Article pubs.acs.org/est

Exposure to Mutagenic Disinfection Byproducts Leads to Increase of Antibiotic Resistance in Pseudomonas aeruginosa Lu Lv, Tao Jiang, Shenghua Zhang, and Xin Yu* Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, People’s Republic of China S Supporting Information *

ABSTRACT: Bacterial antibiotic resistance (BAR) in drinking water has become a global issue because of its risks on the public health. Usually, the antibiotic concentrations in drinking water are too low to select antibiotic resistant strains effectively, suggesting that factors other than antibiotics would contribute to the emergence of BAR. In the current study, the impacts of mutagenic disinfection byproducts (DBPs) on BAR were explored, using four typical DBPs: dibromoacetic acid, dichloroacetonitrile, potassium bromate, and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). After exposure to DBPs, resistances to 10 individual antibiotics and multiple antibiotics were both raised by various levels, norfloxacin and polymycin B resistances were enhanced even greater than 10-fold compared with control. MX increased the resistance most observably in the selected DBPs, which was consistent with its mutagenic activity. The resistant mutants showed hereditary stability during 5-day culturing. The increase of BAR was caused by the mutagenic activities of DBPs, since mutation frequency declined by adding ROS scavenger. Mutagenesis was further confirmed by sequencing of the related genes. Our study indicated that mutagenic activities of the selected DBPs could induce antibiotic resistance, even multidrug resistance, which may partially explain the lack of agreement between BAR and antibiotic levels in drinking water.

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INTRODUCTION The occurrence and spread of bacterial antibiotic resistance (BAR) has become a serious threat to human health. Antibiotic resistance genes (ARGs) are widely regarded as a group of emerging contaminants.1,2 The presence of BAR or ARGs has been documented in drinking water systems for over 30 years.3−5 For example, in Oregon State, 36.7% and 67.8% of standard plate count bacteria from two treated water for two different communities were multiply antibiotic resistant (MAR) bacteria; while the MAR populations in respective raw water were only 20.4% and 18.6%.6 In a drinking water treatment plant in northern Colorado, both tet(M) and tet(O) were detected in the prechlorination and postchlorination treated water compared to none detected in source water. 1 Observations also showed that, although the level of bacteria was decreased in the finished drinking water after treatment, the quantities of both antibiotic resistant bacteria (ARB) and ARGs were greater in the tap water than that in finished water.7 It is generally accepted that the extra-organism reason for generation of antibiotic resistance is antibiotic selective pressure.8 However, concentrations of antibiotics in drinking water are generally low, typically in the ng/L level, and perhaps less than 10 ng/L or even under the detection limit.9,10 It is regarded that antibiotics at concentrations of 10 ng/L or lower would not be able to select antibiotic resistant bacteria.11 Thus, the question remains: how do the bacteria in distribution systems and tap waters mentioned above gain their resistance? © 2014 American Chemical Society

Reasons internal of the organism maybe (1) horizontal gene transfer;12 (2) cross- or coresistance to heavy metals or other antibacterial agents;13,14 and (3) chromosomal mutation.15 However, the premises of horizontal gene transfer and cross- or coresistance are the existences of ARGs in bacterial habitats and heavy metal or other antibacterial agent resistance, respectively. Therefore, chromosomal mutation is the fundamental mechanism for the acquisition of BAR, which refers to the induction of antibiotic resistance via base substitution or frameshift in specific genes. The expression of these mutated genes would result in (1) changes in permeability of cellular membranes, (2) modification of antibiotic target sites, (3) the structural change or degradation of antibiotics, and (4) the activation of efflux pumps, so that bacterial antibiotic resistance is achieved.16 Disinfection of drinking water causes a remarkable decline in both mortality and morbidity rates by waterborne microbial diseases. Nevertheless, the well-known disadvantage of disinfection is the formation of disinfection byproducts (DBPs), such as trihalomethanes (THMs), haloacetic acids (HAAs), MX, and nitrosoamines, which have been identified since the 1970s.17−19 Both DBPs and concentrated drinking water containing them have been demonstrated to be Received: Revised: Accepted: Published: 8188

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control, when IR > 2, the mutagenic activity is considered positive. The test results were considered valid since IR of the positive controls was more than 2 under the test conditions (Data not shown). Exposure to M-DBPs and the Subsequent Antibiotic Resistance Determination. After cultivation for 15 h, PAO1 was diluted 1:500 into 5 mL LB broth in a 50 mL flask containing DBPs (the concentrations referred to the umu test results and shown in Table 1) or no drugs as control, and grown for 24 h at 37 °C and 180 rpm. These cultures of each treatment then were diluted 5 folds with 0.9% NaCl solution and took 100 μL plate on LB agar containing different antibiotics with appropriate concentrations (SI Table S1). For multidrug resistance determination, the diluted cell suspensions were plated on LB agar containing two kinds of antibiotics as the same concentration as the above. These cultures were also diluted in 10-fold series with 0.9% NaCl solution and took 100 μL plate on LB agar to determine the total bacterial count. All of the plates were grown for 48 h at 37 °C, and then the colonies were counted. The colonies formed in the antibiotic-containing plates were considered to have resistance to the corresponding antibiotics. A variable termed “mutation frequency” was introduced to quantify the mutation, which was calculated by dividing the number of colonies on the antibiotic plate by the total bacterial count. (The total bacterial count was enumerated from the plate count on the LB agar. And the detailed calculation can be seen in the SI) (SI Tables S2 and S3) It was regarded that the colony amounts beyond control were attributed to the mutation induced by M-DBPs. In order to represent clearly the change of mutation frequency between exposure to DBPs and control, fold change of mutation frequency was calculated, by dividing all treated mutation frequencies by the control one. Three replicates were taken for each treatment condition, and the averages of the fold change of mutation frequency are shown. Hereditary Stability of the Mutants. Mutants were screened from Cip, Gen, and Tet resistance colonies after exposure to DCAN, MX, and DBAA, respectively. Then they were cultivated in 5 mL LB broth in the absence of antibiotics at 37 °C and 180 rpm for 24 h. The cultures were diluted 1:1000 in 5 mL fresh drug-free LB and incubated again. Each mutant had undergone five such rounds of growth, the initial mutants and the cultures at the end of each round were taken for antimicrobial susceptibility test to see whether the resistance was still well kept. The susceptibility was determined using the discs purchased from Oxoid (U.K.), according to the Kirby− Bauer Disk Diffusion Method.28 Zones of inhibition were measured after 18 h of incubation at 37 °C. The diameters of inhibitory zones were calculated by averaging triplicates. Evaluation of the Effects of Reactive Oxygen Species (ROS). Whether the effects of DBPs on BAR are mutagenesis, which is in the form of ROS formation, was tested with thiourea (TU, Sigma-Aldrich, U.S.A.), a ROS scavenger. After cultivation for 15 h, PAO1 was diluted 1:500 into 5 mL LB broth containing no drugs or MX, DCAN (the concentrations were the same as mentioned above) as control group. And the experimental group added TU on the basis of control group, whose final concentration was 100 mM. The subsequent operations were the same as determining the effect of DBPs on Cip resistance. Sequencing of the Mutants. The related genes of Cip, Gen, Tet, and Rif resistant isolates after exposed to MX were

mutagenic through the Ames test, umu test and other tests of mutagenicity.20−22 It is anticipated more mutagenic DBPs (MDBPs) are likely to be identified in drinking water with the development of novel analysis approaches and instruments.23−26 In distribution pipelines, bacteria in disinfected drinking water have the chance to contact the M-DBPs for a period from a few hours to several days. Thus, it is necessary to explore whether the mutagenic activity of M-DBPs will contribute to the rise of BAR in drinking water. No studies, however, have been reported on the relationship between M-DBPs and BAR in drinking water. In this study, the opportunistic pathogen Pseudomonas aeruginosa PAO1 was exposed to four typical MDBPs and the resistances to 10 antibiotics were measured. The effects of M-DBPs on BAR and the associated mechanism(s) were investigated. The results of this study are expected to reveal a neglected pathway for the bacterial acquisition of antibiotic resistance and potentially provide a novel explanation for the lack of agreement between BARs and concentrations of antibiotics in drinking water.

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MATERIALS AND METHODS DBPs, Antibiotics, and Tested Strains. The selected DBPs: dibromoacetic acid (DBAA, Br2C2H2O2) and dichloroacetonitrile (DCAN, C2HCl2N) were purchased from Supelco (Sigma-Aldrich, U.S.A.); potassium bromate (KBrO3) was obtained from Sinopharm Chemical (China); 3-chloro-4(dichloro methyl)-5-h ydroxy-2(5H)-furanone (MX, C5H3Cl3O3) was from TRC (Canada). The involved antibiotics: carbenicillin (Car), chloramphenicol (Chl), clarithromycin (Cla), gentamicin (Gen), polymycin B (Pol), and tetracycline (Tet) were purchased from Solarbio (China); cefotaxime (Cef), norfloxacin (Nor), and rifampin (Rif) were obtained from Sigma-Aldrich (U.S.A.); ciprofloxacin (Cip) was from LKT Laboratories (U.S.A.). In determination of antibiotic resistance in strain PAO1, appropriate concentrations of these antibiotics were contained in LB agar, which are shown in Table S1, Supporting Information (SI). The opportunistic pathogen P. aeruginosa PAO1 (wild typestrain), which was used to test the resistance change, was obtained from Dr. Feng Guo, Xiamen University, China. The engineered bacterium Salmonella typhimurium TA1535/ pSK1002, which served for the umu test, was obtained from Professor Wenjun Liu, Tsinghua University, China. LB broth and LB nutrient agar for cultivation of PAO1 and Mueller− Hinton agar for the antimicrobial susceptibility test were purchased from Qingdao Hope Bio-Technology (China). Mutagenic Activity Test. The mutagenic activities of the selected DBPs were surveyed using the umu test without S9 activation, according to ISO 13829.27 The umu test evaluates the genotoxic activity of chemical compounds, using the expression of the SOS repair mechanism associated with the umuC-gene. As the solution volume in ISO 13829 might lead to overflow from the wells of 96-well microplate, 0.8 fold solution volume was applied in our protocol instead. Each concentration of DBPs and control were tested in triplicate. The results are represented as the growth factor G and the induction ratio IR, which were calculated based on the absorption values at 600 and 420 nm. And the growth factor G is the bacterial growth rate of DBPs treated bacteria to that of the distilled water control, when G < 0.5, IR cannot be evaluated. The induction ratio IR is the ratio of β-galactosidase activity of DBPs treated bacteria to that of the distilled water 8189

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Table 1. List of Category, Disinfectants, Occurrence and the Test Concentrations of the Selected DBPs chemical

abbreviation

category

dibromoacetic acid

DBAA

HAAs

dichloroacetoni-trile

DCAN

potassium bromate

KBrO3

Haloacetonitriles Oxyhalides

3-chloro-4-(dic-hloromethyl)-5hydroxy-2[5H]-furanone

MX

a

disinfectants

Halofuranones

ref.

chlorine, chloramines, chlorine dioxide and ozone chloramines, chlorine, chlorine dioxide and ozone ozone, chlorine dioxide,

34

chlorine

37

33 34−36

occurrence33

concentration (ppm)a

low- to midμg/L sub- to lowμg/L sub- to lowμg/L ng/L to subμg/L

200, 400, 600, 800, 1600 20, 40, 80, 160, 240 334, 668, 1336, 2672, 4008 0.1, 1, 5, 10, 20

The concentration series of DBPs used to test the effect of DBPs on BAR, which were base on the results of the umu test.

100 ppm, respectively. For MX, the IR value was 7.44 when its concentration was only 0.08 ppm, suggesting that MX had the highest mutagenic activity among the four selected DBPs. These results are consistent with other results reported in the literatures.29−32 The exposure dosages of the DBPs in this study (Table 1) were decided by these results. Effects of the Selected DBPs on Antibiotic Resistance. Figure 1 shows the effects on BAR by exposing the tested strain to the four selected DBPs. The mutation frequency of the control without exposure to DBPs was considered as the spontaneous mutation frequency, which varied from 10−7 to 10−8 for different antibiotics. The spontaneous mutation frequency fold change was defined as one after normalization. The maximum fold change of mutations induced by each selected DBP and its corresponding concentration are summarized in Table 3. All the four DBPs could stimulate the resistances of PAO1 to broad-spectrum antibiotics as all the fold change values were greater than 1. Specifically, 35.90%, 23.08%, 15.38%, and 25.64% of the total fold changes were in the range of 1−2, 2−3, 3−4, and >4, respectively. The corresponding DBPs dosages were mainly the highest or the second highest in the tested series, suggesting that below the cytotoxic concentration of DBPs for PAO1, higher concentration would lead to higher increase of antibiotic resistance. MX showed the most evident increase in almost all of the antibiotic resistance, and an obvious dose−response relationship was observed between mutation frequency and MX concentration (Figure 1D).The Pol and Nor resistances were increased most significantly, reaching 12.07 and 10.99 folds at 20 and 10 ppm MX, respectively. While the Cla resistance had the lowest increase, reaching 2.44 folds at 10 ppm MX. The mutation frequencies of the remaining antibiotics were all increased more than three-fold (Table 3). The resistances to 10 antibiotics with respect to the exposure to DBAA, DCAN, and KBrO3 were also enhanced in varying degrees, as illustrated in Figure 1A−C. Potentials of the selected M-DBPs in terms of their capabilities in BAR enhancement, could be ranked as MX > KBrO3> DCAN > DBAA, since the average increases of 10 antibiotic resistances were 5.95, 3.16, 2.67, and 1.95 folds, respectively (Table 3). This order was consistent with their mutagenic potentials except KBrO3. Although this typical ozonation DBP performed the weakest mutagenic activity (Table 2), it stimulated the strain to obtain the highest Cla and Gen resistance. Table 3 could also provide another rank, the maximum mutation frequency of each antibiotic resistance. The Nor and Pol resistances were more than 10-fold. The Car, Chl, Gen, and Tet were higher than 5. While the Cef, Cip, Cla, and Rif were in the range of 3−5. This diverse profile might be attributed to the various interactions between the DBPs and the

sequenced. Six colonies for each antibiotic were picked up randomly from plates, and grew in LB broth for 15 h, the same as the wild strain PAO1. Genomic DNA was extracted using BioTeke Genomic DNA Extraction Kit following the manufacturer’s instructions. DNA samples were amplified by PCR with the primers shown in SI Table S4. The temperature profile consisted of one cycle for 5 min at 94 °C, followed by 35 cycles at 94 °C for 1 min, annealing (SI Table S4) for 40 s, and 72 °C for 1 min, with a final 7 min at 72 °C. PCR amplicons were tested with agarose gel electrophoresis and purified using AxyPrep DNA Gel Extraction Kit, then sequenced on ABIPRISM 3730 DNA sequencer at Sangon Biotech (China). The obtained gene sequences were compared with the wild strain sequences using the BLAST network service (http://www.ncbi. nlm.nih.gov/blast).

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RESULTS AND DISCUSSION Mutagenic Activity of the Selected DBPs. The four selected DBPs represent typical HAAs, haloacetonitriles, ozonation byproducts, and halogenated furanones, respectively (Table 1). Their mutagenic activities were measured and confirmed by the umu test (Table 2). The mutagenic activity of KBrO3 was the weakest, with a lowest positive concentration of 334 ppm. DBAA and DCAN had stronger mutagenic activities than KBrO3, having lowest positive concentrations of 187.5 and Table 2. Mutagenic Activity of the Selected DBPs by umu Test DBPs

concentration (ppm)

DBAA

1500 750 375 187.5 200 100 50 25 1336 668 334 167 0.63 0.31 0.16 0.08

DCAN

KBrO3

MX

G ± SDa 0.60 0.84 1.00 1.03 0.43 0.57 0.73 0.86 0.58 0.73 0.71 0.82 0.11 0.17 0.45 0.68

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.01 0.04 0.03 0.11 0.13 0.14 0.08 0.21 0.07 0.05 0.06 0.02 0.03 0.09 0.07

IR ± SDb 6.47 4.57 3.33 2.45 4.68 2.79 1.98 1.45 1.84 2.03 2.00 1.87 27.67 28.61 14.75 7.44

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.37 0.27 0.13 0.13 1.42 0.80 0.36 0.08 0.45 0.45 0.35 0.41 3.66 2.65 1.10 1.31

a

The growth factor G: the bacterial growth rate of DBPs treated bacteria to that of the distilled water control; if G < 0.5, IR cannot be evaluated. SD: Standard Deviation. bInduction ratio IR: the ratio of βgalactosidase activity of DBPs treated bacteria to that of the distilled water control; if IR > 2, the mutagenic activity is considered positive. 8190

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The above results provided solid evidence that BAR can also be induced by some compounds other than antibiotics, MDBPs, in drinking water. Hence, this finding in fact reveals a possible pathway for the bacteria to acquire antibiotic resistance in drinking water and gives a partial explanation for the occurrence of relatively higher BAR levels in this environmental media. Effects of the Selected DBPs on Multidrug Resistance. The multidrug resistance is more problematic than resistance to individual antibiotic because multidrug resistance will make clinical treatment more difficult. A recent instance is the superbug carrying NDM-1 multidrug resistant plasmid, which was detected in drinking water in New Delhi, India.38 Herein, the resistances to five pairs combined from four typical antibiotics were determined after exposing to DBAA to test whether the multidrug resistance could be induced by the mutagenic DBPs. The results (Figure 2) show that all of the resistances to five antibiotic pairs were increased from 1.49 to 2.92 folds. The increase may be caused by two approaches. First, M-DBPs could induce mutations randomly in different base sites in the antibiotic resistance related genes, leading to independent resistances to different antibiotics simultaneously. Moreover, different resistances may share the same mechanism and the same resistant gene. For instance, both the β-lactam antibiotic Cef and tetracycline travel through the outer membrane with porin-mediated permeability.39 Therefore, their multidrug resistance may result from the same mutation in a regulatory gene or to decrease ompF expression,40,41 which would lead to the reduction of the Cef and Tet uptake. Hereditary Stability of the Observed Resistance Strains. Since DBPs could increase individual or multiple antibiotic resistances of bacteria, their hereditary stability of the antibiotic resistance has great significance for environmental safety and human health. This was tested using the Kirby− Bauer Disk Diffusion Method,28 and the results were characterized by the variation of inhibitory zone diameters (Figure 3). A test duration of 5 days was used considering the typical retention time of drinking water in the distribution system. The Cip resistant strains exposed to DCAN, the Gen resistant strains exposed to MX and the Tet resistant strains to DBAA could maintain their resistance rather well, since their inhibitory zone diameters were almost unchanged. The results suggested the mutants could transfer the resistance to their offspring stably. Since the bacteria can proliferate in the distribution system, this hereditary stability may lead to continuous increase of the antibiotic resistance. This result releases a dangerous signal that BAR in drinking water can not only be generated, but also has the potential to be multiplied. Effects of ROS elimination on BAR. The exposure to oxidative stress is the main reason for mutagenesis.42 This stress is performed by ROS such as hydroxyl radical (•OH), superoxide radical (O2•) and nonradicals hydrogen peroxide (H2O2).43 The effects of ROS were studied to confirm that enhanced of antibiotic resistance mentioned above was achieved by the mutagenic activity of the selected DBPs. Thiourea (TU), a hydroxyl radical scavenger protecting organisms from hydroxyl radical damage,44 was added to the cultures when the strain was exposed to DBPs. Compared with the control group, mutation frequencies of all tests were reduced significantly upon addition of TU (Independent Sample t test, p ≤ 0.05), except 80 ppm DCAN (Figure 4). For instance, the mutation frequencies rose from 1.63 × 10−7 to

Figure 1. Fold change of mutation frequency for ten antibiotics by exposure to DBAA (A), DCAN (B), KBrO3 (C), and MX (D), relative to untreated control (zero concentration). To better present the data, the results of DCAN, KBrO3, and MX are exhibited on the basis of the value of vertical axis, while DBAA results are exhibited because of DBAA concentrations (absence of Gen). The experiments were taken in triplicate, and error bars represent ± standard error of the mean (SEM).

strain, and various resistance mechanisms for different antibiotics. After exposure to DBPs for 24 h, the total bacterial counts were at the same levels with the controls (SI Tables S2 and S3), which indicated DBPs had no cytotoxicity in the tested concentrations. In addition, M-DBPs could increase mutation frequency of antibiotic resistance with obviously dose− response relationships, suggesting that this increase was not due to selection by killing other bacteria. Moreover, the four DBPs are halo-generated compounds except KBrO3, which are typical refractory organic compounds. Until now, there have been few reports on utilization of halo-generated DBPs by P.aeruginosa. Therefore, these DBPs should have little effect on selecting spontaneous mutants but induce antibiotic resistance due to mutagenesis. 8191

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Table 3. Summary of Effects of Four Selected DBPs on Ten Antibiotic Resistances maximum fold change of mutation frequencyb, corresponding concentration of DBPs/ppm antibiotics Car Cef Chl Cip Cla Gen Nor Pol Rif Tet ave.c

DBAA 1.59 ± 1.83 ± 1.35 ± 1.72 ± 2.71 ± N.M.a 2.85 ± 1.72 ± 2.05 ± 1.75 ± 1.95

DCAN

0.27, 0.23, 0.15, 0.26, 0.74,

400 1600 400 1600 400

0.27, 0.63, 0.35, 0.50,

800 200 400 600

1.40 1.74 1.28 1.47 2.85 3.65 1.69 4.26 3.14 5.19 2.67

± ± ± ± ± ± ± ± ± ±

0.29, 0.23, 0.28, 0.16, 0.27, 0.45, 0.34, 1.78, 0.38, 1.53,

KBrO3 240 240 240 160 240 240 240 240 240 240

2.01 1.82 1.74 2.37 3.27 5.26 1.61 8.46 2.47 2.54 3.16

± ± ± ± ± ± ± ± ± ±

0.95, 0.80, 0.17, 0.86, 0.48, 0.36, 0.36, 5.98, 1.30, 1.46,

MX 2672 2672 4008 4008 4008 4008 4008 2672 2672 2672

5.65 3.44 6.10 4.53 2.44 3.85 10.99 12.07 3.49 6.93 5.95

± ± ± ± ± ± ± ± ± ±

3.04, 1.63, 3.31, 0.67, 0.75, 0.49, 6.78, 8.26, 0.99, 2.39,

10 10 10 20 10 20 10 20 10 20

N.M.: Not measured. bThe maximum fold change of mutation frequency data represent mean ± SEM. cThe average of maximum fold change of mutation frequency of 10 antibiotics treated by each DBP.

a

Figure 2. Maximum fold change of mutation frequency for multidrug resistance by exposure to DBAA, relative to untreated control. The corresponding DBAA concentrations were 200, 400, 800, 800, and 200 ppm, respectively. The error bars represent ± SEM.

Figure 4. Effects of TU on Cip resistant mutation frequency following exposure to MX and DCAN. The inset at the top left corner represents the effects of TU on culture concentrations after treatment with MX and DCAN. The error bars represent ± SEM. Figure 3. Hereditary stability of the mutants was characterized by the variation of inhibitory zone diameters of four parallel isolates. The error bars represent ± SEM.

significant difference from zero concentration of DBPs (P > 0.05), suggesting that the effects of DBPs on antibiotic resistance were eliminated by TU completely. Moreover, TU had no physiological toxicity on PAO1 (see inset of Figure 4), suggesting that decline in mutation frequency was caused by ROS elimination only. It is thus

5.59 × 10−7 as MX concentrations increased without TU; while all of the mutation frequencies were declined to be below 1.06 × 10−7 in the presence of TU. For both MX and DCAN tests, the mutation frequencies of the experimental group had no 8192

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Table 4. Nucleotide and Amino Acid Mutations by Treated with MX strains

gene

nucleotide sequencing data

r

Cip isolates

gyrB parC

Rifr isolates

rpoB

Tetr isolates

mexR

TCC→TAC TAG→AAG TCC→GTCC GAC→AAC GAC→GGC GAC→GAG TCC→TTC GCA→CCA GCA→GTA GAG→TAG ACC→CCC GAG→GATG CGC→AGC CGG→CAG GGC→AGC GCC→GC_ CTG→GTGe CTG→_AG CTG→GTGe CTG→_AG CTG→_TG

Cipr isolates

r

nfxB

Tet isolates

mexZ

Genr isolates

mexZ

amino acid substitutions

misc. mutations

Ser464→Tyr Ile 93→Phe Insb Asp516→Asn Asp516→Gly Asp516→Glu Ser 574→Phe Ala66→Pro Ala66→Val Glu74→termination codon Thr130→Pro Insc Arg42→Ser Arg163→Gln Gly180→Ser Deld Leu70→Val Del&Subf Leu70→Val Del&Subf Delg

no. of mutated isolatesa

ref. for the same mutation

1 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 4

47−49

50, 51 50, 51 51 52 53

54 54

a The number of isolates harbored mutations among the 6 selected ones. bInsertion 1 bp at nt 328 bp, leading to frameshift. cInsertion 1 bp at nt 80 bp, leading to frameshift. dDeletion nt 495−564 bp of the nf xB gene, frame restored at P.aeruginosa PAO1 chromosome, complete genome (NC_002516.2) position 5156144 after the nf xB gene. eConversion of base C208 → G of the PA2021 gene. fDeletion 1 bp at nt 208 of the PA2021 gene, leading to frameshift. And a base substitution T209 → A. gDeletion 1 bp at nt 208 of the PA2021 gene, leading to frameshift.

concluded that the raise of antibiotic resistance was related with ROS formation, i.e., mutagenic activity of the M-DBPs. Sequencing of the Mutant Genes. The genes related to Cip, Gen, Tet, and Rif resistance in the isolates treated by MX were sequenced and compared with the wild strain to provide direct evidence for mutagenesis. These genes included gyrA, gyrB, parC, and parE in the “quinolone resistance-determining region” (QRDR); rpoB and rpoBNC encoding for the β subunit of RNA polymerase, the known target of rifampicin; mexR, nfxB, and mexZ, the multidrug efflux pump repressor genes, whose mutations would lead to overproduction of MexABOprM, MexCD-OprJ, and MexXY-OprM, respectively. Those multidrug efflux pumps are associated with fluoroquinolones, aminoglycosides, and tetracyclines resistance in P.aeruginosa.45,46 The detected mutations, their specific substitution or shiftframe mutation sites, the corresponding amino acid changes and others are listed in Table 4 (No mutations were found in gyrA, parE, and rpoBNC genes, data not shown). For example, one Cip resistance isolate contained point mutation in gyrB, which resulted in a substitution of Tyr-464 for Ser. Identical genetic events have been previously observed in fluoroquinolone resistant mutants of P.aeruginosa and Salmonella typhimurium.47−49 Since the β-subunit domain, the target of fluoroquinolone, was modified, the affinity of Cip would decrease, leading to partial or complete loss of the susceptibility of the subunit. The results showed that most of the Cip, Gen, Rif, and Tet resistance strains harbored mutations in the determined genes, indicating that the rise of BAR resulted from the formation of mutations due to DBPs treatment, mutations were also observed in the multidrug efflux pump repressor genes, which could cause overexpression of efflux pump and emergence of multidrug resistance. The results reaffirmed that broad-

spectrum antibiotic resistance and multidrug resistance were increased by the mutagenesis of the selected DBPs.

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ENVIRONMENTAL IMPLICATIONS

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ASSOCIATED CONTENT

This study demonstrated that exposure of opportunistic pathogen P.aeruginosa PAO1 to mutagenic DBPs could cause the increase of individual and multiple antibiotic resistances, due to their mutagenic activities. Generally, the stronger the mutagenic activity was, the higher the antibiotic resistance was enhanced. Furthermore, this acquired mutation had hereditary stability. This work revealed a possible source for the occurrence of bacterial antibiotic resistance in drinking water without antibiotics or with antibiotics at extremely low levels. So far, over 1000 DBPs species have been identified.55 At present, the total concentration of DBPs can already reach to sub-μg/L or low- to mid-μg/L, in addition more and more DBPs have been proved mutagenic.19 Therefore, the health risk brought by their effects on BAR should not be underestimated, especially when the resistance could be vertically transferred stably. This is an even more severe problem for some economic booming regions with pervasive source water pollution, where the disinfectant is often overused to guarantee the drinking water quality with much more DBPs produced, including the M-DBPs.

S Supporting Information *

The information on the detailed antibiotic concentrations of the antibiotic plates (Table S1), the raw colony count data (Table S2 and Table S3), and primers used in the study (Table S4). This material is available free of charge via the Internet at http://pubs.acs.org. 8193

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86-592-6190780; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the financial support from the National High-Tech R&D(863) Program of China (2012AA062607), National Natural Science Foundation of China (51278482 and 51078343), and the Science and Technology Project of Xiamen (3502Z20132013).

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on June 26, 2014. Due to production error, it was published with an incorrect entry in Table 4 and an incomplete reference (48). The corrected version was reposted on July 1, 2014.

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