, R.; London, N.; Top, J.; Stobberingh, E. E. Antibiotic resistance of faecal enterococci in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother. 2002, 49, 497–505. (10) Alexander, T. W.; Yanke, L. J.; Topp, E.; Olson, M. E.; Read, R. R.; Morck, D. W.; McAllister, T. A. Effect of subtherapeutic administration of antibiotics on the prevalence of antibioticresistant Escherichia coli bacteria in feedlot cattle. Appl. Environ. Microbiol. 2008, 74, 4405–4416. (11) Asai, T.; Harada, K.; Ishihara, K.; Kojima, A.; Sameshima, T.; Tamura, Y.; Takahashi, T. Association of antimicrobial resistance in Campylobacter isolated from food-producing animals with antimicrobial use on farms. Jpn. J. Infect. Dis. 2007, 60, 290– 294. (12) Chee-Sanford, J. C.; Aminov, R. I.; Krapac, I. J.; GarriguesJeanjean, N.; Mackie, R. I. Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities. Appl. Environ. Microbiol. 2001, 67, 1494–1502. (13) Gibbs, S. G.; Green, C. F.; Tarwater, P. M.; Mota, L. C.; Mena, K. D.; Scarpino, P. V. Isolation of antibiotic-resistant bacteria from the airplume downwind of a swine confined or concentrated animal feeding operation. Environ. Health Perspect. 2006, 114, 1032–1037.

(14) Pei, R.; Kim, S.-C.; Carlson, K. H.; Pruden, A. Effects of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res. 2006, 40, 2427–2435. (15) Bo¨nemann, G.; Stiens, M.; Pu ¨ hler, A.; Schlu ¨ ter, A. Mobilizable IncQ-related plasmid carrying a new quinolone resistance gene, qnrS2, isolated from the bacterial community of a wastewater treatment plant. Antimicrob. Agents Chemother. 2006, 50, 3075– 3080. (16) Schlu ¨ ter, A.; Szczepanowski, R.; Pu ¨ hler, A.; Top, E. M. Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol. Rev. 2007, 31, 449–477. (17) Szczepanowski, R.; Linke, B.; Krahn, I.; Gartemann, K.-H.; Go¨tzkow, T.; Eichler, W.; Pu ¨ hler, A.; Schlu ¨ ter, A. Detection of 140 clinically relevant antibiotic resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiol. 2009, 155, 2306–2319. (18) Novais, C.; Coque, T. M.; Ferreira, H.; Sousa, J. C.; Peixe, L. Environmental contamination with vancomycin-resistant enterococci from hospital sewage in Portugal. Appl. Environ. Microbiol. 2005, 71, 3364–3368. (19) Zhang, Y.; Marrs, C. F.; Simon, C.; Xi, C. Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Sci. Total Environ. 2009, 407, 3702–3706. (20) Lili, L. N.; Britton, N. F.; Feil, E. J. The persistence of parasitic plasmids. Genetics 2007, 177, 399–405. (21) Sandaa, R.-A.; Enger, Ø. Transfer in marine sediments of the naturally occurring plasmid pRAS1 encoding multiple antibiotic resistance. Appl. Environ. Microbiol. 1994, 60, 4234–4238. (22) Yoshida, H.; Bogaki, M.; Nakamura, M.; Nakamura, S. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob. Agents Chemother. 1990, 34, 1271– 1272. (23) Yoshida, H.; Bogaki, M.; Nakamura, M.; Yamanaka, L. M.; Nakamura, S. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli. Antimicrob. Agents Chemother. 1991, 35, 1647–1650. (24) Nordmann, P.; Poirel, L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J. Antimicrob. Chemother. 2005, 56, 463–469. (25) Strahilevitz, J.; Jacoby, G. A.; Hooper, D. C.; Robiscek, A. Plasmidmediated quinolone resistance: a multifaceted threat. Clin. Microbiol. Rev. 2009, 22, 664–689. (26) Martı´nez-Martı´nez, L.; Pascual, A.; Jacoby, G. A. Quinolone resistance from a transferrable plasmid. Lancet 1998, 351, 797– 799. (27) Tran, J. H.; Jacoby, G. A. Mechanism of plasmid-mediated quinolone resistance. Proc. Natl. Acad. Sci. 2002, 99, 5638–5642. (28) Robicsek, A.; Strahilevitz, J.; Jacoby, G. A.; Macielag, M.; Abbanat, D.; Park, C. H.; Bush, K.; Hooper, D. C. Fluoroquinolonemodifying enzyme: a new adaptation of a common aminoglycoside actyltransferase. Nat. Med. 2006, 12, 83–88. (29) Kim, H. B.; Wang, M.; Park, C. H.; Kim, E.-C.; Jacoby, G. A.; Hooper, D. C. oqxAB encoding a multidrug efflux pump in human clinical isolates of Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 3582–3584. (30) Poole, K. Efflux-mediated resistance to fluoroquinolones in Gram-negative bacteria. Antimicrob. Agents Chemother. 2000, 44, 2233–2241. (31) Poole, K. Efflux-mediated resistance to fluoroquinolones in Gram-positive bacteria and the mycobacteria. Antimicrob. Agents Chemother. 2000, 44, 2595–2599. (32) Yamane, K.; Wachino, J.; Suzuki, S.; Kimura, K.; Shibata, N.; Kato, H.; Shibayama, K.; Konda, T.; Arakawa, Y. New plasmidmediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 2007, 51, 3354–3360. (33) Zedler, J. B.; Nordby, C. S.; Kus, B. E. The ecology of Tijuana Estuary: A National Estuarine Research Reserve; NOAA Office of Coastal Resource Management, Sanctuaries and Reserves Division: Washington, DC, 1992. (34) Conway, J. B.; Salgado, H. F.; Sandall, C. L.; Kobisher, E. A.; Nuno, L. R. An investigation of water quality in the Tijuana River. J. Border Health 1985, 1, 28–33. (35) Gersberg, R. M.; Rose, M. A.; Robles-Sikisaka, R.; Dhar, A. K. Quantitative detection of hepatitis A virus and enteroviruses near the United States-Mexico border and correlation with levels of fecal indicator bacteria. Appl. Environ. Microbiol. 2006, 72, 7438–7444.

(36) Cummings, D. E.; Zimmerman, A. E.; Unruh, K. R.; Spring, S. Influence of microbially reducible Fe(III) on the bacterial community structure of estuarine surface sediments. Geomicrobiol. J. 2010, 27, 292–302. (37) Kim, H. B.; Park, C. H.; Kim, C. J.; Kim, E.-C.; Jacoby, G. A.; Hooper, D. C. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrob. Agents Chemother. 2009, 53, 639–645. (38) Rozen, S.; Skaletzky, H. J. Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics methods and protocols: Methods in molecular biology; Krawetz, S., Misener, S., Eds.; Humana Press: Totowa, NJ, 2000; pp 365386. (39) Benson, D. A.; Karsch-Mizrachi, I.; Lipman, D. J.; Ostell, J.; Wheeler, D. L. GenBank. Nucleic Acids Res. 2008, 36 (database issue), D25–D30. (40) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403– 410. (41) Rand, K. H.; Houck, H. Taq polymerase contains bacterial DNA of unknown origin. Mol. Cell. Probes 1990, 4, 445–450. (42) Yamane, K.; Wachino, J.-i.; Suzuki, S.; Arakawa, Y. Plasmidmediated qepA gene among Escherichia coli clinical isolates from Japan. Antimicrob. Agents Chemother. 2008, 52, 1564– 1566. (43) Kim, E. S.; Jeong, J.-Y.; Choi, S.-H.; Lee, S.-O.; Kim, S.-H.; Kim, M.-N.; Woo, J. H.; Kim, Y. S. Plasmid-mediated fluoroquinolone efflux pump gene, qepA, in Escherichia coli clinical isolates in Korea. Diagn. Microbiol. Infect. Dis. 2009, 65, 335–338. (44) Park, Y.-J.; Yu, J. K.; Kim, S.-I.; Lee, K.; Arakawa, Y. Accumulation of plasmid-mediated fluoroquinolone resistance genes, qepA and qnrS1, in Enterobacter aerogenes co-producing RmtB and class A β-lactamase LAP-1. Annals Clin. Lab. Sci. 2009, 39, 55– 59. (45) Poirel, L.; Rodriguez-Martinez, J.-M.; Mammeri, H.; Liard, A.; Nordmann, P. Origin of plasmid-mediated resistance determinant QnrA. Antimicrob. Agents Chemother. 2005, 49, 3523– 3525. (46) Coulliette, A. D.; Noble, R. T. Impacts of rainfall on the water quality of the Newport River Estuary (Eastern North Carolina, USA). J. Water Health 2008, 6, 473–482. (47) He, L. M.; He, Z. L. Water quality prediction of marine recreational beaches receiving watershed baseflow and stormwater runoff in southern California, USA. Water Res. 2008, 42, 2563–2573. (48) Jiang, S.; Noble, R.; Chu, W. Human adenoviruses and coliphages in urban runoff-impacted coastal waters of southern California. Appl. Environ. Microbiol. 2001, 67, 179–184. (49) Given, S.; Pendleton, L. H.; Boehm, A. B. Regional public health cost estimates of contaminated coastal waters: A case study of gastroenteritis at southern California beaches. Environ. Sci. Technol. 2006, 40, 4851–4858. (50) Boehm, A. B.; Yamahara, K. M.; Love, D. C.; Peterson, B. M.; McNeill, K.; Nelson, K. L. Covariation and photoinactivation of traditional and novel indicator organisms and human viruses at a sewage-impacted marine beach. Environ. Sci. Technol. 2009, 43, 8046–8052. (51) Sommer, M. O. A.; Dantas, G.; Church, G. M. Functional characterization of the antibiotic resistance reservoir in the human microflora. Science 2009, 325, 1128–1131. (52) Jindal, A.; Kocherginskaya, S.; Mehboob, A.; Robert, M.; Mackie, R. I.; Raskin, L.; Zilles, J. L. Antimicrobial use and resistance in swine waste treatment systems. Appl. Environ. Microbiol. 2006, 72, 7813–7820. (53) Bonnedahl, J.; Drobni, M.; Gauthier-Clerc, M.; Hernandez, J.; Granholm, S.; Kayser, Y.; Melhus, A.; Kahlmeter, G.; Waldenstro¨m, J.; Johansson, A.; Olsen, B. Dissemination of Escherichia coli with CTX-M-type ESBL between humans and yellow-legged gulls in the south of France. PLoS One 2009, 4 (6), e5958, DOI: 10.1371/journal.pone.0005958. (54) Martinez, J. L. The role of natural environments in the evolution of resistance traits in pathogenic bacteria. Proc. R. Soc. B 2010, 276, 2521–2530. (55) Cattoir, V.; Poirel, L.; Mazel, D.; Soussy, C.-J.; Nordmann, P. Vibrio splendidus as the source of plasmid-mediated qnrS-like quinolone resistance determinants. Antimicrob. Agents Chemother. 2007, 51, 2650–2651. (56) Poirel, L.; Liard, A.; Rodriguez-Martinez, J.-M.; Nordmann, P. Vibrionaceae as a possible source of Qnr-like quinolone resistance determinants. J. Antimicrob. Chemother. 2005, 56, 1118–1121. VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

453

(57) Rodrı´guez-Martı´nez, J. M.; Pascual, A.; Garcı´a, I.; Martı´nezMartı´nez, L. Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type β-lactamase. J. Antimicrob. Chemother. 2003, 52, 703–706. (58) Cattoir, V.; Poirel, L.; Rotimi, V.; Soussy, C. J.; Nordmann, P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J. Antimicrob. Chemother. 2007, 60, 394–397. (59) Park, C. H.; Robicsek, A.; Jacoby, G. A.; Sahm, D.; Hooper, D. C. Prevalence in the United States of aac(6′)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob. Agents Chemother. 2006, 50, 3953–3955.

454

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 45, NO. 2, 2011

(60) Nadkarni,M.A.;Martin,F.E.;Jacques,N.A.;Hunter,N.Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiol. 2002, 148, 257–266. (61) Lane, D. J. 16S/23S rRNA sequencing. In Nucleic acid techniques in bacterial systematics; Stackebrandt, E., Ed.; Academic Press: Chichester, England, 1991; pp 115-175. (62) Engberg, J.; On, S. L. W.; Harrington, C. S.; Gerner-Smidt, P. Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples as estimated by a reevaluation of isolation methods for Campylobacters. J. Clin. Microbiol. 2000, 38, 286–291.

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