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Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate
Accepted Manuscript Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate Yaru Hu, Tianyang Zhang, Lei Jiang, Shijie Yao, Hui Ye, Kuangfei Lin, Changzheng Cui PII: DOI: Reference:
S1385-8947(19)30455-3 https://doi.org/10.1016/j.cej.2019.02.207 CEJ 21120
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
29 November 2018 25 February 2019 27 February 2019
Please cite this article as: Y. Hu, T. Zhang, L. Jiang, S. Yao, H. Ye, K. Lin, C. Cui, Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.02.207
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Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate Yaru Hua, Tianyang Zhanga,b, Lei Jiangc, Shijie Yaoa, Hui Yec, Kuangfei Lina, Changzheng Cui*a,b a. State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China, 200237 b. Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, PR China c. National Engineering Research Center of Urban Water Resources, Shanghai, China, 200082
*Corresponding author, Changzheng Cui, Tel: +86 21 64253988; Fax: +86 21 64253988; E-mail: [email protected]
Abstract The inactivation of an isolated sulfonamide antibiotic resistant bacteria (ARB) HLS-6 and reduction of intracellular sul1 and intI1 in its genome by UVC irradiation, PMS oxidation and UVC-activated peroxymonosulfate (UVC/PMS) treatments were investigated in this study. The UVC/PMS treatment was superior to the other two methods in the inactivation of ARB and reduction of qPCR-sul1 and qPCR-intI1. The HLS-6 ARB (108 CFU/mL) could be effectively inactivated 5.3 log by UVC (100 μW/cm2) /PMS (1 mg/L), and the reduction rates of qPCR-sul1 and qPCR-intI1 by UVC (100 μW/cm2) /PMS (20 mg/L), reached up to 2.9 log and 3.4 log, respectively within 30 min. qPCR-intI1 reacted faster than qPCR-sul1 in all methods. Sulfate radical was responsible for the reduction of target genes, while hydroxyl radical had negligible effect on that. The dosage of PMS positively affected the reduction of both genes during UVC/PMS, while the initial concentration of ARB could negatively influence the reduction of target genes. The pH 5 of reaction solution was most beneficial to the reduction of ARGs. The reduction rates at pH 5 reached up to 3.1 log (sul1) and 3.3 log (intI1). The reduction of target genes was slightly facilitated in the initial 5 min and suppressed after 5 min with the co-existence of sulfamethoxazole. This study will provide a potential alternative method for controlling the antibiotic resistance in aquatic environment. Keywords: UVC-activated peroxymonosulfate; antibiotic resistant bacteria; intracellular antibiotic resistance genes; sulfate radical, water treatment.
1 Introduction Antibiotic resistance has been a serious issue worldwide recently, which is constantly challenging the clinical therapeutic authority of antibiotics and threatening the human health, causing social panic [1]. The rapid propagation of antibiotic resistant bacteria (ARB) and vertical, horizontal gene transfer of antibiotic resistance genes (ARGs) enable the antibiotic resistance to spread quickly among different hosts [2,3], even in human [4] and animal bodies [5]. Recently, a variety of studies have reported the extensive detection of ARB and ARGs in the different environmental media [6,7]. The aquatic environment always serves as the terminal recipient of the majority contaminants, which is a hot study spot of ARB and ARGs [8.9]. The antibiotic-resistant E. coli in the Tama River, Japan, presented an ascending trend along the studied stream with a concentration range of 2 to 195 CFU/100 mL, and 32% of them were multiple resistant E. coli [10]. The concentration of multiple resistant bacteria in Jiulongjiang River, South China, ranged from 2.9 to 5.9×104 CFU/mL, and 212 ARG subtypes were detected in all samples with the concentration range from 9.7×107 to 1.0×108 copies/mL [11]. Varieties of ARG subtypes were detected in the surface water [7,12,13]. The antibiotic resistance rates of sulfonamide and tetracycline were 77.3% and 43.1%, respectively, which were positively related to the concentration of tetracycline [14]. What’s more, 7.1×101 to 5.9 ×106 copies/mL of ARGs was also detected in the same water samples [14]. The average relative
abundance of extracellular ARGs (eARGs) detected in Bohai Bay reached up to 4.3×10-1 (ARGs/16S RNA) and was greater than that of intracellular ARGs (iARGs) [15]. Although multiple water treatment methods were adopted to remove the common environmental contaminants, which could partially reduce ARB and ARGs simultaneously, ARB and ARGs still presented high detection frequencies and concentration in the effluent and primary water body [7,9]. Sixteen types of ARB belonging to Proteobacteria and Firmicutes phylums were isolated from a water treatment plant in Shanghai, 75% of which were resistant to more than one types of antibiotics [16]. What’s more, the resistance rates of ARB increased during the water treatment process with the antibiotic concentration range from 1 to 10 mg/L [16]. To control antibiotic resistance and preserve the drinking water quality, as well as obtain an optimal water treatment method that is able to effectively eliminate ARB and ARG, the recent studies focusing on individual drinking water treatment procedures, such as ozonation, UV irradiation, chlorination, as well as advanced oxidation processes, became more and more popular [17-19]. Due to the strong oxidation ability, only 0 - 0.2 mg/L of ozone could reduce about 4.0 log E. coli by damaging the integrality of cell and higher ozone dose (> 0.2 mg/L) was required to inactivate ARGs [20]. The UVC irradiation could obtain 1 log loss of transformation efficiency of ARGs requiring 20 - 25 mJ/cm2 [21]. The UVA/H2O2 treatment could successfully inactivate the antibiotic resistant E. coli under detection limit in 90 min, while it had no obvious effect on the blaTEM, qnrS, tetW genes [22]. What’s more, the
inactivation of eARGs and iARGs were also investigated by UVC/H2O2 treatment to get rid of the organic compounds in cytoplasm, which revealed that UVC/H2O2 could eliminate eARGs [23]. Furthermore, the combination of UV irradiation with TiO2 material was also applied to study the inactivation of both ARB and ARGs. The reduction rate of ARGs was amplified by adding TiO2 thin film under UVC irradiation, and also had effect on the eARGs and iARGs [24]. An ultrafiltration membrane could reduce 98% of ARGs and perfectly remove integrase genes, which impeded the potential horizontal gene transfer of ARGs [25]. In our previous study, ARB and ARGs could be effectively removed by UVC/chlorination treatment [19]. However, the generation of disinfection by-products during the water treatment by chlorination or UV/chlorination would also threat the human health [26,27]. Therefore, a more effective and safer disinfection processes need to be further explored. Previous studies showed that the UVC/PMS treatment was a safe nontoxic environmental friendly water treatment method [28,29]. What’s more, based on our previous study on the effectively removal of twelve sulfonamides by UVC-activated peroxymonosulfate [28] and the potential capacity of sulfate, peroxymonosulfate, and hydroxyl radicals of causing DNA modification [30,31], the inactivation of a typical gram-negative sulfonamide ARB and the reduction of intracellular sulfonamide ARG by UVC irradiation, PMS oxidation and UVC/PMS were further investigated in this study. Several available factors that would affect the reduction rate were taken into consideration to further optimize this process, including the dosage of PMS, UVC
intensity, the pH of ARB solution, the initial ARB concentration and the real wastewater treated by secondary treatment process. Furthermore, a set of quenching experiments was conducted to identify the primary radical species during UVC/PMS. The co-reduction of ARB and iARG with sulfamethoxazole (SMX) was analyzed under optimal conditions of UVC/PMS. The results could not only give a better understanding of the elimination of ARB and ARG, but also provide a potential alternative method for controlling the antibiotic resistance in aquatic environment.
2 Materials and methods 2.1 Chemicals and reagents PMS (2KHSO5·KHSO4·K2SO4); H2SO4, HCl, NaCl, Na2HPO4∙12H2O, KH2PO4, KCl, NaOH, Na2S2O3 and tert-butyl alcohol (TBA, analytical-grade) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Sulfamethoxazole (SMX, C10H11N3O3S, BP grade) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Ultrapure water was prepared using a Milli-Q water system (Millipore, USA). An isolated ARB pseudomonas sp. HLS-6 (CCTCC AB 2017269) was selected to investigate the inactivation of ARB and the reduction of ARGs by different disinfection processes. Sulfonamide ARGs sul1 and class I integrase gene intI1 are harbored in the genome of the HLS-6 strain (NCBI accession number: NZ_CP024478.1). The ARB was cultivated in liquid lysogeny broth (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, pH= 7.0 ± 0.2) with 200 mg/L SMX at 30 °C, 150
rpm overnight. The ARB cells was collected by centrifuging at 8,000 rpm for 5 min and washed three times with 1:10 diluted sterile phosphate-buffered saline solution (PBS, pH = 7.0), then, was re-suspended in PBS [18,20]. The initial concentration of the obtained ARB cells in PBS was ~108 CFU/mL. 2.2 Experimental approach Because the inactivation of ARB was easier than the break of DNA [23], the dosage of PMS during these two experiments were different. PMS solution was added at 1 mg/L (for ARB inactivation) and 20 mg/L (for ARGs reduction), respectively. The UVC dose (254 nm) was adapted using a UVC intensity radiometer (Photoelectric Instrument Factory of Beijing Normal University, Beijing, China). The pH of ARB solution was adjusted using HCl and NaOH solutions. All reactions were quenched by stopping UVC irradiation and adding Na2S2O3 solution (Na2S2O3/PMS = 2 mol/mol). Before the reactions, the UV dose was adjusted to 100 μW/cm2 using a simple elevator (Fig. 1). The effective PMS was calibrated according to a previous study [28]. Suspended HLS-6 cultures (50 mL) were transferred into a petri dish (Diameter×Height: 90 mm×12 mm), and gently mixed with a magnetic stir bar (Fig. 1). Then the solution was exposed to direct UVC irradiation, PMS oxidation and UVC/PMS, respectively (Fig. 1). 1 mL solution was sampled at the pre-determined intervals. The factors affecting the reduction of ARGs by UVC/PMS including the PMS dosage, UVC dose, pH of the solution and initial concentration of ARB cell were also analyzed. The sampling time was all 10 min. To identify the major radical
species during UVC/PMS, a set of quenching experiments was performed. The experimental procedures were same as the previous methods with the additional reagents of methanol (EtOH) and tert-butyl alcohol (TBA). The amount of each alcohol was added based on the previous studies [31]. 2.3 Analytical approach 2.3.1 ARB survival The different-time treated samples were processed using gradient dilution by 1:10 PBS [19]. Then 0.1 mL of each diluted sample was smeared evenly in duplicates on the surface of prepared LB solid medium, which contained 200 mg/L of SMX, and then incubated at 37 oC. After 24 h’s incubation, the number of ARB colony was counted and recorded as CFU/0.1 mL, which was further normalized CFU/mL and represented the surviving number of ARB. 2.3.2 DNA extraction and ARGs quantification, residual SMX and water parameters analysis The prepared ARB solutions (HLS-6) were directly treated with UVC irradiation, PMS oxidation or UVC/PMS processes. Then, the inactivation of ARB was analyzed by dilution method of plate counting and recorded as CFU/mL. The DNA in the disinfected samples was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany), and the short fragments of target genes (<500 bp, qPCR-sul1; qPCR-intI1) were subsequently quantified using a LightCycler 480II (Roche, Swiss) basing on the quantification method as previously described [32]. The breakage of the long segment
of target genes (about 500 bp: PCR-sul1; PCR-intI1) were amplified by a Thermo Cycler (Biorad S1000, USA) and further analyzed by 1.2 % gel electrophoresis (DYY-12C, Beijing, China) at 1400mV for 30 min. The detailed information of PCR, qPCR primers and parameters of PCR condition were shown in Section S1 and Table S1. The residual SMX concentration was analyzed according to previous studies [33] using a high performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS, Agilent, USA) under the multiple reaction monitoring (MRM) mode and the method parameters and HPLS-MS/MS details were shown in Section S2 and the detail mass spectrometric parameters, recoveries, and limits of quantitation of SMX were shown in Table S2. The detail analysis of water quality characterization are shown in Section S3.
3 Results and discussion 3.1 Inactivation of sulfonamide ARB HLS-6 The inactivation of ARB by UVC irradiation, PMS oxidation, and UVC/PMS was plotted in Fig. 2. The UVC intensity was 100 μW/cm2 and the equivalent UVC dose was calculated as UVC intensity (100 μW/cm2) × sampling time (0.5 min, 1.0 min…… 30 min) = UVC dose (mJ/cm2). It could be easily sorted by degradation rate and efficiency of ARB followed: UVC/PMS > UVC irradiation > PMS oxidation. The single PMS oxidation showed no obvious effect on the ARB inactivation with the PMS dosage of 1 mg/L and the inactivation ratio was only 0.28 ± 0.04 log. The inactivation ratios of ARB by UVC irradiation and UVC/PMS showed an increasing
trend with the increase of UVC dose. The UVC irradiation (100 µW/cm2, 10 min: 60 mJ/cm2) could effectively inactivate ARB from 108 to 103 - 104 CFU/mL. Furthermore, the inactivation ratios were empilfied from 4.1 ± 0.0 log to 5.3 ± 0.1 log with the additional dosage of 1 mg/L PMS. This result demonstrated that the combination of UVC irradation and PMS oxidation was able to reinforce the inactivation efficiency of ARB. That UV irradaition is well-known to be able to sterilize, which is due to it’s capacity to induce DNA damages by hydration of the base or base dimerization, causing DNA mutations and holding up the DNA replication [34,35]. A varity of studies have investigated the inactivation of ARB by individual UV irradiation [17,19]. An ampicillin-resistant E. coli CGMCC 1.1595 (intital ARB concentration 107 CFU/mL) was inactivated by 5 - 20 mJ/cm2 UVC irradiation with the ratio of 2.0 log, and the ratio increased to 5.5 log at the UVC dose of 40 mJ/cm2 [18]. The same HLS-6 ARB was sterilized by 4 log at the UVC dose of 12 mJ/cm2 with the intital ARB concentration 107 CFU/mL [19]. Both results were slightly higher than that of this study (1.0 log at 12 mJ/cm2 and 4.1 log at 60 mJ/cm2), which could explained by the higher ARB concentration used in this study. The breakage of PMS peroxide bond occurred under UV radiation, which produced one hydroxyl radical (HO•) and one sulfate radical (SO4•−) (HSO5-/ SO52- + hv → SO4• − + HO•) [36,37]. The high redox potentials of HO• enables it to rapidly react with most compounds like proteins [38,39], which are the main components of cell walls and DNA double strands. The
SO4•− generated by transition metal-activated processes was responsible for the inactivation of Escherichia coli [40,41], which confirmed the excellent sterilization effect of SO4•−. Therefore, the inactivation of ARB was not a difficult thing for the combination of UVC irradiation, HO• and SO4•− during UVC/PMS treatment. 3.2 Reduction of ARG sul1 and integrase gene intI1 The reduction and of sul1 and intI1 genes by UVC irradiation, PMS oxidation, and UVC/PMS was plotted in Fig. 3. Preliminary experiments showed that the DNA damage occurred more slowly than the inactivation of ARB [23], so the dosage of PMS was adjusted from 1 to 20 mg/L and the sampling time was extended to 30 min. and in these conditions ARB was completely removed within 1 min by UVC/PMS (Fig. S1). The reduction rates of both genes were represented by the log change of the short amplicons (qPCR-sul1 and qPCR-intI1). The reduction of both genes followed the same trend in the initial 2 min and differed in the following time. The reduction of target genes occurred slowly during UVC irradiation and the losses of qPCR-sul1 and qPCR-intI1 were 1.2 ± 0.5 log and 0.8 ± 0.1 log in 30 min (130 mJ/cm2), respectivley, which were lower than the reduction of amp and kan genes (1.1 - 1.6 log at 40 mJ/cm2) in a previous study [23]. The reduction rate of individual PMS oxidation exceeded that of UVC irradiation before 10 min, which eventually acquired 2.0 ± 0.1 log and 2.3 ± 0.1 log in 30 min. The UVC/PMS treatment still presented the best reduction rate of both genes with the reduction rates being 2.9 ± 0.1 log (qPCR-sul1) and 3.4 ± 0.1 log (qPCR-intI1) within 30 min, respectively, which was slghtly inferior to the
reduction time of the same genes by UVC/chlorination at the same UVC dose [19]. Apparently, the reduction of both genes by these three treatment methods followed a first-order kinetics with respect to the sampling time (referring to the UVC dose), ignoring some deviation of the last 30 min sampling, and this was similar to the results of previous studies [21,23]. The calculated reduction rate constants were summarised in Table S3. The k value also proved that the combinated reduction of UVC irradiation and PMS oxidation exceeded both single treatment (kUVC/PMS > kUVC + kPMS > kUVC ≈ kPMS), and the reaction rate of UVC/PMS were 2.8 - 2.9 times higher than that of UVC irradiation and PMS oxidation. Same superiority of the combined method could be explained by that the UVC irradiation could decrease the bioactivity of ARB, thus made it easily for free radicals to enter into the cells and inactivate ARGs [42]. The values of k’UVC in this study were lower than those reported previously for the amp and kan genes [23] and tetA and blaTEM-1 genes [21], but higher than that of the same gene reduction in another previous study [19]. A longer gene is expected to react faster with UVC irradiation due to the presence of more UVC target sites than a small one [17,43], while the values of k’UVC only had moderate correlations with the gene length (r = 0.43). The DNA lesions induced by UVC irradiation almost occurred between two adjacent pyrimidines with the thymine-thymine cyclobutane-pyimidine dimers (TT CPDs) being the primary product [43], which furnish evidence of the values of k’UV strongly being related to the number of adjacent TT (r = 0.79). Interestingly, the k’UV values of the same gene
reduction by higher UVC intensity irradiation (200 μW/cm2) in a previous study [19] was lower than that in this study (UVC intensity: 100μW/cm2), the reason of which was still not clear. The k’PMS values of both genes in this study were greater than that of chlorination [19]. This might be attributed to the oxidation reduction potential of PMS (1.85 V), which was higher than most oxidizing agents, including sodium hypochlorite. The k’UVC/PMS values of both genes in this studies were lower than those of the amp and kan genes by UVC/H2O2 treatment [23]. Theoretically, HO• is one of the most active reactive oxide species (ROS), which would attack the C5, C6 sites of pyrimidine bases and the C4, C5 and C8 sites of purine bases [45], causing the direct DNA damage. However, due to the non-selectivity of reaction with the organic compounds in the cytoplasm, HO• would be consumed quickly, which would result in its noneffectiveness against ARGs [22]. The reduction of amp and kan genes by UVC/H2O2 treatment was mediated by a plasmid [23], which contained less organic substance than a whole cell in this study. However, SO4• − was proved to be responsible for the modification of DNA by reacting with guanine [30,46]. Since sul1 and intI1 genes have the similar guanine content, so the k values of them were similar too. It is noteworthy that the k value of intI1 gene was always greater than that of sul1 no matter by UVC irradiation, PMS oxidation and UVC/PMS. 3.3 The roles of HO• and SO4• − in the removal of ARGs during UVC/PMS Since there were two kinds of reactive oxygen species (HO• and SO4• −) existing during the UVC/PMS system, a set of quenching studies was conducted to identify the
primary radical species (Fig. 5). Two kinds of alcohols (TBA and EtOH) were chosen based on their different reactivity and rates with different radical species and added into the UVC/PMS system at certain amount ratios to PMS [31]. The presence of EtOH and TBA only resulted in a moderate decrease in the reduction of target genes (less than 4% - 25%), furthermore, a slight increase of sul1 gene (5%) occurred with the existance of EtOH (250:1). The reaction rates between EtOH with HO• was approximately 50-fold greater than that with SO4• − [31] and the reaction rate between TBA with HO• was about 1000 times higher than that with SO4• − [47,48]. Due to relatively chemical inertness of SO4• − toward alcohols, the added quenching agents in the system would preferentially consume HO• [31]. However, the reduction of ARGs was not seriously affected. Therefore, it could be concluded that SO4• − are responsible for the reduction of target genes. SO4• − would attack the guanine heterocycle on the π face, which was responsible for the modification of DNA [30]. However, HO• was not effective in the reduciton of ARGs [19,22]. 3.4 Gene breakage and potential gene transfer loss Although the qPCR could detect the minor base modification of DNA damage [49] and the qPCR results also showed a declining trend of target genes’ concentrations, the cleavage of whole sul1 and intI1 genes could occur at any gene site along the target gene sequence and couldn’t be fully delegated by only a 200 bp fragments. Therefore, two longer routine PCR amplicons (PCR-sul1: 433 bp; PCR -intI1: 484 bp) was presented by gel electrophoresis (Fig. 4). The brightness of both
gene stripes showed a declining trend over time during UVC irradiation, PMS oxidation and UVC/PMS and UVC/PMS was also the best treatment method to destroy the integrity of PCR-sul1 and PCR-intI1. Although the qPCR results showed that the qPCR-intI1 gene still present ~ 105 copies/mL, the PCR-intI1 couldn’t be visualized by gel gelectrophoresis, which indicated the intermediate breakage of PCR-intI1. The co-existance of intI1 and sul1 gene analyzed in this study, is indentified as the genetic marker of class I integron [50], which always confers the horizonal gene transfer of gene cattases among different hosts. The interval open reading frame gene between intI1 and sul1 is aadA2 gene (780 bp), which encodes the resistance to aminoglycoside antibiotic. Once this integron is successfully assimilated by pathogens, it will eventually aggravates the antibiotic resistance. However, the whole gene fragment can’t be fully quantified by common qPCR due to the limit of gene length. Resorting to the extrapolated equation reported previously [17], the damage of whole intI1-aadA2-sul1 and intI1-aadA2 gene fragments could be predicted.and the results were shown in Fig. S2. The predicted reduction of these two gene fragments were between 43 - 56 log and 32 - 42 log, respectively, which accounting for approximately 3.0×1040 - 1.8×1053 and 2.4×1029 -7.9×1038 copies/mL. Therefore, the gene transfer potential was greatly suppressed. 3.5 Impact factors on the reduction of target genes by UVC/PMS Since UVC/PMS treatment was superior to UVC irradiation and PMS oxidation
in the reduction of target genes, several available factors that would affect the reduction rate were taken consideration to further optimize this process. All these impact factors were investigated under the same condition as previous described, unless changing the unique variable parameters, including the PMS dosage, UVC intensity, pH and initial ARB concentration. The dosage of PMS could significantly affect the reduction of both genes (Fig. 6(a)). An increase of PMS dosage resulted in a greater reduction of both genes. The reduction rate ascended from 1.1 log to 2.8 log in 10 min with the PMS dosage increased from 5 to 30 mg/L. The log reduction of both genes was positively related to the dosage of PMS (sul1: r2=0.996; intI1: r2 = 0.945), which contributed most to the more SO4• − generated by greater dosage of oxidant [51]. Fig. 6(b) showed the effect of another important factor UVC intensity on the gene reduction during UVC/PMS. The log reduction of sul1 gene increased slowly as the UVC intensity increased from 50 to 300 μW/cm2, while for intI1 gene, the reduction change didn’t follow regularly with the increase of UVC intensity. Otherwise, the reduction of intI1 showed a falling trend at high intensity UVC. Though long gene intI1 have more reaction sites than short gene sul1, the sul1 gene was more sensitive than intI1 gens to UVC intensity. Previous studies demonstrated that the pH of the reaction solution could significantly impact the decomposition of PMS [28] and the pH under 5 would induce the PMS decomposition (SO4• − + HSO5 HSO4
SO5•
+
< 104 M-1 s-1) [52], so the pH range of 5 - 9 was analyzed. As shown in
Fig. 6(c), it benefited the greater reduction of both genes in acidic condition than that
in alkalescent condition. The PMS couldn’t generate SO4• − under low pH, and partial SO4• − could react with OH- to generate HO• (SO4• −+ HO− → HO• + SO42−; k = 6.5×107 M-1 s-1) [52], which was not effective in eliminating ARGs. Fig. 6(d) showed the influence of the initial ARB concentration on the reduction of both genes. It could easily be concluded that the lower the initial concentration was, the higher the reduction rate would be. During the death of ARB cells, higher concentration of organic compounds was released from the cytoplasm than the lower one, which was the prior ROS consumer. In order to explore the impact of the real environment media on the reduction of ARGs by UVC/PMS, an additional experiment were conducted in the wastewater from the secondary treatment process. The water quality parameters and the reduction of ARGs were presented in Table S2 and Fig. S3, respectively. The reduction of ARGs increased gradually with the sample time increasing, and the reduction rates reached up to 0.9 log (sul1) and 1.0 log (intI1), which were higher than that in a previous study [53]. The organic matters also decreased after treated by UVC/PMS, whose reduction efficiencies were consistent with that of the real wastewater disinfection process (chlorination). However, the reduction efficiencies of organic matters were lower than that of the previous study [53]. This discrepancy might be explained by the wastewater type and the reagent dosage. The water quality parameters in this study were 0.1 - 189 times lower than that in the previous study, and additive H2O2 and iron (II) sulfate heptahydrate were added into the treatment system to generate more HO·,
which would preferentially react with organic matters, not with ARGs [39]. This would induce the low reduction efficiency of organic matters and high reduction rate of ARGs. 3.6 Co-reduction of SMX and ARGs by UVC/PMS To investigate the real effect of UV/PMS, an organic substance was chosen to explore the co-reduction. The HLS-6 strain shows resistance to 200 mg/L SMX, which is a frequently detected antibiotic in the drinking water source [9,33]. Therefore, the co-reduction of SMX, ARB and target genes (sul1 and intI1) by UVC/PMS under the optimal condition was also investigated in this study. As for the single reduction of SMX, it was completely eliminated within 1 min by 30 mg/L PMS and 100 μW/cm2 (data not shown). When the SMX was co-existing with ARB solution, it was also degraded immediately (Fig. 7). However, the reduction of target genes was slightly facilitated in the initial 5 min, and was inhibited after 5 min. The reduction rate was 1.3 log (qPCR-sul1) and 1.8 log (qPCR-intI1). These radicals rapidly attacked many organic compounds with rate constants ranging from 106 to 1010 M−1·s−1 [54,55], and 0.2 μg/L SMX could be completely removed in 5 min with 5 mg/L PMS [28]. The initial slight facilitation of ARGs reduction could be explained the solvent effect. Because the HO• was consumed quickly by the organic compounds due to its non-selectivity, more SO4• − was generated, which was the dominate free radicals to remove ARGs, thus enhanced the reduction of ARGs temporarily. Once SMX was completely degraded, the dissociation equilibrium of PMS might recover, the
secondary products of SMX would competitively consume free radicals with ARGs, thus inhibited the reduction of ARGs after 5 min.
4. Conclusion The inactivation of an isolated sulfonamide ARB HLS-6 and reduction of sul1 and intI1 in its genome by UVC irradiation, PMS oxidation and UVC/PMS treatments were investigated in this study. The UVC/PMS treatment was superior to the other two methods in the inactivation of ARB and reduction of qPCR-sul1 and qPCR-intI1, which acquired 5.3 log of ARB inactivation (PMS: 1 mg/L), 2.9 log and 3.4 log reduction of ARGs (PMS: 20 mg/L) and qPCR-intI1 gene reacted faster than qPCR-sul1 in all three methods. The gel gelectrophoresis of two long fragments PCR-sul1 and PCR-intI1 gene revealed that the potential horizontal gene transfer could successfully suppressed. The quenching study showed that SO4• − was responsible for the reduction of target genes. However, HO• was not remarkable effect on the reduciton of ARGs. The dosage of PMS positively affected the reduction of both genes due to the more ROS generated by greater PMS dosage. However, the initial concentration of ARB would negatively affect the reduction of ARGs due to the orgainc compounds in cytoplasm, which was the competitive consumer of ROS. Sul1 gene was more sensetive than intI1 to the UVC intensity. The slightly acidic of reaction solution was beneficial to the hydrolysis of PMS and the generation of SO4• −. The reduction of target genes was slightly facilitated in the initial 5 min and suppressed after 5 min with the existence of SMX. To our knowledge, this was the
first study on the inactivation of ARB and reduction of ARGs by UVC/PMS, which provides a new potential treatment method to control antibiotic resistance in aquatic environment.
Acknowledgements This work was supported by the National Water Pollution Control and Management Technology Major Projects (No. 2017ZX07402003), Shanghai Municipal Science and Technology Commission (No. 16XD1420500) and Open Project of State Key Laboratory of Urban Water Resource and Environment (No. QA201612), Shanghai Sailing Program (No. 18YF1406000), China Postdoctoral Science Foundation (No. 2017M621391) and the Fundamental Research Funds for the Central Universities (No. 222201814055).
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Fig. 1 Scheme of three treatment methods in this study Fig. 2 Inactivation of ARB by UVC, PMS and UVC/PMS treatments Fig. 3 Fig. 3 Removal of ARGs by UV, PMS and UVC/PMS treatments Fig. 4 Quenching analysis for target gene by UVC/PMS oxidation Fig. 5 DNA electrophoresis gel of intracellular genes treated by UVC/PMS Fig. 6 Impact factors on the removal of target ARGs during UVC/PMS Fig. 7 Co-reduction of SMX and target ARGs by UVC/PMS
Fig. 1 Scheme of three treatment methods in this study (a) PMS oxidation; (b) UVC/PMS; (c) UVC irradiation
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Fig. 3 Removal of ARGs by UV, PMS and UVC/PMS treatments (UVC intensity: 100 μW/cm2, the UVC doses at 2.0, 5.0, 10, 30 min are 12, 30, 60, 180 mJ/cm2, respectively; Effective PMS: 20 mg/L, pH: 7.0) (a)sul1; (b)intI1
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Fig. 5 DNA electrophoresis gel of intracellular genes treated by UVC/PMS (M: maker, NC: negative control; UVC intensity: 100 μW/cm2, PMS dosage: 20 mg/L, pH: 7.0).
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(a) PMS dosage; (b) UVC intensity (50, 100, 200, 300 μW/cm2: 30, 60, 120, 180 mJ/cm2); (c) pH; (d) initial ARB concentration
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Graphical abstract
UVC
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M -interme iate o i ation com o n ro cts
Highlights
The combination of UVC and PMS greatly improved the ARB and ARGs reduction.
SO4• − was responsible for the target genes reduction by UVC/PMS.
qPCR-intI1 reactes faster than qPCR-sul1 by all methods.
Sul1 gene was more sensitive to UVC than intI1.
The co-existence of SMX would partially influence the co-reduction of ARB and ARGs.
S1385-8947(19)30455-3 https://doi.org/10.1016/j.cej.2019.02.207 CEJ 21120
To appear in:
Chemical Engineering Journal
Received Date: Revised Date: Accepted Date:
29 November 2018 25 February 2019 27 February 2019
Please cite this article as: Y. Hu, T. Zhang, L. Jiang, S. Yao, H. Ye, K. Lin, C. Cui, Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.02.207
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Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate Yaru Hua, Tianyang Zhanga,b, Lei Jiangc, Shijie Yaoa, Hui Yec, Kuangfei Lina, Changzheng Cui*a,b a. State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, China, 200237 b. Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, PR China c. National Engineering Research Center of Urban Water Resources, Shanghai, China, 200082
*Corresponding author, Changzheng Cui, Tel: +86 21 64253988; Fax: +86 21 64253988; E-mail: [email protected]
Abstract The inactivation of an isolated sulfonamide antibiotic resistant bacteria (ARB) HLS-6 and reduction of intracellular sul1 and intI1 in its genome by UVC irradiation, PMS oxidation and UVC-activated peroxymonosulfate (UVC/PMS) treatments were investigated in this study. The UVC/PMS treatment was superior to the other two methods in the inactivation of ARB and reduction of qPCR-sul1 and qPCR-intI1. The HLS-6 ARB (108 CFU/mL) could be effectively inactivated 5.3 log by UVC (100 μW/cm2) /PMS (1 mg/L), and the reduction rates of qPCR-sul1 and qPCR-intI1 by UVC (100 μW/cm2) /PMS (20 mg/L), reached up to 2.9 log and 3.4 log, respectively within 30 min. qPCR-intI1 reacted faster than qPCR-sul1 in all methods. Sulfate radical was responsible for the reduction of target genes, while hydroxyl radical had negligible effect on that. The dosage of PMS positively affected the reduction of both genes during UVC/PMS, while the initial concentration of ARB could negatively influence the reduction of target genes. The pH 5 of reaction solution was most beneficial to the reduction of ARGs. The reduction rates at pH 5 reached up to 3.1 log (sul1) and 3.3 log (intI1). The reduction of target genes was slightly facilitated in the initial 5 min and suppressed after 5 min with the co-existence of sulfamethoxazole. This study will provide a potential alternative method for controlling the antibiotic resistance in aquatic environment. Keywords: UVC-activated peroxymonosulfate; antibiotic resistant bacteria; intracellular antibiotic resistance genes; sulfate radical, water treatment.
1 Introduction Antibiotic resistance has been a serious issue worldwide recently, which is constantly challenging the clinical therapeutic authority of antibiotics and threatening the human health, causing social panic [1]. The rapid propagation of antibiotic resistant bacteria (ARB) and vertical, horizontal gene transfer of antibiotic resistance genes (ARGs) enable the antibiotic resistance to spread quickly among different hosts [2,3], even in human [4] and animal bodies [5]. Recently, a variety of studies have reported the extensive detection of ARB and ARGs in the different environmental media [6,7]. The aquatic environment always serves as the terminal recipient of the majority contaminants, which is a hot study spot of ARB and ARGs [8.9]. The antibiotic-resistant E. coli in the Tama River, Japan, presented an ascending trend along the studied stream with a concentration range of 2 to 195 CFU/100 mL, and 32% of them were multiple resistant E. coli [10]. The concentration of multiple resistant bacteria in Jiulongjiang River, South China, ranged from 2.9 to 5.9×104 CFU/mL, and 212 ARG subtypes were detected in all samples with the concentration range from 9.7×107 to 1.0×108 copies/mL [11]. Varieties of ARG subtypes were detected in the surface water [7,12,13]. The antibiotic resistance rates of sulfonamide and tetracycline were 77.3% and 43.1%, respectively, which were positively related to the concentration of tetracycline [14]. What’s more, 7.1×101 to 5.9 ×106 copies/mL of ARGs was also detected in the same water samples [14]. The average relative
abundance of extracellular ARGs (eARGs) detected in Bohai Bay reached up to 4.3×10-1 (ARGs/16S RNA) and was greater than that of intracellular ARGs (iARGs) [15]. Although multiple water treatment methods were adopted to remove the common environmental contaminants, which could partially reduce ARB and ARGs simultaneously, ARB and ARGs still presented high detection frequencies and concentration in the effluent and primary water body [7,9]. Sixteen types of ARB belonging to Proteobacteria and Firmicutes phylums were isolated from a water treatment plant in Shanghai, 75% of which were resistant to more than one types of antibiotics [16]. What’s more, the resistance rates of ARB increased during the water treatment process with the antibiotic concentration range from 1 to 10 mg/L [16]. To control antibiotic resistance and preserve the drinking water quality, as well as obtain an optimal water treatment method that is able to effectively eliminate ARB and ARG, the recent studies focusing on individual drinking water treatment procedures, such as ozonation, UV irradiation, chlorination, as well as advanced oxidation processes, became more and more popular [17-19]. Due to the strong oxidation ability, only 0 - 0.2 mg/L of ozone could reduce about 4.0 log E. coli by damaging the integrality of cell and higher ozone dose (> 0.2 mg/L) was required to inactivate ARGs [20]. The UVC irradiation could obtain 1 log loss of transformation efficiency of ARGs requiring 20 - 25 mJ/cm2 [21]. The UVA/H2O2 treatment could successfully inactivate the antibiotic resistant E. coli under detection limit in 90 min, while it had no obvious effect on the blaTEM, qnrS, tetW genes [22]. What’s more, the
inactivation of eARGs and iARGs were also investigated by UVC/H2O2 treatment to get rid of the organic compounds in cytoplasm, which revealed that UVC/H2O2 could eliminate eARGs [23]. Furthermore, the combination of UV irradiation with TiO2 material was also applied to study the inactivation of both ARB and ARGs. The reduction rate of ARGs was amplified by adding TiO2 thin film under UVC irradiation, and also had effect on the eARGs and iARGs [24]. An ultrafiltration membrane could reduce 98% of ARGs and perfectly remove integrase genes, which impeded the potential horizontal gene transfer of ARGs [25]. In our previous study, ARB and ARGs could be effectively removed by UVC/chlorination treatment [19]. However, the generation of disinfection by-products during the water treatment by chlorination or UV/chlorination would also threat the human health [26,27]. Therefore, a more effective and safer disinfection processes need to be further explored. Previous studies showed that the UVC/PMS treatment was a safe nontoxic environmental friendly water treatment method [28,29]. What’s more, based on our previous study on the effectively removal of twelve sulfonamides by UVC-activated peroxymonosulfate [28] and the potential capacity of sulfate, peroxymonosulfate, and hydroxyl radicals of causing DNA modification [30,31], the inactivation of a typical gram-negative sulfonamide ARB and the reduction of intracellular sulfonamide ARG by UVC irradiation, PMS oxidation and UVC/PMS were further investigated in this study. Several available factors that would affect the reduction rate were taken into consideration to further optimize this process, including the dosage of PMS, UVC
intensity, the pH of ARB solution, the initial ARB concentration and the real wastewater treated by secondary treatment process. Furthermore, a set of quenching experiments was conducted to identify the primary radical species during UVC/PMS. The co-reduction of ARB and iARG with sulfamethoxazole (SMX) was analyzed under optimal conditions of UVC/PMS. The results could not only give a better understanding of the elimination of ARB and ARG, but also provide a potential alternative method for controlling the antibiotic resistance in aquatic environment.
2 Materials and methods 2.1 Chemicals and reagents PMS (2KHSO5·KHSO4·K2SO4); H2SO4, HCl, NaCl, Na2HPO4∙12H2O, KH2PO4, KCl, NaOH, Na2S2O3 and tert-butyl alcohol (TBA, analytical-grade) were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Sulfamethoxazole (SMX, C10H11N3O3S, BP grade) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Ultrapure water was prepared using a Milli-Q water system (Millipore, USA). An isolated ARB pseudomonas sp. HLS-6 (CCTCC AB 2017269) was selected to investigate the inactivation of ARB and the reduction of ARGs by different disinfection processes. Sulfonamide ARGs sul1 and class I integrase gene intI1 are harbored in the genome of the HLS-6 strain (NCBI accession number: NZ_CP024478.1). The ARB was cultivated in liquid lysogeny broth (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, pH= 7.0 ± 0.2) with 200 mg/L SMX at 30 °C, 150
rpm overnight. The ARB cells was collected by centrifuging at 8,000 rpm for 5 min and washed three times with 1:10 diluted sterile phosphate-buffered saline solution (PBS, pH = 7.0), then, was re-suspended in PBS [18,20]. The initial concentration of the obtained ARB cells in PBS was ~108 CFU/mL. 2.2 Experimental approach Because the inactivation of ARB was easier than the break of DNA [23], the dosage of PMS during these two experiments were different. PMS solution was added at 1 mg/L (for ARB inactivation) and 20 mg/L (for ARGs reduction), respectively. The UVC dose (254 nm) was adapted using a UVC intensity radiometer (Photoelectric Instrument Factory of Beijing Normal University, Beijing, China). The pH of ARB solution was adjusted using HCl and NaOH solutions. All reactions were quenched by stopping UVC irradiation and adding Na2S2O3 solution (Na2S2O3/PMS = 2 mol/mol). Before the reactions, the UV dose was adjusted to 100 μW/cm2 using a simple elevator (Fig. 1). The effective PMS was calibrated according to a previous study [28]. Suspended HLS-6 cultures (50 mL) were transferred into a petri dish (Diameter×Height: 90 mm×12 mm), and gently mixed with a magnetic stir bar (Fig. 1). Then the solution was exposed to direct UVC irradiation, PMS oxidation and UVC/PMS, respectively (Fig. 1). 1 mL solution was sampled at the pre-determined intervals. The factors affecting the reduction of ARGs by UVC/PMS including the PMS dosage, UVC dose, pH of the solution and initial concentration of ARB cell were also analyzed. The sampling time was all 10 min. To identify the major radical
species during UVC/PMS, a set of quenching experiments was performed. The experimental procedures were same as the previous methods with the additional reagents of methanol (EtOH) and tert-butyl alcohol (TBA). The amount of each alcohol was added based on the previous studies [31]. 2.3 Analytical approach 2.3.1 ARB survival The different-time treated samples were processed using gradient dilution by 1:10 PBS [19]. Then 0.1 mL of each diluted sample was smeared evenly in duplicates on the surface of prepared LB solid medium, which contained 200 mg/L of SMX, and then incubated at 37 oC. After 24 h’s incubation, the number of ARB colony was counted and recorded as CFU/0.1 mL, which was further normalized CFU/mL and represented the surviving number of ARB. 2.3.2 DNA extraction and ARGs quantification, residual SMX and water parameters analysis The prepared ARB solutions (HLS-6) were directly treated with UVC irradiation, PMS oxidation or UVC/PMS processes. Then, the inactivation of ARB was analyzed by dilution method of plate counting and recorded as CFU/mL. The DNA in the disinfected samples was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany), and the short fragments of target genes (<500 bp, qPCR-sul1; qPCR-intI1) were subsequently quantified using a LightCycler 480II (Roche, Swiss) basing on the quantification method as previously described [32]. The breakage of the long segment
of target genes (about 500 bp: PCR-sul1; PCR-intI1) were amplified by a Thermo Cycler (Biorad S1000, USA) and further analyzed by 1.2 % gel electrophoresis (DYY-12C, Beijing, China) at 1400mV for 30 min. The detailed information of PCR, qPCR primers and parameters of PCR condition were shown in Section S1 and Table S1. The residual SMX concentration was analyzed according to previous studies [33] using a high performance liquid chromatographic tandem mass spectrometry (HPLC-MS/MS, Agilent, USA) under the multiple reaction monitoring (MRM) mode and the method parameters and HPLS-MS/MS details were shown in Section S2 and the detail mass spectrometric parameters, recoveries, and limits of quantitation of SMX were shown in Table S2. The detail analysis of water quality characterization are shown in Section S3.
3 Results and discussion 3.1 Inactivation of sulfonamide ARB HLS-6 The inactivation of ARB by UVC irradiation, PMS oxidation, and UVC/PMS was plotted in Fig. 2. The UVC intensity was 100 μW/cm2 and the equivalent UVC dose was calculated as UVC intensity (100 μW/cm2) × sampling time (0.5 min, 1.0 min…… 30 min) = UVC dose (mJ/cm2). It could be easily sorted by degradation rate and efficiency of ARB followed: UVC/PMS > UVC irradiation > PMS oxidation. The single PMS oxidation showed no obvious effect on the ARB inactivation with the PMS dosage of 1 mg/L and the inactivation ratio was only 0.28 ± 0.04 log. The inactivation ratios of ARB by UVC irradiation and UVC/PMS showed an increasing
trend with the increase of UVC dose. The UVC irradiation (100 µW/cm2, 10 min: 60 mJ/cm2) could effectively inactivate ARB from 108 to 103 - 104 CFU/mL. Furthermore, the inactivation ratios were empilfied from 4.1 ± 0.0 log to 5.3 ± 0.1 log with the additional dosage of 1 mg/L PMS. This result demonstrated that the combination of UVC irradation and PMS oxidation was able to reinforce the inactivation efficiency of ARB. That UV irradaition is well-known to be able to sterilize, which is due to it’s capacity to induce DNA damages by hydration of the base or base dimerization, causing DNA mutations and holding up the DNA replication [34,35]. A varity of studies have investigated the inactivation of ARB by individual UV irradiation [17,19]. An ampicillin-resistant E. coli CGMCC 1.1595 (intital ARB concentration 107 CFU/mL) was inactivated by 5 - 20 mJ/cm2 UVC irradiation with the ratio of 2.0 log, and the ratio increased to 5.5 log at the UVC dose of 40 mJ/cm2 [18]. The same HLS-6 ARB was sterilized by 4 log at the UVC dose of 12 mJ/cm2 with the intital ARB concentration 107 CFU/mL [19]. Both results were slightly higher than that of this study (1.0 log at 12 mJ/cm2 and 4.1 log at 60 mJ/cm2), which could explained by the higher ARB concentration used in this study. The breakage of PMS peroxide bond occurred under UV radiation, which produced one hydroxyl radical (HO•) and one sulfate radical (SO4•−) (HSO5-/ SO52- + hv → SO4• − + HO•) [36,37]. The high redox potentials of HO• enables it to rapidly react with most compounds like proteins [38,39], which are the main components of cell walls and DNA double strands. The
SO4•− generated by transition metal-activated processes was responsible for the inactivation of Escherichia coli [40,41], which confirmed the excellent sterilization effect of SO4•−. Therefore, the inactivation of ARB was not a difficult thing for the combination of UVC irradiation, HO• and SO4•− during UVC/PMS treatment. 3.2 Reduction of ARG sul1 and integrase gene intI1 The reduction and of sul1 and intI1 genes by UVC irradiation, PMS oxidation, and UVC/PMS was plotted in Fig. 3. Preliminary experiments showed that the DNA damage occurred more slowly than the inactivation of ARB [23], so the dosage of PMS was adjusted from 1 to 20 mg/L and the sampling time was extended to 30 min. and in these conditions ARB was completely removed within 1 min by UVC/PMS (Fig. S1). The reduction rates of both genes were represented by the log change of the short amplicons (qPCR-sul1 and qPCR-intI1). The reduction of both genes followed the same trend in the initial 2 min and differed in the following time. The reduction of target genes occurred slowly during UVC irradiation and the losses of qPCR-sul1 and qPCR-intI1 were 1.2 ± 0.5 log and 0.8 ± 0.1 log in 30 min (130 mJ/cm2), respectivley, which were lower than the reduction of amp and kan genes (1.1 - 1.6 log at 40 mJ/cm2) in a previous study [23]. The reduction rate of individual PMS oxidation exceeded that of UVC irradiation before 10 min, which eventually acquired 2.0 ± 0.1 log and 2.3 ± 0.1 log in 30 min. The UVC/PMS treatment still presented the best reduction rate of both genes with the reduction rates being 2.9 ± 0.1 log (qPCR-sul1) and 3.4 ± 0.1 log (qPCR-intI1) within 30 min, respectively, which was slghtly inferior to the
reduction time of the same genes by UVC/chlorination at the same UVC dose [19]. Apparently, the reduction of both genes by these three treatment methods followed a first-order kinetics with respect to the sampling time (referring to the UVC dose), ignoring some deviation of the last 30 min sampling, and this was similar to the results of previous studies [21,23]. The calculated reduction rate constants were summarised in Table S3. The k value also proved that the combinated reduction of UVC irradiation and PMS oxidation exceeded both single treatment (kUVC/PMS > kUVC + kPMS > kUVC ≈ kPMS), and the reaction rate of UVC/PMS were 2.8 - 2.9 times higher than that of UVC irradiation and PMS oxidation. Same superiority of the combined method could be explained by that the UVC irradiation could decrease the bioactivity of ARB, thus made it easily for free radicals to enter into the cells and inactivate ARGs [42]. The values of k’UVC in this study were lower than those reported previously for the amp and kan genes [23] and tetA and blaTEM-1 genes [21], but higher than that of the same gene reduction in another previous study [19]. A longer gene is expected to react faster with UVC irradiation due to the presence of more UVC target sites than a small one [17,43], while the values of k’UVC only had moderate correlations with the gene length (r = 0.43). The DNA lesions induced by UVC irradiation almost occurred between two adjacent pyrimidines with the thymine-thymine cyclobutane-pyimidine dimers (TT CPDs) being the primary product [43], which furnish evidence of the values of k’UV strongly being related to the number of adjacent TT (r = 0.79). Interestingly, the k’UV values of the same gene
reduction by higher UVC intensity irradiation (200 μW/cm2) in a previous study [19] was lower than that in this study (UVC intensity: 100μW/cm2), the reason of which was still not clear. The k’PMS values of both genes in this study were greater than that of chlorination [19]. This might be attributed to the oxidation reduction potential of PMS (1.85 V), which was higher than most oxidizing agents, including sodium hypochlorite. The k’UVC/PMS values of both genes in this studies were lower than those of the amp and kan genes by UVC/H2O2 treatment [23]. Theoretically, HO• is one of the most active reactive oxide species (ROS), which would attack the C5, C6 sites of pyrimidine bases and the C4, C5 and C8 sites of purine bases [45], causing the direct DNA damage. However, due to the non-selectivity of reaction with the organic compounds in the cytoplasm, HO• would be consumed quickly, which would result in its noneffectiveness against ARGs [22]. The reduction of amp and kan genes by UVC/H2O2 treatment was mediated by a plasmid [23], which contained less organic substance than a whole cell in this study. However, SO4• − was proved to be responsible for the modification of DNA by reacting with guanine [30,46]. Since sul1 and intI1 genes have the similar guanine content, so the k values of them were similar too. It is noteworthy that the k value of intI1 gene was always greater than that of sul1 no matter by UVC irradiation, PMS oxidation and UVC/PMS. 3.3 The roles of HO• and SO4• − in the removal of ARGs during UVC/PMS Since there were two kinds of reactive oxygen species (HO• and SO4• −) existing during the UVC/PMS system, a set of quenching studies was conducted to identify the
primary radical species (Fig. 5). Two kinds of alcohols (TBA and EtOH) were chosen based on their different reactivity and rates with different radical species and added into the UVC/PMS system at certain amount ratios to PMS [31]. The presence of EtOH and TBA only resulted in a moderate decrease in the reduction of target genes (less than 4% - 25%), furthermore, a slight increase of sul1 gene (5%) occurred with the existance of EtOH (250:1). The reaction rates between EtOH with HO• was approximately 50-fold greater than that with SO4• − [31] and the reaction rate between TBA with HO• was about 1000 times higher than that with SO4• − [47,48]. Due to relatively chemical inertness of SO4• − toward alcohols, the added quenching agents in the system would preferentially consume HO• [31]. However, the reduction of ARGs was not seriously affected. Therefore, it could be concluded that SO4• − are responsible for the reduction of target genes. SO4• − would attack the guanine heterocycle on the π face, which was responsible for the modification of DNA [30]. However, HO• was not effective in the reduciton of ARGs [19,22]. 3.4 Gene breakage and potential gene transfer loss Although the qPCR could detect the minor base modification of DNA damage [49] and the qPCR results also showed a declining trend of target genes’ concentrations, the cleavage of whole sul1 and intI1 genes could occur at any gene site along the target gene sequence and couldn’t be fully delegated by only a 200 bp fragments. Therefore, two longer routine PCR amplicons (PCR-sul1: 433 bp; PCR -intI1: 484 bp) was presented by gel electrophoresis (Fig. 4). The brightness of both
gene stripes showed a declining trend over time during UVC irradiation, PMS oxidation and UVC/PMS and UVC/PMS was also the best treatment method to destroy the integrity of PCR-sul1 and PCR-intI1. Although the qPCR results showed that the qPCR-intI1 gene still present ~ 105 copies/mL, the PCR-intI1 couldn’t be visualized by gel gelectrophoresis, which indicated the intermediate breakage of PCR-intI1. The co-existance of intI1 and sul1 gene analyzed in this study, is indentified as the genetic marker of class I integron [50], which always confers the horizonal gene transfer of gene cattases among different hosts. The interval open reading frame gene between intI1 and sul1 is aadA2 gene (780 bp), which encodes the resistance to aminoglycoside antibiotic. Once this integron is successfully assimilated by pathogens, it will eventually aggravates the antibiotic resistance. However, the whole gene fragment can’t be fully quantified by common qPCR due to the limit of gene length. Resorting to the extrapolated equation reported previously [17], the damage of whole intI1-aadA2-sul1 and intI1-aadA2 gene fragments could be predicted.and the results were shown in Fig. S2. The predicted reduction of these two gene fragments were between 43 - 56 log and 32 - 42 log, respectively, which accounting for approximately 3.0×1040 - 1.8×1053 and 2.4×1029 -7.9×1038 copies/mL. Therefore, the gene transfer potential was greatly suppressed. 3.5 Impact factors on the reduction of target genes by UVC/PMS Since UVC/PMS treatment was superior to UVC irradiation and PMS oxidation
in the reduction of target genes, several available factors that would affect the reduction rate were taken consideration to further optimize this process. All these impact factors were investigated under the same condition as previous described, unless changing the unique variable parameters, including the PMS dosage, UVC intensity, pH and initial ARB concentration. The dosage of PMS could significantly affect the reduction of both genes (Fig. 6(a)). An increase of PMS dosage resulted in a greater reduction of both genes. The reduction rate ascended from 1.1 log to 2.8 log in 10 min with the PMS dosage increased from 5 to 30 mg/L. The log reduction of both genes was positively related to the dosage of PMS (sul1: r2=0.996; intI1: r2 = 0.945), which contributed most to the more SO4• − generated by greater dosage of oxidant [51]. Fig. 6(b) showed the effect of another important factor UVC intensity on the gene reduction during UVC/PMS. The log reduction of sul1 gene increased slowly as the UVC intensity increased from 50 to 300 μW/cm2, while for intI1 gene, the reduction change didn’t follow regularly with the increase of UVC intensity. Otherwise, the reduction of intI1 showed a falling trend at high intensity UVC. Though long gene intI1 have more reaction sites than short gene sul1, the sul1 gene was more sensitive than intI1 gens to UVC intensity. Previous studies demonstrated that the pH of the reaction solution could significantly impact the decomposition of PMS [28] and the pH under 5 would induce the PMS decomposition (SO4• − + HSO5 HSO4
SO5•
+
< 104 M-1 s-1) [52], so the pH range of 5 - 9 was analyzed. As shown in
Fig. 6(c), it benefited the greater reduction of both genes in acidic condition than that
in alkalescent condition. The PMS couldn’t generate SO4• − under low pH, and partial SO4• − could react with OH- to generate HO• (SO4• −+ HO− → HO• + SO42−; k = 6.5×107 M-1 s-1) [52], which was not effective in eliminating ARGs. Fig. 6(d) showed the influence of the initial ARB concentration on the reduction of both genes. It could easily be concluded that the lower the initial concentration was, the higher the reduction rate would be. During the death of ARB cells, higher concentration of organic compounds was released from the cytoplasm than the lower one, which was the prior ROS consumer. In order to explore the impact of the real environment media on the reduction of ARGs by UVC/PMS, an additional experiment were conducted in the wastewater from the secondary treatment process. The water quality parameters and the reduction of ARGs were presented in Table S2 and Fig. S3, respectively. The reduction of ARGs increased gradually with the sample time increasing, and the reduction rates reached up to 0.9 log (sul1) and 1.0 log (intI1), which were higher than that in a previous study [53]. The organic matters also decreased after treated by UVC/PMS, whose reduction efficiencies were consistent with that of the real wastewater disinfection process (chlorination). However, the reduction efficiencies of organic matters were lower than that of the previous study [53]. This discrepancy might be explained by the wastewater type and the reagent dosage. The water quality parameters in this study were 0.1 - 189 times lower than that in the previous study, and additive H2O2 and iron (II) sulfate heptahydrate were added into the treatment system to generate more HO·,
which would preferentially react with organic matters, not with ARGs [39]. This would induce the low reduction efficiency of organic matters and high reduction rate of ARGs. 3.6 Co-reduction of SMX and ARGs by UVC/PMS To investigate the real effect of UV/PMS, an organic substance was chosen to explore the co-reduction. The HLS-6 strain shows resistance to 200 mg/L SMX, which is a frequently detected antibiotic in the drinking water source [9,33]. Therefore, the co-reduction of SMX, ARB and target genes (sul1 and intI1) by UVC/PMS under the optimal condition was also investigated in this study. As for the single reduction of SMX, it was completely eliminated within 1 min by 30 mg/L PMS and 100 μW/cm2 (data not shown). When the SMX was co-existing with ARB solution, it was also degraded immediately (Fig. 7). However, the reduction of target genes was slightly facilitated in the initial 5 min, and was inhibited after 5 min. The reduction rate was 1.3 log (qPCR-sul1) and 1.8 log (qPCR-intI1). These radicals rapidly attacked many organic compounds with rate constants ranging from 106 to 1010 M−1·s−1 [54,55], and 0.2 μg/L SMX could be completely removed in 5 min with 5 mg/L PMS [28]. The initial slight facilitation of ARGs reduction could be explained the solvent effect. Because the HO• was consumed quickly by the organic compounds due to its non-selectivity, more SO4• − was generated, which was the dominate free radicals to remove ARGs, thus enhanced the reduction of ARGs temporarily. Once SMX was completely degraded, the dissociation equilibrium of PMS might recover, the
secondary products of SMX would competitively consume free radicals with ARGs, thus inhibited the reduction of ARGs after 5 min.
4. Conclusion The inactivation of an isolated sulfonamide ARB HLS-6 and reduction of sul1 and intI1 in its genome by UVC irradiation, PMS oxidation and UVC/PMS treatments were investigated in this study. The UVC/PMS treatment was superior to the other two methods in the inactivation of ARB and reduction of qPCR-sul1 and qPCR-intI1, which acquired 5.3 log of ARB inactivation (PMS: 1 mg/L), 2.9 log and 3.4 log reduction of ARGs (PMS: 20 mg/L) and qPCR-intI1 gene reacted faster than qPCR-sul1 in all three methods. The gel gelectrophoresis of two long fragments PCR-sul1 and PCR-intI1 gene revealed that the potential horizontal gene transfer could successfully suppressed. The quenching study showed that SO4• − was responsible for the reduction of target genes. However, HO• was not remarkable effect on the reduciton of ARGs. The dosage of PMS positively affected the reduction of both genes due to the more ROS generated by greater PMS dosage. However, the initial concentration of ARB would negatively affect the reduction of ARGs due to the orgainc compounds in cytoplasm, which was the competitive consumer of ROS. Sul1 gene was more sensetive than intI1 to the UVC intensity. The slightly acidic of reaction solution was beneficial to the hydrolysis of PMS and the generation of SO4• −. The reduction of target genes was slightly facilitated in the initial 5 min and suppressed after 5 min with the existence of SMX. To our knowledge, this was the
first study on the inactivation of ARB and reduction of ARGs by UVC/PMS, which provides a new potential treatment method to control antibiotic resistance in aquatic environment.
Acknowledgements This work was supported by the National Water Pollution Control and Management Technology Major Projects (No. 2017ZX07402003), Shanghai Municipal Science and Technology Commission (No. 16XD1420500) and Open Project of State Key Laboratory of Urban Water Resource and Environment (No. QA201612), Shanghai Sailing Program (No. 18YF1406000), China Postdoctoral Science Foundation (No. 2017M621391) and the Fundamental Research Funds for the Central Universities (No. 222201814055).
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Fig. 1 Scheme of three treatment methods in this study Fig. 2 Inactivation of ARB by UVC, PMS and UVC/PMS treatments Fig. 3 Fig. 3 Removal of ARGs by UV, PMS and UVC/PMS treatments Fig. 4 Quenching analysis for target gene by UVC/PMS oxidation Fig. 5 DNA electrophoresis gel of intracellular genes treated by UVC/PMS Fig. 6 Impact factors on the removal of target ARGs during UVC/PMS Fig. 7 Co-reduction of SMX and target ARGs by UVC/PMS
Fig. 1 Scheme of three treatment methods in this study (a) PMS oxidation; (b) UVC/PMS; (c) UVC irradiation
9
10
8
ARB survival (CFU/mL)
10
7
10
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UVC PMS UVC/PMS
4
10
3
10
0
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4 6 Time/min
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Fig.2 Inactivation of ARB by UVC, PMS and UVC/PMS treatments (UVC intensity: 100 μW/cm2; the UVC doses at 0.5, 1.0, 2.0, 5.0, 10 min are 3, 6, 12, 30, 60 mJ/cm2, respectively; Effective PMS: 1 mg/L; pH: 7.0)
6.5
(a)
6.0
6.0
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Log concentration of ARGs
Log concentration of ARGs
6.5
5.0 4.5 4.0 3.5
UVC PMS UVC/PMS
3.0 2.5
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5
(b)
5.0 4.5 4.0 3.5 UVC PMS UVC/PMS
3.0
10
15 20 Time/min
25
30
2.5
0
5
10
15 Time/min
20
25
30
Fig. 3 Removal of ARGs by UV, PMS and UVC/PMS treatments (UVC intensity: 100 μW/cm2, the UVC doses at 2.0, 5.0, 10, 30 min are 12, 30, 60, 180 mJ/cm2, respectively; Effective PMS: 20 mg/L, pH: 7.0) (a)sul1; (b)intI1
sul1 intI1
Log reduction of target genes
1.0
0.8
0.6
0.4
0.2
0.0 UVC
PMS
UVC
/PMS
EtOH
:1) :1) :1) :1) (250 (500 (250 (500 TBA TBA EtOH
Fig. 4 Quenching analysis for target gene by UVC/PMS oxidation
Fig. 5 DNA electrophoresis gel of intracellular genes treated by UVC/PMS (M: maker, NC: negative control; UVC intensity: 100 μW/cm2, PMS dosage: 20 mg/L, pH: 7.0).
(a)
3.5
sul1 intI1
3.0
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Log(C0/C)
Log(C0/C)
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10 20 Dosage of PMS (mg/L)
(c)
30
50 3.5
sul1 intI1
3.0 2.5 2.0 1.5
100 200 2 UVC(W/cm )
300
(d)
sul1 intI1
3.0
Log(C0/C)
Log(C0/C)
sul1 intI1
1.5
1.0 3.5
(b)
2.5 2.0 1.5
1.0
1.0 5
6
7 pH
8
9
6
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10 initial ARB (CFU/mL)
10
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Fig. 6 Impact factors on the removal of target ARGs during UVC/PMS
(a) PMS dosage; (b) UVC intensity (50, 100, 200, 300 μW/cm2: 30, 60, 120, 180 mJ/cm2); (c) pH; (d) initial ARB concentration
without SMX with SMX SMX
5.5
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without SMX with SMX SMX
5.5 5.0
0.6
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4.0 0.2 3.5 0.0
3.0 0
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Fig. 7 Co-reduction of SMX and target ARGs by UVC/PMS
(a) sul1; (b) intI1
1.0 0.8
0.6
5.0
(b)
6.0 0.8
C/C 0
Log C of target genes
6.0
6.5
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15 20 Time/min
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C/C 0
(a)
Log C of target genes
6.5
Graphical abstract
UVC
ant
■■ ■ ■
nt
5´C
nt int
3´C M
M -interme iate o i ation com o n ro cts
Highlights
The combination of UVC and PMS greatly improved the ARB and ARGs reduction.
SO4• − was responsible for the target genes reduction by UVC/PMS.
qPCR-intI1 reactes faster than qPCR-sul1 by all methods.
Sul1 gene was more sensitive to UVC than intI1.
The co-existence of SMX would partially influence the co-reduction of ARB and ARGs.