Distinguishing Effects of Ultraviolet Exposure and Chlorination on the

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Distinguishing Effects of Ultraviolet Exposure and Chlorination on the Horizontal Transfer of Antibiotic Resistance Genes in Municipal Wastewater Mei-Ting Guo,*,† Qing-Bin Yuan,† and Jian Yang State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, 200092 Shanghai, China S Supporting Information *

ABSTRACT: Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities; however, the disinfection processes, as microbial control technologies, have not been evaluated for their impacts on ARGs transfer. In this study, the effects of ultraviolet (UV) disinfection and chlorination on the frequency of ARGs transfer have been explored based on the conjugative transfer model between Gram-negative strains of E. coli. The results indicated that UV disinfection and chlorination exhibit distinct influences on the conjugative transfer. Low UV doses (up to 8 mJ/cm2) had little influence on the frequency of conjugative transfer, and UV exposure only decreased the bacterial number but did not change the cell permeability. By comparison, low chlorine doses (up to 40 mg Cl min/L) significantly promoted the frequency of conjugative transfer by 2−5-fold. The generated chloramine stimulated the bacteria and improved the cell permeability. More pilus were induced on the surface of conjugative cells, which acted as pathways for ARGs transfer. The frequency of ARG transfers was greatly suppressed by high doses of UV (>10 mJ/cm2) or chlorine (>80 mg Cl min/L).

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INTRODUCTION The constant emergence of antibiotic resistance is becoming a major threat to global public health. Over the years, almost all kinds of antibiotic resistance genes (ARGs) have been detected in various environments, including soil, sewage, drinking water, and even air.1−3 Therefore, ARGs are treated as emerging contaminants, and a global strategy is called for to face the problem.4 Wastewater treatment plants (WWTPs) directly receive wastewater containing many antibiotics and ARGs and are thus considered as important reservoirs of antibiotic resistance in the environment.5−9 Horizontal gene transfer (HGT) is an essential step for competitive bacterial survival in the environment and is also considered to be one of major drivers for ARGs transfer.10 The three major mechanisms by which bacteria transfer genes horizontally are conjugation within bacteria, natural transformation, and transduction,11 with conjugative transfer within bacteria being the most studied. The ARGs acquisition rate by HGT can possibly be affected by various environmental conditions, such as temperature, pH, antibiotic concentration, etc.12,13 Many studies have been conducted to explore the ARGs transfer characteristics in laboratory broth media.12,14,15 Nevertheless, evidence of their transfer in wastewater is not frequently reported probably because of complex environmental matrices.16 © XXXX American Chemical Society

WWTPs provide abundant nutrients and antibiotics residuals, high bacterial concentrations, and excellent mixing conditions, which are believed suitable for plasmids containing ARGs transfer. Marcinek et al. reported that gene transfer by conjugation could take place under actual conditions in WWTPs, and the transfer events were up to 105−108 in 4 h.17 The transfer between bacteria in activated sludge and laboratory strains or pathogens was also observed.18,19 Because of a high bacterial community density, activated sludge in WWTPs was reported to promote significantly the frequency of ARGs conjugative transfer.20,21 By contrast, other treatment processes, especially disinfection units, are rarely explored for their effects on the ARGs transfer. Wastewater treatment processes are not able to remove effectively antibiotic resistant bacteria (ARB) and ARGs in wastewater;22−24 therefore, disinfection facilities are considered fairly important to promote effluent security after traditional treatment processes.25−27 The effects of ultraviolet (UV) disinfection and chlorination on the removal of ARB and ARGs in various WWTPs have been widely investigated.25,26,28,29 Although probably causing the reduction of Received: February 5, 2015 Revised: April 1, 2015 Accepted: April 7, 2015

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DOI: 10.1021/acs.est.5b00644 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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mg/L, respectively. The total viable bacteria number density was 3 × 103−6 × 105 CFU/mL. The wastewater was filtered by a 0.22 μm filter and adopted as the mating medium for conjugative transfer. To establish the conjugative transfer model for the following experiment, four influencing factors, including bacterial concentration, donor/recipient ratio, mating time, and nutrients [organics (expressed by COD), nitrogen (N), and phosphate (P)], were optimized by single factor experiment. The single factor experimental scheme is shown in Table S1 (Supporting Information). Conjugation Experiment Treated with Disinfection. A broth mating method was modified and adopted in the conjugation experiment, according to a previous publication.12 In detail, the donor and recipient cultures (109 CFU/mL each) were mixed in the same volume (1.5 mL each), centrifuged at 4500 × g for 10 min, and then resuspended in 3 mL of phosphate-buffered saline (PBS, pH = 7.4). The mixtures were added to 150 mL filtered sewage containing nutrients at a specific concentration and then fully mixed. For UV disinfection, each 20 mL sample was transferred to a Petri dish (diameter: 90 mm). A low-pressure (LP) collimated beam apparatus was employed to conduct the UV disinfection process. This apparatus contains a LP (120 W, 30% UVC, TL 120W/01, Philips) mercury UV lamp. More details of the disinfection apparatus used are described in our previous publication.25 The irradiance values were fixed at 0.17 mW/cm2 throughout the experiment, while the exposure time was changed for various plates to reach UV doses of 1, 2, 4, 8, 10, 20, and 40 mJ/cm2, respectively. For chlorination, each 20 mL volume was transferred to sterilized Erlenmeyer flasks (100 mL), and 3.38 mM sodium hypochlorite was added to achieve different initial concentrations of chlorine (0.5, 1, 2, 4, 8, and 16 mg active Cl/L) for a contact time of 10 min. The CT value (the product of initial active chlorine concentration and contact time) was used to express the chlorine dose, with values of 5, 10, 20, 40, 80, and 160 mg active Cl min/L adopted in the study, respectively. The pH was monitored during the process to maintain it at 7.0. Five mL of each chlorinated sample was then applied for the determination of residual chlorine (Residual chlorine analyzer, HACH), while 44, 88, 176, 352, and 704 μL of a sodium thiosulfate solution (0.5%) were added respectively to the remainder to terminate chlorination. Three mL of disinfected samples by UV or chlorine were removed to determine the numbers of donors and recipients, while the remaining were then incubated at 25 °C and shaken at 150 rpm to mate. Samples without disinfection were also processed as controls. After a 12 h mating period, the cultures were appropriately diluted and then plated on LB medium containing the appropriate antibiotics to determine the numbers of donors, recipients, and transconjugants. In detail, after 24 h of incubation at 37 °C, the numbers of donors and recipients were determined by counting colonies on LB agar [tryptone: 10 g/L, yeast extract: 5 g/L, NaCl: 10 g/L, agar: 10 g/L, pH: 7.4] supplemented with 32 mg/L tetracycline (for donors) or 160 mg/L rifampicin and 50 mg/L nalidixic acid (for recipients), respectively. Transconjugants were selected and counted on LB agar plates containing 32 mg/L tetracycline, 160 mg/L rifampicin, and 50 mg/L nalidixic acid. All of the conjugation experiments were conducted in triplicates. Analytical Methods. The determinations of COD, NH4+N, and TP were conducted in accordance with the Standard

ARB or ARGs concentrations, the general observation is that UV or chlorine cannot completely eliminate antibiotic resistance,5,7,24,29 so that the risk of ARGs transfer in the final effluent still exists. Therefore, it is essential to explore how the disinfection process affects the ARGs transfer efficiency, so as to better evaluate the subsequent potential risk of ARGs spread and transfer. Unfortunately, to the best of our knowledge, studies in this area are rather sparse. The object of this study is to investigate the effects of UV disinfection and chlorination on the ARG conjugative transfer in wastewater. First, antibiotic resistant E. coli strains were isolated from wastewater and tested for their availability as potential ARG donors, and then the model of conjugative transfer in wastewater was established by optimizing four keyinfluencing factors, including bacterial concentration, donor/ recipient ratio, mating time, and nutrient. Second, the frequency of the conjugative transfer within bacteria was studied in wastewater after treatment by UV disinfection or chlorination at various doses. Third, the mechanisms that underlie this process were explored with regard to inactivation of donor/recipient and cell membrane changes caused by UV disinfection and chlorination.

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MATERIALS AND METHODS Bacterial Strains and Plasmids. To obtain a potential donor from wastewater for conjugative transfer, three kinds of candidate E. coli strains resistant to tetracycline, gentamycin, and erythromycin, were isolated from Q WWTP in Shanghai. After antibiotic resistance exploration, the strain resistant to tetracycline was used as the donor strain (Supporting Information, Text 1). The strain was then identified to be E. coli K12 (accession number: NR 102804.1), harboring the plasmid RP4 (accession number: L27758). The presence of the plasmid RP4 in E. coli K12 and transconjugants was confirmed as follows. Pure cultured E. coli K12 and transconjugant colonies (5−10 colonies) were randomly selected from plates of maximal dilution. The plasmid DNA was extracted and purified using the MiniBest Plasmid Purification Kit (TaKaRa, Japan) and then digested with the restriction enzyme SphI, and fragments were separated by horizontal gel electrophoresis on a 0.8% (w/v) agarose gel. To further identify the presence of RP4, the RP4-specific traGlike gene in the extracted plasmid DNA was detected by PCR; and the details are described in a previous publication.18 The recipient strain was E. coli NK5449 [CGMCC NO. 1.1437, obtained from China General Microbiological Culture Collection Center (CGMCC)], which encodes high-level resistance to rifampicin (>160 mg/L) and nalidixic acid (>50 mg/L), and is commonly adopted as an ARGs recipient.30 The recipient strain bacteria were first tested for their inability to grow on tetracycline (32 mg/L) containing plates, as the antibiotic was used as a selective marker to detect transconjugants. Optimization of Influencing Factors on Conjugative Transfer. To simulate the disinfection process, the wastewater prior to UV disinfection in Q WWTP in Shanghai was collected. The principal biological process of the plant is the anaerobic-anoxic-oxic (A2/O) process. Biological aerated filter (BAF) is adopted as an advanced treatment process before the final treatment of UV disinfection. More details of the WWTP were introduced in our previous publication.25 The pH, A254, COD, SS, and NH4+-N of the collected wastewater were 6.8− 7.2, 0.12−0.15, 28.3−71.5 mg/L, 8.2−13.4 mg/L, and 3.2−11.0 B

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Figure 1. Effects of a single factor on the conjugative transfer. (A) The effect of bacterial concentration and donor/recipient ratio on the conjugative transfer at 18-h mating time and no extra nutrients added. (B) The effect of nutrient (organics) on the conjugative transfer at 107 CFU/mL bacteria, donor/recipient ratio = 1:1, and 18-h mating time. Glucose, α-lactose, and NaAc represented three kinds of organic nutrients adopted for conjugative transfer. (C) The effect of ammonia nitrogen and phosphate on the conjugative transfer at 107 CFU/mL bacteria, donor/recipient ratio = 1:1, 18-h mating time, and glucose added as the organic nutrient. (D) The effect of mating time on the conjugative transfer at 107 CFU/mL bacteria, donor/ recipient ratio = 1:1, and no extra nutrients added.

Methods.24 Lactate dehydrogenase (LDH) release of the donor and recipient was used to evaluate the change in cell permeability; the method is detailed in the Supporting Information (Text 2). Transmission electron microscopy (TEM, JEOL 2011, Japan) was used to analyze the effect of chlorination on the microstructure and morphology of the conjugative cells, and the detail is described in the Supporting Information (Text 3). Data Analysis. Referring to previous publications,13,21 the frequency of conjugative transfer was expressed as the numbers of transconjugants cells per recipient cells, as shown in eq 1.

conjugative transfer model for the following experiments (Figure 1). The frequency of conjugative transfer was not significantly influenced by high donor or recipient concentration over 105 CFU/mL, but it was greatly suppressed when the bacterial concentration was below 104 CFU/mL, where almost no transconjugant was detected. It indicated that a threshold of the bacterial number for conjugation might exist. The general bacterial concentration adopted in previous conjugative transfer experiments was generally up to 107−109 CFU/mL.31,32 Apparently a high level of bacterial concentration in wastewater is a benefit for conjugative transfer. This seemed to be reasonable, since conjugation was reported to require cell-to-cell contact to form a pilus or pore that allows for the passage of plasmids.11 Higher bacterial concentration means more opportunities for the contact of bacteria. In WWTPs, the highest bacterial concentration is generally found in activated sludge, which was basically 105−108 CFU/mL, indicating a high possibility of plasmid transfer, just as some researchers had explored.18,19,21 Nevertheless, in real wastewater a bacterial concentration below 104 CFU/mL (e.g., the effluent after biological treatment or disinfection) does not signify impossibility of conjugative transfer, since many potential donors and recipients exist in actual wastewater,21 which might increase the pathways of conjugative transfer and then decrease the threshold concentration. By comparison, the donor/recipient ratio at any level (from 1:1 to 1:7) had no significant effect on the conjugative transfer (Figure 1A).

frequency of conjugative transfer =

number of transconjugants (CFU/mL) number of recipients (CFU/mL)

(1)

The student t test (SPSS 19.0 for Windows) was used to assess statistically significant differences (p < 0.05) among the values of the frequency. The null hypothesis that the frequency was not different between different samples was rejected at a pvalue less than or equal to 0.05.

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RESULTS AND DISCUSSION Establishment of the Conjugative Transfer Model between Gram-Negative Strains of E. coli. First, four single-factors (bacteria concentration, donor/recipient ratio, mating time, and nutrient) were optimized to establish the C

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Figure 2. Effects of UV disinfection (A) and chlorination (B) on the frequency of conjugative transfer. Significant differences between the frequency with 5−40 mg active Cl min/L chlorine added and the control (no chlorine added) were tested with the student test, *p < 0.05.

(2.5−5.0) × 10−3, approximately a 2−5-fold promotion compared to the control sample (Figure 2B). Further increase of the chlorine dose significantly suppressed the conjugative transfer when treated by chlorination higher than 80 mg active Cl min/L. The data indicated that UV disinfection and chlorination do have impacts on the conjugative transfer but act differently according to the dose applied. A lower UV dose imposed very little influence on the frequency, while chlorination at lower doses significantly amplified the conjugative transfer. On the other hand, both UV and chlorine at higher doses greatly suppressed the frequency of conjugative transfer. Concerning the inactivation mechanisms of UV disinfection and chlorination, two likely possible aspects are proposed for their different effects on the conjugative transfer. One is that the numbers of donor and recipient strains were changed during disinfection, thus leading to a variation of conjugative transfer, or that the bacteria were damaged and thus changed the cell permeability, which promotes ARGs transfer. To explore the mechanisms of UV disinfection and chlorination affecting the conjugative transfer, these two assumptions were both checked in the following. Exploration of the Mechanism of UV Disinfection on ARGs Conjugative Transfer. The inactivation of donor and recipient strains by UV disinfection was first explored (Figure 3). UV disinfection effectively inactivated both the donor and

Nutrients, including carbon source, N, and P, were considered essential for the conjugative transfer as either the ARGs transfer by donor or the receipt of the recipient required energy.33 The addition of glucose and α-lactose as nutritive substances significantly enhanced the frequency of conjugative transfer, while NaAc had almost no effect since neither the donor nor the recipient could use it as carbon source. It implied that the type of carbon source could greatly influence the conjugative transfer. Furthermore, the addition of glucose higher than 5000 mg/L greatly promoted the frequency of conjugative transfer by more than 4 orders of magnitude compared to the transfer with no nutrient added (Figure 1B). N and P nutrients also show significant effect on the conjugative transfer. The frequency of conjugative transfer with glucose and N and P nutrients added simultaneously was approximately 2-fold of the transfer with no N or P nutrients added and over 100-fold higher than the transfer with only glucose added (Figure 1C). Similar results had been reported that the conjugative transfer was amplified significantly in the nutrient-abundant environments.31,34,35 The conjugative transfer frequency increased with the prolongation of mating time within 6 h, whereas the frequency was very slightly promoted by a further increase of mating hours from 6 to 24 h (Figure 1D). Thus, the conjugative model for Gram-negative strains of E. coli was optimized under conditions of (i) bacterial concentration above 104 CFU/mL and donor/recipient ratio of 1:1; (ii) addition of glucose (5000 mg/L COD), N and P nutrients, and the ratio COD:NH4+:TP of 100:5:1, and (iii) conjugation for 12 h. The optimized conditions were adopted in the following to explore the effects of wastewater disinfection on the conjugative transfer. Effect of UV Disinfection and Chlorination on ARGs Conjugative Transfer. The ARGs conjugative transfer was explored during UV disinfection and chlorination (Figure 2). It was observed that the conjugative transfer frequency was not significantly affected by low UV doses up to 8 mJ/cm2 (p > 0.05), with the value maintained at (3.0 ± 1.4) × 10−2, while the frequency was significantly reduced at higher UV doses (Figure 2A). Actually, no transconjugant could be detected when the UV dose was higher than 10 mJ/cm2. By comparison, chlorination at low doses of 5−40 mg active Cl min/L significantly improved the frequency, with values of

Figure 3. Donor and recipient survival numbers in wastewater versus UV dose. D

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to see that the transfer was significantly amplified, thus the change of the cell permeability after chlorination was also explored. The LDH release of the donor and recipient was determined in Figure 4. LDH exists in all bacterial cytoplasma, which

the recipient strains. The bacteria numbers decreased significantly from 107 to 104 CFU/mL when the UV dose was increased to 10 mJ/cm2. Bacterial survivals at a UV dose higher than 10 mJ/cm2 were all below 104 CFU/mL. The effect of initial bacterial concentration on conjugative transfer has been explored above (Figure 1A). The frequency of conjugative transfer was not significantly reduced when the bacterial concentration was higher than 104 CFU/mL (p > 0.05), while the frequency was greatly suppressed when bacterial concentration was below 104 CFU/mL. The results indicate that there might be a bacterial concentration threshold for conjugative transfer. The conjugative transfer can happen when initial bacterial concentration is higher than the threshold, and the conjugative transfer frequency does not vary significantly above the threshold. The bacterial concentration was always higher than 104 CFU/mL when the UV dose is less than 10 mJ/cm2, so it is easy to understand that conjugative transfer did not vary significantly (Figure 2A). By comparison, a low bacterial concentration meant a much smaller opportunity for the contact, which was considered as the major possibility for the decrease of the conjugative transfer frequency at UV doses higher than 10 mJ/cm2. To confirm that the bacterial concentration is the key issue for conjugative transfer during UV disinfection, the frequency of conjugative transfer at similar bacterial concentration level treated by (i) UV disinfection and (ii) direct dilution was compared (Table S2, Supporting Information). It was observed that the frequency of conjugative transfer treated by UV disinfection coincided essentially with the samples diluted directly (p > 0.05). The result could arise mainly by the mechanism of UV disinfection. UV irradiation inactivates bacteria mainly by attacking DNA and causing DNA deactivation via pyrimidine dimerization.36 Cho et al. reported that UV had no any measurable effect on the cell membrane, including protein release, lipid peroxidation, and cell permeability.26 The data implies that the decrease of bacterial numbers by low UV doses (up to 8 mJ/cm2) did not affect the frequency of conjugative transfer because UV disinfection only decreased the bacterial number, while it did not damage bacterial properties (e.g., the cell membrane). By comparison, the effective inactivation of bacteria by high UV doses (>10 mJ/cm2) resulted in the complete inhibition of conjugative transfer. Exploration of the Mechanism of Chlorination on ARGs Conjugative Transfer. Chlorination at low doses (up to 40 mg active Cl min/L) was not so effective in bacterial inactivation compared to UV disinfection, with log removals of 0.09−0.30 for the donor strain and 0.34−0.55 for the recipient strain, respectively (Figure S1, Supporting Information). By comparison, further increase of the chlorine dose (>80 mg active Cl min/L) greatly reduced the bacterial concentrations, with bacteria surviving at 104 CFU/mL or below. Apparently, effective inactivation of bacterial concentrations (80 mg active Cl min/L) could be the major mechanism resulting in the decrease of the ARGs transfer frequency (Figures 1A and 2B), similar to UV disinfection at doses higher than 10 mJ/cm2. When a chlorine dose was lower than 40 mg active Cl min/L, the bacterial concentration was maintained at the level of 107 CFU/mL. According to Figure 1A, the bacterial concentration could not affect conjugative transfer as long as cell concentration was higher than 104 CFU/mL. It was surprising

Figure 4. Relative LDH release of the donor and recipient after treatment by two chlorine doses.

cannot be released from the cell until the membrane gets damaged and cell permeability is increased.37 To some extent, LDH release represents an index assessing the cell permeability. It was found that chlorination (e.g., 10 and 20 mg active Cl min/L) significantly increased the LDH release of the bacteria. Especially, the LDH release of the donor improved to nearly 2fold after chlorination (p < 0.05), implying that the cell permeability was significantly amplified. We postulate that the amplification of ARGs transfer by chlorination arises mainly from changes in the cell permeability, which improves the ARGs release from the donor, and also the acceptance of the recipient. TEM micrographs (Figure 5)

Figure 5. TEM micrographs of the mating cells during conjugative transfer. (A) The matting cells during conjugative transfer in the control group (no chlorination). (B) The matting cells during conjugative transfer after chlorination (20 mg Cl min/L), the partially magnified images (Inset) marked with a “x” in B indicated more potential pore and higher cell permeability of matting cells after treated by chlorination. The white circles represent the background Formvar coated grid (scale bars, 1000 nm.).

indicated that more potential pilus or pores emerged on the surface of the conjugative cells after treatment by chlorination at a chlorine dose of 20 mg Cl min/L (Figure 5B). By comparison, the surface of bacterial cell without chlorination was smooth and intact (Figure 5A). The potential pilus probably improved the cell permeability and transfer pathways of ARGs. This seems to be understandable, since chlorination has been reported for its ability to react strongly with the lipids E

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Table 1. Effect of Different Chlorination Conditions on Recipient Numbers, Residual Chlorine Concentration, and Frequency of Conjugative Transfera I

II

chlorination condition and dose (mg Cl min/L)

0

10

20

2

5

recipient numbers (CFU/mL) free residual chlorine (mg/L) combined residual chlorine (mg/L) frequency of conjugative transfer

1.6 × 107 0 0 2.6 × 10−2

0.92 × 107 0 0.6 11 × 10−2

0.85 × 107 0.02 2.8 14 × 10−2

1.0 × 107 0 0 2.8 × 10−2

0.95 × 107 0 0 3.0 × 10−2

a

Condition I was the method of chlorination used in this study, which is described in the section entitled Conjugation Experiment Treated with Disinfection. Condition II is described in the Supporting Information (Text 4).

Figure 6. Comparison of the mechanisms of UV disinfection (A) and chlorination (B) affecting the ARGs conjugation transfer.

of the membrane and other components of the membranes.26,38 This finding indicates that low chlorination doses promote bacterial conjugation. As is known, ammonia nitrogen (250 mg/L) was used in the study as the necessary nutrient for ARGs transfer. Chlorine reacts with ammonia first and generates NH2Cl and then NHCl2 and NCl3 (eqs 2−4).39 Actually, a rather low concentration of free residual chlorine was detected (10 mJ/cm2) or chlorination (>80 mg active Cl min/L) is more effective for the control of ARGs transfer. Actually, no transconjugant could be detected at these doses in this study. Bacterial concentration was considered as the key factor determining the probability of ARGs transfer. A too low bacterial number (below 104 CFU/ mL) might not be conducive to the occurrence of conjugative transfer. Disinfection processes are considered essential for the control of antibiotic resistance in WWTPs. However, this study indicates that neither chlorination nor UV disinfection at low doses can reduce the frequency of the plasmid transfer. According to our investigation on the effectiveness of UV lamps in several of WWTPs of Shanghai, the effective dose of UV disinfection in most WWTPs was generally below 10 mJ/cm2. Similar ineffectiveness of disinfection procedures was also reported in other WWTPs.5,7,43 The doses needed in our study to terminate ARGs transfer are difficult to reach in most WWTPs. In addition, concerning many more bacterial species present in real wastewater, which might decrease the threshold concentration as mentioned above, significantly higher UV doses (>10 mJ/cm2) or chlorine doses (>80 mg Cl min/L) than those used in the study might be needed to reduce the ARGs transfers. Previous publications also reported that UV or chlorine disinfection could not effectively reduce ARB numbers or even cause their selection.7,44−46 This implies that the disinfection process has limited effect on reducing the potential risk of ARGs discharge and transfer. The risk of ARGs transfer to other bacteria in WWTPs effluents, especially to human pathogens,19 probably exists and thus needs much more attention. Further research on improving the effectiveness of UV and chlorine disinfection on ARB reduction is urgent. Development of other disinfection processes as potential alternatives, such as ozone and advanced oxidation, is also proposed.

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†

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was founded by the National Natural Science Foundation of China (51308399) and the Shanghai Natural Science Foundation (13ZR1443300). The authors would like to thank the engineers of the WWTP for their assistance in obtaining the wastewater samples. The authors also express their gratitude to Prof. James Bolton for writing improvement.

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(1) de Oliveira, A. J.; Pinhata, J. M. Antimicrobial resistance and species composition of Enterococcus spp. isolated from waters and sands of marine recreational beaches in Southeastern Brazil. Water Res. 2008, 42 (8−9), 2242−2250. (2) Falcone-Dias, M. F.; Vaz-Moreira, I.; Manaia, C. M. Bottled mineral water as a potential source of antibiotic resistant bacteria. Water Res. 2012, 46 (11), 3612−3622. (3) Su, J. Q.; Wei, B.; Xu, C. Y.; Qiao, M.; Zhu, Y. G. Functional metagenomic characterization of antibiotic resistance genes in agricultural soils from China. Environ. Int. 2014, 65C, 9−15. (4) Pruden, A.; Pei, R. T.; Storteboom, H.; Carlson, K. H. Antibiotic resistance genes as emerging contaminants: studies in Northern Colorado. Environ. Sci. Technol. 2006, 40 (23), 7445−7450. (5) Borjesson, S.; Melin, S.; Matussek, A.; Lindgren, P. E. A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant. Water Res. 2009, 43 (4), 925−932. (6) Castiglioni, S.; Pomati, F.; Miller, K.; Burns, B. P.; Zuccato, E.; Calamari, D.; Neilan, B. A. Novel homologs of the multiple resistance regulator marA in antibiotic-contaminated environments. Water Res. 2008, 42 (16), 4271−4280. (7) Munir, M.; Wong, K.; Xagoraraki, I. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan. Water Res. 2011, 45 (2), 681−693. (8) Zhang, X. X.; Zhang, T. Occurrence, abundance, and diversity of tetracycline resistance genes in 15 sewage treatment plants across China and other global locations. Environ. Sci. Technol. 2011, 45 (7), 2598−2604. (9) Szczepanowski, R.; Linke, B.; Krahn, I.; Gartemann, K. H.; Gutzkow, T.; Eichler, W.; Puhler, A.; Schluter, A. Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology 2009, 155 (Pt 7), 2306−2319. (10) Aminov, R. I. Horizontal gene exchange in environmental microbiota. Front. Microbiol. 2011, 2, 158. (11) Huddleston, J. R. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infect. Drug Resist. 2014, 7, 167−176. (12) He, X.; Ahn, J. Assessment of conjugal transfer of antibiotic resistance genes in Salmonella Typhimurium exposed to bile salts. J. Microbiol. 2014, 52 (8), 716−719. (13) Qiu, Z.; Yu, Y.; Chen, Z.; Jin, M.; Yang, D.; Zhao, Z.; Wang, J.; Shen, Z.; Wang, X.; Qian, D.; Huang, A.; Zhang, B.; Li, J. W. Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (13), 4944−4949. (14) Stewart, G. J.; Sinigalliano, C. D. Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic Sediments. Appl. Environ. Microbiol. 1990, 56 (6), 1818−1824. (15) Remigi, P.; Capela, D.; Clerissi, C.; Tasse, L.; Torchet, R.; Bouchez, O.; Batut, J.; Cruveiller, S.; Rocha, E. P.; Masson-Boivin, C.

ASSOCIATED CONTENT

S Supporting Information *

Four texts (exploration of the potential donor for the conjugative transfer, Text 1; lactate dehydrogenase (LDH) release assay, Text 2; transmission electron microscopy imaging, Text 3; and the chlorination process of condition 2, Text 4), two tables (single factor experimental scheme, Table S1; and the frequency of conjugative transfer treated by UV disinfection and direct dilution, Table S2) and two figures (donor and recipient survival numbers versus chlorine dose, Figure S1; and residual chlorine concentration after treated by chlorination, Figure S2) addressing additional experimental data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86 21-6598-4275. Fax: 86 21-6598-4275. E-mail: [email protected]. G

DOI: 10.1021/acs.est.5b00644 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.5b00644 Environ. Sci. Technol. XXXX, XXX, XXX−XXX