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Influence of Dissolved Organic Matter on Tetracycline Bioavailability to an Antibiotic-Resistant Bacterium Zeyou Chen,†,‡ Yingjie Zhang,† Yanzheng Gao,†,‡ Stephen A. Boyd,† Dongqiang Zhu,§ and Hui Li*,† †

Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, Michigan 48824, United States Institute of Organic Contaminant Control and Soil Remediation, College of Resource and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China § State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210093, People’s Republic of China Downloaded by SUNY UPSTATE MEDICAL UNIV on September 4, 2015 | http://pubs.acs.org Publication Date (Web): August 26, 2015 | doi: 10.1021/acs.est.5b02158

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S Supporting Information *

ABSTRACT: Complexation of tetracycline with dissolved organic matter (DOM) in aqueous solution could alter the bioavailability of tetracycline to bacteria, thereby alleviating selective pressure for development of antibiotic resistance. In this study, an Escherichia coli whole-cell bioreporter construct with antibiotic resistance genes coupled to green fluorescence protein was exposed to tetracycline in the presence of DOM derived from humic acids. Complexation between tetracycline and DOM diminished tetracycline bioavailability to E. coli, as indicated by reduced expression of antibiotic resistance genes. Increasing DOM concentration resulted in decreasing bioavailability of tetracycline to the bioreporter. Freely dissolved tetracycline (not complexed with DOM) was identified as the major fraction responsible for the rate and magnitude of antibiotic resistance genes expressed. Furthermore, adsorption of DOM on bacterial cell surfaces inhibited tetracycline diffusion into the bioreporter cells. The magnitude of the inhibition was related to the amount of DOM adsorbed and tetracycline affinity for the DOM. These findings provide novel insights into the mechanisms by which the bioavailability of tetracycline antibiotics to bacteria is reduced by DOM present in water. Agricultural lands receiving livestock manures commonly have elevated levels of both DOM and antibiotics; the DOM could suppress the bioavailability of antibiotics, hence reducing selective pressure on bacteria for development of antibiotic resistance.

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INTRODUCTION Tetracycline antibiotics have been extensively used as therapeutic medicine for treatment of human and animal infections and as feed supplements at subtherapeutic levels for promoting livestock growth. According to a report of the U.S. Food and Drug Administration (FDA), in 2012, more than 5.9 million kg of tetracyclines were sold and distributed in the U.S. for use as therapeutic medicine and feed supplements in livestock, equivalent to 41% of the total antibiotics administered to animals.1 Most tetracyclines administered to animals are partially metabolized, and these metabolites along with parent compounds are discharged in the excreta. Land application of livestock wastes to agricultural fields introduces large quantities of tetracyclines into agroecosystems; tetracyclines are also directly sprayed into aquaculture ponds and onto fruit trees for disease control.2−9 As a result, tetracyclines are frequently found in multiple environmental media, including soil, surface water, and groundwater.4,10,11 Exposure of bacteria to tetracyclines in the environment poses threats to the ecosystem and human health. A major concern is that low levels of antibiotics in the environment © XXXX American Chemical Society

could exert selective pressure on indigenous microbial communities for development and proliferation of antibioticresistant bacteria.12−18 Tetracycline resistance genes were more frequently detected at sites where tetracyclines were present as a result of land application of livestock wastes and biosolids from wastewater treatment plants (WWTPs).19−22 Positive correlations between the abundance of sulfonamide resistance genes and concentrations of sulfonamides in WWTPs and rivers have been reported.23−26 However, the existence of such relations between the levels of tetracyclines and tetracycline resistance genes is less clear23,25−28 as a result of multiple confounding factors, such as horizontal gene transfer, coselection, and differential bioavailability of tetracyclines. The levels of tetracycline found in the environment are well below the thresholds required to exhibit apparently inhibitory effects on susceptible bacterial populations. However, the selective Received: April 29, 2015 Revised: August 6, 2015 Accepted: August 14, 2015

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DOI: 10.1021/acs.est.5b02158 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Downloaded by SUNY UPSTATE MEDICAL UNIV on September 4, 2015 | http://pubs.acs.org Publication Date (Web): August 26, 2015 | doi: 10.1021/acs.est.5b02158

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EXPERIMENTAL SECTION The Supporting Information provides the details about chemicals and materials used in this study. Elliott soil humic acid, Pahokee peat humic acid, and Waskish peat humic acid used to prepare the DOM solutions were purchased from the International Humic Substances Society (Table S1). Bacterial Cultivation. The Gram-negative E. coli bioreporter MC4100/pTGM used in this study was constructed by inserting tet(M) gene into plasmid pTGM, which contains transcriptional fusion between tetR-regulated Ptet promoter and flow cytometry-optimized gf p gene (gf pmut3) encoding green fluorescence protein (GFP).43 Tetracycline in the E. coli bioreporter cells could deactivate the tetR repressor protein and activate gf p gene transcription. The GFP translated from the expression of gf p gene emits a fluorescence signal, which could be measured by spectrofluorometer or flow cytometry. The E. coli bioreporter was incubated in lysogeny broth (LB) medium amended with 100 mg L−1 ampicillin at pH of 7.0 [in 50 mM 3(N-morpholino)propanesulfonic acid (MOPS) buffer] and 30 ± 0.2 °C for approximately 8 h to reach mid-log growth phase with an optical density at 600 nm (OD600) of ∼0.7. Measurement of Expression of Antibiotic Resistance. Tetracycline and humic acid were mixed in 10.0 mL of solution in autoclave-sterilized triangular flasks at room temperature (22 ± 0.5 °C) on a shaker (50 rpm) for 24 h in the dark, and then 10.0 mL of LB medium was added to the flask. Negligible amounts of tetracycline (if any) were found to be adsorbed by the flasks. The humic acid, tetracycline, and LB medium solutions passed through 0.22 μm Millex-GS membrane (Merck Millipore, Carrigtwohill, Ireland) prior to mixing. This operation was carried out in a superclean bench to minimize the potential of bacterial contamination. The mixtures were shaken for another 24 h to approach equilibration with tetracycline concentration of 12.5, 50, 100, and 200 μg L−1. The limit of quantification of the E. coli bioreporter was ∼5 μg L−1 for tetracycline. The DOM concentrations were 0, 5.0, 25, and 50 mg of C L−1 (prepared using the humic acids in this study). Then, the E. coli culture (0.2 mL) was added to the mixture of tetracycline−DOM−LB medium amended with 100 mg L−1 ampicillin and incubated on a shaker (150 rpm) at 30 ± 0.2 °C. All samples were prepared in triplicate. To measure the expression rate of antibiotic resistance at steady state, promoter activity was calculated for the E. coli bioreporter exposed to tetracycline in the presence and absence of DOM. To do so, 1 mL of the samples was collected from each treatment at 30 min intervals for 4 h, and the OD600 of the culture and the emitted fluorescence (excitation wavelength = 488 nm, and emission wavelength = 511 nm) were measured using a SpectraMax M2 spectrofluorometer (Molecular Devices, Sunnyvale, CA). The promoter activity (P, relative unit of GFP per OD unit per hour, RU OD−1 h−1) was quantified using P = fssμ(1 + μ/m), in which fss (RU OD−1) is the measured fluorescence emitted from the bioreporter at steady state, μ (h−1) is the bacterial growth rate constant, and m is the maturation constant for GFP (1.54 h−1 for gf pmut3).44 The fss values were estimated from the slope of the linear range of measured fluorescence intensity against OD600 (steady state). The growth rate constant was estimated from the slope of the natural logarithm of OD600 value versus time. This model for calculating promoter activity can circumvent the effects of dilution of GFP contents and GFP maturation during bacterial

pressure on indigenous bacterial populations exposed to these antibiotics could still result in an increase of antibiotic resistance genes and an enrichment of antibiotic-resistant bacterial strains, most likely via horizontal gene transfer between microorganisms and co-selection.29−34 Diminished bioavailability of tetracyclines to bacteria could lower the selective pressure and, hence, reduce their efficacy in promoting antibiotic resistance. Zhang et al. recently demonstrated that the same total tetracycline concentration in aqueous solutions with varying chemistries (e.g., different pH, metal cations, and organic ligands) could manifest differential expression of antibiotic resistance genes in Escherichia coli.35,36 These studies demonstrated that solution chemistry altered the fractional distribution of tetracycline species in solution, and thus modulated the bioavailability of tetracycline for bacterial uptake. Zwitterionic tetracycline was identified as being most favorable for bacterial uptake and most effectively evoked the expression of antibiotic resistance genes. Tetracycline sorption by soils reduces its bioavailability to bacteria, thereby lowering selective pressure on microbial communities. In one recent study, the presence of chlortetracycline did not enhance the abundance or preservation of tetracycline resistance genes in the soils amended with cattle feces, which is likely due to the strong sorption of tetracyclines by soils and/or the lack of direct contact between bacteria and soil-sorbed antibiotics.28 In natural environments, dissolved organic matter (DOM) is ubiquitous in water at the levels of milligram per liter, which can interact with antibiotics, such as tetracyclines, via cation bridging, complexation, cation exchange, and hydrogen bonding.37−41 It was estimated that, at pH 8.0, the presence of 5 mg L−1 humic acid could complex ca. 26% tetracycline (50 μg L−1) in water,42 which could significantly modulate the bioavailability to the surrounding microorganisms. Freely soluble versus DOM-bound tetracycline in aqueous solution could therefore exert varying selective pressure on bacteria for the development and preservation of antibiotic resistance in microbial communities. The objective of this study was to investigate the influence of DOM on the bioavailability of tetracycline by comparing the ability of freely soluble versus DOM-bound fractions to elicit the expression of antibiotic resistance genes in an E. coli bioreporter. We hypothesize that naturally occurring humic acid-derived DOM can modulate bioavailability of tetracycline by the formation of complexes. The adsorption of DOM on the E. coli outer membrane could reduce the rate of tetracycline uptake by the bacteria. Three humic acids were used as sources of DOM at environmentally relevant concentrations to examine their impact on bioavailability of tetracycline to the E. coli bioreporter. The magnitude of expression of antibiotic resistance genes was quantified as the mean fluorescence intensity per cell using flow cytometry, and the rate at steady state was estimated as promoter activity. These biological responses were then related to freely soluble versus DOMbound tetracycline in solution as well as tetracycline associated with the bioreporter. The results reveal that the presence of humic acids in water reduces the bioavailability of tetracycline to E. coli by formation of DOM−tetracycline complexes, and DOM adsorption on cell surfaces further retards tetracycline diffusion into the bacteria. B




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RESULTS AND DISCUSSION Tetracycline can enter the E. coli bioreporter cells, activate the antibiotic resistance genes, and evoke fluorescence emission by the GFP. Response of the bacterial antibiotic resistance genes to tetracycline was evaluated by promoter activity at steady state and emission of fluorescence. The effects of the three humic acids on the promoter activity and mean fluorescence emitted per E. coli bioreporter cell are shown in Figure 1. In

growth, which enables comparison of antibiotic resistance responses among different experimental settings.44 To measure the absolute fluorescence emitted by the bioreporter, after 4 h of incubation, the bacterial suspension (∼10 μL) was diluted in 3.0 mL of filtration-sterilized 10 mM phosphate-buffered solution (PBS, pH 7.4) and the emission of GFP fluorescence was quantified using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). The flow cytometer was equipped with an argon ion laser (488 nm) for excitation of GFP. Voltage was set at 400 V for side scatter (SSC), 575 V for detector FL1, and 650 V for forward scatter (FSC). A threshold of 400 was set on the SSC detector to eliminate small nonbacterial particles. Only cells with the defined sizes determined by tetracycline-free control were analyzed for GFP emission by rectangular sort gate in bivariate SSC versus FSC dot plots. The mean fluorescence was the average of emission of 10 000 cells within the range of the FL1 detector. Data analysis was performed with the BD FACSSDiva software. Tetracycline Analysis in Solution and in Bacteria. The tetracycline concentration in DOM solution was determined using a solid-phase extraction method, which could separate freely dissolved tetracycline from DOM-complexed tetracycline.42 A hydrophilic−lipophilic balance (HLB) cartridge (Waters Corporation, Milford, MA) was preconditioned by sequential rinses with methanol (3 mL), 0.1 M HCl (3 mL), and water (6 mL). After the sample was collected for analysis by a flow cytometer, the bacteria were separated from the culture media by centrifugation at 15 000 g for 30 min at 4 °C. The supernatant (5.0 mL) passed through the preconditioned HLB cartridge to extract freely soluble tetracycline in solution (sorption by the cartridge), and the DOM−tetracycline complexes eluted through the cartridge with humic acids.42 Tetracycline retained by the cartridge was eluted with 5.0 mL of methanol/water solution (1:1, v/v) containing 150 mg L−1 ethylenediaminetetraacetic acid (EDTA) and then with an additional 5.0 mL of methanol containing 1% (v/v) formic acid. The eluted solutions were combined and quantified for the tetracycline concentration using matrix-matched standards by a Shimadzu high-performance liquid chromatography fully integrated with a Sciex 3200 triple quadrupole mass spectrometer (LC−MS/MS). In the LC−MS/MS, a C18 column (Gemini, 5 μm, 50 × 2.0 mm, Phenomenex, Inc., Torrance, CA) was used with a flow rate at 0.35 mL min−1. Tetracycline was quantified in multiple reaction monitoring mode with a precursor/product ion transition of m/z 445.4/ 410.0, and two other pairs of precursor/product transitions (m/ z 445.4/428.2 and 445.4/339.3) were used for confirmation of tetracycline fingerprints. No apparent matrix effect was observed in the quantification of tetracycline in DOM−LB media after the treatment with the HLB cartridge. The settled bacterial cell pellets after centrifugation were collected and rinsed twice with PBS solution. The bacterial cells were resuspended in 10 mL of 0.1 M McIlvaine buffer/0.1 M EDTA (pH 4.0) by vortex for 1 min, followed by sonication for 10 min and centrifugation at 150 000 g for 15 min. McIlvaine buffer/EDTA could disrupt bacterial membranes and, hence, dissolve tetracycline in the extracts. This extraction step was repeated, and the extracts were combined, cleaned up using HLB cartridge, and quantitated using LC−MS/MS (described above). The measured tetracycline was normalized on the basis of bacterial biomass obtained using the relationship between OD600 and dry bacterial biomass.

Figure 1. Bacterial antibiotic resistance response from E. coli bioreporter exposed to tetracycline in the presence of different types of dissolved humic acids. The DOM concentration was 50 mg of C L−1, and pH was 7.0. At the same tetracycline concentration, an asterisk (∗) represents a significant difference between DOM versus DOM-free treatments (p < 0.05). Error bars represent standard deviations of triplicate samples.

these experiments, the LB media contained 50 mg of C L−1 (from humic acids) and different levels of tetracycline. Both mean fluorescence and promoter activity increased with increasing the tetracycline concentration. For example, in the absence of DOM, the promoter activity was 177 ± 6, 519 ± 29, 931 ± 32, and 1295 ± 161 RU OD−1 h−1 when the bioreporter was exposed to 12.5, 50, 100, and 200 μg L−1 tetracycline. The corresponding mean emitted fluorescence per cell was 1943 ± 135, 17 165 ± 984, 34 500 ± 213, and 49 029 ± 1765. These results establish that increasing tetracycline concentrations manifest enhanced bacterial antibiotic resistance responses. The presence of DOM (as humic acid) at 50 mg of C L−1 substantially reduced the bacterial antibiotic resistance response (p < 0.05). In media containing 200 μg L−1 tetracycline, the promoter activity decreased from 1295 ± 161 (DOM-free C


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Environmental Science & Technology control) to 941 ± 19, 748 ± 57, and 553 ± 69 RU OD−1 h−1 in the presence of Waskish Peat, Pahokee Peat, and Elliott soil humic acids, respectively. The corresponding mean fluorescence values also significantly decreased from 49 029 ± 1765 to 46 149 ± 477, 42 834 ± 243, and 41 209 ± 259 (p < 0.05). The reduction in the promoter activity and mean fluorescence emitted from the E. coli bioreporter exposed to the DOM− tetracycline mixture followed the order of DOM-free > Waskish Peat > Pahokee Peat > Elliott soil humic acids. It was noted that, in the tetracycline-free control, the estimated promoter activity was 130 RU OD−1 h−1, which could be due to bacterial background fluorescence. A similar phenomenon has been reported for this E. coli bioreporter construct when used to quantify bioavailable tetracycline in the environment.43 The presence of humic acids in tetracycline-free controls reduced fluorescence intensity as a result of the quenching effects from humic acids. The contribution of such quenching effects was minimal compared to the GFP florescence emitted from the E. coli bioreporter when exposed to tetracycline. Furthermore, the fss value in the calculation of promoter activity was obtained from the slope of linear fitting between fluorescence intensity and OD600, which negates the quenching effects of DOM in the background. Decreased tetracycline bioavailability to E. coli also occurred with increasing the humic acid concentration. Using the Elliott soil humic acid as a representative DOM (Figure 2), increasing DOM concentrations of 5.0, 25, and 50 mg of C L−1 in media containing 200 μg L−1 tetracycline resulted in reductions of mean emitted fluorescence intensity per cell by 5.3, 8.9, and 12% and promoter activity by 16, 39, and 60% compared to the humic acid-free control. These results indicate that DOM at the levels relevant to natural waters (i.e., ≤50 mg L−1) reduced tetracycline bioavailability to bacteria, which potentially diminishes selective pressure for development and enrichment of antibiotic-resistant bacteria. Decreased tetracycline bioavailability can be attributed to the complexation of tetracycline with DOM. In comparison to tetracycline freely dissolved in bulk solution, DOM−tetracycline complexes are plausibly less bioavailable for bacterial uptake, thereby diminishing the risks of tetracycline (when associated with humic acids) from the perspective of propagation of antibiotic resistance. To further evaluate whether DOM−tetracycline complexation induced reductions in the bioavailability of tetracycline to E. coli, the freely soluble and DOM-bound tetracycline fractions in LB medium were quantified using a recently developed solidphase extraction method.42 Freely soluble tetracycline is defined as the fraction of tetracycline dissolved in water and not bound to humic acid. Tetracycline (100 μg L−1) and the three humic acids (at 5.0, 25, and 50 mg of C L−1) were mixed and allowed to approach equilibrium (48 h) in LB medium. The mixtures were then filtered through HLB solid-phase cartridges, which retain freely soluble tetracycline while allowing for humic-acid-bound tetracycline to pass through the cartridges.42 The cartridge-retained tetracycline was eluted and quantified by LC−MS/MS, which is referred to as freely dissolved tetracycline. Tetracycline bound to DOM was calculated by the difference between tetracycline initially added and freely soluble tetracycline. All three humic acids used as the sources of DOM manifested apparent affinity for tetracycline in the order: Waskish peat < Pahokee peat < Elliott soil (Figure S1). The corresponding sorption coefficients were 2750, 6732, and 12 036 L kg−1 of humic acid. Increasing humic acid concentration in LB media rendered more tetracycline

Figure 2. Bacterial antibiotic resistance response from E. coli bioreporter exposed to tetracycline in the presence of different concentrations of Elliott soil humic acid at pH of 7.0. At the same tetracycline concentration, an asterisk (∗) represents a significant difference between DOM versus DOM-free treatments (p < 0.05). Error bars represent standard deviations of triplicate samples.

bound to DOM. For the Elliott soil humic acid mixed with 100 μg L−1 tetracycline solution, approximately 3.4, 26.2, and 40.2% of the total tetracycline was bound to DOM at the DOM concentrations of 5.0, 25, and 50 mg of C L−1, respectively. The Waskish peat humic acid demonstrated a comparatively lower affinity for tetracycline, with Pahokee peat > Waskish peat humic acids (Figure 4A). For the Elliott soil humic acid, decreasing DOM concentration from 50 to 5 mg of C L−1 manifested an apparent increase in promoter activity partially as a result of the presence of more freely soluble tetracycline in the solution (Figure 4B). For the Waskish peat humic acid with the least capability to complex with tetracycline, the increasing DOM concentration did not substantially alter the freely dissolved tetracycline concentration. However, reduced promoter activity was observed (Figure 4C). As the humic acid concentration decreased, the promoter activity gradually approached the curve in the absence of humic acid (Figure 4C). This could be due to adsorption of humic acids from water on the outer membrane

Figure 4. Relationship between the freely dissolved tetracycline concentration (unbound to DOM) and promoter activity in the systems of (A) three tested humic acids at 50 mg of C L−1, (B) Elliott soil humic acid, and (C) Waskish peat humic acid at varying DOM concentrations (pH 7.0).

of the bioreporter bacterial cells, which could diminish the uptake rate of tetracycline. The promoter activity obtained using the model by Leveau and Lindow44 represents the overall rate of tetracycline diffusion into bacterial cells, interaction with ribosomal protection proteins, and activation of gf p gene transcription during steady-state bacterial growth. The steady state is referred to the period during which the emitted fluorescence is linearly related to bacterial numbers (OD600 values in this study). Among these processes, diffusion is the rate-limiting step for intracellular accumulation of tetracycline and interaction with tetR in E. coli.45 The intracellular tetracycline concentration determines the extent of evoked antibiotic resistance bioresponse.35 Adsorption of DOM on the bacterial outer membrane could function as a barrier to tetracycline diffusion into the bioreporter. In addition, the humic acids adsorbed on bacterial surfaces can also sorb tetracycline, which further retards the movement of tetracycline into cells. These factors could function collectively, leading to the decreased promoter activity. To test this hypothesis, adsorption of the three humic E


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accumulation of tetracycline in bacteria and, hence, does not affect the fluorescence intensity. To further assess this interpretation, the amount of tetracycline associated with the E. coli bioreporter cells was measured in the cultures with and without humic acid. In the experimental systems with humic acids, the measured tetracycline concentration included both intracellular tetracycline and tetracycline complexed with the humic acids adsorbed on the cell surfaces. The measurements revealed that, at a given freely soluble tetracycline concentration, a greater amount of tetracycline was associated with the bacteria in the presence of DOM (50 mg of C L−1) compared to the DOM-free controls (Figure 6A). Among the three DOMs tested, the Elliott soil


acids by the bioreporter cells was measured at pH 7.0 (detailed in the Supporting Information). It was observed that adsorption by the bioreporter followed the order: Elliott soil > Pahokee peat > Waskish peat humic acids (Figure S2). The adsorbed humic acids could interact with tetracycline and slow its penetration into the cells. Recall that, among the three humic acids studied, the Elliott soil humic acid was both adsorbed strongest by bacterial cells and demonstrated the greatest complexation with tetracycline, followed by Pahokee peat and Waskish peat humic acids (Figure S1). The mean fluorescence measured by flow cytometry represents the average bioresponse normalized on the basis of one cell. These mean fluorescence values, including those obtained from the combinations of different tetracycline concentrations and the three sources of DOMs at several concentrations, were plotted against the corresponding freely soluble tetracycline concentration (Figure 5). These data

Figure 5. Relationship between the freely dissolved tetracycline concentration and mean fluorescence per cell in the systems of all tetracycline and DOM concentrations tested in this study.

showed that the mean fluorescence intensity increased with increasing the freely soluble tetracycline concentration. The mean fluorescence intensity emitted from the bioreporter in the presence of DOM was almost coincident with the relation for DOM-free samples when plotted as a function of the freely dissolved tetracycline concentration (Figure 5), suggesting that DOM did not influence the resultant mean fluorescence. These results again confirm that freely soluble tetracycline is the predominant bioavailable species evoking the response of tetracycline resistance genes in the bioreporter. Unlike the promoter activity, which diminished in the presence of DOM, a specific concentration of freely soluble tetracycline resulted in the similar fluorescence emission, regardless of the presence or absence of DOM. It should be noted that the mean fluorescence intensity was measured at the end of the experiment, which reflects the collective GFP content produced by the E. coli bioreporter during the experimental period. The promoter activity primarily describes the overall rate of activation of antibiotic resistance genes during steady state. Adsorption of DOM on bacterial outer membranes could retard the diffusion of tetracycline into bioreporter cells, as reflected by the decrease in promoter activity during steady state. However, it might not diminish the overall intracellular

Figure 6. Tetracycline concentration associated with E. coli cells in the presence of (A) humic acid at 50 mg of C L−1 and (B) Elloitt soil humic acid at 5.0, 25, and 50 mg of C L−1. Error bars represent standard deviations of triplicate samples.

humic acid generally demonstrated the greatest amount of tetracycline associated with the bioreporter, followed by Pahokee peat and Waskish peat humic acids (Figure 6A). Decreasing the Elliott soil humic acid concentration reduced the amount of tetracycline associated with the bioreporter (Figure 6B). The strong affinity between tetracycline and Elliott soil humic acid (Figure S1) and the greater adsorption by bacteria (Figure S2) are responsible for the enhanced tetracycline associated with the bacteria. However, the higher F


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(5) Hamscher, G.; Sczesny, S.; Hoper, H.; Nau, H. Determination of persistent tetracycline resiues in soil fertilized with liquiud manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Anal. Chem. 2002, 74, 1509−1518. (6) Fahrenfeld, N.; Knowlton, K.; Krometis, L. A.; Hession, W. C.; Xia, K.; Lipscomb, E.; Libuit, K.; Green, B. L.; Pruden, A. Effect of manure application on abundance of antibiotic resistance genes and their attenuation rates in Soil: Field-scale mass balance approach. Environ. Sci. Technol. 2014, 48, 2643−2650. (7) Marti, R.; Tien, Y.-C.; Murray, R.; Scott, A.; Sabourin, L.; Topp, E. Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure? Appl. Environ. Microbio. 2014, 80, 3258−3265. (8) McManus, P. S.; Stockwell, V. O.; Sundin, G. W.; Jones, A. L. Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 2002, 40, 443−465. (9) Seyfried, E. E.; Newton, R. J.; Rubert, K. F.; Pedersen, J. A.; McMahon, K. D. Occurrence of tetracycline resistance genes in aquaculture facilities with varying use of oxytetracycline. Microb. Ecol. 2010, 59, 799−807. (10) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999− 2000: A national reconnaissance. Environ. Sci. Technol. 2002, 36, 1202−1211. (11) Gottschall, N.; Topp, E.; Metcalfe, C.; Edwards, M.; Payne, M.; Kleywegt, S.; Russell, P.; Lapen, D. R. Pharmaceutical and personal care products in groundwater, subsurface drainage, soil, and wheat grain, following a high single application of municipal biosolids to a field. Chemosphere 2012, 87, 194−203. (12) Laxminarayan, R.; Duse, A.; Wattal, C.; Zaidi, A. K.; Wertheim, H. F.; Sumpradit, N.; Vlieghe, E.; Hara, G. L.; Gould, I. M.; Goossens, H. Antibiotic resistance-the need for global solutions. Lancet Infect. Dis. 2013, 13, 1057−1098. (13) Davies, J.; Davies, D. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. R 2010, 74, 417−433. (14) Landers, T. F.; Cohen, B.; Wittum, T. E.; Larson, E. L. A Review of antibiotic use in food animals: Perspective, policy, and potential. Public Health Rep. 2012, 127, 4−22. (15) Rysz, M.; Mansfield, W. R.; Fortner, J. D.; Alvarez, P. J. J. Tetracycline resistance gene maintenance under varying bacterial growth rate, substrate and oxygen availability, and tetracycline concentration. Environ. Sci. Technol. 2013, 47, 6995−7001. (16) Ghosh, S.; LaPara, T. M. The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria. ISME J. 2007, 1, 191−203. (17) Knapp, C. W.; Engemann, C. A.; Hanson, M. L.; Keen, P. L.; Hall, K. J.; Graham, D. W. Indirect evidence of transposon-mediated selection of antibiotic resistance genes in aquatic systems at low-level oxytetracycline exposures. Environ. Sci. Technol. 2008, 42, 5348−5353. (18) Munoz-Aguayo, J.; Lang, K. S.; LaPara, T. M.; González, G.; Singer, R. S. Evaluating the effects of chlortetracycline on the proliferation of antibiotic-resistant bacteria in a simulated river water ecosystem. Appl. Environ. Microbio. 2007, 73, 5421−5425. (19) Schmitt, H.; Stoob, K.; Hamscher, G.; Smit, E.; Seinen, W. Tetracyclines and tetracycline resistance in agricultural soils: Microcosm and field studies. Microb. Ecol. 2006, 51, 267−276. (20) Peak, N.; Knapp, C. W.; Yang, R. K.; Hanfelt, M. M.; Smith, M. S.; Aga, D. S.; Graham, D. W. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ. Microbiol. 2007, 9, 143−151. (21) 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. Microbio. 2001, 67, 1494−1502. (22) Pei, R. T.; Kim, S. C.; Carlson, K. H.; Pruden, A. Effect of river landscape on the sediment concentrations of antibiotics and

tetracycline concentration (associated with the bioreporter) did not lead to increased bioresponse. Rather, reduced promoter activity in the treatments containing humic acids (Figure 4) indicates that considerable fractions of tetracycline could be complexed with the humic acids on the bacterial outer membrane. Humic acids have been shown to be adsorbed on E. coli cell surfaces in many previous studies.46,47 To evoke fluorescence emission, tetracycline has to enter the bacterial cells and activate the resistance genes. The DOM adsorbed on bioreporter cells could function as a barrier to the transport of tetracycline into the cells, thereby diminishing uptake rate by E. coli, as evidenced by the decreased promoter activity. The mean emitted fluorescence was dependent upon the freely soluble tetracycline concentration rather than the amount or type of DOM present in solution, suggesting that humic acid in the solution may not affect the intracellular tetracycline concentration. The complexation of tetracycline with humic acid diminishes the fraction of freely soluble tetracycline in solution, thereby decreasing the bioavailability.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b02158. Details of chemicals and humic acids used in this study, percentage of tetracycline bound to humic acids, and adsorption of humic acids by the E. coli bioreporter (Table S1 and Figures S1 and S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 517-775-9894. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by the National Science Foundation (CBET-1438105), the National Science Foundation of China (21428701 and 21225729), and Michigan AgBioResearch. The authors thank Dr. Søren J. Sørensen at the University of Copenhagen for generously providing the E. coli strain MC4100/pTGM bioreporter.

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REFERENCES

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