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Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination
Water Research 91 (2016) 331e338
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Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination Wenfang Lin a, Shuai Li a, Shuting Zhang b, Xin Yu a, * a b
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China College of Environment and Safety Engineering, Shenyang University of Chemical Technology, Shenyang, 110142, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 September 2015 Received in revised form 29 December 2015 Accepted 11 January 2016 Available online 12 January 2016
The widespread presence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the drinking water system facilitates their horizontal gene transfer among microbiota. In this study, the conjugative gene transfer of RP4 plasmid after disinfection including ultraviolet (UV) irradiation and lowlevel chlorine treatment was investigated. It was found that both UV irradiation and low-level chlorine treatment reduced the conjugative gene transfer frequency. The transfer frequency gradually decreased from 2.75 103 to 2.44 105 after exposure to UV doses ranging from 5 to 20 mJ/cm2. With higher UV dose of 50 and 100 mJ/cm2, the transfer frequency was reduced to 1.77 106 and 2.44 108. The RP4 plasmid transfer frequency was not significantly affected by chlorine treatment at dosages ranging from 0.05 to 0.2 mg/l, but treatment with 0.3e0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 105 or below the detection limit. The mechanisms underlying these phenomena were also explored, and the results demonstrated that UV irradiation and chlorine treatment (0.3 and 0.5 mg/l) significantly reduced the viability of bacteria, thereby lowering the conjugative transfer frequency. Although the lower chlorine concentrations tested (0.05e0.2 mg/l) were not sufficient to damage the cells, exposure to these concentrations may still depress the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport-related gene (TraG). Additionally, fewer pili were scattered on the bacteria after chlorine treatment. These findings are important in assessing and controlling the risk of ARG transfer and dissemination in the drinking water system. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Conjugative gene transfer Antibiotic resistant genes UV irradiation Chlorination
1. Introduction Antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) have been extensively detected in drinking water systems (Zhang et al., 2009; Bouki et al., 2013; Bergeron et al., 2015), and their widespread presence in drinking water constitutes a major public health issue. Flemming and Ridgway (2009) suggested that most microorganisms (95%) can aggregate as biofilms on the surface of pipelines despite the presence of chlorine. The ubiquitous presence of bacteria in a planktonic state or in biofilms found in both water treatment processes and in pipelines easily transmit resistance through horizontal gene transfer (HGT) (Madsen et al., 2012). Additionally, some of the microorganisms can be pathogens, which further increases the risks and dangers posed to human beings.
* Corresponding author. E-mail address: [email protected] (X. Yu). http://dx.doi.org/10.1016/j.watres.2016.01.020 0043-1354/© 2016 Elsevier Ltd. All rights reserved.
Bacterial antibiotic resistance can be conferred either by spontaneous mutation or by horizontal transfer. Mutagenic factors, such as disinfection by-products, are believed to induce antibiotic resistance in water treatment processes (Lv et al., 2014). However, in many cases, bacteria cannot adapt to such harsh conditions, and instead, they receive mobile genetic elements (MGEs) that carry the resistance genes from other bacteria, which is known as HGT (Ojala et al., 2014). Three genetic mechanisms are responsible for HGT of ARGs: 1) conjugative transfer by MGEs such as plasmids, transposons, and integrons; 2) transformation of naked DNA in either naturally competent bacteria or bacteria with competency induced by environmental factors; and 3) transduction by bacteriophages (Zhang et al., 2009; Dodd, 2012). Bacterial conjugation promotes the horizontal transfer of genetic materials when donor and recipient cells are in physical contact (Ghigo, 2001). Plasmid-based conjugative transfer has been widely recognized to occur in environmental microbial communities and in laboratory conditions (Anjum et al., 2011; Yang et al., 2013; Bellanger et al., 2014). Therefore, conjugative transfer may be particularly important in
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disseminating ARGs in drinking water system. Disinfection technologies (such as UV irradiation and chlorination disinfection), are widely applied to ensure microbial safety in water systems. Over the past few decades, UV treatment has been an increasingly popular technology for water disinfection because microorganism inactivation is achieved without the production of genotoxic byproducts (Guo et al., 2013; Lee et al., 2015). In addition, chlorine, as a common disinfectant, was introduced into urban water supplies in the early 19th century to eliminate waterborne pathogens based on its effectiveness, easy of use, and low cost (Ferey et al., 2000; Simoes et al., 2010). However, despite the application of these different disinfection processes, bacterial regrowth is a common problem during water distribution. As a result, maintaining residual chlorine (>0.05 mg/l) is a common practice for controlling microbial regrowth in pipelines. Previous studies have typically focused on the direct influence of disinfection on ARB and ARGs, and have revealed that disinfection processes (e.g., chlorination and UV disinfection) can substantially remove ARB and ARGs during water treatment (Huang et al., 2011; Guo et al., 2013). However, it has been shown that some of the ARB or ARGs perform resistance against disinfectants (Xi et al., 2009; Shi et al., 2013). Consequently, these persistent bacteria can survive and spread resistance to downstream communities, especially in water supply pipelines. Moreover, intact remnants of DNA may be transferred to surrounding populations via horizontal transfer. Thus, transfer of ARGs among bacteria after disinfection in drinking water terminal systems is a major public concern with direct consequences for consumers. To date, there have been no reports regarding the effects of UV irradiation and low-level chlorine treatment (0.05e0.5 mg/l, simulating residual chlorine in pipelines) on the risk of HGT of ARGs. In this study, we established a conjugative model system under laboratory conditions and investigated changes in the conjugative transfer frequency after UV irradiation and low-level chlorination as well as the underlying mechanisms. 2. Materials and methods 2.1. Bacteria strains and plasmid The donor strain used in this study was Escherichia. coli HB101, which harbored the RP4 plasmid that confers resistance to kanamycin, tetracycline, and ampicillin. These resistances were encoded by the aphA, tetA and tetR, and bla genes, respectively. This broadhost-range plasmid RP4 was classified as medium-copy plasmid, and could have a copy number of about 4e7 in each cell (Priefer et al., 1985). Another E. coli K12 strain resistant to rifampicin was used as the recipient bacteria. Their resistance was conferred by mutations in rpoB. Both the donor and recipient strains were gifted by Professor Junwen Li and Dr. Zhigang Qiu of the Institute of Health and Environmental Medicine. The donor and recipient strains were pre-cultured separately in LuriaeBertani (LB) broth supplemented with appropriate antibiotics (80 mg/l kanamycin, 50 mg/l tetracycline, and 60 mg/l ampicillin for donor strains, and 100 mg/l rifampicin for recipient strains) on a 180 rpm shaking incubator at 37 C. After overnight incubation, the cell density reached approximately 109 CFU/ml, and the cells were washed twice with 0.9% sterile saline. Cultures were centrifuged at 4500 g for 10 min. Samples of donor and recipient strains were re-suspended and then diluted to approximately 108 CFU/ml with pure or 1/500 LB broth. 2.2. Conjugation and disinfection experiment For UV treatment, donor cells resuspended in 0.9% sterile saline were exposed to the UV irradiation at different fluences (0, 5, 10, 15,
20, 50, or 100 mJ/cm2) according to the standard collimated beam test protocol described by Guo et al. (2013) and Zhang et al. (2015). After UV irradiation, donor cells were mixed with recipient cells (resuspended by pure LB broth) at a 1:1 ratio (10 ml each). All steps were carried out in the dark to prevent photoreactivation. The mixtures were grown at 37 C with shaking at 80 rpm for 24 h. During chlorination, LB broth was diluted 500-fold (total organic carbon (TOC) ¼ 10 mg/l) to avoid chlorine consumption by a high concentration of organic compounds. Solutions of resuspended donor and recipient cells (10 ml each) were mixed together at a 1:1 ratio and then cultured under the same conditions applied for UV treatment. Sodium hypochlorite was introduced to establish different doses of free chlorine (0, 0.05, 0.1, 0.2, 0.3, and 0.5 mg/l Cl2), and cells were cultured for 0e48 h (Huang et al., 2011, 2013). The medium was replaced by fresh medium (1/500 LB) supplemented with the appropriate chlorine concentration every 12 h. Finally, chlorination was terminated with the addition of 1.5% sodium thiosulfate solution before plating. 2.3. Transconjugant identification and mechanism analysis After mating periods of 6, 12, 24, or 48 h, 1 ml of the donor and recipient mixture was collected from each disinfection system. The cultures were serially diluted and spread on LB medium plates containing different antibiotics. The numbers of donors and recipients were determined by counting colonies on LB agar supplemented with 80 mg/l kanamycin, 50 mg/l tetracycline, and 60 mg/l ampicillin or 100 mg/l rifampicin. The recipients possessing the RP4 plasmid were recognized as transconjugants. The transconjugants were selected and counted on LB agar plates containing 80 mg/l kanamycin, 50 mg/l tetracycline, 60 mg/l ampicillin, and 100 mg/l rifampicin. To confirm that the donor and recipient strains could be distinguished, they were cross plated. Transconjugant colonies were randomly selected, and then confirmed using colony polymerase chain reaction (PCR)-based detection technology. The PCR reactions and amplification conditions recommended by Yang et al. (2013) were used. PCR products were detected by electrophoresis in 0.8% agarose gel. Electrophoresis was carried out at 25 C at 180 V for 20 min in Tris-acetate-EDTA (TAE) buffer. Finally, the PCR products (140 bp) were sent to Sangon Biotech (Shanghai, China) for sequencing. The retrieved sequences were aligned with those found in the NCBI database using the Blast tool (http://www. ncbi.nlm.nih.gov/blast). To analyze the mechanisms responsible for the effects of UV irradiation and chlorine treatment on conjugative gene transfer, different donor/recipient rates (e.g., 103, 104, 105, 106, 107, and 108 CFU/ml donor cells mixed with 108 CFU/ml recipient cells) were inoculated, and the corresponding transfer frequencies were calculated. Additionally, the expression of three genes, i.e., a flagllum gene (FlgC), an outer membrane protein gene (ompF), and a transfer regulated gene (TraG) were assessed. FlgC encodes a flagellar protein. For conjugation to occur, the donor and recipient cells must be in physical contact (Dodd, 2012). Bacterial mobility is regulated by polar flagella, which affects bacterial collision and attachment. Thus, the transfer frequency is influenced by the expression of FlgC. OmpF is a common porin protein, and occlusion of ompF may interrupt substance import, including nucleic acid intake during conjugative transfer (Spector et al., 2010). TraG is essential for the conjugative process, because it directly determines the mating between the donors and recipients (Hamilton et al., 2000). RNA extraction was carried out using the EasyPure™ RNA kit (TransGen Biotech, China) according to the manufacturer's instructions. Because RNAs with higher turnover rates would disappear more rapidly, reverse transcription was performed immediately using a reverse transcription (RT)-PCR kit (Promega,
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USA) following the manufacturer's protocols. Finally, the cDNA was stored at 80 C until use. RT-qPCR products were amplified with primers based on previous studies (Ren et al., 2004; Jaktaji and Heidari, 2013; Yuan, 2014; Zhang et al., 2015) (Table S.1, see Supplementary data). The PCR mixtures consisted of 60 ng template cDNA, 2 SYBR Green Mix, and 200 mM each primer in a total volume of 20 ml. In the relative qPCR analysis, target gene expression was normalized to that of a reference gene (16S rRNA gene). Scanning electron microscopy (SEM, Hitachi S-4800, Japan) was used to observe the morphology of the conjugative cells before/ after chlorination. In brief, 1 ml of the cell mixture was collected after 6 h of mating and fixed in 2.5% glutaraldehyde for 2 h. Subsequently, it was washed twice with water, and then dried in an oven at 60 C overnight. 2.4. Statistical analysis The colony averages were calculated using the triplicate plates. The transfer frequency of the RP4 plasmid was calculated using the following formula (Yang et al., 2013; Guo et al., 2015): Conjugative transfer frequency ¼ number of transconjugants (CFU/ml)/number of recipients (CFU/ml). The relationship between UV doses and log conjugative transfer frequency was analyzed using a linear fitting model. In addition, the mRNA expression levels of conjugation related-genes were calculated using the 2-△△CT method as follows (Zhu et al., 2013):
DCt ¼ CtðFunctional genesÞ Ctð16s rRNA geneÞ DDCt ¼ DCtðChlorinationÞ DCtðControlÞ FCðfold changeÞ ¼ 2DDCt Where the Ct value is the cycle threshold. The functional genes represent FlgC, ompF, and TraG. No chlorine was added in the control group. 3. Results and discussion 3.1. Conjugative transfer model construction From three plates on which 100, 187, and 65 colonies grew, 79, 70, and 65 transconjugants were randomly chosen respectively. No false-positive colony was identified by PCR and gel electrophoresis (Fig S.1a). To further confirm the occurrence of plasmid transfer, PCR products were sent to sequencing, and analysis of the data indicated that the amplified fragments shared 97% similarity with the aphA gene of the RP4 plasmid (Fig. S1b). This result showed that all colonies acquired RP4 plasmids, and thus, we successfully constructed the conjugative transfer model. 3.2. Impact of UV irradiation on RP4 plasmid conjugative transfer UV disinfection is being increasingly used in water treatment to inactivate waterborne pathogens. It was evident that the donor strain was significantly affected by UV disinfection before it was mixed with recipient bacteria (Fig. 1a). The number of donor colonies decreased with increasing UV fluence. In fact, 4.23-log and 4.18-log reductions were observed after 5 and 10 mJ/cm2 UV treatments, respectively. In addition, when the fluence was increased to 20 mJ/cm2, the donor number decreased by 5.47 log. Furthermore, 6.29-log and 7.04-log reductions were induced by 50 and 100 mJ/cm2 UV irradiation, respectively. Similarly, Guo et al. (2013) revealed that UV disinfection at a dose of 20 mJ/cm2
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could result in an approximately 4-log reduction. They also found all ARB were eliminated to below the detection limit when the fluence was increased to 50 mJ/cm2. However, after mating for 24 h, the donor cell concentrations again increased significantly after exposure to UV irradiation. At doses ranging from 5 to 20 mJ/ cm2, the donor cell concentrations increased by 1.81e2.91 log. Particularly, increases of 1.31 log and 1.54 log were found at respective doses of 50 and 100 mJ/cm2. Finally, without exposure to UV irradiation, the number of donor cells tended to be more stable. Fig. 1b shows the reduction in cell numbers for both donor and transconjugant cells with increasing UV fluence. Simultaneously, the RP4 plasmid conjugative transfer frequency decreased greatly as the UV irradiation dose increased. The transfer frequency was determined to be 8.63 103 without UV treatment. With exposure to UV doses of 5e20 mJ/cm2, the transfer frequency gradually decreased from 2.75 103 to 2.44 105. The transfer frequency further dropped down to 1.77 106 and 2.44 108 after exposure to UV doses of 50 and 100 mJ/cm2, respectively. As the number of recipient cells was relatively similar in all mating conditions, the transfer frequencies were mainly determined by the numbers of donor cells, with a higher donor density providing more transferable plasmids. Moreover, a high bacterial density was conducive for close contact between cells, which facilitated plasmid transfer. For conjugation to occur in liquid environments, donor and recipient cells must collide, attach, and then conjugate before detachment occurs (Zhong et al., 2010). Thus, the initial cell density significantly affected the conjugative frequency. 3.3. Impact of low-dose chlorination on RP4 plasmid conjugative transfer UV treatment is effective for microbial inactivation in drinking water, but UV irradiation cannot provide a residual disinfectant effect in the drinking water distribution system. Thus, maintaining residual chlorine after chlorination disinfection is a practical approach to repress bacterial regrowth in pipelines. To date, little has been known about how a low dosage of chlorine (0.05e0.5 mg/ l) impacts conjugative plasmid transfer in bacteria. Our study showed that RP4 plasmid transfer was not significantly affected by chlorine treatment at doses ranging from 0.05 to 0.2 mg/l. After mating for 6 h, only a slight decrease (from 2.31 104 to 2.2 104) was observed after exposure to 0.05e0.2 mg/l chlorine. However, a dosage of 0.3e0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 105 or below detection limit (Fig. 2a). We observed similar trends in decreasing transfer frequency after cultivation for 12 h and 24 h (Fig. 2b and c), with frequencies of conjugative transfer of 9.05 106 and 1.01 106, respectively, after treatment with 0.3 mg/l chlorine. No transfer frequency was observed when cells were exposed to 0.5 mg/l chlorine. One explanation for the effect of chlorine treatment on conjugative transfer could be the reduction of donor and recipient cells. After mating for 24 h, donor and recipient strains were decreased to about 1.19 104 and 1.97 104 CFU/ml after treatment with 0.5 mg/l chlorine, respectively (Fig. 2c). Up to 48 h, there was no transfer frequency when the concentration of chlorine was 0.3 and 0.5 mg/l. Moreover, the transfer frequency decreased gradually as the chlorine dosage increased from 0.05 to 0.2 mg/l (Fig. 2d). Therefore, the transfer of RP4 plasmid from the donor strain to recipient bacteria was decreased by low-level chlorine treatment. In addition, the decreasing trend in transfer frequency induced by chlorine was gradually enhanced with prolonged mating time.
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Fig. 1. Impact of UV irradiation on the frequency of RP4 plasmid conjugative transfer. (a) Donor cell density immediately after UV irradiation and after mating for 24 h. (b) The changes in density of donors, recipients, transconjugants, and transfer frequency upon irradiation at different UV fluences.
Fig. 2. Impact of low-level chlorination on the frequency of RP4 plasmid conjugative transfer after mating for 6 h (a), 12 h (b), 24 h (c), and 48 h (d).
3.4. Mechanisms underlying the effects of UV irradiation on RP4 plasmid conjugative transfer To analyze the potential mechanisms responsible for the reduction of conjugative transfer frequency upon UV treatment, we exposed bacteria at different donor/recipient (D:R) rates, to UV irradiation and then calculated the corresponding transfer frequencies. For instance, we mixed 103, 104, 105, 106, 107, and 108 CFU/ ml donor cells with 108 CFU/ml recipient cells. After mating for 24 h, we found that with the same density of recipient cells (108 CFU/ml), higher density of donor cells induced higher numbers of transconjugants and caused higher transfer frequencies (Fig. 3a). We fitted the log concentrations of initial donors (0 h) and log concentrations of final donors (24 h) with log transfer frequency and obtained two linear fitting equations (Fig. 3b and c). The fitted transfer frequency was calculated based on the initial donor density once exposed to UV irradiation and the donor density after mating 24 h. Therefore, theoretical transfer values were obtained (Fig. 3d).
By comparing three groups of transfer frequency (0 h fitting, 24 h fitting, and 24 h mating), it was found that the fitted transfer frequency at 0 h was lower than that at 24 h. This might be explained by bacterial proliferation, regrowth, and repair. First, a low fluence of UV irradiation (<50 mJ/cm2) was not enough to inactive the whole bacteria, and thus, the remaining cells were still alive and could proliferate during the 24 h mating time. Live donors could transfer the plasmid to the recipients. Additionally, it was reported that UV light was directly absorbed by the nucleic acids, causing damage to the DNA of the microorganisms and thereby resulting in the formation of pyrimidine dimers (Lee et al., 2015). Moreover, according to Dodd (2012), UV light directly penetrated the cell membrane to be absorbed by the nucleobases comprising DNA and RNA, and thus, the cell membrane remained intact. Süß et al. (2009) demonstrated that bacteria could recover after UV treatment, but the recovery frequency appeared to be slower with higher UV doses and longer exposure times. Therefore, the bacterial inactivation frequency after UV
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Fig. 3. Analysis of the mechanisms responsible for the impact of UV irradiation on RP4 plasmid conjugative transfer frequency. (a) Impact of different bacterial density ratios on transfer frequency. (b) Fitting equation with log donor (0 h) and log transfer frequency (24 h). (c) Fitting equation with log donor (24 h) and log transfer frequency (24 h). (d) Comparison of theoretical and actual transfer frequencies.
irradiation may be underestimated. Previous studies suggested that thymine dimers induced by UV irradiation were efficiently removed by photoreactivation and dark repair systems/nucleotide excision repair (Jungfer et al., 2007; Giannakis et al., 2014; Bugay et al., 2015). Microorganisms can recover from the damage induced by UV disinfection processes, which can lead to extensive bacterial regrowth. Similarly, Lee et al. (2015) also suggested that the injured DNA could be repaired by photoreactivation or dark repair. Notably, Guo et al. (2012) indicated that the initial antibiotic resistance could be retained or recovered through photoreactivation, which provoked health safety concerns about UV disinfection. In addition, UV irradiation may induce the donors entering into a state characterized as viable but not culturable (VBNC) (Trevors, 2011; Zhang et al., 2015). Although these bacteria cannot be detected by conventional culture methods, they retain metabolic activity. Moreover, certain cellular functions, such as respiratory activity, membrane integrity, glucose incorporation, etc. may decrease rapidly but continue at low stable levels after UV treatment (Villarino et al., 2000). We inferred that conjugative transfer might occur with VBNC bacteria. Accordingly, the transfer frequency that actually occurred after 24 h of mating time was higher than the other fitted values (Fig. 3d). However, additional research is required to further investigate this hypothesis (Fig. 4a). Overall, conjugative transfer after UV irradiation in this study was achieved with high nutrient concentrations and bacterial cell densities than the real ones in drinking water conditions. This is because it is impossible to obtain statistically significant data if real drinking water conditions are applied. However, the results could be extended to drinking water environment to a certain degree. For example, the linear relationship in Fig. 3b was so good that the transfer frequency with very low bacterial density could be inferred. Further, the experimental results can still be applied in
assessing maximum risks and can provide useful references for researchers and policy-makers of water treatment. 3.5. Mechanisms underlying the effects of chlorination on RP4 plasmid conjugative transfer Chlorination is a widely used technology for microbial control in both drinking water and wastewater processing, and a number of mechanisms responsible for the inhibitory effects of chlorine on RP4 plasmid conjugative transfer. First, microorganisms that survive chlorination may be injured rather than inactivated at low chlorine levels (0.05e0.2 mg/l). In addition, the injured cells may repair cellular damage and recover under suitable conditions (Virto et al., 2005). Previous studies have also shown that bacterial regrowth is a common problem in drinking water systems (van der Kooij and van der Wielen, 2013). Consequently, no apparent decrease in conjugative transfer was observed upon exposure to 0.05e0.2 mg/l chlorine (Fig. 1). Conversely, a dosage of 0.3e0.5 mg/l chlorine could significantly reduce the density of donor and recipient cells, leading to lower transfer frequencies. At higher concentrations, chlorine could directly penetrate and break the cell wall (Fig. 4b). Thus, bacteria lost cell membrane integrity and cellular culturability (Nescerecka et al., 2014). In addition, chlorine reacted with nucleotides and lipids, leading to DNA strand breakage and lipid peroxidation (Gray et al., 2012). In our study, the densities of donor and recipient cells decreased simultaneously at dosages of 0.3 and 0.5 mg/l chlorine, although these two types of bacteria had different susceptibilities towards chlorine. Significant reductions in conjugative transfer occurred under these conditions. Similarly, Dodd (2012) also suggested that conventional water disinfection processes generally provide a highly effective means for mitigating the transport of live
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Fig. 4. Analysis of the mechanisms responsible for the impact of UV irradiation (a) and low-level chlorine treatment (b) on RP4 plasmid conjugative transfer frequency.
ARB, thereby minimizing the risks of conjugative gene transfer. Furthermore, Christie and Vogel (2000) suggested that the conjugation mechanism of Gram-negative bacteria is related to bacterial surface structures, such as the conjugal pili for cellecell contacts. The pilus is the mating channel through which DNA are transferred intermediately. In addition, Zhong et al. (2010) also indicated that bacterial attachment and detachment rates can be influenced by pilus length and flexibility. Fig. 5 shows that fewer pili were scattered on bacteria treated by chlorination compared with the control bacteria, and this finding further demonstrated that bacterial conjugation was significantly repressed by chlorine. In another study, Guo et al. (2015) found that treatment with 0.5e4 mg/l chlorine significantly promoted the frequency of conjugative transfer by 2e5 fold. This might be due to differences in the chlorination treatment. They added different initial concentrations of chlorine (<4 mg/l) for 10 min contact in filtered sewage system containing ammonia nitrogen used as necessary nutrition for ARGs transfer. Chlorine reacted with the ammonia first and generated NH2Cl and then NHCl2 and NH3. The formation of chloramine in wastewater promoted the conjugative transfer. However, in our experiment, chlorination was performed with a low dose (0.05e0.5 mg/l) for a longer time (0e48 h) without chloramine. Furthermore, approximately 104e105 CFU/ml donor cells remained alive after treatment with a dosage of 0.5 mg/l chlorine
during mating for 24 h. Based on our theoretical results (Fig. 3a), the transfer frequency was 106e107 with the donor cell densities of 104e105 CFU/ml. However, the real transfer frequency was below the detection limit in this case. There might be other reasons for the inhibitory effects of chlorine on RP4 plasmid conjugative transfer. According to Camper et al. (1979), the physiological functions of cells, such as respiration, growth rate, etc., were reduced by chlorine treatment. Therefore, the expression of three genes related to the conjugative transfer process, i.e., a flagllum gene (FlgC), an outer membrane protein gene (ompF), and a transfer regulated gene (TraG), were quantified in our study. Bacterial mobility is regulated by surface organelles such as polar flagella. Upon exposure to chlorine, the expression of FlgC was suppressed by 0.04e0.34 fold (Fig. 6). Thus, the incidence of bacterial collision and attachment was reduced indirectly. As a result, conjugative transfer was inhibited by chlorine treatment. The outer membrane porin proteins are the major regulators of the permeability of the cell membrane. For instance, ompF is a common porin protein in E. coli that mediates bacterial defense to adverse environments (Cramer et al., 2011; Stenkova et al., 2011). According to Jaktaji and Heidari (2013), this protein exhibits inhibited expression under stress conditions, such as in the presence of chlorine. In our study, the relative expression of ompF was decreased significantly by 0.07e0.44 fold upon chlorine treatment (Fig. 6), which resulted in
Fig. 5. SEM images showing the pili during conjugative transfer with (a) no chlorination or (b) 0.5 mg/l chlorine after mating for 12 h.
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(0.3e0.5 mg/l) treatment is necessary to decrease the HGT frequency in the drinking water distribution system. Furthermore, after UV irradiation bacteria may still transfer plasmid to surrounding communities, which should raise more concerns among both the public and researchers. Acknowledgments This study was financially supported by National Natural Science Foundation of China (51478450, 51278482, and 51408372). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2016.01.020. References Fig. 6. Relative expression of FlgC, OmpF, and TraG after exposure to different doses of chlorine and mating for 6 h.
the formation of small membrane pores. Transient, occlusion of ompF channels may interrupt substance import and membrane mobility, including nucleic acid intake (Spector et al., 2010). As a result, the conjugative transfer frequency was depressed with reduced expression of ompF. The genetic information required for conjugation is encoded by plasmids, which can be classified into two basic groups. One group is non self-transmissible, and an example of non self-transmissible plasmids is RSF1010. The other group is self-transmissible plasmids, such as RP4. According to Cabezon et al. (1997), the TraG protein family is responsible for delivering nicked DNA to the DNA transport for self-transmissible plasmid conjugation. In addition, Hamilton et al. (2000) also suggested that TraG of the RP4 plasmid was thought to couple the relaxosome with the mating bridge. Based on our study, the expression of TraG was inhibited by 0.30e0.37 fold compared to that in the control (Fig. 6), which further explains the potential mechanism by which conjugative transfer was inhibited. 4. Conclusions In the present study, we systematically studied the ability of the common water treatment processes UV irradiation and chlorination to dim the dissemination of ARGs by horizontal transfer. Our experiments showed that UV irradiation reduced the number of live donor cells, and thus, lowered the conjugative transfer frequency. In addition, the transfer frequency was higher than the theoretic values calculated from fitting equations, which may be explained by bacterial regrowth or recovery after UV irradiation. Moreover, we inferred that plasmid transfer might also occur in VBNC bacteria. Of course, further research needed to test this hypothesis. Chlorination also reduced the conjugative plasmid transfer and the inhibitory effect increased with increasing doses of chlorine. The mechanisms behind this phenomenon were also studied. A dosage of 0.3e0.5 mg/l chlorine significantly decreased the density of donor and recipient cells, leading to reductions in conjugative transfer frequency. Additionally, the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport related gene (TraG) was depressed by 0.05e0.5 mg/l chlorine. Furthermore, fewer pili appeared on cells in the chlorination treatment groups. Overall, these findings are important for evaluating the risks of ARB and ARGs in water treatment systems and some management measures can be suggested. For example, low-level chlorine
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of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex. Biophys. J. 99 (12), 3880e3886. Stenkova, A.M., Isaeva, M.P., Shubin, F.N., Rasskazov, V.A., Rakin, A.V., 2011. Trends of the major porin gene (ompF) evolution: insight from the genus Yersinia. PloS one 6 (5). Trevors, J.T., 2011. Viable but non-culturable (VBNC) bacteria: gene expression in planktonic and biofilm cells. J. Microbiol. Methods 86 (2), 266e273. Van der Kooij, D., van der Wielen, P.W., 2013. Microbial growth in drinking water supplies: a continuing challenge. Water 21 (15.6), 18e19. Villarino, A., Bouvet, O.M.M., Regnault, B., Martin-Delautre, S., Grimont, P.A.D., 2000. Exploring the frontier between life and death in Escherichia coli: evaluation of different viability markers in live and heat- or UV-killed cells. Res. Microbiol. 151 (9), 755e768. Virto, R., Manas, P., Alvarez, I., Condon, S., Raso, J., 2005. Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorinedemanding substrate. Appl. Environ. Microbiol. 71 (9), 5022e5028. Xi, C.W., Zhang, Y.L., Marrs, C.F., Ye, W., Simon, C., Foxman, B., Nriagu, J., 2009. Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Appl. Environ. Microbiol. 75 (17), 5714e5718. Yang, D., Wang, J., Qiu, Z., Jin, M., Shen, Z., Chen, Z., Wang, X., Zhang, B., Li, J.-W., 2013. Horizontal transfer of antibiotic resistance genes in a membrane bioreactor. J. Biotechnol. 167 (4), 441e447. Yuan, H., 2014. Effects of Sub-minimum Inhibitory Concentrations Antibiotics on Conjugation between Escherichia coli and Pseudomonas aeruginosa. Guangzhou University of Chinese Medicine (Thesis In chinese). Zhang, S., Ye, C., Lin, H., Lv, L., Yu, X., 2015. UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa. Environ. Sci. Technol. 49 (3), 1721e1728. Zhang, X.-X., Zhang, T., Fang, H.P., 2009. Antibiotic resistance genes in water environment. Appl. Microbiol. Biotechnol. 82 (3), 397e414. Zhong, X., Kro_l, J.E., Top, E.M., Krone, S.M., 2010. Accounting for mating pair formation in plasmid population dynamics. J. Theor. Biol. 262 (4), 711e719. Zhu, Y.-G., Johnson, T.A., Su, J.-Q., Qiao, M., Guo, G.-X., Stedtfeld, R.D., Hashsham, S.A., Tiedje, J.M., 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proc. Natl. Acad. Sci. 110 (9), 3435e3440.
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Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination Wenfang Lin a, Shuai Li a, Shuting Zhang b, Xin Yu a, * a b
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China College of Environment and Safety Engineering, Shenyang University of Chemical Technology, Shenyang, 110142, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 September 2015 Received in revised form 29 December 2015 Accepted 11 January 2016 Available online 12 January 2016
The widespread presence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the drinking water system facilitates their horizontal gene transfer among microbiota. In this study, the conjugative gene transfer of RP4 plasmid after disinfection including ultraviolet (UV) irradiation and lowlevel chlorine treatment was investigated. It was found that both UV irradiation and low-level chlorine treatment reduced the conjugative gene transfer frequency. The transfer frequency gradually decreased from 2.75 103 to 2.44 105 after exposure to UV doses ranging from 5 to 20 mJ/cm2. With higher UV dose of 50 and 100 mJ/cm2, the transfer frequency was reduced to 1.77 106 and 2.44 108. The RP4 plasmid transfer frequency was not significantly affected by chlorine treatment at dosages ranging from 0.05 to 0.2 mg/l, but treatment with 0.3e0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 105 or below the detection limit. The mechanisms underlying these phenomena were also explored, and the results demonstrated that UV irradiation and chlorine treatment (0.3 and 0.5 mg/l) significantly reduced the viability of bacteria, thereby lowering the conjugative transfer frequency. Although the lower chlorine concentrations tested (0.05e0.2 mg/l) were not sufficient to damage the cells, exposure to these concentrations may still depress the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport-related gene (TraG). Additionally, fewer pili were scattered on the bacteria after chlorine treatment. These findings are important in assessing and controlling the risk of ARG transfer and dissemination in the drinking water system. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Conjugative gene transfer Antibiotic resistant genes UV irradiation Chlorination
1. Introduction Antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) have been extensively detected in drinking water systems (Zhang et al., 2009; Bouki et al., 2013; Bergeron et al., 2015), and their widespread presence in drinking water constitutes a major public health issue. Flemming and Ridgway (2009) suggested that most microorganisms (95%) can aggregate as biofilms on the surface of pipelines despite the presence of chlorine. The ubiquitous presence of bacteria in a planktonic state or in biofilms found in both water treatment processes and in pipelines easily transmit resistance through horizontal gene transfer (HGT) (Madsen et al., 2012). Additionally, some of the microorganisms can be pathogens, which further increases the risks and dangers posed to human beings.
* Corresponding author. E-mail address: [email protected] (X. Yu). http://dx.doi.org/10.1016/j.watres.2016.01.020 0043-1354/© 2016 Elsevier Ltd. All rights reserved.
Bacterial antibiotic resistance can be conferred either by spontaneous mutation or by horizontal transfer. Mutagenic factors, such as disinfection by-products, are believed to induce antibiotic resistance in water treatment processes (Lv et al., 2014). However, in many cases, bacteria cannot adapt to such harsh conditions, and instead, they receive mobile genetic elements (MGEs) that carry the resistance genes from other bacteria, which is known as HGT (Ojala et al., 2014). Three genetic mechanisms are responsible for HGT of ARGs: 1) conjugative transfer by MGEs such as plasmids, transposons, and integrons; 2) transformation of naked DNA in either naturally competent bacteria or bacteria with competency induced by environmental factors; and 3) transduction by bacteriophages (Zhang et al., 2009; Dodd, 2012). Bacterial conjugation promotes the horizontal transfer of genetic materials when donor and recipient cells are in physical contact (Ghigo, 2001). Plasmid-based conjugative transfer has been widely recognized to occur in environmental microbial communities and in laboratory conditions (Anjum et al., 2011; Yang et al., 2013; Bellanger et al., 2014). Therefore, conjugative transfer may be particularly important in
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disseminating ARGs in drinking water system. Disinfection technologies (such as UV irradiation and chlorination disinfection), are widely applied to ensure microbial safety in water systems. Over the past few decades, UV treatment has been an increasingly popular technology for water disinfection because microorganism inactivation is achieved without the production of genotoxic byproducts (Guo et al., 2013; Lee et al., 2015). In addition, chlorine, as a common disinfectant, was introduced into urban water supplies in the early 19th century to eliminate waterborne pathogens based on its effectiveness, easy of use, and low cost (Ferey et al., 2000; Simoes et al., 2010). However, despite the application of these different disinfection processes, bacterial regrowth is a common problem during water distribution. As a result, maintaining residual chlorine (>0.05 mg/l) is a common practice for controlling microbial regrowth in pipelines. Previous studies have typically focused on the direct influence of disinfection on ARB and ARGs, and have revealed that disinfection processes (e.g., chlorination and UV disinfection) can substantially remove ARB and ARGs during water treatment (Huang et al., 2011; Guo et al., 2013). However, it has been shown that some of the ARB or ARGs perform resistance against disinfectants (Xi et al., 2009; Shi et al., 2013). Consequently, these persistent bacteria can survive and spread resistance to downstream communities, especially in water supply pipelines. Moreover, intact remnants of DNA may be transferred to surrounding populations via horizontal transfer. Thus, transfer of ARGs among bacteria after disinfection in drinking water terminal systems is a major public concern with direct consequences for consumers. To date, there have been no reports regarding the effects of UV irradiation and low-level chlorine treatment (0.05e0.5 mg/l, simulating residual chlorine in pipelines) on the risk of HGT of ARGs. In this study, we established a conjugative model system under laboratory conditions and investigated changes in the conjugative transfer frequency after UV irradiation and low-level chlorination as well as the underlying mechanisms. 2. Materials and methods 2.1. Bacteria strains and plasmid The donor strain used in this study was Escherichia. coli HB101, which harbored the RP4 plasmid that confers resistance to kanamycin, tetracycline, and ampicillin. These resistances were encoded by the aphA, tetA and tetR, and bla genes, respectively. This broadhost-range plasmid RP4 was classified as medium-copy plasmid, and could have a copy number of about 4e7 in each cell (Priefer et al., 1985). Another E. coli K12 strain resistant to rifampicin was used as the recipient bacteria. Their resistance was conferred by mutations in rpoB. Both the donor and recipient strains were gifted by Professor Junwen Li and Dr. Zhigang Qiu of the Institute of Health and Environmental Medicine. The donor and recipient strains were pre-cultured separately in LuriaeBertani (LB) broth supplemented with appropriate antibiotics (80 mg/l kanamycin, 50 mg/l tetracycline, and 60 mg/l ampicillin for donor strains, and 100 mg/l rifampicin for recipient strains) on a 180 rpm shaking incubator at 37 C. After overnight incubation, the cell density reached approximately 109 CFU/ml, and the cells were washed twice with 0.9% sterile saline. Cultures were centrifuged at 4500 g for 10 min. Samples of donor and recipient strains were re-suspended and then diluted to approximately 108 CFU/ml with pure or 1/500 LB broth. 2.2. Conjugation and disinfection experiment For UV treatment, donor cells resuspended in 0.9% sterile saline were exposed to the UV irradiation at different fluences (0, 5, 10, 15,
20, 50, or 100 mJ/cm2) according to the standard collimated beam test protocol described by Guo et al. (2013) and Zhang et al. (2015). After UV irradiation, donor cells were mixed with recipient cells (resuspended by pure LB broth) at a 1:1 ratio (10 ml each). All steps were carried out in the dark to prevent photoreactivation. The mixtures were grown at 37 C with shaking at 80 rpm for 24 h. During chlorination, LB broth was diluted 500-fold (total organic carbon (TOC) ¼ 10 mg/l) to avoid chlorine consumption by a high concentration of organic compounds. Solutions of resuspended donor and recipient cells (10 ml each) were mixed together at a 1:1 ratio and then cultured under the same conditions applied for UV treatment. Sodium hypochlorite was introduced to establish different doses of free chlorine (0, 0.05, 0.1, 0.2, 0.3, and 0.5 mg/l Cl2), and cells were cultured for 0e48 h (Huang et al., 2011, 2013). The medium was replaced by fresh medium (1/500 LB) supplemented with the appropriate chlorine concentration every 12 h. Finally, chlorination was terminated with the addition of 1.5% sodium thiosulfate solution before plating. 2.3. Transconjugant identification and mechanism analysis After mating periods of 6, 12, 24, or 48 h, 1 ml of the donor and recipient mixture was collected from each disinfection system. The cultures were serially diluted and spread on LB medium plates containing different antibiotics. The numbers of donors and recipients were determined by counting colonies on LB agar supplemented with 80 mg/l kanamycin, 50 mg/l tetracycline, and 60 mg/l ampicillin or 100 mg/l rifampicin. The recipients possessing the RP4 plasmid were recognized as transconjugants. The transconjugants were selected and counted on LB agar plates containing 80 mg/l kanamycin, 50 mg/l tetracycline, 60 mg/l ampicillin, and 100 mg/l rifampicin. To confirm that the donor and recipient strains could be distinguished, they were cross plated. Transconjugant colonies were randomly selected, and then confirmed using colony polymerase chain reaction (PCR)-based detection technology. The PCR reactions and amplification conditions recommended by Yang et al. (2013) were used. PCR products were detected by electrophoresis in 0.8% agarose gel. Electrophoresis was carried out at 25 C at 180 V for 20 min in Tris-acetate-EDTA (TAE) buffer. Finally, the PCR products (140 bp) were sent to Sangon Biotech (Shanghai, China) for sequencing. The retrieved sequences were aligned with those found in the NCBI database using the Blast tool (http://www. ncbi.nlm.nih.gov/blast). To analyze the mechanisms responsible for the effects of UV irradiation and chlorine treatment on conjugative gene transfer, different donor/recipient rates (e.g., 103, 104, 105, 106, 107, and 108 CFU/ml donor cells mixed with 108 CFU/ml recipient cells) were inoculated, and the corresponding transfer frequencies were calculated. Additionally, the expression of three genes, i.e., a flagllum gene (FlgC), an outer membrane protein gene (ompF), and a transfer regulated gene (TraG) were assessed. FlgC encodes a flagellar protein. For conjugation to occur, the donor and recipient cells must be in physical contact (Dodd, 2012). Bacterial mobility is regulated by polar flagella, which affects bacterial collision and attachment. Thus, the transfer frequency is influenced by the expression of FlgC. OmpF is a common porin protein, and occlusion of ompF may interrupt substance import, including nucleic acid intake during conjugative transfer (Spector et al., 2010). TraG is essential for the conjugative process, because it directly determines the mating between the donors and recipients (Hamilton et al., 2000). RNA extraction was carried out using the EasyPure™ RNA kit (TransGen Biotech, China) according to the manufacturer's instructions. Because RNAs with higher turnover rates would disappear more rapidly, reverse transcription was performed immediately using a reverse transcription (RT)-PCR kit (Promega,
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USA) following the manufacturer's protocols. Finally, the cDNA was stored at 80 C until use. RT-qPCR products were amplified with primers based on previous studies (Ren et al., 2004; Jaktaji and Heidari, 2013; Yuan, 2014; Zhang et al., 2015) (Table S.1, see Supplementary data). The PCR mixtures consisted of 60 ng template cDNA, 2 SYBR Green Mix, and 200 mM each primer in a total volume of 20 ml. In the relative qPCR analysis, target gene expression was normalized to that of a reference gene (16S rRNA gene). Scanning electron microscopy (SEM, Hitachi S-4800, Japan) was used to observe the morphology of the conjugative cells before/ after chlorination. In brief, 1 ml of the cell mixture was collected after 6 h of mating and fixed in 2.5% glutaraldehyde for 2 h. Subsequently, it was washed twice with water, and then dried in an oven at 60 C overnight. 2.4. Statistical analysis The colony averages were calculated using the triplicate plates. The transfer frequency of the RP4 plasmid was calculated using the following formula (Yang et al., 2013; Guo et al., 2015): Conjugative transfer frequency ¼ number of transconjugants (CFU/ml)/number of recipients (CFU/ml). The relationship between UV doses and log conjugative transfer frequency was analyzed using a linear fitting model. In addition, the mRNA expression levels of conjugation related-genes were calculated using the 2-△△CT method as follows (Zhu et al., 2013):
DCt ¼ CtðFunctional genesÞ Ctð16s rRNA geneÞ DDCt ¼ DCtðChlorinationÞ DCtðControlÞ FCðfold changeÞ ¼ 2DDCt Where the Ct value is the cycle threshold. The functional genes represent FlgC, ompF, and TraG. No chlorine was added in the control group. 3. Results and discussion 3.1. Conjugative transfer model construction From three plates on which 100, 187, and 65 colonies grew, 79, 70, and 65 transconjugants were randomly chosen respectively. No false-positive colony was identified by PCR and gel electrophoresis (Fig S.1a). To further confirm the occurrence of plasmid transfer, PCR products were sent to sequencing, and analysis of the data indicated that the amplified fragments shared 97% similarity with the aphA gene of the RP4 plasmid (Fig. S1b). This result showed that all colonies acquired RP4 plasmids, and thus, we successfully constructed the conjugative transfer model. 3.2. Impact of UV irradiation on RP4 plasmid conjugative transfer UV disinfection is being increasingly used in water treatment to inactivate waterborne pathogens. It was evident that the donor strain was significantly affected by UV disinfection before it was mixed with recipient bacteria (Fig. 1a). The number of donor colonies decreased with increasing UV fluence. In fact, 4.23-log and 4.18-log reductions were observed after 5 and 10 mJ/cm2 UV treatments, respectively. In addition, when the fluence was increased to 20 mJ/cm2, the donor number decreased by 5.47 log. Furthermore, 6.29-log and 7.04-log reductions were induced by 50 and 100 mJ/cm2 UV irradiation, respectively. Similarly, Guo et al. (2013) revealed that UV disinfection at a dose of 20 mJ/cm2
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could result in an approximately 4-log reduction. They also found all ARB were eliminated to below the detection limit when the fluence was increased to 50 mJ/cm2. However, after mating for 24 h, the donor cell concentrations again increased significantly after exposure to UV irradiation. At doses ranging from 5 to 20 mJ/ cm2, the donor cell concentrations increased by 1.81e2.91 log. Particularly, increases of 1.31 log and 1.54 log were found at respective doses of 50 and 100 mJ/cm2. Finally, without exposure to UV irradiation, the number of donor cells tended to be more stable. Fig. 1b shows the reduction in cell numbers for both donor and transconjugant cells with increasing UV fluence. Simultaneously, the RP4 plasmid conjugative transfer frequency decreased greatly as the UV irradiation dose increased. The transfer frequency was determined to be 8.63 103 without UV treatment. With exposure to UV doses of 5e20 mJ/cm2, the transfer frequency gradually decreased from 2.75 103 to 2.44 105. The transfer frequency further dropped down to 1.77 106 and 2.44 108 after exposure to UV doses of 50 and 100 mJ/cm2, respectively. As the number of recipient cells was relatively similar in all mating conditions, the transfer frequencies were mainly determined by the numbers of donor cells, with a higher donor density providing more transferable plasmids. Moreover, a high bacterial density was conducive for close contact between cells, which facilitated plasmid transfer. For conjugation to occur in liquid environments, donor and recipient cells must collide, attach, and then conjugate before detachment occurs (Zhong et al., 2010). Thus, the initial cell density significantly affected the conjugative frequency. 3.3. Impact of low-dose chlorination on RP4 plasmid conjugative transfer UV treatment is effective for microbial inactivation in drinking water, but UV irradiation cannot provide a residual disinfectant effect in the drinking water distribution system. Thus, maintaining residual chlorine after chlorination disinfection is a practical approach to repress bacterial regrowth in pipelines. To date, little has been known about how a low dosage of chlorine (0.05e0.5 mg/ l) impacts conjugative plasmid transfer in bacteria. Our study showed that RP4 plasmid transfer was not significantly affected by chlorine treatment at doses ranging from 0.05 to 0.2 mg/l. After mating for 6 h, only a slight decrease (from 2.31 104 to 2.2 104) was observed after exposure to 0.05e0.2 mg/l chlorine. However, a dosage of 0.3e0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 105 or below detection limit (Fig. 2a). We observed similar trends in decreasing transfer frequency after cultivation for 12 h and 24 h (Fig. 2b and c), with frequencies of conjugative transfer of 9.05 106 and 1.01 106, respectively, after treatment with 0.3 mg/l chlorine. No transfer frequency was observed when cells were exposed to 0.5 mg/l chlorine. One explanation for the effect of chlorine treatment on conjugative transfer could be the reduction of donor and recipient cells. After mating for 24 h, donor and recipient strains were decreased to about 1.19 104 and 1.97 104 CFU/ml after treatment with 0.5 mg/l chlorine, respectively (Fig. 2c). Up to 48 h, there was no transfer frequency when the concentration of chlorine was 0.3 and 0.5 mg/l. Moreover, the transfer frequency decreased gradually as the chlorine dosage increased from 0.05 to 0.2 mg/l (Fig. 2d). Therefore, the transfer of RP4 plasmid from the donor strain to recipient bacteria was decreased by low-level chlorine treatment. In addition, the decreasing trend in transfer frequency induced by chlorine was gradually enhanced with prolonged mating time.
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Fig. 1. Impact of UV irradiation on the frequency of RP4 plasmid conjugative transfer. (a) Donor cell density immediately after UV irradiation and after mating for 24 h. (b) The changes in density of donors, recipients, transconjugants, and transfer frequency upon irradiation at different UV fluences.
Fig. 2. Impact of low-level chlorination on the frequency of RP4 plasmid conjugative transfer after mating for 6 h (a), 12 h (b), 24 h (c), and 48 h (d).
3.4. Mechanisms underlying the effects of UV irradiation on RP4 plasmid conjugative transfer To analyze the potential mechanisms responsible for the reduction of conjugative transfer frequency upon UV treatment, we exposed bacteria at different donor/recipient (D:R) rates, to UV irradiation and then calculated the corresponding transfer frequencies. For instance, we mixed 103, 104, 105, 106, 107, and 108 CFU/ ml donor cells with 108 CFU/ml recipient cells. After mating for 24 h, we found that with the same density of recipient cells (108 CFU/ml), higher density of donor cells induced higher numbers of transconjugants and caused higher transfer frequencies (Fig. 3a). We fitted the log concentrations of initial donors (0 h) and log concentrations of final donors (24 h) with log transfer frequency and obtained two linear fitting equations (Fig. 3b and c). The fitted transfer frequency was calculated based on the initial donor density once exposed to UV irradiation and the donor density after mating 24 h. Therefore, theoretical transfer values were obtained (Fig. 3d).
By comparing three groups of transfer frequency (0 h fitting, 24 h fitting, and 24 h mating), it was found that the fitted transfer frequency at 0 h was lower than that at 24 h. This might be explained by bacterial proliferation, regrowth, and repair. First, a low fluence of UV irradiation (<50 mJ/cm2) was not enough to inactive the whole bacteria, and thus, the remaining cells were still alive and could proliferate during the 24 h mating time. Live donors could transfer the plasmid to the recipients. Additionally, it was reported that UV light was directly absorbed by the nucleic acids, causing damage to the DNA of the microorganisms and thereby resulting in the formation of pyrimidine dimers (Lee et al., 2015). Moreover, according to Dodd (2012), UV light directly penetrated the cell membrane to be absorbed by the nucleobases comprising DNA and RNA, and thus, the cell membrane remained intact. Süß et al. (2009) demonstrated that bacteria could recover after UV treatment, but the recovery frequency appeared to be slower with higher UV doses and longer exposure times. Therefore, the bacterial inactivation frequency after UV
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Fig. 3. Analysis of the mechanisms responsible for the impact of UV irradiation on RP4 plasmid conjugative transfer frequency. (a) Impact of different bacterial density ratios on transfer frequency. (b) Fitting equation with log donor (0 h) and log transfer frequency (24 h). (c) Fitting equation with log donor (24 h) and log transfer frequency (24 h). (d) Comparison of theoretical and actual transfer frequencies.
irradiation may be underestimated. Previous studies suggested that thymine dimers induced by UV irradiation were efficiently removed by photoreactivation and dark repair systems/nucleotide excision repair (Jungfer et al., 2007; Giannakis et al., 2014; Bugay et al., 2015). Microorganisms can recover from the damage induced by UV disinfection processes, which can lead to extensive bacterial regrowth. Similarly, Lee et al. (2015) also suggested that the injured DNA could be repaired by photoreactivation or dark repair. Notably, Guo et al. (2012) indicated that the initial antibiotic resistance could be retained or recovered through photoreactivation, which provoked health safety concerns about UV disinfection. In addition, UV irradiation may induce the donors entering into a state characterized as viable but not culturable (VBNC) (Trevors, 2011; Zhang et al., 2015). Although these bacteria cannot be detected by conventional culture methods, they retain metabolic activity. Moreover, certain cellular functions, such as respiratory activity, membrane integrity, glucose incorporation, etc. may decrease rapidly but continue at low stable levels after UV treatment (Villarino et al., 2000). We inferred that conjugative transfer might occur with VBNC bacteria. Accordingly, the transfer frequency that actually occurred after 24 h of mating time was higher than the other fitted values (Fig. 3d). However, additional research is required to further investigate this hypothesis (Fig. 4a). Overall, conjugative transfer after UV irradiation in this study was achieved with high nutrient concentrations and bacterial cell densities than the real ones in drinking water conditions. This is because it is impossible to obtain statistically significant data if real drinking water conditions are applied. However, the results could be extended to drinking water environment to a certain degree. For example, the linear relationship in Fig. 3b was so good that the transfer frequency with very low bacterial density could be inferred. Further, the experimental results can still be applied in
assessing maximum risks and can provide useful references for researchers and policy-makers of water treatment. 3.5. Mechanisms underlying the effects of chlorination on RP4 plasmid conjugative transfer Chlorination is a widely used technology for microbial control in both drinking water and wastewater processing, and a number of mechanisms responsible for the inhibitory effects of chlorine on RP4 plasmid conjugative transfer. First, microorganisms that survive chlorination may be injured rather than inactivated at low chlorine levels (0.05e0.2 mg/l). In addition, the injured cells may repair cellular damage and recover under suitable conditions (Virto et al., 2005). Previous studies have also shown that bacterial regrowth is a common problem in drinking water systems (van der Kooij and van der Wielen, 2013). Consequently, no apparent decrease in conjugative transfer was observed upon exposure to 0.05e0.2 mg/l chlorine (Fig. 1). Conversely, a dosage of 0.3e0.5 mg/l chlorine could significantly reduce the density of donor and recipient cells, leading to lower transfer frequencies. At higher concentrations, chlorine could directly penetrate and break the cell wall (Fig. 4b). Thus, bacteria lost cell membrane integrity and cellular culturability (Nescerecka et al., 2014). In addition, chlorine reacted with nucleotides and lipids, leading to DNA strand breakage and lipid peroxidation (Gray et al., 2012). In our study, the densities of donor and recipient cells decreased simultaneously at dosages of 0.3 and 0.5 mg/l chlorine, although these two types of bacteria had different susceptibilities towards chlorine. Significant reductions in conjugative transfer occurred under these conditions. Similarly, Dodd (2012) also suggested that conventional water disinfection processes generally provide a highly effective means for mitigating the transport of live
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Fig. 4. Analysis of the mechanisms responsible for the impact of UV irradiation (a) and low-level chlorine treatment (b) on RP4 plasmid conjugative transfer frequency.
ARB, thereby minimizing the risks of conjugative gene transfer. Furthermore, Christie and Vogel (2000) suggested that the conjugation mechanism of Gram-negative bacteria is related to bacterial surface structures, such as the conjugal pili for cellecell contacts. The pilus is the mating channel through which DNA are transferred intermediately. In addition, Zhong et al. (2010) also indicated that bacterial attachment and detachment rates can be influenced by pilus length and flexibility. Fig. 5 shows that fewer pili were scattered on bacteria treated by chlorination compared with the control bacteria, and this finding further demonstrated that bacterial conjugation was significantly repressed by chlorine. In another study, Guo et al. (2015) found that treatment with 0.5e4 mg/l chlorine significantly promoted the frequency of conjugative transfer by 2e5 fold. This might be due to differences in the chlorination treatment. They added different initial concentrations of chlorine (<4 mg/l) for 10 min contact in filtered sewage system containing ammonia nitrogen used as necessary nutrition for ARGs transfer. Chlorine reacted with the ammonia first and generated NH2Cl and then NHCl2 and NH3. The formation of chloramine in wastewater promoted the conjugative transfer. However, in our experiment, chlorination was performed with a low dose (0.05e0.5 mg/l) for a longer time (0e48 h) without chloramine. Furthermore, approximately 104e105 CFU/ml donor cells remained alive after treatment with a dosage of 0.5 mg/l chlorine
during mating for 24 h. Based on our theoretical results (Fig. 3a), the transfer frequency was 106e107 with the donor cell densities of 104e105 CFU/ml. However, the real transfer frequency was below the detection limit in this case. There might be other reasons for the inhibitory effects of chlorine on RP4 plasmid conjugative transfer. According to Camper et al. (1979), the physiological functions of cells, such as respiration, growth rate, etc., were reduced by chlorine treatment. Therefore, the expression of three genes related to the conjugative transfer process, i.e., a flagllum gene (FlgC), an outer membrane protein gene (ompF), and a transfer regulated gene (TraG), were quantified in our study. Bacterial mobility is regulated by surface organelles such as polar flagella. Upon exposure to chlorine, the expression of FlgC was suppressed by 0.04e0.34 fold (Fig. 6). Thus, the incidence of bacterial collision and attachment was reduced indirectly. As a result, conjugative transfer was inhibited by chlorine treatment. The outer membrane porin proteins are the major regulators of the permeability of the cell membrane. For instance, ompF is a common porin protein in E. coli that mediates bacterial defense to adverse environments (Cramer et al., 2011; Stenkova et al., 2011). According to Jaktaji and Heidari (2013), this protein exhibits inhibited expression under stress conditions, such as in the presence of chlorine. In our study, the relative expression of ompF was decreased significantly by 0.07e0.44 fold upon chlorine treatment (Fig. 6), which resulted in
Fig. 5. SEM images showing the pili during conjugative transfer with (a) no chlorination or (b) 0.5 mg/l chlorine after mating for 12 h.
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(0.3e0.5 mg/l) treatment is necessary to decrease the HGT frequency in the drinking water distribution system. Furthermore, after UV irradiation bacteria may still transfer plasmid to surrounding communities, which should raise more concerns among both the public and researchers. Acknowledgments This study was financially supported by National Natural Science Foundation of China (51478450, 51278482, and 51408372). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2016.01.020. References Fig. 6. Relative expression of FlgC, OmpF, and TraG after exposure to different doses of chlorine and mating for 6 h.
the formation of small membrane pores. Transient, occlusion of ompF channels may interrupt substance import and membrane mobility, including nucleic acid intake (Spector et al., 2010). As a result, the conjugative transfer frequency was depressed with reduced expression of ompF. The genetic information required for conjugation is encoded by plasmids, which can be classified into two basic groups. One group is non self-transmissible, and an example of non self-transmissible plasmids is RSF1010. The other group is self-transmissible plasmids, such as RP4. According to Cabezon et al. (1997), the TraG protein family is responsible for delivering nicked DNA to the DNA transport for self-transmissible plasmid conjugation. In addition, Hamilton et al. (2000) also suggested that TraG of the RP4 plasmid was thought to couple the relaxosome with the mating bridge. Based on our study, the expression of TraG was inhibited by 0.30e0.37 fold compared to that in the control (Fig. 6), which further explains the potential mechanism by which conjugative transfer was inhibited. 4. Conclusions In the present study, we systematically studied the ability of the common water treatment processes UV irradiation and chlorination to dim the dissemination of ARGs by horizontal transfer. Our experiments showed that UV irradiation reduced the number of live donor cells, and thus, lowered the conjugative transfer frequency. In addition, the transfer frequency was higher than the theoretic values calculated from fitting equations, which may be explained by bacterial regrowth or recovery after UV irradiation. Moreover, we inferred that plasmid transfer might also occur in VBNC bacteria. Of course, further research needed to test this hypothesis. Chlorination also reduced the conjugative plasmid transfer and the inhibitory effect increased with increasing doses of chlorine. The mechanisms behind this phenomenon were also studied. A dosage of 0.3e0.5 mg/l chlorine significantly decreased the density of donor and recipient cells, leading to reductions in conjugative transfer frequency. Additionally, the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport related gene (TraG) was depressed by 0.05e0.5 mg/l chlorine. Furthermore, fewer pili appeared on cells in the chlorination treatment groups. Overall, these findings are important for evaluating the risks of ARB and ARGs in water treatment systems and some management measures can be suggested. For example, low-level chlorine
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