Ionic Liquid Facilitates the Conjugative Transfer of Antibiotic

Article pubs.acs.org/est

Ionic Liquid Facilitates the Conjugative Transfer of Antibiotic Resistance Genes Mediated by Plasmid RP4 Qing Wang,‡ Daqing Mao,*,† and Yi Luo*,‡ †

School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin 300071, China

‡

S Supporting Information *

ABSTRACT: The dissemination and propagation of antibiotic resistance genes (ARGs) is an emerging global health concern. In our previous study, the ionic liquid (IL) 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]) had been proven to facilitate the dissemination of ARGs via horizontal gene transfer. In this study, we further confirm that this compound facilitates the horizontal transfer of plasmid RP4 through a conjugation mechanism and not by natural transformation. The mechanisms for [BMIm][PF6] promoting conjugative transfer are attributable to enhancing the mRNA expression levels of conjugative and global regulatory genes, as well as by inhibiting the genes that are responsible for the vertical transfer of cell growth. [BMIm][PF6] significantly enhanced the expression of the outer membrane porin proteins (OMPs) OmpC and OmpA and the corresponding mRNA expression levels of ompC and ompA genes in recipient bacteria, which contributed to pore formation and increased cell membrane permeability. The increased expression of pilin and pili allowed the donor pilus to attach to and access the recipient cells, thereby assisting cell-to-cell contact to facilitate the conjugative transfer of plasmid RP4. To the best of our knowledge, this is the first insightful exploration of [BMIm][PF6] facilitating the conjugative transfer of ARGs mediated by plasmid RP4 and of several other ILs with different cations or anions that are capable of promoting plasmid transfer. It is therefore suggested that the application of some ILs in industrial processes should be carefully evaluated before their bulk emission into the environment.

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applied in modern industry.17,18 There has not yet been any report of ILs in the environment.19,20 However, the growing interest in the various applications and high stability of ionic liquids results in their presence and persistence in the environment. The application of ILs from industrial processes makes their introduction into wastewater treatment plants (WWTPs) conceivable, as is their discharge into the receiving surface water through the effluent. Meanwhile, ILs may be enriched into sediment through adsorption and a resistance to degradation.20,21 To prepare for the possible environmental release of ILs, researchers have made recent efforts toward the potential ecological and environmental risks of ILs.21,22 Imidazolium ionic liquids have critical inhibitory effects on the growth of a variety of bacteria and fungi23−25 and have higher toxicological activity with increasing alkyl chain length.26,27 Additionally, a previous study investigated the Enterobacter lignolyticus strain genome profile treated with an IL (1-ethyl-3-methylimidazolium chloride), including the upregu-

INTRODUCTION Because antibiotic resistance genes (ARGs) are currently considered environmental pollutants, the propagation of ARGs is an emerging global health concern.1−5 ARGs can be disseminated among microorganisms through horizontal gene transfer, which includes conjugation (the transfer of mobile genetic elements from a donor cell to a recipient cell), transformation (the uptake of naked DNA), and transduction (the use of bacteriophages as transporters of genetic information).6−8 Conjugation is considered the principal mode for antibiotic resistance transfer via mobile genetic elements (such as plasmids, integrons, and transposons).9−14 Conjugation occurs more frequently between closely related strains (within genera) or species of bacteria and occurs at a relatively low frequency across genera.8,15 In our previous study, we demonstrated that an ionic liquid (IL) 1-butyl-3methylimidazolium hexafluorophosphate ([BMIm][PF6]) is capable of facilitating the dissemination of ARGs via horizontal gene transfer.16 However, whether it induces horizontal gene transfer through bacterial conjugation or through transformation mechanisms is not understood. Ionic liquids, considered as “environmentally friendly” replacements for industrial organic solvents, have been widely © XXXX American Chemical Society

Received: December 8, 2014 Revised: June 5, 2015 Accepted: June 29, 2015

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DOI: 10.1021/acs.est.5b01129 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology lation of osmoprotectant transporters and drug efflux pumps.28 [BMIm][PF6], as one of the common ILs, possesses numerous fascinating properties and is of fundamental interest to modern chemistry and the chemical industry.18,20 The incompatibility group P1 (Inc.P) plasmid RP4 conjugative transfer process has been grouped into two functional subsets belonging to the mating pair formation (Mpf) and the DNA transfer and replication (Dtr) systems, which require a series of conjugation genes and the corresponding involvement of regulatory genes and entry exclusion.29,30 The mechanism by which exposure to [BMIm][PF6] changes the regulated gene expression to influence the conjugative transfer of plasmid RP4 remains unknown. Additionally, conjugation requires the cell-to-cell contact of donor and recipient bacteria and the crossing of bacterial membranes.31,32 Gram-negative bacteria possess a number of outer membrane porin proteins (OMPs), which play an important role in specific and nonspecific pore formation and membrane transport.33,34 The mechanism by which treatment with [BMIm][PF6] alters cell membrane permeability and the expression of OMPs to promote the conjugative transfer of plasmid RP4 is also unknown. In this study, based on our prior findings, we further investigate the mechanism of an IL, [BMIm][PF6], facilitating the dissemination of ARGs, whether through plasmid RP4 conjugation or through natural transformation. We also explore the molecular mechanisms that assist the conjugative transfer of ARGs with regard to cell membrane permeability, the mRNA expression levels of conjugative transfer genes, and the expression of OMPs in the recipient and pilin in the donor. In addition to [BMIm][PF6], the possibility of other ILs carrying the same cation or the same anion facilitating the horizontal transfer of plasmid RP4 are tested. To our knowledge, this is the first study to explore how IL [BMIm][PF6] promotes the conjugative transfer of the environmentally widely distributed plasmid RP4, thereby facilitating the propagation of ARGs.

0.01, 0.1, 0.5, and 2.5 g/L. The IL concentrations in this study were comparable or lower than those in previous toxicity tests.27,28 After mating progressed for 12 h at pH 7.0 and 30 °C, the colony-forming units per milliliter of culture (cfu/mL) of the transconjugants (AmpR, KmR, TcR, and StrR) were counted on LB-selective plates containing 100 mg/L of Amp, 50 mg/L of Km, 10 mg/L of Tc, and 30 mg/L of Str. PCR and sequencing were used to verify that the plasmid RP4 had been transferred into the recipient strain. For details on conjugative transfer experiments, please refer to section 1 in the Supporting Information. To rule out the natural transformation from the naked DNA to the recipient bacteria in the conjugative transfer process, we used natural transformation experiments35 as the negative controls for the transfer from the naked DNA (plasmid RP4) to the recipient E. coli HB101 and S. enterica treated with [BMIm][PF6]. The donor plasmid RP4 was used, and plasmid RP4 extraction was performed in E. coli DH5α using a bacterial DNA kit according to the manufacturer’s instructions (Omega, USA). Donor plasmid RP4 was added to the recipient E. coli HB101 and S. enterica at final concentrations of 5 μg/mL (this concentration is the equivalent of the donor E. coli DH5α harboring plasmid RP4 at a concentration indicative of conjugative transfer) and 50 μg/mL, and it was added to [BMIm][PF6] at initial concentrations of 0, 0.001, 0.01, 0.1, 0.5, and 2.5 g/L. For details of these transformations, please refer to section to in the Supporting Information. Flow Cytometry Detection of Cell Membrane Permeability Induced by [BMIm][PF6]. To explore the mechanisms by which [BMIm][PF6] stimulates horizontal gene transfer, we employed flow cytometry (FCM; BD FACSCalibur, USA) to differentiate and quantify the [BMIm][PF6]exposed bacterial cells and control cells. In these experiments, fluorescence intensity was a function of cell membrane permeability, with higher fluorescence intensity signifying enhanced cell membrane permeability. The FCM was equipped with an excitation wavelength of 488 nm. A sample containing 1 mL of bacterial suspension ([BMIm][PF6]-treated or control) was stained with 10 μL of propidium iodide (PI; 1 mg/mL, Omega, USA) and incubated in darkness for 8 min before measurement.36 The concentration of bacterial suspension was always less than 106 cells/mL. All data were processed with CellQuest Pro software. Detection of the mRNA Expression of Genes Responsible for Conjugative Transfer. Bacterial cell pellets were collected by centrifugation (10 000 rpm for 10 min), and the total bacterial RNA was extracted using an RNA extraction kit (Omega, USA) according to the manufacturer’s instructions. The quality and quantity of the extracted RNA were determined by agarose gel electrophoresis and analysis with a spectrophotometer (JENWAY Genova, British). RNA was transcribed to cDNA by reverse transcription polymerase chain reaction (RT-PCR) with a reverse transcription kit (Omega, USA). Polymerase chain reaction (PCR) assays were used to target the global regulatory genes (trfAp, trbBp, korA, korB, and trbA), conjugative genes (traF, kilA, and kilB), and an entryexclusion-system gene (trbK) of the plasmid RP4, as well as ompC and ompA porin genes. Qualitative PCR assays were conducted in a Biometra T100 gradient cycler (Biometra, Germany), and quantitative PCR (qPCR) analyses were performed on a Bio-Rad iQ5 instrument (Bio-Rad Company, CA, USA) to quantify the 16S rRNA, trfAp, trbBp, korA, korB, trbA, traF, kilA, kilB, trbK, ompC, and

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MATERIALS AND METHODS IL Chemicals. A disparate class of IL compounds including the same cation of imidazolium (1-butyl-3-methylimidazolium hexafluorophosphate, [BMIm][PF6]; 1-butyl-3-methylimidazolium tetrafluoroborate, [BMIm][BF4]) or the same anion of hexafluorophosphate (PF6−) and bromide (1-butyl-3-methylimidazolium hexafluorophosphate, [BMIm][PF6]; N-ethylpyridinium hexafluorophosphate, [EPy][PF6]; N-butyl-Nmethylpyrrolidinium bromide, [BMPr][Br]; tetraethylammonium bromide, [NEt4][Br]) were selected as the tested compounds in this study. All ILs were purchased from Chinese Academy of Sciences (>99% pure). Plasmid RP4 Conjugative Transfer System. Plasmid RP4 conjugative transfer was used to determine the effects of the IL [BMIm][PF6] on the transfer of plasmid RP4 from Escherichia coli DH5α (E. coli DH5α, as the donor) to E. coli HB101 (streptomycin resistance (StrR) as the recipient within the same species) and Salmonella enterica (StrR as the recipient across genera). The strain E. coli DH5α, harboring the plasmid RP4 carrying the ampicillin, kanamycin, and tetracycline resistance genes (AmpR, KmR, and TcR) but no StrR, was used as the donor. The E. coli HB101 strain and S. enterica carrying StrR in the genome were used as the recipients and lacked AmpR, KmR, and TcR. The system was spiked with the IL [BMIm][PF6], resulting in initial concentrations of 0, 0.001, B


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RESULTS AND DISCUSSION [BMIm][PF6] Facilitation of the Conjugative Transfer of Plasmid RP4. The horizontal transfer frequency of plasmid RP4 was significantly increased (ANOVA, p < 0.05) by the tested ILs, such as [BMIm][PF6], 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]), and N-ethylpyridinium hexafluorophosphate ([EPy][PF6]) (Figure 1), tetraethylam-

ompA genes. The PCR primers and conditions, details of the PCR and qPCR protocols, and the standard curves are listed in Table S1 and in sections 3 and 4 of the Supporting Information. Extraction and Detection of Outer Membrane Porin Proteins in the Recipient. To study the expression of [BMIm][PF6]-induced proteins, we prepared OMPs by a method previously described with modification.37,38 Triplicates of the culture media with the addition of ILs were run in this study. Briefly, recipient bacteria were grown and [BMIm][PF6]-treated as described earlier. Cell pellets were harvested at 10 000 rpm for 10 min and suspended in 20 mM Tris-HCl buffer (pH 7.6) containing 5 mM MgCl2. Cells were washed twice and suspended in the same buffer containing 2 mM phenyl methyl sulfonyl fluoride (PMSF, Sigma, USA). Cells were then disrupted by ultrasonication (SCIENTZ-II D, China) (20 min of 4 s with 4 s intervals), and the undisrupted material was removed by low-speed centrifugation (3000g, 4 °C, 20 min). Supernatant was then ultracentrifuged (BECKMAN COULTER, Optima MAX-XP Ultracentrifuge, USA) at 120000g for 60 min at 4 °C, and the pellet was suspended in 1% sodium lauryl sarcosyl (Sigma, USA) in 20 mM Tris-HCl buffer at pH 7.6. After incubation for 2 h at 37 °C, the fraction of detergent-insoluble OMPs was collected by ultracentrifugation at the same speed. The recovered outer membrane fraction was suspended in 20 mM Tris buffer containing 2 mM PMSF and stored at −20 °C. Protein analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously.37,39 Protein estimation was performed with bovine serum albumin as a standard.40 Extraction of Pilin from the Donor. Pilins were isolated from the donor strain DH5α by mechanical disruption as previously described.41 The donor bacteria were cultured for 48 h at 37 °C with shaking (160 rpm) in Trypticase soy broth (TSB). A sample of 80 mL of bacterial culture was centrifuged at 4 °C for 10 min at 5000g, and the bacterial pellet was washed twice with phosphate-buffered saline (PBS; 10 g/L NaCl, 0.25 g/L KCl, 1.8 g/L Na2HPO4, and 0.3 g/L KH2PO4). The pellet was resuspended in 10 mL of PBS and oscillated in a water bath on a shaking table at 60 °C for 30 min, then centrifuged at 4 °C for 20 min at 10000g after high-speed mixing for 20 min. Saturated ammonium sulfate solution was added to the supernatant, and the solution was incubated overnight at 4 °C. Subsequently, the solution was precipitated by centrifuging at 4 °C for 15 min at 10000g and was dialyzed for 48 h. The supernatant was centrifuged at 4 °C for 15 min at 8000g, and the precipitate was resuspended in 2 mL of PBS. This solution was analyzed by SDS-PAGE, and protein estimation was performed with bovine serum albumin as a standard, as described previously.37,39 Phage M13 Infection of Donor E. coli DH5α. Pilin expression with the donor E. coli DH5α induced by [BMIm][PF6] was tested with infection by phage M13 (a host in the E. coli ER 2738). Phage M13 infection of E. coli DH5α was a function of the pilus, with a greater expression of the pilus signifying enhanced infection.42 Donor bacteria were grown and [BMIm][PF6]-treated as described earlier. Donor bacteria were adjusted to specific microbial concentrations (OD600 = 0.15), and the final phage concentrations were adjusted to 107 plaque-forming units per milliliter (pfu/mL).43 After phage infection, the bacterial concentration was determined with a spectrophotometer before saturated infection occurred (approximately 4 h).

Figure 1. Effect of ILs on the transfer frequency of the horizontal transfer of plasmid RP4 from E. coli DH5α to E. coli HB101 for 12 h at pH 7.0 and 30 °C. The ILs had a significant effect on the conjugative transfer of RP4 (ANOVA, P < 0.05); significant differences between ionic liquids with the same cation or anion were tested with the S−N− K test (*, P < 0.05 and **, P < 0.01). [BMIm][PF6]:1-butyl-3methylimidazolium hexafluorophosphate (CAS: 174501−64−5); [BMIm][BF4]: 1-butyl-3-methylimidazolium tetrafluoroborate (CAS: 174501−65−6); [EPy][PF6]: N-ethylpyridinium hexafluorophosphate (CAS: 103173−73−5); Na[PF6]: sodium hexafluorophosphate (CAS: 21324−39−0).

monium bromide ([NEt4][Br]), and N-butyl-N-methylpyrrolidinium bromide ([BMPr][Br]) (Figure S2 in the Supporting Information). Compared with the results of the blank control groups (without ILs and Na[PF6]), the inorganic salt Na[PF6] significantly facilitated the conjugative transfer of RP4, implicating that the [PF6]− anion was the reactive group to accelerate the transfer process (Figure 1); the transfer frequency facilitated by [BMIm][PF6] treatment was significantly higher than that of Na[PF6] treatment, indicating that the cation of [BMIm]+ played a significant role in the promotion of the conjugative transfer of RP4. Furthermore, the increasing effect of the [BMIm]+ cation on RP4 transfer was confirmed by the higher transfer frequency facilitated by the [BMIm][PF6] treatment than that of the [EPy][PF6] treatment (p < 0.05) using the Student−Newman−Keuls (S− N−K) test, as shown in Figure 1. The promoting role that the [PF6]− anion played on RP4 transfer was also consolidated with the higher transfer frequency of [BMIm][PF6] treatment than that of [BMIm][BF4] treatment (p < 0.05, S−N−K test) as shown in Figure 1, implicating the better effect exerted by [PF6]− than by the [BF4]− anion in low concentrations (0.04 and 0.4 mmol/L). The conjugative transfer frequency increased with increasing [BMIm][PF6] concentrations (0.001 to 0.5 g/L) and was up to 88-fold higher [(1.82 ± 0.38) × 10−3 per recipient cell] than that of the control groups [(2.07 ± 0.62) × 10−5 per recipient cell] under 0.5 g/L of [BMIm][PF6] treatment within the same C


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[BMIm][PF6]-Enhanced Cell Membrane Permeability. FCM assays of both E. coli HB101 and S. enterica treated with [BMIm][PF6] showed that the percentage of PI-positive cells increased with increasing concentrations of [BMIm][PF6], indicating that bacterial cell membrane permeability increases with increasing concentrations of [BMIm][PF6] (Figure 3). These concentrations were up to 25-fold higher with the 0.5 g/ L [BMIm][PF6]-treated groups than with the [BMIm][PF6]untreated controls (p < 0.01, S−N−K test). During plasmid conjugation, plasmid DNA were transferred from the donor to the recipient bacteria, crossing bacterial membranes.30 However, the bacterial cell membrane serves as a barrier against the conjugation transfer of genetic information between different species or genera of bacteria.9 Increased cell membrane permeability induced by [BMIm][PF6] may suppress cell membrane barriers, driving plasmid RP4 transfer across the bacterial membrane and facilitating the conjugative transfer of genetic information. Interestingly, there was no significant difference in cell membrane permeability between recipients within the same species and cross-genera (Figure 3b). These findings indicate that the observed differences in conjugative transfer frequency within the same species and across genera were not attributed to the cause of enhanced cell membrane permeability. Gram-negative bacteria adapt their permeability by modulating the expression of outer membrane porin proteins (OMPs), including OmpC (molecular mass 36 kDa) and OmpA (molecular mass 34 kDa), which are considered classical porins.33,44 The OmpC and OmpA porins are known to play important roles in the membrane transport of genetic information between the inside of the cell and the outer environment, and they are known to control the permeability of the cellular membrane.34,44 OmpC and OmpA porin expression increased in recipient bacteria (Figure 4a,c), contributing to the observed enhanced cell membrane permeability (Figure 3) and facilitating communication between the recipient cell and the outer environment, particularly the donor cell, which in turn facilitated gene transfer of the resistance plasmid, increasing the risk of ARG propagation in the environment. [BMIm][PF6]-Enhanced MRNA Expression Levels of Conjugative Transfer Genes. Conjugation is the formation of conjugative bridges between the donor strain and the recipient strain, a process under the regulation of the mating pair formation (Mpf) system gene trbBp and the plasmid transfer and replication (Dtr) system gene trfAp.30,31 A previous study investigated the Enterobacter lignolyticus strain genome profile treated with an IL (1-ethyl-3-methylimidazolium chloride), including the up-regulation of osmoprotectant transporters and drug efflux pumps.28 However, the regulation of gene expression related to plasmid conjugation under exposure to ILs has not yet been reported. In this study, we first reported [BMIm][PF6]-enhanced conjugative transfer through the regulation of conjugative gene expression. The mRNA expression levels of Mpf gene trbBp (Figure 5a) treated with 0.5 g/L [BMIm][PF6] increased significantly, up to 18-fold [(1.33 ± 0.06) × 10−4/16S rRNA in E. coli HB101] and 12-fold [(9.18 ± 0.86) × 10−5/16S rRNA in S. enterica] higher than did the controls in the same species and across genera, respectively, which were conducive to the formation of more conjugants. The Dtr gene trfAp mRNA expression levels (Figure 5b) also increased significantly with increasing [BMIm][PF6] concentrations, both in the same species and across genera, which is conducive to the transfer and replication of RP4. [BMIm][PF6]

species (Figure 2). [BMIm][PF6] also promoted cross-genera conjugative transfer (Figure 2), with a conjugative transfer

Figure 2. Conjugation of plasmid RP4 from E. coli DH5α to E. coli HB101 and from E. coli DH5α to S. enterica affected by [BMIm][PF6] concentration mated for 12 h at pH 7.0 and 30 °C. The concentration of [BMIm][PF6] had a significant effect on the transfer frequency of the plasmid RP4 (ANOVA, P < 0.05). Significant differences between each of the IL-treated groups and the control (0 g/L of IL) were tested with the S−N−K test and shown with * (P < 0.05) and ** (P < 0.01). The differences in conjugative transfer frequencies between bacteria of the same species and across genera were conducted by ANOVA and shown with ** (P < 0.01) above the polygonals. Significant differences between each of the IL-treated groups and the control (0 g/L of IL) were tested with the S−N−K test and shown with ** (P < 0.01).

frequency that increased with increasing [BMIm][PF6] concentrations and reached a peak value of 0.5 g/L of [BMIm][PF6], approximately 16-fold [(5.27 ± 0.14) × 10−5 per recipient cell] higher than that of the control groups [(3.33 ± 0.67) × 10−6 per recipient cell]. In contrast, the conjugative transfer frequency was decreased by the very high concentration of [BMIm][PF6] (2.5 g/L), shown in Figure 2, which is largely due to the lower bacteria survival for both donors (19.65% of survival compared to control) and recipients (19.90% of survival compared to control); details are shown in Table S3 in the Supporting Information). These results were identical to those of Qiu et al., who reported that the decreased conjugative transfer was attributable to lower donors and recipients concentration affected by nanoalumina.29 Notably, the conjugative transfer frequency was significantly lower across genera than within the same species. The natural transformation frequency from naked DNA to the recipient strain was very low [(1.1 ± 0.5) × 10−8 per recipient cell].35 In this study, when donor plasmid RP4 was enhanced at a 50 μg/mL concentration, the transformation frequency reached peak values [(2.52 ± 0.88) × 10−8 per recipient cell in E. coli HB101 and (2.30 ± 0.73) × 10−8 per recipient cell in S. enterica] at 0.5 g/L of [BMIm][PF6], which is 5-fold higher than that of the [BMIm][PF6]-untreated group (Figure S1 in the Supporting Information). However, natural transformation was not observed when the donor plasmid RP4 concentration (5 μg/mL) was equivalent to the donor plasmid RP4 concentration of the conjugative transfer in [BMIm][PF6]-treated groups and the untreated groups. It is therefore reasonable to conclude that [BMIm][PF6] exerted selective pressure, promoting the spread of plasmid RP4 (both within genera and cross-genera) by conjugation but not by natural transformation. D


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Figure 3. Cell membrane permeability of recipients of E. coli HB101 and S. enterica treated with [BMIm][PF6] quantified using FCM. (a) Flow cytometry data in a dot plot. Quadrant D: negative signal (normal cells). Quadrant C: PI-positive (cells with increased membrane permeability). (b) Percentage of cells with increased membrane permeability (PI-positive) treated with [BMIm][PF6]. [BMIm][PF6] concentration had a significant effect on the cell membrane permeability of microorganisms (ANOVA, P < 0.05). Significant differences between single concentration groups and the control groups (0 g/L group) were tested with the S−N−K test (*, P < 0.05 and **, P < 0.01).

facilitated conjugative transfer by enhancing the expression levels of horizontal transfer genes. [BMIm][PF6] was found to inhibit the genes responsible for vertical transfer. The TraF gene mRNA expression levels increased significantly, up to 80fold [(1.47 ± 0.68) × 10−3/16S rRNA in E. coli HB101] and 65-fold [(9.22 ± 0.33) × 10−4/16S rRNA in S. enterica] after treatment with 0.5 g/L [BMIm][PF6] as compared with those of the controls (Figure 5f), which led to enhanced conjugative transfer. Our previous study observed that [BMIm][PF6] (2.5 g/L) exposure inhibited bacterial growth.16 In this study, kilA and kilB mRNA expression levels known to negatively impact vertical transfer both increased significantly with increasing [BMIm][PF6] concentrations (Figure 5g,h). This explained the contribution of [BMIm][PF6] to the repression of vertical transfer by inhibiting cell growth. Entry exclusion was previously defined as the inhibition of plasmid transfer into a recipient cell.49 A single gene, trbK, is responsible for the entry exclusion phenotype of plasmid

promoted conjugative transfer by repressing the expression of global regulatory genes on plasmid RP4. We found that the mRNA expression levels of the major global regulatory genes korA, korB, and trbA were significantly repressed with increasing concentrations of [BMIm][PF6] compared with those of the control groups, both in the same species and across genera (Figure 5c−e). Both korA and korB repress trfAp expression, and korB and trbA expression severely repress the trbBp promoter.45,46 Alleviation of the repression of the korA, korB, and trbA genes significantly triggered the expression of the promoters trbBp and trfAp, promoting the conjugative transfer of plasmid RP4. Widespread dissemination of the resistance plasmid by vertical transfer (i.e., by cell growth) and horizontal gene transfer processes was found throughout bacterial populations.7 The TraF gene was positively related to horizontal transfer, and the kilA and kilB genes were negatively related to plasmid vertical transfer.47,48 We confirmed that [BMIm][PF6] E


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Figure 4. (a) SDS-PAGE of outer membrane porin proteins (OMP) with recipients of E. coli HB101 and S. enterica for varying [BMIm][PF6] concentrations. Lanes: M, markers; 1, E. coli HB101 (0 g/L of [BMIm][PF6]); 2, S. enterica (0 g/L of [BMIm][PF6]; 3, E. coli HB101 (0.5 g/L of [BMIm][PF6]); 4, S. enterica (0.5 g/L of [BMIm][PF6]). (b) SDS-PAGE of pilin with a donor of E. coli DH5α. Lanes: 5, E. coli DH5α (0.5 g/L of [BMIm][PF6]); 6, E. coli DH5α (0 g/L of [BMIm][PF6]); M, markers. (c) OMP concentrations in E. coli HB101 and S. enterica recipients for varying [BMIm][PF6] concentrations. (d) Pilin concentrations in E. coli DH5α donors. Significant differences between single concentration groups and the control groups (0 g/L group) were tested with the S−N−K test (*, P < 0.05 and **, P < 0.01).

Figure 5. MRNA expression levels of horizontally transferred genes. The concentration of [BMIm][PF6] had a significant effect on the expression levels of the global regulatory genes (a−e), conjugative genes (f−h), and entry exclusion system genes (i) of the plasmid RP4 in E. coli DH5α to E. coli HB101 and E. coli DH5α to S. enterica transfers. (ANOVA, P < 0.05); a significant difference in the expression levels of the entry exclusion system genes (i) was induced by [BMIm][PF6] between E. coli DH5α to E. coli HB101 and E. coli DH5α to S. enterica. Significant differences between the single concentration groups and the control groups (0 g/L group) were tested with the S−N−K test (*, P < 0.05 and **, P < 0.01). F


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Figure 6. MRNA expression levels of the ompC and ompA porin genes. The concentration of [BMIm][PF6] had a significant effect on the expression levels of the ompC and ompA porin genes in the transfers from E. coli DH5α to E. coli HB101 and E. coli DH5α to S. enterica. Significant differences between the single concentration groups and the control groups (0 g/L group) were tested with the S−N−K test (*, P < 0.05 and **, P < 0.01).

Figure 7. (a) The microbial concentrations (OD600) with donors of [BMIm][PF6]-treated E. coli DH5α infected by phage M13 via the pilus. (■): E. coli DH5α only; (●): E. coli DH5α with 0 g/L of [BMIm][PF6] treatment and infected by phage M13; (▲): E. coli DH5α with 0.1 g/L of [BMIm][PF6] treatment, infected by phage M13; (▼): E. coli DH5α with 0.5 g/L of [BMIm][PF6] treatment, infected by phage M13. (b) Donor E. coli DH5α growth curve in response to 0.1 g/L of [BMIm][PF6] (●) and 0.5 g/L of [BMIm][PF6] (▲).

RP4.49,50 In this study, the mRNA expression levels of trbK gene were found to decrease significantly with increasing [BMIm][PF6] concentrations, up to 94-fold [(1.68 ± 0.32) × 10−6/16S rRNA] less than those of control groups in the same species (Figure 5i). However, we observed only a 5-fold [(1.81 ± 0.09) × 10−4/16S rRNA] decrease compared to those of the control groups across genera (Figure 5i). This significantly lower mRNA expression level of the trbK gene within the same species explains why the conjugative transfer frequency is much higher within the same species than across genera. [BMIm][PF6]-Increased Expression of OmpC and OmpA Porins and Pilin. Gram-negative bacteria possess a number of OMPs (OmpA and OmpC porins, etc.) related to conjugative transfer from a recipient that play important roles in concert with specific and nonspecific pore-forming proteins and membrane transport.33,34 The mRNA levels of ompC and ompA genes were significantly enhanced under exposure to 0.5 g/L of [BMIm][PF6] (Figure 6). The expression of OmpA is tightly regulated at the post-transcriptional level, as has been previously confirmed in E. coli.51 Furthermore, the increased

post-transcriptional level of OmpA and OmpC expression in E. coli HB101 and the S. enterica strain that was observed as an increased protein amount (Figure 4c) under exposure of 0.5 g/ L of [BMIm][PF6] was involved in the present research. In contrast, a down-regulation of the mRNA ompA gene in E. lignolyticus strain treated with another IL (1-ethyl-3-methylimidazolium chloride) was found with Khudyakov et al.28 This difference might be attributed to the time scale (12 h in this study versus 72 h in the Khudyakov et al. study).28 Meanwhile, the complex structure of the ILs family may contribute to the different regulations of genome profile expressions. In this study, exposure to 0.5 g/L of [BMIm][PF6] significantly increased the concentrations of OMPs (OmpA and OmpC) in the E. coli HB101 and S. enterica recipients, demonstrating a 4.7-fold increase compared to that of untreated bacteria (Figure 4c). Additionally, the concentration (13.55 μg/mL) of pilin in E. coli DH5α that was [BMIm][PF6]-treated (0.5 g/L) increased 4.2-fold compared to that of untreated bacteria (3.24 μg/mL) (Figure 4d). The enhanced expression of pilus in donor bacteria under treatment with [BMIm][PF6] was further G



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confirmed by determining the efficiency of the RP4-pilusspecific phage M13. Phage M13 can only infect plasmid-bearing donor cells by the pili,52 and the E. coli host continues to grow and slow the following infection with M13.53 As observed with the infection by phage M13, the lower bacterial concentration (OD600) with [BMIm][PF6] treatment (0.1 and 0.5 g/L) was attributable to enhanced infection by phage M13 via the pilus (Figure 7), indicating the increased expression of pili in bacteria that were [BMIm][PF6]-treated, because phage M13 infecting the RP4-bearing E. coli DH5α was a function of the pilus, and the greater expression of pili signified enhanced infection.42 Increased OmpC and OmpA porin expression in the recipient bacteria contributes to pore-forming, which allowed a donor pilus to attach to and access the recipient cell.32,34 The increased expression of the donor pilin and pili allowed the donor pili to attach to and access recipient cells,54 facilitating cell-to-cell contact and the conjugative transfer of plasmid RP4. This study first demonstrated that the IL [BMIm][PF6] facilitates the conjugative transfer of plasmid RP4 so as to promote the dissemination of ARGs between the same species of bacteria and across genera. It is not limited only to this compound; four other classes of tested ILs with the same cation or the same anion also showed the ability to promote the horizontal transfer of plasmid RP4 (Figure 1), and the cation [BMIm]+ and anion [PF6]− investigated in the present research both have influence on plasmid horizontal transfer. Conjugative transfer frequency was observed to be much higher within the same species than across genera, which may be attributable to the significantly lower mRNA expression levels of the entry exclusion gene trbK in the same species. The mechanisms by which ILs facilitate the conjugative transfer of plasmid RP4 may involve enhanced cell membrane permeability, the elevated expression of horizontal transfer genes and an entry exclusion gene, the repression of global regulatory genes, and the increased expression of OmpC and OmpA porins and pilin. These findings suggest that some ILs have the potential to facilitate the propagation and proliferation of AGRs among bacteria. Environmental Implications. ILs, as the safe and environmentally friendly alternative to conventional solvents, have recently been proposed to replace volatile organic solvents in industrial reactions. In this study, we confirmed that several classes of tested ILs are capable of facilitating the dissemination of ARGs mediated by an environmentally widely distributed plasmid RP4, and an IL [BMIm][PF6] caused that effect via conjugative transfer in environmental microorganisms, e.g., E. coli and S. enterica, which are pathogenic and opportunistic pathogens in the environment. These two strains could acquire antibiotic resistance, therefore increasing the risk of ARG dissemination to human pathogens and posing great threat to public health. Meanwhile, excellent chemical and thermal stability make their entry into wastewater treatment plants conceivable, as is their discharge into the receiving environment through an effluent or sludge landfill. As a consequence, ILs can represent a continuous selective pressure for the environmental bacteria and facilitate the dissemination and propagation of ARGs.21 On the basis of this study, we propose that the application of some ILs in industrial processes should be further carefully evaluated by more toxicological tests before their bulk emission into the environment.

Article

ASSOCIATED CONTENT

S Supporting Information *

Details of the conjugation and transformation system, PCR conditions and primers, statistical analysis, the effect of [BMIm][PF6] on transformation, and supplementary references. Figures showing the transformation of DNA affected with [BMIm][PF6] and the effect of ILs on transfer frequency. Tables showing PCR primers and conditions, system pH values of the [BMIm][PF6], the conjugation of plasmid RP4, and IL concentrations. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.est.5b01129.

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AUTHOR INFORMATION

Corresponding Authors

*D.M. Phone: +86 (22) 87402072; e-mail: [email protected]. *Y.L. Phone: +86 (22) 23508535; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Education of the People’s Republic of China as an innovative research team project (grant no. IRT13024) and the National Natural Science Foundation of China (grants 31470440 and 41473085). Meanwhile, we express our sincerest thanks to Professor Zhangyong Hong (The College of Life Sciences in Nankai University, Tianjin, China) for the provision of the phage M13 (host in the E. coli ER 2738).

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REFERENCES

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Ionic Liquid Facilitates the Conjugative Transfer of Antibiotic

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