Enhanced Horizontal Transfer of Antibiotic Resistance Genes in Freshwater Microcosms Induced by an Ionic Liquid

The spread and propagation of antibiotic resistance genes (ARGs) is a worldwide public health concern. Ionic liquids (ILs), considered as “environmentally friendly” replacements for industrial organic solvents, have been widely applied in modern industry. However, few data have been collected regarding the potential ecological and environmental risks of ILs, which are important for preparing for their potential discharge into the environment. In this paper, the IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6]) (0.001-5.0 g/L) was tested for its effects on facilitating ARGs horizontal transfer mediated by plasmid RP4 in freshwater microcosms. In the horizontal transfer microcosms, the transfer frequency of plasmid RP4 was significantly enhanced (60-fold higher than untreated groups) by the IL [BMIm][PF6] (1.0 g/L). Meanwhile, two strains of opportunistic pathogen Acinetobacter spp. and Salmonella spp. were isolated among the transconjugants, illustrating plasmid RP4 mediated horizontal transfer of ARGs occurred in pathogen. This could increase the risk of ARGs dissemination to human pathogens and pose great threat to public health. The cause that [BMIm[PF6] enhanced the transfer frequency of plasmid RP4 was proposed by suppressed cell membrane barrier and enhanced cell membrane permeability, which was evidenced by flow cytometry (FCM). This is the first report that some ILs facilitate horizontal transfer of plasmid RP4 which is widely distributed in the environment and thus add the adverse effects of the environmental risk of ILs.


Introduction
The emergence and persistence of clinically relevant antibiotic resistance genes (ARGs) in natural and engineered environments has become a major global health issue [1][2][3][4]. Antibiotic resistance in bacteria can be intrinsic or acquired by antibiotic selective pressure [5][6][7]. Acquired resistance is the main reason for ARGs proliferation and it occurs between individual cells or species mainly by horizontal transfer processes [8][9][10]. The primary mechanisms of horizontal transfer are conjugation (plasmids are transferred from a donor cell to a recipient cell), horizontal transfer of plasmid RP4. This is the first study showing the IL-induced promotion of horizontal transfer of ARGs mediated by plasmid RP4 in a freshwater environment.

Setup of plasmid RP4 horizontal transfer in microcosm
Setup of plasmid RP4 horizontal transfer in microcosms was based on the OECD 308 test [56] to test the effects of the IL [BMIm][PF6] on the plasmid RP4 horizontal transfer to indigenous bacteria in the microcosm. The freshwater sample was collected from the Water Parke (39°6 '13"N, 117°9'21"E) in Tianjin, China on Sep 2013. No specific permission was required as this is a public park and is open to visit and scientific research. In addition, the authors confirm that no endangered or protected species were involved in this study. Water properties are described in S1 Table and detection method of water samples in SI-5 in S1 Text. The strain of rifampicin resistance (Rif R ) E.coli K12 (ATCC 47076) harboring the plasmid RP4, carrying ampicillin, kanamycin and tetracycline resistance (Ap R , Km R , and Tc R ), was used as the RP4 donor strain. The recipients were indigenous bacteria in freshwater microcosms. Meanwhile, in microcosms, recipient isolates were negative in the Rif R screening and PCR screening, traF gene (indicator for RP4) and aphA gene (kanamycin-resistance gene on RP4) were negative also in the PCR screening.
The water samples (1800 mL in 2000 mL break) were stabilized for 3 hours at 4°C in a refrigerator to remove sediment. Supernatant (1500 mL) was collected and supplemented with 1% (V:V) Luria-Bertani (LB) media, incubated overnight on a shaker incubator (160 rpm) at 30°C, and diluted to reach microbial concentrations (the optical densities) of OD 600 = 0.400. Water samples (200 mL) were then supplemented with 1% (V:V) E.coli K12 donor strains (approximately plasmid RP4 concentration of 5 μg/mL), making microcosms. Donor-free microcosms also used as controls (no horizontal transfer in donor-free microcosms in preexperiment). The microcosms were spiked with an IL [BMIm][PF6] (>99% pure, Chinese Academy of Science), at initial concentrations of 0, 0.001, 0.01, 0.1, 1.0, and 5.0 g/L. Each concentration was set up in triplicate in flask (500 mL) and the experiments lasted for 48 h at 30°C, as described in SI-1 in S1 Text. During mating, the transconjugants were counted and calculated as colony-forming units per milliliter culture (CFU/mL) on Luria-Bertani (LB)-selective plates (containing 100 mg/L of Ap, 60 mg/L of Km, and 10 mg/L of Tc) [19,30]. Periodic sampling (10 mL) was collected at 0, 4,8,12,16,20,32,48 hours and used for plate counting and DNA extraction.

Plasmid RP4 horizontal transfer microcosms
The counting plate method [32] was used to detect changes of plasmid RP4 horizontal transfer in freshwater microcosms treated with 1.0 g/L of [BMIm][PF6]. The number of total cultivable indigenous recipients (Nr) was determined using culturing on LB agar plates. The number of donor strains (N 4 ) (Ap R , Km R , Tc R and Rif R ) was enumerated on LB tetra-antibiotic plates containing 60 mg/L Km, 100 mg/L of Ap, 10 mg/L of Tc and 40 mg/L of Rif. Meanwhile, the colonies (N 3 ) (Ap R , Km R and Tc R ), which contained donor strains and transconjugants, were counted on LB plates containing antibiotics (containing 100 mg/L of Ap, 60 mg/L of Km, and 10 mg/L of Tc). Furthermore, in parallel, samples from microcosms were plated onto LB tri-antibiotic plates as negative controls to rule out spontaneous mutation of the recipient strains.
Horizontal transfer frequency (f) was calculated using the formula: where

Identification of transconjugants
Transconjugants were isolated by replica plating [57]. Microcosm samples were plated to triantibiotic plates and quad-antibiotic plates by replica plating to insure the same strains are at the same location in these two plates. Transconjugants are those that can grow on tri-antibiotic plates but not on quad-antibiotic plates at the same point. Meanwhile, PCR and DNA sequencing were used to verify that the plasmid RP4 had been transferred into the recipient strains; PCR primers were specially designed and are shown in S2 Table. Transconjugants were incubated in liquid LB tri-antibiotic media containing 100 mg/L of Ap, 50 mg/L of Km, and 10 mg/ L of Tc overnight on a shaker incubator (160 rpm) at 37°C. Plasmid DNA was extracted using a plasmid extraction kit (OMEGA, USA) according to the manufacturer's instructions. Furthermore, the characteristics of transconjugant morphology were recorded, and the isolated transconjugants were also identified by 16S rRNA sequencing.
Quantification of the aphA gene and traF gene Samples from microcosms (8 mL) were centrifuged and the total DNA extraction was performed using a bacterial DNA Kit according to manufacturer's instructions (OMEGA, USA). Polymerase chain reaction (PCR) assays were used to target the 16S rRNA gene, aphA gene, and traF gene. Qualitative PCR assays were conducted in a Biometra T100 gradient thermal cycler (Biometra). The PCR mixture (25 μL) contained 1 μL diluted DNA extract as the template, 1.5 μmol/L of each primer (S2 Table), 2 μL 250 μmol/L of each deoxyribonucleoside triphosphate (dNTP), 1.25 U Taq DNA polymerase (TransGen), and 2.5μL of 10xPCR buffer (TransGen). DNA products were diluted from 20 to 50-fold and used in 16S rRNA gene PCR to optimize the template concentration. For each DNA extract, duplicate PCR tubes were analyzed for the presence of each target gene. The negative controls, both the reaction mixture without DNA and the reaction mixture with DNA from the microcosm without the donor were included in each of the PCR run. The PCR procedure for 16S rRNA, aphA gene, and traF gene started with an initial DNA denaturation (95°C for 5 min), followed by 30 cycles of 30 sec at 95°C (denaturing), 30 sec of annealing at the temperatures specified in S2 Table, and 1 min at 72°C (extension), followed by a final extension of 7 min at 72°C. The size (S2 Table) and specificity (unique band) of PCR products were determined by comparison with DNA standards (Marker I, TransGen) after 1.5% (w/v) agarose gel electrophoresis [6].
qPCR analyses were performed on a Bio-Rad iQ5 instrument to quantify bacteria 16S rRNA, aphA gene, and traF gene. Calibration standard curves for positive controls were generated as previously described. The establishment of negative and positive controls and qPCR reactions were described previously. The primers, amplification details, and standard curves of the 16S rRNA, aphA gene, and traF gene are listed in S2 Table.

Detection of cell membrane permeability effects of [BMIm][PF6]
Flow cytometry (FCM) (BD FACSCalibur) was applied to differentiate and quantify the [BMIm][PF6]-treated group (0.001-5.0 g/L) and control group cells in freshwater microcosms as a function of the percentage of positive cells based on the extent of cell membrane permeability. A higher percentage of positive cells signifies enhanced cell membrane permeability. The FCM was equipped with an excitation wavelength of 488 nm. Propidium iodide (PI) (OMEGA, USA) was used to determine increased membrane permeability cells and control cells, respectively. Microcosm samples (4 mL) were centrifuged and washed 3x with phosphate buffer solution (PBS) (10 g/L NaCl, 0.25 g/L KCl, 1.8 g/L Na 2 HPO4, and 0.3 g/L KH 2 PO 4 ). Bacterial cells (the concentration was always less than 10 6 cells/mL) were then stained with PI (0.01 mg/mL) and incubated in the dark for 8 min before measurement [58]. All data were processed using CellQuest Pro software (USA).

[BMIm][PF6] promoted the horizontal transfer of plasmid RP4
In the [BMIm][PF6] (1.0 g/L)-treated microcosm, changes of plasmid RP4 abundance were tracked by traF gene (Fig 1a). The abundance of the traF gene increased up to 56 (Fig 1a). These facts suggest that In the plasmid RP4 horizontal transfer microcosm, the natural transfer frequency of plasmid RP4 in the water environment was very low [30,59], only (5.10±0.71)×10 -5 per recipient cell in control groups ([BMIm][PF6] untreated groups) in the present study (Fig 2). This low transfer frequency was similar to those described by previous studies, where the transfer frequency of plasmid RP4 from the Pseudomonas putida donor strain to indigenous bacteria in activated sludge was in the range of 4.0×10 -6 to 1.0×10 -5 transconjugants per recipient [60]. For all treatments of [BMIm][PF6], plasmid RP4 horizontal transfer was enhanced and plateaued by 16 hours. In the horizontal transfer microcosms, after inoculation of the donor bacteria, a decrease in cultivated donor bacterial numbers was observed in the initial phase of the horizontal transfer (Fig 3). The donor strains acclimatized to the indigenous bacteria community by decreasing their abundance before stabilizing. In contrast, there was no significant variance (p = 0.861) of the growth of cultivable recipients under 1.0 g/L of [BMIm][PF6] exposure during plasmid RP4 horizontal transfer (Fig 3). Meanwhile, the cultivated bacterial number of transconjugants significantly increased and reached the plateau by 16 hours (Fig 3), suggesting the plasmid RP4 horizontal transfer occurred. The increasing transfer frequency of plasmid RP4 is in accordance with the increasing abundance of the traF gene (indicator of plasmid

RP4) under [BMIm]
[PF6] treatment, suggesting that proliferation of plasmid RP4 in the microcosms was mainly caused by horizontal transfer of plasmid RP4, which is promoted by [BMIm][PF6] treatment. Widespread dissemination of ARGs was throughout bacterial populations by vertical transfer and horizontal transfer processes [12]. Acquired resistance occurs between individual cells or species mainly by horizontal transfer [8][9][10]. In this study, the IL [BMIm][PF6] increased plasmid RP4 transfer frequency is mainly via horizontal transfer from resistant bacteria to indigenous bacteria, which is fundamental for the dissemination of ARGs. Meanwhile, during the gene horizontal transfer process, transconjugant reproduction or replication (defined as the gene vertical transfer) may have simultaneously occurred with horizontal transfer, and this should not be excluded. The calculated of horizontal transfer frequency based on the transconjugants in plasmid mediated horizontal transfer was similar to previous publications [12,23].
Additionally, there was no significant difference (p = 0.344) in the abundance of 16S rRNA gene (as a surrogate for total bacteria) between [BMIm][PF6] (1.0 g/L) treated groups and controls (Fig 1c). This result was comparable with previous report that the growth of E.coli, P.pastoris, and B.cereus was unaffected by 1. [Cl] as "environmentally friendly" solvents, essentially have non-toxic effect on bacterial growth. Although some ILs under certain concentrations exert neglected effects on bacterial growth both observed in previous studies and our study, due to their remarkable effects on facilitating the horizontal transfer of the plasmid RP4 which is the multi-drug resistance carrier, further consideration on the their application and discharges into the environment should be carefully evaluated.  (Table 1). Compared with the cultivable indigenous bacteria (recipients in microcosm) (S3 Table), Gram-negative bacteria of Acinetobacter spp., Alcaligenes spp., Achromobacter spp. and Pseudomonas spp. were identified as dominant plasmid RP4 recipients, as summarized in Table 1

Genus
Strains  The horizontal transfer frequency is much higher (3-fold) in recipient of Salmonella spp. which is Gram-negative bacteria than microbacterium spp. suggesting that Gram-negative bacteria are more likely to acquire ARGs during the horizontal transfer of plasmid RP4, resulting in many indigenous bacteria have to be resistant to several antibiotics simultaneously and capable of transferring their resistance among environmental microorganisms of different genera [61,62]. This increases the threat of Gram-negative bacteria to public health and poses challenges for developing a new generation of antibiotics against them [13,63]. Particularly notable, two strains of opportunistic pathogen Acinetobacter spp. and Salmonella spp. were both Gramnegative bacteria and they were isolated among the transconjugants, increasing the risk of ARGs dissemination to human pathogens and posing great threat to public health.
[BMIm][PF6]-enhanced cell membrane permeability FCM assay showed that cell membrane permeability of bacteria treated with [BMIm][PF6] (Fig 5a, quadrant B and C) were significantly increased up to 38-fold higher (16.49%) in  [PF6] exerted selective pressure promoting the spread of plasmid RP4 by conjugation but not by transformation. The cell membrane serves as barrier to cell permeation and acts as a substantial barrier against penetration [19,64], preventing any compounds (e.g., antibiotics, nutrients, environmental pollutants and genetic exchange) from entering the cell, maintaining the physiological equilibrium [65]. The bacterial cell membranes constitute an important barrier for horizontal transfer of genetic material among bacteria of different species or genera [19]. Bacteria adapt their permeability by modulating the expression of outer membrane porin proteins (OMPs), which are known to play important roles in the membrane transport and to control the permeability of the cellular membrane [66,67]. Pore-forming with modulation of porin proteins allow donor pilus to attach to and access the recipient cell [68,69], facilitating cell-to-cell contact and the conjugative transfer of plasmid RP4. Meanwhile, enhanced cell membrane permeability facilitate communication between recipients and the outer environment, particularly donors, which in turn promote gene transfer of the resistance plasmid [70]. Other chemicals, such as antibiotics [53,55] and nanomaterials [23] are capable of inducing oxidative stress which is a general adaptation of the organism in exposure of chemicals that can damage cell membranes and may promote the horizontal transfer of genes [71]. The [BMIm][PF6], as chemical stressor and it is possible that they induce stress responses in the bacteria and therefore enhanced cell membrane permeability. Previous studies have reported fluidity of bacterial cell membrane and interference with membrane-bound enzyme activity with exposure to some ILs [48,55,72]. This study showed that [BMIm][PF6] suppresses the cell membrane barrier possibly by enhanced cell membrane permeability that is likely to facilitate the transfer of ARGs between indigenous bacteria.
[BMIm][PF6] enhanced propagation of ARGs mediated by plasmid RP4 Plasmids, as the main vector for resistance genes and carrying multiple resistance genes, play a critical role in horizontal transfer and contribute to the spread and dissemination of ARGs in the environment [24]. In the present study, changes of the aphA gene (kanamycin resistance gene which is located on plasmid RP4) followed a similar trend as those of the traF gene (indicator for the abundance of plasmid RP4) (Fig 1b). A positive correlation (p<0.01) was found between relative abundance of the aphA (aphA gene/16S rRNA gene) and traF (traF gene/16S rRNA gene) genes (Fig 1d), implying that the proliferation and propagation of aphA gene could be attributed to [BMIm][PF6]-promoted plasmid RP4 horizontal transfer. This study illustrated that [BMIm][PF6] increased ARGs propagation mediated by plasmid RP4 in the water environment. To date, there have been no reports of ILs discharged into the environment; however, the ever increasing production and applications of ILs may accelerate ILs entering the environment and make a variety of bacterial community exposed to ILs, which increased the risk of antibiotic resistance dissemination [46]. The results of this study suggest that careful evaluation is required before bulk emission of some ILs into the aquatic environment.

Conclusions
This study illustrates that the IL [BMIm][PF6] facilitated the horizontal transfer of ARGs mediated by plasmid RP4 among indigenous bacteria in aquatic environment. Meanwhile, in the horizontal transfer microcosms, two strains of opportunistic pathogen Acinetobacter spp. and Salmonella spp. were isolated among the transconjugants, illustrating plasmid RP4 mediated horizontal transfer of ARGs occurred in pathogen. This could increase the risk of ARGs dissemination to human pathogens and pose great threat to public health.
[BMIm][PF6] compromises the cell membrane barrier by enhanced cell membrane permeability which is likely the mechanism of increased horizontal transfer of plasmid RP4. This study implies that ILs facilitate horizontal transfer of the ARGs, posing great risks to public health. It is suggested that application of ILs in industrial processes should be carefully evaluated before their bulk emission into the aquatic environment.
Supporting Information S1