Fate of Tetracycline Resistance Genes in Aquatic Systems: Migration

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Environ. Sci. Technol. 2008, 42, 5131–5136

Fate of Tetracycline Resistance Genes in Aquatic Systems: Migration from the Water Column to Peripheral Biofilms C H R I S T I N A A . E N G E M A N N , †,# PATRICIA L. KEEN,‡ C H A R L E S W . K N A P P , †,§ K E N N E T H J . H A L L , ‡ A N D D A V I D W . G R A H A M * ,†,§ Department of Civil, Environmental, and Architectural Engineering, University of Kansas, Lawrence, Kansas, 66045, Department of Civil and Environmental Engineering, University of British Columbia, Vancouver, BC, Canada, and School of Civil Engineering and Geosciences, Newcastle University, Newcastle Upon Tyne, UK NE1 7RU

Received January 24, 2008. Revised manuscript received March 29, 2008. Accepted April 01, 2008.

Antibiotic resistance genes (ARGs) are emerging contaminants that are being found at elevated levels in sediments and other aquatic compartments in areas of intensive agricultural and urban activity. However, little quantitative data exist on the migration and attenuation of ARGs in natural ecosystems, which is central to predicting their fate after release into receiving waters. Here we examined the fate of tetracycline-resistance genes in bacterial hosts released in cattle feedlot wastewater using field-scale mesocosms to quantify ARG attenuation rate in the water column and also the migration of ARGs into peripheral biofilms. Feedlot wastewater was added to fifteen cylindrical 11.3-m3 mesocosms (some of which had artificial substrates) simulating five different receiving water conditions (in triplicate), and the abundance of six resistance genes (tet(O), tet(W), tet(M), tet(Q), tet(B), and tet(L)) and 16S-rRNA genes was monitored for 14 days. Mesocosm treatments were varied according to light supply, microbial supplements (via river water additions), and oxytetracycline (OTC) level. Firstorder water column disappearance coefficients (kd) for the sum of the six genes (tetR) were always higher in sunlight than in the dark (-0.72 d-1 and -0.51 d-1, respectively). However, water column kd varied among genes (tet(O) < tet(W) < tet(M) < tet(Q); tet(B) and tet(L) were below detection) and some genes, particularly tet(W), readily migrated into biofilms, suggesting that different genes be considered separately and peripheral compartments be included in future fate models. This work provides the first quantitative field data for modeling ARG fate in aquatic systems.

Introduction Antibiotic resistance genes (ARGs) recently have been identified as emerging contaminants (1); however, very little * Corresponding author phone: (44)-0-191-222-7930; fax: (44)-0191-222-6502; e-mail: [email protected]. † University of Kansas. ‡ University of British Columbia. § Newcastle University. # Currently at Tetra Tech EM Inc, 415 Oak Street, Kansas City, MO, USA 64106. 10.1021/es800238e CCC: $40.75

Published on Web 06/06/2008

 2008 American Chemical Society

is known about their disappearance kinetics or fate mechanisms after release into aquatic systems. Although elevated ARG levels have been seen in river sediments downstream of intensive agricultural and urban areas (2), how the genes move into the sediments, what proportion of genes that enter the river end up in peripheral compartments, and how cocontaminating antibiotics influence in situ ARG retention is largely unknown. Clearly, antibiotic resistance is increasing in bacteria across nature due to apparent overuse in medical and veterinary applications (3, 4), and alternative approaches to antibiotic management are needed. However, optimal mitigation strategies, either by changing antibiotic use patterns and-or by improving water management and treatment practices, are only possible once we understand better what regulates ARG fate and disappearance after release. A first step in establishing best management practices (BMPs) for ARGs is to determine what actually happens to ARGs after they enter receiving waters and then calibrate observed mechanisms for predictive modeling. This study was undertaken to quantify the fate of selected ARGs under different receiving water conditions, including the extent to which ARGs released into the water column migrate to peripheral compartments in aquatic systems. ARGs are believed to primarily enter receiving waters via fecal or related matter within bacterial hosts that have been previously exposed to elevated antibiotic levels, usually in higher organisms (3, 5, 6). However, it is not known whether bacterial hosts die in the water column, share ARGs with environmental species (e.g., by horizontal gene transfer), or migrate into peripheral compartments where similar fate processes occur. Engemann et al. (7) previously showed that tetracyclineresistance genes disappear rapidly in light-exposed aquatic systems in laboratory studies, but their systems did not have peripheral compartments nor were the results field-validated, which is the focus of this work. This issue is fundamental because, although rapid ARG disappearance appears to occur in water columns, genes simply may be moving into peripheral compartments where they might be harbored for long durations, which has been suggested by field monitoring data (1, 2). To address this issue, a novel experimental approach was employed using field mesocosms (containing retrievable artificial substrates for biofilm development), which were manipulated to simulate different receiving water conditions (8, 9). Mesocosm treatments varied according to light supply, microbial supplements (via river water additions), and oxytetracycline (OTC) supply, which are factors suspected to influence ARG fate in aquatic systems. Tetracycline resistance genes were chosen as biomarkers for resistance migration in this study because quantitative detection systems already exist for this class of genes (10, 11) and tetracyclines are often used in therapeutic and nontherapeutic applications in animal operations (12, 13). The specific genes quantified included tet(O), tet(W), tet(M), tet(Q), tet(B), and tet(L) because they are the six most common tetracycline resistance genes in current databases, being found in over 90% of identified resistant species (14, 15). Furthermore, this broad coverage allows the sum of these genes (tetR) to be used as a general marker for quantifying resistance in the environment. Finally, ambient water chemistry conditions were monitored to relate gene levels with water conditions, permit comparisons with past laboratory studies, and calibrate future models. VOL. 42, NO. 14, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Materials and Methods Experimental Design. The experiment design was based on previous laboratory data and mesocosm studies (7–9), and is summarized in Table S1 (see Supporting Information, SI). The mesocosms were flat-bottomed, fiberglass cylindrical tanks (1.3 m working depth, 11.3 m3) placed into a single shallow “host” pond (analogous to a water bath) located at Nelson Environmental Studies Area (NESA) located near Lawrence, Kansas. 15 tanks were used that represented five receiving water conditions (in triplicate), including: sunlight with no amendments (light), sunlight with 250 µg OTC/L levels (lightsOTC), sunlight with supplemental water from the Kansas River (lightsriver), “dark” conditions with no amendments (dark), and dark with river water addition (darksriver). The different light conditions were created by either draping porous window screen (light) or black vinyl plastic (dark) lids ∼20 cm above the water surface at the top of each tank (9). Screens were used to reduce light intensities in the light treatments (the tanks were rather shallow) and to minimize invasion problems with birds and other wildlife at the site. Mesocosm Preparation and Treatment Details. Mesocosms preparation was initiated by placing three 39 × 53 cm plastic trays containing uncontaminated sediments onto the bottom of each tank (solely to provide microbial inocula to the units (8)), and filling the tanks with water from a protected reservoir at NESA that had no previous direct exposure to anthropogenic ARG sources. The surrounding host pond was then filled with water, and the mesocosms were allowed to condition for five weeks. Three weeks before waste addition, 40 presterilized, 1.0 cm2 600 grit sand paper disks (attached to 12.5 × 8.5 cm plates) were suspended by fishing line near the bottom of four tanks (two each in the light and lightsOTC units) to provide retrievable surfaces for biofilm development. The lids were then placed on the tanks one week prior to waste addition with each lid being provided a resealable flapopening to allow access for sampling and other water manipulations. The experiment was initiated at dusk on June 22, 2005 by adding 18.9 L of river water to the light- and darksriver treatment tanks, and OTC to 250 ug/L to the lightsOTC units (additions were provided every 3 days thereafter; see Table SI1 for details). The following morning, 22.9 L of cattle feedlot lagoon wastewater was added to all 15 tanks at 9:00 a.m., tank waters were rapidly mixed with dedicated paddles, and the waters were allowed to equilibrate for one hour prior to initial sampling. Sampling proceeded for 14 days. A 21 day sampling program was originally planned (based on previous results (7)), but gene abundances had returned to near baseline levels after 14 days and the experiment was stopped early. Determining Lagoon Waste, River Water, and OTC Addition Rates. The amounts of lagoon waste and river water added to targeted units were determined by direct analysis of the wastewater feed and river water contents, whereas OTC additions were based on measured ambient OTC levels and OTC disappearance data (16, 17); i.e., OTC was provided as needed every 3 days to achieve a pseudo-steady level of 250 µg/L OTC (see Table S1). Wastewater addition was based on measured ARG and 16S-rRNA gene abundances (collected the previous day from the Kansas State University cattle research facility), which averaged: tet(O), 4.2 × 104;tet(W), 1.34 × 106; tet(M), 3.31 × 105; tet(Q), 6.2 × 104; tet(B), 2.9 × 103; tet(L) < 101; tetR, 1.78 × 106; and 16S-rRNA, 5.15 × 107 genes/mL, respectively. A 1:500 wastewater-to-tank water volume ratio (or 22.9 L of wastewater) was chosen to achieve initial tetR levels of ∼1.0 × 104 genes/mL, which experience indicated was adequate to observe differences among experimental treatments. The rate of river water addition was determined by quantifying tetR, 16S-rRNA gene, and tetracyclines levels in 5132

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a subset of Kansas River samples (n ) 10). The purpose of river water addition was to provide new organisms to some units (see Table S1) to assess how altering the gene pool within a mesocosm affected ARG fate. River water tetR and 16S-rRNA gene levels averaged 76.5 and 1.8 × 107 genes/mL, respectively, and no tetracyclines were detected in any river sample. Therefore, a supply rate of 6.3 L/day (i.e., 18.9 L every 3 days; see Table S1) was chosen because this rate provided 2.8 × 104 new 16S-rRNA genes/mL/day while only adding 0.1 additional tetR genes/mL/day. Sample Collection and Processing. Mesocosm Sampling and Processing: Water Chemistry. Samples for water chemistry were collected on Days 0, 1, 7, and 14 after waste addition. Dissolved oxygen (DO), pH, and water temperature were determined at three depths (0.3, 0.7, and 1.2 m) using a Water Checker Field Monitor (Horiba Instruments), and weighted averages were calculated for each tank per sample-day. Water column light intensities were quantified at depths 0.0, 0.5, and 1.0 m for each unit at midday (LI-COR spherical quantum sensor; LI185A, Lincoln, NE); however, only values from the 0.5 m depth reading were used for comparing treatments. Depth-integrated water column samples were collected for analysis of dissolved organic carbon (DOC), total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and chlorophyll a using 1.2 m length, 25 mm diameter PVC samplers (18). The collected samples were stored in sterile 500 mL acid-washed, amber glass bottles equipped with Teflon-lined caps, and temporarily placed on ice. Upon return to the laboratory, 100 mL of each sample was removed immediately for TOC, TN, and TP analysis; the remainder was used for other analyses. Specifically, 250 mL was separated and filtered through prerinsed Whatman GF/F glass-fiber filters (particle retention of >0.7 µm) for chlorophyll a, DOC, and OTC analyses. The filtrate was then divided into two 100 mL portions for duplicate OTC analysis and a 10 mL portion for DOC analysis, and the filter was retained for the analysis of chlorophyll a. Mesocosm Sampling and Processing: Molecular Biology. Samples for molecular biological analysis were collected on days 0, 1, 4, 7, and 14 for the water column, and days 0, 1, 4, 7, 12, and 14 for biofilms. Water column samples were collected separately from samples for chemical analysis; however, the same PVC samplers were used. Samples for molecular analysis were always collected first among samples each day. Typically, the samplers were rinsed with ethanol three times and once with tank water from the tank to be sampled. Then, 500 mL samples were rapidly withdrawn, transferred to sterile acid-washed 250 mL amber bottles, and duplicate 100 mL aliquots were filtered immediately through 47 mm diameter, presterilized 0.20 µm Nalgene filters (NNI, Rochester, NY). The filters were placed in sterile centrifuge tubes, frozen immediately on dry ice, and stored at -80 °C before further processing. Biofilm samples also were collected in duplicate by carefully drawing the disk-plates to the surface and rapidly, but aseptically (with sterile forceps), transferring the disks to presterilized 2 mL microcentrifuge tubes, which were immediately frozen on dry ice similar to the watercolumn filters. Analytical Methods. Oxytetracycline and Other Chemical Analysis. Free OTC (i.e., soluble) was measured using RIDASCREEN ELISA tetracycline detection kits (R-Biopharm, Darmstadt, Germany) and verified by LC-MS-MS as reported previously (11). Duplicate samples, spiked tank water samples (to determine matrix effects), and neat standards were performed routinely to verify the efficacy of the method. DOC and TOC were determined using a Dohrmann organic carbon analyzer. TP and TN were analyzed spectrophotometrically (Shimadzu UV-160) following wet digestion with potassium persulfate (19, 20) and alkaline persulfate (20, 21),

TABLE 1. Disappearance-rate coefficients (kd in d-1) for tetracycline resistance genes under the five field treatments compared with equivalent rate coefficients from previous flask experiments (7). tet(O) treatment light lightsOTC lightsriver dark darksriver

field -0.41 (0.06) -0.41 (0.07) -0.49 (0.06) -0.33 (0.06) -0.30 (0.10)

laboratory c

-0.27 (0.06) -0.30 (0.05) N/Ad -0.18 (0.09) N/A

tet(W) b

field -0.76 (0.09) -0.88 (0.11) -0.85 (0.09) -0.35 (0.10) -0.57 (0.11)

laboratory -0.66 (0.09) -0.69 (0.13) N/A -0.38 (0.09) N/A

tet(Q) field

laboratory

-0.79 (0.12) -0.62 (0.12) -0.72 (0.10) -0.57 (0.10) -0.57 (0.11)

-1.2 (0.09) -1.2 (0.09) N/A -0.66 (0.13) N/A

tetRa

tet(M) field -0.74 (0.07) -0.75 (0.07) -0.70 (0.08) -0.53 (0.06) -0.63 (0.07)

laboratory -0.99 (0.17) -0.81 (0.09) N/A -0.48 (0.10) N/A

field -0.71 (0.07) -0.67 (0.09) -0.77 (0.07) -0.47 (0.02) -0.55 (0.10)

laboratory -0.84 (0.10) -0.82 (0.12) N/A -0.49 N/A

a tetR is the sum of the four detected tet genes. tet(B) and tet(L) were below detection limits ( 0.10). Figure S1 presents mean abundance levels of individual genes within each treatment over the 14 day sampling period. tet(Q) was the most prominent detected ARG in the wastewater feed; however, it disappeared rapidly after release with a consistent shift in dominance to tet(W) as the dominant gene after four days. Table 1 indicates that tet(W) and tet(Q) had the highest kd among individual genes when light was VOL. 42, NO. 14, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Distribution of detected resistance genes over time in the water column and peripheral biofilms in the light treatment. Water column abundance equals water column concentration (genes/mL) multiplied by mesocosm volume, whereas biofilm abundance equals biofilm concentration (genes/cm2 disk area) multiplied by the sum the tank bottom and wall surface areas. Percentages refer to the proportion of detected genes in the biofilms (i.e., tetR) relative to all genes in the mesocosm. No water column samples were collected on day 12.

FIGURE 1. Water-column tetracycline resistance (tetR) and 16S-rRNA gene abundances over time for the five treatments. Note: (O), tetR is the sum of tet(M), tet(O), tet(Q), tet(W), tet(B), and tet(L) genes/mL per sample-day; (b), 16S-rRNA genes/mL per sample-day. Error bars show the range of measured values at each point. Trendlines indicate estimated disappearance curves assuming a first-order model. present, whereas tet(Q) and tet(M) had the highest kd under dark conditions. The lowest kd were seen for tet(O), which also had the least pronounced difference in kd between light and dark treatments. No rate data are provided for tet(B) and tet(L) because gene levels were always below detection limits (0.5 genes/mL). Migration of ARGs from the Water Column to Peripheral Biofilms. Sandpaper disks were suspended near the bottom of four of the light and lightsOTC treatment tanks, and biofilms were harvested over time from the disks for characterization of ARGs and 16S-rRNA genes. Figure 2 presents extrapolated ARG gene abundances for biofilms in a whole tank (calculated by multiplying detected gene levels per cm2 on the disks by the surface area of the tank walls and bottom) and equivalent abundances for the water column (determined by multiplying water column concentrations in genes/mL by the tank volume) for the two light units (lightsOTC results were similar, but not shown). The figure shows movement of ARG genes from the water column to biofilms, especially tet(W), which peaked four days after release. Interestingly, despite this distinct peak, only 2.8% of total tetR in the mesocosms were in the biofilms on day 4, and it was not until day 14 that >50% of total tetR genes in the system were biofilm-associated. 5134

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FIGURE 3. Absolute tetR abundances in the water column and peripheral biofilms in the light treatment over time. Water column gene levels were the calculated product of tank volume and water column gene concentration, whereas the biofilm gene level was the product of the tank surface area and aerial biofilm gene concentration. Trendlines indicate estimated disappearance curves for water column data only (- - - -) and water column minus biofilm data (ss), and assume a firstorder model. Associated first-order rate coefficients are inset. Although the migration of genes into the biofilms is noteworthy, it also was desired to assess whether gene migration altered previous kd estimates from water column data only (i.e., Table 1). Therefore, Figure 3 was developed to compare kd with and without gene migration to biofilms. The figure shows that absolute tetR disappearance is dominated by events prior to entering the biofilms. Specifically, kd changed by only ∼15% when biofilms were considered in the calculation. This does not mean that ARGs in biofilms are insignificant (field evidence indicates that genes can be harbored in sediments (1, 2)). However, it does suggest that water column events might dominate absolute ARG disappearance rate, which is important for modeling absolute ARG levels in receiving waters.

Discussion There is strong evidence that ARGs are accumulating in the environment due to anthropogenic activity (2, 11, 26); however, little quantitative data exist on the attenuation and

fate of such genes in aquatic systems (1). Recent studies have shown that ARGs can be elevated in sediments in areas of intensive agricultural and-or urban activity (2), although who their bacterial hosts are, and the rates and mechanism(s) by which ARGs enter and attenuate in such settings have not been defined. As such, determining the proportion of ARGs that enter a receiving water that degrade within the water column versus migrate to peripheral compartments is important because it influences modeling ARG fate in aquatic systems. It also will guide BMPs aimed at minimizing the impact of anthropogenic ARGs in the environment. Table 1 and Figure 1 corroborate past laboratory results, which showed that ambient light conditions dominate resistance gene disappearance rates in the water column (i.e., average kd for tetR were -0.72 and -0.51 d-1 in sunlight and dark, respectively), although we show here that other factors also influence rate. For example, although watercolumn disappearance rates were consistently higher in sunlight, the highest kd were actually seen when river water was also provided to the system (i.e., systems with microbial supplementation). Further, although dark conditions produced the lowest kd, river water addition in the dark also resulted in relatively elevated kd. In total, these data imply that light exposure with microbial supplementation results in the highest rates of tetR disappearance, which is consistent with previous speculation that photosynthesis-driven ecological pressure might controls tetracycline-resistance gene fate in the water column (7). Interestingly, OTC addition had no impact on the field kd over the exposure times assessed here, which is also consistent with previous laboratory data (7). Despite these general observations, kd patterns did differ among individual resistance genes. For example, kd for tet(W) was most impacted by differing light-dark and river water conditions relative to other genes, whereas kd of tet(O) was least affected (Table 1). Additionally, kd values were quite similar between the laboratory and field studies for tet(W), tet(M), tet(Q), and tetR. When one combines all laboratory and field data (Table 1), one can see clear differences in kd among genes; i.e., kd sort as tet(O) < tet(W) < tet(M) < tet(Q) with mean values of -0.32, -0.64, -0.70, and -0.79 d-1, respectively, where kd for tet(O) is significantly lower than for tet(W) (p < 0.05), and tet(M) and tet(Q) (p < 0.01). Whereas, kd for tet(W), tet(M), and tet(Q) do not differ significantly among each other, although trends are apparent. tet(W) became most prevalent in all treatments over time after release, implying it disappears least rapidly; whereas, tet(Q) always declined most rapidly relative to original levels in the lagoon waste (see ref 7 and Figure S1). Although specific reasons cannot be provided for all observations, some speculation is possible. For example, Figures S1 and 2 show that tet(W) is preferentially retained under all treatments and also tends to migrate to biofilms. Interestingly, the extended survival of tet(W) genes has been noted elsewhere (26, 27), especially where tet(W) gene-bearing organisms have moved from nutritionally rich to more dilute environments (7, 11). Therefore, our observation of tet(W) being relatively retained and tet(Q) being relatively lost is consistent with past work, and implies that different ARGs and their hosts are not equally promiscuous. In this case, the dominant gene switched from tet(Q) to tet(W) when the bacterial hosts moved from a concentrated waste environment to a dilute receiving water, suggesting that different conditions may favor one gene versus the other. Regardless, the apparent selection of tet(W) in some receiving waters might partially explain why this gene is among the most rapidly increasing resistance genes detected in all systems (6). The final implication of this study is presented in Figure 3, which shows that despite evidence of gene migration from

the water column to biofilms, the majority of the ARGs are still found in the water column up to 14 days after release. As such, we suggest that kd from water column data are likely good, but simple, markers for estimating ARG disappearance rates in receiving waters. This does not necessarily mean that peripheral compartments are insignificant as harbors for ARGs in the environment because field data suggest that ARGs can accumulate over time in sediments with repeated anthropogenic ARG exposure (2). However, our data suggest that gene “destruction” largely occurs in the water column prior to reaching peripheral compartments, which should be considered in BMPs for minimizing anthropogenic ARG levels prior to release. These results provide the first quantitative data for modeling ARG fate in the environment, which are corroborated by consistent laboratory and field data. Further, the results suggest new directions for work aimed at assessing ARG fate under different environmentally relevant scenarios, including the more realistic case where waste inputs are semicontinuous. It is possible that semicontinuous waste inputs might result in peripheral compartments becoming pseudo-steady harbors for ARG genes, although this must be proven. Regardless, field-validated data are now available that should underpin future work, hopefully suggesting techniques for minimizing ARG accumulation in the environment, including relatively simple methods such as maximizing light penetration in exposed systems.

Acknowledgments This research was supported by U.S. Environmental Protection Agency STAR Grant no. R82900801-0. We thank Teresa Lane, Katie Bloor, Marilyn Smith, and Susan Okalebo who assisted in different aspects of the project, and Jim Drouillard at Kansas State University for providing lagoon waste and general advice related to cattle feedlots. C.A.E. and P.L.K. contributed equally to this work.

Supporting Information Available Additional information on the experimental design and schedule (Table S1), water conditions in the five experimental treatments (Table S2), and the abundances of individual genes of time in the five treatments (Figure S1). This is material available free of charge via the Internet at http://pubs.acs.org.

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