Tetracycline Resistance and Class 1 Integron Genes Associated with

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Tetracycline Resistance and Class 1 Integron Genes Associated with Indoor and Outdoor Aerosols Alison L. Ling,† Norman R. Pace,‡ Mark T. Hernandez,† and Timothy M. LaPara*,§ †

Department of Civil, Environmental, and Agricultural Engineering, University of Colorado, Boulder, Colorado 80309, United States Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, United States § Department of Civil Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡

S Supporting Information *

ABSTRACT: Genes encoding tetracycline resistance and the integrase of Class 1 integrons were enumerated using quantitative PCR from aerosols collected from indoor and outdoor environments. Concentrated animal feeding operations (CAFOs) and human-occupied indoor environments (two clinics and a homeless shelter) were found to be a source of airborne tet(X) and tet(W) genes. The CAFOs had 10- to 100-times higher concentrations of airborne 16S rRNA, tet(X), and tet(W) genes than other environments sampled, and increased concentrations of aerosolized bacteria correlated with increased concentrations of airborne resistance genes. The two CAFOs studied had statistically similar concentrations of resistance genes in their aerosol samples, even though antibiotic use was markedly different between the two operations. Additionally, tet(W) genes were recovered in outdoor air within 2 km of livestock operations, which suggests that antibiotic resistance genes may be transported via aerosols on local scales. The integrase gene (intI1) from Class 1 integrons, which has been associated with multidrug resistance, was detected in CAFOs but not in human-occupied indoor environments, suggesting that CAFO aerosols could serve as a reservoir of multidrug resistance. In conclusion, our results show that CAFOs and clinics are sources of aerosolized antibiotic resistance genes that can potentially be transported via air movement.

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encodes resistance.4 Of these resistance mechanisms, antibiotic resistance genes (ARGs) are of greatest concern because of their potential for widespread dissemination,6−9 and indeed ARGs are now recognized as a serious environmental “pollutant” of concern.9 ARGs have been detected in groundwater, surface water, and soil near CAFOs10−15 as well as in treated wastewater16−19 and in residual wastewater solids.18,20−22 Compared to the existing literature on ARGs in soils and in water, relatively little is known regarding ARGs in aerosols and their associated airborne transmission.7,23 Several studies have isolated antibiotic resistant bacteria11,17,18,24,25 or directly detected ARGs from aerosols in animal confinement buildings.26−28 In clinical environments, a cohort of studies have suggested that airborne transmission may be the primary method of transfer for several important diseases caused by bacteria known to carry relatively high levels of antibiotic resistance.7,23,29,30 There are limited data that address the presence of resistance genes in outdoor aerosols, although two

INTRODUCTION The discovery and exploitation of antibiotic compounds produced by microbes have been enormously beneficial for public health. In the early years of the antibiotic era, most bacterial infections were susceptible to antibiotic chemotherapy; however, it is now increasingly common that pathogens resist antibiotic treatment.1,2 Because many antibiotics and antibiotic resistance genes are natural phenomena,1,2 the generally accepted explanation for the proliferation of antibiotic resistant bacteria is that the extensive use (and misuse) of antibiotics imposes a selective pressure in favor of resistant strains compared to sensitive strains.3 The widespread subtherapeutic use of antibiotics in animal feed has been controversial in the United States, since it has been considered a major source of antibiotic resistance to nearby soil and water, and is often considered to be unnecessary or avoidable.4,5 In general, hospitals and confined animal feeding operations (CAFOs) are assumed to be the most significant sources of antibiotic resistance because of their extremely high utilization of antibiotics.1 Bacteria can be resistant to antibiotic chemotherapy because they are intrinsically resistant (i.e., lack the appropriate antibiotic target), they become resistant following mutation, or they become resistant by acquiring a gene (or genes) that © 2013 American Chemical Society

Received: Revised: Accepted: Published: 4046

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(method 4). For agricultural and urban outdoor samples, 24-h samples were collected onto sterilized 47-mm quartz filters using a medium-flow dichotomous filter sampler with a 50 L min−1 PM10 inlet and virtual impactor38,39 (method 5). On the basis of size distribution for aerosol particles associated with bacterial DNA collected from indoor and outdoor spaces43 and reported particle capture efficiencies for the sampling methods used,39−41 the expected sample collection efficiency for Omni (method 1), SKC impinger (method 2), and virtual impactor methods (methods 3−5) are 61%, 69%, and 84%, respectively (Table S3). Given that concentrations of gene numbers detected among different environments spanned several orders of magnitude, we expect that the variation in aerosol sampling methods imparts insignificant variation in the quantity of DNA collected. DNA Extraction and Recovery. Polycarbonate or quartz filters were bead-beaten for 2 min in a buffered phenol− chloroform solution, and DNA was precipitated using 7.5 M ammonium acetate and isopropanol.44 Ethanol-rinsed DNA pellets were resuspended in RNase and DNase-free water and stored at −80 °C. Quantitative PCR. tet(W) and tet(X) genes were chosen to target two of the three types of tetracycline resistance genes. tet(W) inhibits the effect of tetracycline by interfering with its ribosome-binding,45 whereas the tet(X) gene codes for an NADPH-dependent oxidoreductase that inactivates tetracycline.46 We also assayed the integrase gene (intI1) of Class 1 integrons because of its implications for intercellular transfer of resistance and association with multidrug resistance.33,36,47 qPCR was performed for bacterial-specific 16S rRNA, tet(X), tet(W), and intI1 genes as previously described16 using an Eppendorf ep Realplex Thermocycler (Eppendorf, Hamburg, Germany). Target sequences ranged in length from 168 to 280 nt. Reaction mixtures (total volume 25 μL) contained 1X iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA), 1 μg/μL bovine serum albumin (Roche Applied Science, Indianapolis, IN), and optimized quantities of forward and reverse primers (Table S2). Template was melted at 95 °C for 1 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. PCR products were melted on a constant gradient from 60 to 95 °C for 20 min, and nonspecific amplification was not observed. All qPCR reactions were performed in triplicate, and PCR efficiency was between 95% and 105%. Extraction negative controls were always below qPCR detection limits. Results were quantified using standard curves generated from 10-fold serial dilutions of a known quantity of plasmid template (R2 > 0.99). Plasmid standards were produced by amplifying positive controls and ligating into the pGEM-T Easy Cloning Vector (Promega, Madison, WI) and transforming into E. coli JM109 cells. Plasmids were purified using an alkaline lysis procedure and quantitated by staining with Hoechst 33258 dye. Fluorescence was measured with a TD-700 fluorometer (Turner Designs, Sunnyvale, CA) using calf thymus DNA as a standard. The qPCR detection limit was determined by the minimum of the lowest gene number on the standard curve that yielded an exponential fit with an R2 > 0.99 and the average gene number of the qPCR negative controls for an assay (no template) (Table S2). Samples with more than one bacterial 16S rRNA gene qPCR reaction below detection limit were removed from analysis. Data Analysis. For each gene assayed, gene number was normalized to volume of air collected and subjected to a logarithmic transformation. These data were normally dis-

studies have recovered antibiotic-resistant isolates downwind of CAFOs.24,31 In this study, quantitative polymerase chain reaction (qPCR) was used to determine the abundance of tetracycline resistance and Class 1 integrase genes in aerosol samples collected from livestock farms, human-occupied indoor spaces, and outdoor air along the Rocky Mountain Front Range in Colorado, USA. We hypothesized that CAFOs and clinics may contribute significant concentrations of aerosolized resistance genes which can potentially spread via aerosol transfer. Two tetracycline resistance genes, tet(W) and tet(X), were chosen for this study because tetracycline is the most widely used antibiotic in animal agriculture32 and because these genes have been detected in previous studies of livestock environments.10,12,27,28 We also assayed the integrase gene (intI1) of Class 1 integrons, which have been associated with multidrug resistance and animal manure.33−36

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MATERIALS AND METHODS Sample Collection. Samples were collected between 2007 and 2011 from indoor and outdoor environments in Colorado. Sites were chosen to span a range of subtypes in each type of environment, with indoor sites chosen in environments where antibiotics are heavily used and outdoor sites chosen at varying distances from suspected sources. The indoor environments studied were two CAFOs (one swine, one dairy), and three human-occupied indoor environments (two clinics and a homeless shelter). Outdoor aerosols were collected from urban, semiurban, livestock agriculture, and alpine forest regions. Additional sample and site information is presented in the Supporting Information (SI) Table S1. Aerosol Sampler Descriptions. Aerosol samples were collected using five different methods with comparable sampling efficiencies (all greater than 60% for particle sizes associated with bacterial DNA). The liquid-capture methods (methods 1 and 2) have varied particle collection efficiencies due to physical properties of particle collection into a liquid. The virtual impaction sampling methods (methods 3−5) are >90% efficient for collection of particles smaller than 10 μm.37−39 For CAFO, clinic, and homeless shelter indoor samples, an Omni 3000 wet concentrator sampler (InnovaPrep, Drexel, MO) was used to sample PM10 aerosols into sterile, DNA-free phosphate-buffered saline (PBS) for between 15 and 30 min at a flow rate of 277 L min−1. Collected liquid was filtered through sterile 47-mm polycarbonate filters using aseptic, DNA-free techniques. Omni particle capture efficiency ranges from 40% for 0.5 μm particles and 30% for 8 μm particles, to 93% for 5 μm particles 40 (method 1). For some CAFO samples, glass impingers (BioSampler SKC Inc., Eighty Four, PA) were run for 30 min at a flow rate of 12.5 L min−1 to collect PM10 into sterile DNA-free water, which was then filtered through 47-mm sterile polycarbonate filters. Capture efficiency for SKC impingers is highest (>90%) for 10 μm particles, and drops to 80% for 7 μm particles and 30% for 0.3 μm particles41,42 (method 2). For semiurban outdoor samples, 48-h samples were collected onto 47-mm sterile polycarbonate filters using a Mini-Vol sampler (Air Metrics, Eugene, OR) equipped with a PM10 virtual impaction cone and operated at 7.9 L min−1 (method 3). For forest outdoor samples, 48-h samples were collected onto 47-mm sterile polycarbonate filters using an 8-head medium-volume URG sampler (URG Corp., Chapel Hill, NC) with a PM10 virtual impactor operated at 66 total L min−1. Each PM10 filter saw 8.25 L min−1 of flow 4047

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tributed, and averages, standard deviations, and statistical parameters were calculated from volume-normalized, logtransformed data. A heteroscedastic two-tailed student’s t test for samples with unequal variance was used to assess significance pairwise differences in gene abundance among the environments monitored. The t test alpha value was assigned as 0.01 to account for the increased probability of false positives when using t test for pairwise comparison among more than two groups. A single-factor ANOVA was also performed for each gene to determine whether the expected mean value for gene number per cubic meter of air is different for the three environments. PCR-Amplicon Cloning and Sequencing. For each of tet(X), tet(W), and intI1, qPCR amplicons independently recovered from three environments (one swine, one dairy, and one clinic) were ligated and cloned into electrocompetent E. coli cells using the TOPO TA Cloning Kit (Invitrogen/Life Technologies, Grand Island, NY). Consensus sequences were obtained from bidirectional sequence information using T3 and T7 primers on a MegaBace 1000 capillary sequencer (GE Healthcare, Waukesha, WI). At least 14 nucleotide sequences were obtained for each gene target and compared to nucleotide and protein sequences in the online GenBank database48 using Basic Local Alignment Search Tool (BLAST).49 For each gene, MUSCLE was used to generate multiple sequence alignments for both DNA and translated protein sequences, and Mothur was used determine the degree of sequence variation within the group.50,51 Nucleotide sequences were submitted to GenBank under accession numbers KC132871−KC132913. Meteorological Modeling. Meteorological source tracking for an outdoor air sample with detectable levels of tet(X) and intI1 was conducted using NOAA’s Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) model.52 The HYSPLIT model is further described in the SI.

Figure 1. Measured abundance of bacterial 16S rRNA genes recovered from aerosols collected in the following environments: concentrated animal feeding operations (CAFO (black bar)); clinics and homeless shelters (Indoor (gray bar)); and outdoor air samples (Outdoor (white bar)). All values are normalized to the air volume sampled (m3). Bar heights represent averages of log-transformed CT values from three independent qPCR observations, and error bars represent one standard deviation.

fold higher than tet(X) genes in aerosols recovered from CAFO environments, which is consistent with a previous study that found increased abundance of ribosomal-protection type tetracycline resistance genes over other tetracycline resistance genes in CAFO aerosols.27 Class 1 integrase genes were only detected in CAFO samples and in one outdoor air sample collected in a pristine alpine forest area (Figure 2c). Genes homologous with tet(X) were detected in aerosol samples recovered from both clinics, and tet(W) genes were detected in the homeless shelter and in one clinic. While the clinic and shelter samples contained significantly lower concentrations of 16S rRNA and tetracycline resistance genes, those tet(X) or tet(W) genes observed above the detection limit contributed to approximately 10-fold higher tet(X) or tet(W) to 16S rRNA ratios than observed in CAFOs (t test, P = 0.003 and 0.14 for tet(X) and tet(W), respectively) (Table S4). In contrast, the outdoor samples had markedly lower or nondetectable levels of resistance genes. Two aerosol samples collected outdoors in an agricultural region were found to contain low levels of tet(W) genes. One outdoor forest sample contained intI1 genes, and meteorological source modeling for the site indicates that the air mass in the area on the day the sample was collected came from the direction of western and southern Colorado, which has areas of large cattle farms. Four of seven outdoor air samples (1/1 urban, 2/2 semi-urban, and 1/2 forest) were below detection limit for tet(X), tet(W), and intI1. PCR-Amplicon Cloning and Sequencing. Eighty-three sequences with a minimum length of 102 nucleotides were determined from qPCR products in order to validate primer specificity. All tet(X), tet(W), and intI1 gene sequences matched appropriate nucleotide sequences in the GenBank database with greater than 91% identity over at least 57 nucleotides, and inferred amino acid sequences all matched with greater than 80% amino acid similarity over at least 20 positions (Table S5). The maximum nucleotide variations between sequences within gene sets were 65% (tet(X)), 65% (tet(W)), and 51% (intI1), which is consistent with the known degree of variability for

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RESULTS 16S rRNA Genes. Indoor and CAFO air samples had between 1.5 × 101 and 1.9 × 105 bacterial 16S rRNA gene per cubic meter of air sampled. CAFO aerosols had higher 16S rRNA gene numbers than both human-occupied indoor (t test, P < 0.01) and outdoor aerosols (t test, P < 0.01), and the expected mean gene number per volume of air is different among the three types of environments (ANOVA, P < 0.01). Concentrations in outdoor air were comparable to those in human-occupied indoor spaces (Figure 1). Bacterial 16S rRNA gene counts measured by qPCR in CAFO and outdoor air were comparable to levels previously reported by qPCR and direct epifluorescent microscopy in indoor and outdoor air (104−106 and 103−105 bacteria per m3, respectively).53−56 16S rRNA concentrations in clinics and a homeless shelter were lower than those previously reported indoors.54,55 Tetracycline Resistance and Class 1 Integrase Genes. ANOVA indicates that the expected mean values of gene numbers per volume of air are different among the three types of environments sampled (ANOVA, P < 0.01 for tet(X), tet(W), and intI1 genes). The abundance of tetracycline resistance genes was significantly higher in CAFO, clinic, and shelter samples than in outdoor samples (t test, P < 0.01). tet(W) and tet(X) genes were detected in every farm CAFO sample, and these samples had higher gene numbers of tet(X) (t test, P < 10−4) and tet(W) (t test, P < 10−4) than other environments monitored (Figure 2a and b). The concentrations of airborne tet(W) genes were between 10- and 1004048

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Figure 2. Measured abundance of airborne tet(X) (top), tet(W) (middle), and intI1 (bottom) genes recovered from aerosols in the following environments: concentrated animal feeding operations (CAFO (black bar)); clinics and homeless shelters (Indoor (gray bar)); and outdoor air samples (Outdoor (white bar)). All values are normalized to the air volume sampled (m3). Bar heights represent averages of log-transformed CT values from three independent qPCR observations, and error bars represent one standard deviation. The asterisk indicates that only one qPCR replicate was successful for the indicated sample.

these genes. There was no significant difference in sequence variation between inter- and intraenvironment comparisons.

is of particular interest. While this study cannot definitively determine whether the resistance genes recovered outdoors originated from a nearby agricultural source, it suggests a genetic basis for potential antibiotic resistance transfer via airborne routes across outdoor spaces. Recent reports have confirmed that bacterial cells can undergo interregional as well as intercontinental atmospheric transport,58−60 so bioaerosols could potentially transfer antibiotic resistance genes across large distances. Aerosol samples collected from clinical settings contained both tet(X) and tet(W) genes, indicating that clinics can also be potential sources of airborne antibiotic resistance genes. This is a matter of concern for both healthy and sick individuals, who may serve as both receptors and carriers. We also recovered airborne tet(W) genes in a homeless shelter. This environment cycles through large numbers of people in various levels of physical wellness, and the presence of resistance genes indicates that resistance may travel between source and nonsource human-occupied environments. The relatively low levels of 16S rRNA genes recovered from these environments demonstrate the success of indoor air quality mitigation measures. However, in comparison with CAFO environments, human-occupied indoor spaces had higher tet(X):16S rRNA and tet(W):16S rRNA ratios despite lower levels of both 16S rRNA and tetracycline resistance genes. This indicates that a higher proportion of airborne bacteria may carry resistance potential in these environments.

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DISCUSSION Significantly higher concentrations of tet(X), tet(W), and intI1 genes were measured in aerosol samples collected within CAFOs than in other indoor and outdoor spaces, supporting the hypothesis that CAFOs are a source of bioaerosols carrying resistance genes. When normalized to air volume, the relatively high concentrations of resistance genes observed in this study coincide with comparatively high levels of 16S rRNA gene abundance in the CAFO samples. This indicates that CAFOs are a source of relatively high concentrations of airborne bacteria, a substantial fraction of which carry antibiotic resistance genes. From a practical perspective, this suggests that removing particulate matter and bacteria from CAFO air could decrease their potential as sources of airborne resistance. CAFOs have been reported as “point-sources” of antibiotic resistancetransporting microbes with these genes to nearby groundwater, surface water, and soils. 15,24−28,31,57 This perspective has been primarily limited to terrestrial routes, and the magnitude and scales through which bioaerosols may carry resistance genes through the environment have not yet been considered. We report converging lines of evidence which support our hypothesis that the aerosol transfer of antibiotic resistance genes may be an important mechanism contributing to the spread of antibiotic resistance. In this context, our recovery of tet(W) genes in outdoor air in an agricultural region 4049

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aerosal loads and thus an increased likelihood of detecting antibiotic resistance genes. In CAFO and indoor sites, the source of antibiotic resistance genes (humans and animals) is contained indoors, so abundances of these genes are not expected to vary significantly by season. In conclusion, CAFOs and clinics are sources of aerosolized tetracycline resistance genes in particle sizes that can remain aloft on time scales relevant to environmental transport and thereby potentially facilitate short- and long-range transmission of antibiotic resistance. This is the first study to recover airborne antibiotic resistance genes from clinical settings, which has implications for public health and the transfer of antibiotic resistant nosocomial diseases (i.e., MRSA). CAFOs are potential reservoirs and sources of aerosolized resistance and Class 1 integrons to a significantly larger degree than clinical settings. Additionally, airborne concentrations of resistance genes in a livestock facility did not correlate to respective antibiotic use in this study. This work sets a baseline for future studies to more comprehensively investigate the occurrence of resistance genes in different airborne environments and to directly evaluate the scales and risks of airborne transfer of resistance between different types of bacteria. More generally, our recovery of antibiotic resistance genes from aerosols across different indoor environments has implications for public health, hospital quarantine measures, and indoor air quality.

Class 1 integrase genes were recovered only from CAFO environments and one outdoor sample, but were not detected in human-occupied indoor spaces. This suggests that CAFOs can be a significant reservoir of Class 1 integrons, which are often linked to multiple antibiotic resistance. This observation is consistent with previous studies that have found intI1 genes associated with animal wastes.34−36 In the environment, an important mechanism for the environmental transmission of antibiotic resistance is horizontal gene transfer, which can be facilitated by integrons, transposons, and other gene transfer cassettes.1,47,61,62 Class 1 integrons, in particular, are frequently associated with cells that are resistant to several classes of antibiotics.33 From this perspective, the apparent absence of intI1 genes from clinical aerosols suggests that hospital procedures to minimize the spread of antibiotic resistance can help limit the transfer of resistance genes. There was no significant difference in airborne concentrations of tet(X), tet(W), or intI1 between swine farm and dairy farms. Farms raising dairy cows typically use antibiotics only for veterinary purposes due to regulations that prohibit antibiotic use among cows producing milk intended for human consumption. In contrast, farms raising swine frequently use antibiotics for growth promotion or prophylaxis at subtherapeutic levels.4,9 Our findings suggest that the amount of antibiotic use at a farm may not have a significant effect on the abundance of aerosolized tetracycline resistance or Class 1 integrase genes. These results are inconsistent with the frequent assumption that subtherapeutic use of antimicrobials in animal feed leads to increased spread of antibiotic resistance.4,5,8,9,33,63 One possible explanation is that animal manure at CAFOs can potentially act as a reservoir and maintain antibiotic resistance in farm animal microbiota regardless of the degree of antibiotic use at the site. Animals that have not been treated with antibiotics can still be exposed to resistance genes through the manure of animals that have been treated. Resistance genes in this manure reservoir could be maintained and spread by the presence of integrons. Most previous studies have used culture-based techniques to assess the presence of antibiotic resistance in aerosols.24−26,31 However, less than 1% of bacteria have been cultured,64 and genes from noncultivable or nonviable cells can be transferred via horizontal gene transfer to other cells.61,62 qPCR is more sensitive than culture-based methods because it targets the genotype of resistance rather than the phenotypes and can detect resistance genes across environments that have different types of bacteria present.65 This method is more indicative of the potential for aerosol-mediated transfer of antibiotic resistance between environments than culture-based methods, and results can be more easily compared among studies. Nonetheless, the method is limited by it its ability to detect only a fragment (about 250 nt) of genes targeted. Truncated sequences and nonexpressed sequences cannot be resolved from expressed gene sequences using qPCR, so the levels reported could overestimate the number of functional, fulllength genes. Samples for this study were collected only once or twice at each site, so temporal variation in aerosol DNA loads was not considered in this study. Recent studies have found high levels of temporal variation in bacterial rRNA genes collected from outdoor air,66,67 so the rRNA gene levels we report for outdoor sites may not be an accurate representation of bacterial loads across seasons. Summer and fall sampling dates were chosen because these seasons experience heightened outdoor bio-

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ASSOCIATED CONTENT

S Supporting Information *

Sample metadata (Table S1); information regarding qPCR conditions, estimated aerosol sampling efficiencies (Tables S2 and S3); meteorological modeling software; and additional results regarding gene number normalized to 16S rRNA copy number and sequence comparison to the GenBank database (Tables S4 and S5). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: (612)624-6028; fax: (612)626-7750. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported in part by the Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF), made possible in part by the American Recovery and Reinvestment Act of 2009, administered by ORISE-ORAU under contract DE-AC05-06OR23100. Indoor air sampling and sequencing was supported by an Alfred P. Sloan Foundation Grant to N.R.P. Outdoor agricultural and urban area sampling was supported by EPA Star Grant R833744. Indoor aerosol samples were collected by Piret Koll, Mari Envagrov-Rodriguez, and Alison Ling. Outdoor urban and agricultural samples were collected by Nicholas Clements and Michael Hannigan as part of the CCRUSH study. Outdoor (pristine) samples were collected by Benjamin Miller and Alina Handorean. HYSPLIT modeling was performed by Nicholas Clements. Thanks to Erin Fletcher, Kevin McCabe, and Roberto Rodriguez for help with data analysis methods, and to Tucker Burch and Katheryn Hope for help with qPCR procedures. 4050

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