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Subinhibitory concentrations of ciprofloxacin induce SOS response and mutations of antibiotic resistance in bacteria

Subinhibitory concentrations of ciprofloxacin induce SOS response and mutations of antibiotic... Ann Microbiol (2010) 60:511–517 DOI 10.1007/s13213-010-0080-x ORIGINAL ARTICLE Subinhibitory concentrations of ciprofloxacin induce SOS response and mutations of antibiotic resistance in bacteria Peng Wang & Xiaoni Zhang & Li wang & Zhang zhen & Mingli Tang & Jiabin Li Received: 20 March 2010 /Accepted: 19 May 2010 /Published online: 16 June 2010 Springer-Verlag and the University of Milan 2010 . . Abstract In this work, Salmonella typhimurium strains Keywords Antibiotic resistance SOS response . . (Sal94) containing plasmid-borne fusions of Vibrio fischeri Ciprofloxacin Sub-inhibitory concentration Mutation lux to the recA promoter was used to test the SOS response induced by sub-minimum inhibitory concentration (sub- Abbreviations MIC) of ciprofloxacin. The SOS response of Sal94 strain MIC Minimum inhibitory concentration was rapidly increased during 20 min when supplemented Sub-MIC Subminimum inhibitory concentration with sub-MIC ciprofloxacin, but came down thereafter. The induction level of 1/2 MIC was higher than that of 1/4 MIC, showing some dose-dependency. Both mutation frequencies (MF) of anti-ciprofloxacin resistance and transformation Introduction frequency of pMD18-T Vector (lacZ ori Amp)caused by the treatment of 1/4 and 1/2 MIC ciprofloxacin in Escher- The application of penicillin as a therapeutic agent in ichia coli AB1157 strain (wild-type recA, SOS inducible) 1942 ushered in the era of antimicrobial chemotherapy were markedly increased, but there were no or only slight and marks a historic milestone in medicine. Many changes in Escherichia coli IC400 strain (recA mutant, additional classes of antibiotics spanning a broad range deficient in SOS response). These results, combining the of chemical structures and targets soon followed, forming positive relationship between the induction factor and the the foundation of the current armory of antibiotics MF of anti-ciprofloxacin induced by sub-MIC ciprofloxacin (Lipsitch and Samore 2002; Peter and Romesberg 2007; in AB1157 strain, indicated that SOS response played an Rice 2006). But in recent years, the use of antibiotics is important role in the acquirement of antibiotic resistance continually being challenged by the emergence of resistant resulting from sub-MIC ciprofloxacin treatment. strains of bacteria, resulting in an worldwide medical, social and economic problem (Diekema et al. 2004; Smolinski et al. 2003), especially in the developing countries where the inappropriate or over use of anti- : : P. Wang L. wang M. Tang biotics is comparatively prevalence. Key Laboratory of Ion Beam Bioengineering, Antibiotic resistance has been mainly considered a Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China consequence of errors (spontaneous and induced muta- tions) that accumulate during replication of the bacterial : : X. Zhang Z. zhen J. Li genome. Chemists at the Scripps Research Institute and Department of Infection Disease, the University of Wisconsin have uncovered evidence The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China that spontaneous mutations are not the only way in which bacteria acquire resistance to antibiotics (Cirz et M. Tang (*) J. Li (*) al. 2005;Cirzand Romesberg 2006). The rapid rate at P. O. Box 1126, Hefei, Anhui 230031, People’s Republic of China which bacteria develop antibiotic resistance is due in large e-mail: tangmingli100@hotmail.com e-mail: lijiabin948@vip.sohu.com part to mutations arising during stress-induced DNA repair 512 Ann Microbiol (2010) 60:511–517 and during the lateral transfer of genes between organisms Materials and methods (Foster 2005; Hersh et al. 2004; Matic et al. 2004). Previous studies showed that the SOS response resulting Bacterial strain and antimicrobial agents from antibiotic therapy played an important role in the acquirement of antibiotic resistance in bacteria (Beaber et All strains used in this work are listed in Table 1. al. 2004; Cirz et al. 2005;Cirzand Romesberg 2006; Salmonella typhimurium strains (Sal94) containing Hastings et al. 2004). plasmid-borne fusions of Vibrio fischeri lux to the recA Customarily, antibiotic therapy is based on achieving promoter was used to test the SOS response induced by and exceeding a minimum inhibitory concentration sub-minimum inhibitory concentration (sub-MIC) of cipro- (MIC) for a sufficient amount of time in infected tissues. floxacin. AB1157 and IC400 are Escherichia coli K12 Butthere aresomeconditionsthatbacteriafaceinsub- strains, and they are different in the recA gene (wild-type in MICs of the antibiotic: inappropriate antibiotic use, AB1157 and deficient in IC400). therapeutic option of sub-MIC of the antibiotic, in wild The antimicrobial agents of ampicillin (Amp), kana- environments, the antibiotics use in farm animals, etc. mycia (Kan), tetracycline (TC), ciprofloxacin (Cip) When an antimicrobial is used inappropriately—for too were obtained as standard reference powders of known short a time, at too low a dose, at inadequate potency— potency for laboratory use (National Institute for the or for the wrong disease, microbes are more likely to Control of the Pharmaceutical and Biological Products, develop resistance to that drug. Therefore, choosing and China) using the right drug at the right dose is an important way to combat drug resistance. The use of antibiotics for Bacteria growth growth promotion has arisen with the intensification of livestock farming, but evidence strongly suggests that it Bacteria were cultured overnight in LB medium results in the development of drug-resistant microbes in (Maniatis et al. 1982), in a shaking incubator with or those animals. Drug resistance in animals may lead to without 50 μg/ml ampicillin and 30 μg/ml kanamycin, drug resistance in humans, because the drug-resistant according to their needs for maintaining the plasmid. bacteria can be transmitted from animals to humans Cells were diluted 100-fold with fresh LB medium and (Teuber 1999, 2001; Witte 1998). Sub-MIC antibiotic grown to mid-log with shaking to a density of about 1× 8 8 therapies can also lead to treatment failure and antibiotic 10 to 2×10 bacteria per ml, the samples were used for resistance (Roe and Pillai 2003). Furthermore, bacteria in the induction of the SOS response and the mutations of wild niches frequently meet sub-MICs of various anti- antibiotic resistance by sub-MIC of ciprofloxacin. Plate biotics. Therefore, careful evaluation of sub-MIC effects cultures were grown on LB agar with or without on bacterial physiology is needed prior to therapeutic use antimicrobial according to their needs. Incubation temper- of sub-MICs and animal growth promotion. atures were 37°C throughout, except for strain Sal94, The mechanisms leading to the development and which was grown at 26°C. transmission of antibiotic resistance by sub-MICs of antibiotics are not fully understood. Recent researchers Susceptibility testing indicated that sub-MICs of various antibiotics changed the expression of many proteins in bacteria, especially Minimal inhibitory concentrations (MICs) of antimicro- proteins related to SOS response (Cirz et al. 2007; bial agents were determined by means of the Agar Nandurietal. 2006, 2008). But the relationship of Dilution Method according to a standard procedure antibiotic resistance mutations and SOS response induced described by the Clinical and Laboratory Standards by sub-MICs of antibiotics, and/or whether sub-MICs of Institute/NCCLS (2005). The antimicrobial agents were antibiotics exerting natural selection for resistance on incorporated into Mueller-Hinton Agar (Oxoid, UK) in these organisms is still not very clear. In this work, we serial twofold concentrations from 0.06 to 128 μg/ml. used Salmonella typhimurium strains (Sal94) containing Quality control strains were E. coli ATCC 25922 and E. plasmid-borne fusions of Vibrio fischeri lux to the recA coli ATCC 35218withevery batchofclinical strains to promoter (Mingli et al. 2009), directly examined the SOS ensure accurate and comparable performance of assays. induction factor induced by 1/2 and 1/4 MIC of cipro- The plates were incubated in ambient air at 35°C for 18 h. floxacin in bacteria, and compared the MF of ciproflox- The inoculating concentration of bacteria was approximate acin resistance at different induction times to evaluate the 1.5×10 CFU/ml, equivalent to a 0.5 McFarland standard. relationship of SOS response and mutations of resistance An inoculum of 10 CFU per spot was delivered with a by sub-MICs of antibiotics. multipoint inoculator (AQS, UK). Ann Microbiol (2010) 60:511–517 513 Table 1 Strains used in this work (all are Escherichia coli K-12 strain except Sal94) Strain/plasmid Genotype Source/reference AB1157 thr-1 araC14 leuB6(Am)Δ (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4 Our laboratory/(Bachmann 1972) (Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(Str ) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1 IC400 AB1157 recA430 E. Botello/(Grompone et al. 2003) C R R Sal94 S. typhimurium strain with pRecA::LuxCDABE tolC Cm Amp S. Belkin/(Davidov et al. 2000) Influence of the bacteria growth by sub-MIC of ciprofloxacin The mutant frequencies were obtained by the mean of the mutant colonies divided by the mean of the survival Overnight cultured cells were diluted 100-fold with fresh colonies. LB medium, incubated with shaking with or without sub- MIC of ciprofloxacin. The cell density at 600 nm was Determination of transformation frequency examined sequentially with a Beckman DU640 spectro- photometer every 20 min. IC400 and AB1157 strain were used as a recipient for pMD18-T Vector (lacZ ori Amp ) (Takara, China). Cells of The SOS response induced by sub-MIC of ciprofloxacin IC400 and AB1157 were cultured to logarithmic phase in Mueller-Hinton medium. Then 1/2 and 1/4 MICs of Mid-log Sal94 cells were supplemented with sub-MIC ciprofloxacin were added to the mixture, respectively, and of ciprofloxacin, and cultured to the desired times. incubated for 1–2 h with surveillance. After the calcium The induced samples were diluted 100-fold with fresh chloride treatment, pMD18-T Vector was introduced into LB medium and continually cultured for 1.5 h for the the mixture, and incubation continued at 30°C for 30 min SOS expressing. and then at 37°C for a further 2 h for transformation. Cells Luminescence assay: 1 ml of expression cells of were plated on Mueller-Hinton agar supplemented with induction or control were directly transferred to a 1.5-ml ampicillin (100 μg /ml) overnight for screening the Eppendorf tube. The emitted luminescence (arbitrary transformer. The frequency of ampicillin-resistant trans- relative light units, RLUs) was monitored using a lumin- formants was determined by colonies on plate with and ometer (GLOMAX 20/20 Luminometer) and the cell without ampicillin (100 μg /ml). density was measured using a Beckman DU640 spectro- photometer at 600 nm. The SOS induction factor of control sample (no Results induction at 0 time) was defined as 1.0. The SOS induction factor of induced samples was calculated by dividing the Antimicrobial susceptibility mean luminescence (RLUs/OD ) of an induced sample displayed at the different times by that of the non-induced The MICs of bacteria strains in this work are shown in sample at 0 time. Table 2. AB1157 and IC400 are E.coli strains but different in recA gene (wild-type in AB1157 and deficient in IC400). Induction of antibiotic resistance by sub-MIC of ciprofloxacin The MIC of the IC400 strain is lower than that of the AB1157 strain. Overnight culture cells of AB1157 and IC400 were diluted 100-fold with fresh LB medium and grown to mid-log with Growth kinetics 8 8 shaking to a density of about 1×10 to 2×10 bacteria per ml, then supplemented with ciprofloxacin at 1/4 or 1/2 MIC The growth of strains Sal94, IC400, AB1157 affected by 1/4 of these strains, and incubated continually with shaking. and 1/2 MIC of ciprofloxacin are shown in Figs. 1, 2 and 3, Samples at desired times were diluted to appropriate concentration with sterile water, then 0.1 ml was spread Table 2 The ciprofloxacin sus- −1 Strain MIC ( μgml ) ceptibility of bacteria on the screen plate containing ciprofloxacin of 5-fold MIC and on the LB agar plate without ciprofloxacin for AB1157 0.125 overnight culture. The colonies on the screen plate were IC400 0.06 ciprofloxacin-resistant mutants, and the colonies on the LB Sal94 0.5 plate without antibiotic represented the total survival cells. 514 Ann Microbiol (2010) 60:511–517 Fig. 1 The growth of S. typhimurium strain Sal94 affected by Fig. 3 The growth of AB1157 strain affected by subinhibitory subinhibitory concentrations of ciprofloxacin. Sub-minimum inhibito- concentrations of ciprofloxacin. Sub-minimum inhibitory concentra- ry concentration doses used are based on MIC values in Table 2. tion doses used are based on MIC values in Table 2. Symbols: ■ Symbols: ■ Control, ● 1/4 MIC, ▲ 1/2 MIC Control, ● 1/4 MIC, ▲ 1/2 MIC respectively. When compared with the control, the growth of Induction factor induced by sub-MIC of ciprofloxacin all strains was influenced by 1/4 and 1/2 MICs of ciprofloxacin treatments over 320 min. The data also showed Bacteria Sal94 with plasmid pRecALux3 was induced by a dose-dependent effect (the influence of 1/2 MIC of sub-MIC of ciprofloxacin, and the luminescence and cell ciprofloxacin on the growth of all these strains was density (arbitrary relative light units, RLUs) at different obviously more than that of 1/4 MIC of ciprofloxacin). times were assayed. RLUs/OD and the SOS induction Meanwhile, when the three strains were compared, the factor was shown in Fig. 4. The data indicated that the SOS growth of strain IC400 with mutant recA gene was more response gene recA could be induced rapidly by sub-MIC impacted by sub-MIC of ciprofloxacin than that of strain of ciprofloxacin in 10 min, but slowly came down to a Sal94 and AB1157 with wild-type recA gene. definite level thereafter. The SOS response induced by 1/2 MIC of ciprofloxacin was relatively higher than that of 1/4 MIC of ciprofloxacin and displayed a dose-dependency. Antibiotic resistance induced by sub-MIC of ciprofloxacin Mutation frequencies (MF) of ciprofloxacin resistance in AB1157 strain induced by sub-MIC of ciprofloxacin were showninFig. 5. The MF induced by sub-MIC of ciprofloxacin was higher than that of spontaneous muta- tions, and its increase was time- and dose-dependent in this work (the MF increased with induced time, and 1/2 MIC of ciprofloxacin resulted in higher mutations than that of the 1/ 4 MIC of ciprofloxacin). Figure 6 compares the spontaneous mutation frequen- cy of anti-ciprofloxacin resistance with that induced by sub-MIC of ciprofloxacin in strains AB1157 and IC400. The anti-ciprofloxacin resistance mutations produced by 1/4 and 1/2 MICs of ciprofloxacin treatment were Fig. 2 The growth of IC400 strain affected by subinhibitory remarkably increased in the AB1157 strain (p<0.05), concentrations of ciprofloxacin. Sub-minimum inhibitory concentra- but in the IC400 strain these changes were not very tion doses used are based on MIC values in Table 2. Symbols: ■ Control, ● 1/4 MIC, ▲ 1/2 MIC distinct (p>0.05). Ann Microbiol (2010) 60:511–517 515 Fig. 4 The expression (right) and induction factor (left) of the recA in as mean values of the experiments. Sub-minimum inhibitory concen- S. typhimurium strain Sal94 induced by subinhibitory concentrations tration doses used are based on MIC values in Table 2. Symbols: ■1/2 of ciprofloxacin. RLUs/OD are expressed as the mean ± SD of at MIC, ● 1/4MIC least three repeated experiments, and induction factors are expressed Transformation frequency induced by sub-MIC Discussion ciprofloxacin Bacteria often encounter sub-MICs of antibiotics in wild The pMD18-T Vector with ampicillin resistance gene niches and therapeutic conditions, and many aspects of the was transformed into AB1157 and IC400 strains which bacteria response to these conditions have been investigated were induced by sub-MIC of ciprofloxacin. Treatment of in recent years (Davies et al. 2006; Fajardo and Martínez sub-MIC of ciprofloxacin significantly increased the 2008; Nanduri et al. 2006; Henderson-Begg et al. 2006). transformation frequency in the AB1157 strain, but The data of growth kinetics in this work showed that sub- barely changed the MF of transformation in the IC400 MIC of ciprofloxacin impaired the bacteria growth in all strain (Fig. 7). Fig. 6 Camparison of the mutation frequency of the AB1157 and IC400 strains induced by subinhibitory concentrations of ciprofloxacin. Mid-log Fig. 5 Mutation frequencies of ciprofloxacin resistance induced by cells of AB1157 and IC400 were incubated with shaking without (C) or subinhibitory concentrations of ciprofloxacin in AB1157. Mid-log with 1/4 and 1/2 MICs of ciprofloxacin for 2 h, anti-ciprofloxacin cells of AB1157 were induced by 1/4 and 1/2 MIC of ciprofloxacin, mutants were selected on 5-fold MIC ciprofloxacin plates. Data were at anti-ciprofloxacin mutants at desired times were selected on 5-fold least three independent experiments. Sub-minimum inhibitory concen- MIC of ciprofloxacin plates. Data were at least three independent tration doses used are based on MIC values in Table 2. Symbols: insert experiments. Sub-minimum inhibitory concentration doses used are IC400, in the two square AB1157. *Statistically significant based on MIC values in Table 2. Symbols: ■ 1/2 MIC, ● 1/4 MIC difference from those of spontaneous mutations (p<0.05) 516 Ann Microbiol (2010) 60:511–517 But this induction was relatively weak because of a lower concentration, and some of the subsequent damage might be rehabilitated by the bacteria repair system stimulated by the response. The mutations of ciprofloxacin resistance of the AB1157 strain in this work were dose-dependent (Fig. 5); the MF of 1/2 MIC induced was comparatively higher than that of 1/4 MIC. Previous studies have shown antibiotics with different concentrations have different effects on bacteria: high concentrations of antibiotics (equal or above inhibitory concentrations) mainly deal with cell growth inhibition (or death), while low concentrations may act as signaling molecules modulating expression of specific genes (Fajardo and Martínez 2008). The data of this work reflected that the different intensity or a different kind of effect also existed Fig. 7 Camparison of the transformation frequency of the AB1157 and IC400 strains induced by subinhibitory concentrations of cipro- below inhibitory concentrations. Combining the data of the floxacin. Data were at least three independent experiments. C Control induction factor resulting from the 1/2 and 1/4 MICs of groups, and sub-minimum inhibitory concentration doses are based on ciprofloxacin (Fig. 4), a positive relationship between the MIC values in Table 2. Symbols: IC400, AB1157. SOS response and the mutations of anti-ciprofloxacin *Statistically significant difference from those of spontaneous muta- tions (p <0.05) resistance was found with co-culture of sub-MIC of ciprofloxacin in the AB1157 strain. Further evidence for this positive relationship was obtained by comparing the strains AB1157, IC400, and Sal94 compared to the control, MF of anti-ciprofloxacin resistance induced by sub-MIC of and the influence level was positively correlated with the ciprofloxacin in the recA mutant strain IC400 (deficient concentration (Figs. 1, 2 and 3). These were consistent with strain in SOS response) with the recA wild strain AB1157 the results obtained in bacteria by sub-MICs of antibiotics (SOS inducible strain) (Fig. 6); the MF of sub-MIC (Lorian 1975; Reeks et al. 2005). The growth of strain ciprofloxacin induction was significantly higher than the IC400 with mutant recA gene was markedly reduced spontaneous mutations in the AB1157 strain (p<0.05), but compared with those of AB1157 and Sal94 strains with only a slight higher in the IC400 strain (p>0.05). The SOS wild-type recA gene by sub-MIC of ciprofloxacin. The response which stimulated the bacteria error prone repair possible reason is that the IC400 cells cannot repair the system has been considered the main reason for the damage induced by sub-MICs of antibiotics because of mutations of antibiotic resistance induced by antibiotics, the deficient recA gene, which results in more cell death especially the type of beta-lactams and quinolones (Lewin than those with the wild-type recA gene. et al. 1989; Miller et al. 2004). The evidence of this work For a growing number of bacteria, the SOS response has indicated that SOS response was one of the main reasons been recognized as a critical component of the response to for mutations of antibiotic resistance induced by sub-MIC environmental stress, in particular to antibiotics such as of ciprofloxacin. ciprofloxacin (Cirz et al. 2007; Miller et al. 2004; Power The mutations were time dependent with the supplement and Phillips 1992). Ciprofloxacin induces double-stranded of sub-MIC of ciprofloxacin in AB1157 (Fig. 5). It is DNA breaks and stalled replication forks, both of which are essential that the high concentrations of antibiotics have processed to single-stranded DNA. RecA forms filaments selective effects for mutations of antibiotic resistance, on the single-stranded DNA, and these nucleoprotein because only the mutants can grow in this condition. The filaments facilitate recombination repair as well as bind data of Fig. 3 showed that cells of AB1157 could be the SOS gene repressor LexA, stimulating its auto- slightly inhibited when sub-MIC of ciprofloxacin was proteolysis. This cleavage inactivates the LexA repressor supplemented. However, it was uncertain the increases of and results in the induction of the SOS genes. In this work, mutations were resulting from the different growth with and sub-MIC of ciprofloxacin markedly induced SOS response without sub-MIC of ciprofloxacin supplement. in Sal94 strain in 10 min (Fig. 4), but slowly came down Gene transfer is an important way for the acquirement of thereafter. The induction factor resulting from 1/2 MIC was antibiotic resistance (Beaber et al. 2004; Hastings et al. comparatively higher than that of the 1/4 MIC. These 2004; Lewin et al. 1989). Generally, antibiotic resistance results indicated that even sub-MIC of ciprofloxacin could genes exist in plasmid, transposon, integron, and other rapidly induce double-stranded DNA breaks and stall transposable elements. In this work, the impact of anti- replication forks, resulting in SOS response in bacteria. biotics on transformation frequency was investigated for the Ann Microbiol (2010) 60:511–517 517 AB1157 and IC400 strains. The transformation frequency Grompone G, Ehrlich SD, Michel B (2003) Replication restart in gyrB Escherichia coli mutants. Mol Microbiol 48:845–854 of pMD18-T Vector was significantly increased in the Hastings PJ, Rosenberg SM, Slack A (2004) Antibiotic-induced lateral presence of subinhibitory concentrations of ciprofloxacin in transfer of antibiotic resistance. Trends Microbiol 12:401–404 the AB1157 strain, but there was almost no change in the Henderson-Begg SK, Livermore DM, Lucinda MCH (2006) Effect of subinhibitory concentrations of antibiotics on mutation frequency IC400 strain (Fig. 7). These results were similar to the in Streptococcus pneumoniae. J Antimicro Chemother 57:849– mutations induced by sub-MIC of ciprofloxacin in these strains (Fig. 6), and suggested that the recA gene played an Hersh MN, Ponder RG, Hastings PJ, Rosenberg SM (2004) Adaptive important role for the transformation of plasmid in bacteria mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress. Res Microbiol 155:352–359 impacted by the stress of antibiotics. Lewin CS, Howard MA, Ratcliffe NT, Smith JT (1989) 4-Quinolones and the SOS response. J Med Microbiol 29:139–144 Acknowledgements We would like to thank Dr Emilia Botello Lipsitch M, Samore MH (2002) Antimicrobial use and antimicrobial (University of Extremadura department of Biochemistry and Molec- resistance: a population perspective. Emerg Infect Dis 8:347–354 ular Biology and Genetics. 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Science 279:996–997 Mutat Res 569:3–11 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Subinhibitory concentrations of ciprofloxacin induce SOS response and mutations of antibiotic resistance in bacteria

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Springer Journals
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Copyright © 2010 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-010-0080-x
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Abstract

Ann Microbiol (2010) 60:511–517 DOI 10.1007/s13213-010-0080-x ORIGINAL ARTICLE Subinhibitory concentrations of ciprofloxacin induce SOS response and mutations of antibiotic resistance in bacteria Peng Wang & Xiaoni Zhang & Li wang & Zhang zhen & Mingli Tang & Jiabin Li Received: 20 March 2010 /Accepted: 19 May 2010 /Published online: 16 June 2010 Springer-Verlag and the University of Milan 2010 . . Abstract In this work, Salmonella typhimurium strains Keywords Antibiotic resistance SOS response . . (Sal94) containing plasmid-borne fusions of Vibrio fischeri Ciprofloxacin Sub-inhibitory concentration Mutation lux to the recA promoter was used to test the SOS response induced by sub-minimum inhibitory concentration (sub- Abbreviations MIC) of ciprofloxacin. The SOS response of Sal94 strain MIC Minimum inhibitory concentration was rapidly increased during 20 min when supplemented Sub-MIC Subminimum inhibitory concentration with sub-MIC ciprofloxacin, but came down thereafter. The induction level of 1/2 MIC was higher than that of 1/4 MIC, showing some dose-dependency. Both mutation frequencies (MF) of anti-ciprofloxacin resistance and transformation Introduction frequency of pMD18-T Vector (lacZ ori Amp)caused by the treatment of 1/4 and 1/2 MIC ciprofloxacin in Escher- The application of penicillin as a therapeutic agent in ichia coli AB1157 strain (wild-type recA, SOS inducible) 1942 ushered in the era of antimicrobial chemotherapy were markedly increased, but there were no or only slight and marks a historic milestone in medicine. Many changes in Escherichia coli IC400 strain (recA mutant, additional classes of antibiotics spanning a broad range deficient in SOS response). These results, combining the of chemical structures and targets soon followed, forming positive relationship between the induction factor and the the foundation of the current armory of antibiotics MF of anti-ciprofloxacin induced by sub-MIC ciprofloxacin (Lipsitch and Samore 2002; Peter and Romesberg 2007; in AB1157 strain, indicated that SOS response played an Rice 2006). But in recent years, the use of antibiotics is important role in the acquirement of antibiotic resistance continually being challenged by the emergence of resistant resulting from sub-MIC ciprofloxacin treatment. strains of bacteria, resulting in an worldwide medical, social and economic problem (Diekema et al. 2004; Smolinski et al. 2003), especially in the developing countries where the inappropriate or over use of anti- : : P. Wang L. wang M. Tang biotics is comparatively prevalence. Key Laboratory of Ion Beam Bioengineering, Antibiotic resistance has been mainly considered a Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031, People’s Republic of China consequence of errors (spontaneous and induced muta- tions) that accumulate during replication of the bacterial : : X. Zhang Z. zhen J. Li genome. Chemists at the Scripps Research Institute and Department of Infection Disease, the University of Wisconsin have uncovered evidence The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China that spontaneous mutations are not the only way in which bacteria acquire resistance to antibiotics (Cirz et M. Tang (*) J. Li (*) al. 2005;Cirzand Romesberg 2006). The rapid rate at P. O. Box 1126, Hefei, Anhui 230031, People’s Republic of China which bacteria develop antibiotic resistance is due in large e-mail: tangmingli100@hotmail.com e-mail: lijiabin948@vip.sohu.com part to mutations arising during stress-induced DNA repair 512 Ann Microbiol (2010) 60:511–517 and during the lateral transfer of genes between organisms Materials and methods (Foster 2005; Hersh et al. 2004; Matic et al. 2004). Previous studies showed that the SOS response resulting Bacterial strain and antimicrobial agents from antibiotic therapy played an important role in the acquirement of antibiotic resistance in bacteria (Beaber et All strains used in this work are listed in Table 1. al. 2004; Cirz et al. 2005;Cirzand Romesberg 2006; Salmonella typhimurium strains (Sal94) containing Hastings et al. 2004). plasmid-borne fusions of Vibrio fischeri lux to the recA Customarily, antibiotic therapy is based on achieving promoter was used to test the SOS response induced by and exceeding a minimum inhibitory concentration sub-minimum inhibitory concentration (sub-MIC) of cipro- (MIC) for a sufficient amount of time in infected tissues. floxacin. AB1157 and IC400 are Escherichia coli K12 Butthere aresomeconditionsthatbacteriafaceinsub- strains, and they are different in the recA gene (wild-type in MICs of the antibiotic: inappropriate antibiotic use, AB1157 and deficient in IC400). therapeutic option of sub-MIC of the antibiotic, in wild The antimicrobial agents of ampicillin (Amp), kana- environments, the antibiotics use in farm animals, etc. mycia (Kan), tetracycline (TC), ciprofloxacin (Cip) When an antimicrobial is used inappropriately—for too were obtained as standard reference powders of known short a time, at too low a dose, at inadequate potency— potency for laboratory use (National Institute for the or for the wrong disease, microbes are more likely to Control of the Pharmaceutical and Biological Products, develop resistance to that drug. Therefore, choosing and China) using the right drug at the right dose is an important way to combat drug resistance. The use of antibiotics for Bacteria growth growth promotion has arisen with the intensification of livestock farming, but evidence strongly suggests that it Bacteria were cultured overnight in LB medium results in the development of drug-resistant microbes in (Maniatis et al. 1982), in a shaking incubator with or those animals. Drug resistance in animals may lead to without 50 μg/ml ampicillin and 30 μg/ml kanamycin, drug resistance in humans, because the drug-resistant according to their needs for maintaining the plasmid. bacteria can be transmitted from animals to humans Cells were diluted 100-fold with fresh LB medium and (Teuber 1999, 2001; Witte 1998). Sub-MIC antibiotic grown to mid-log with shaking to a density of about 1× 8 8 therapies can also lead to treatment failure and antibiotic 10 to 2×10 bacteria per ml, the samples were used for resistance (Roe and Pillai 2003). Furthermore, bacteria in the induction of the SOS response and the mutations of wild niches frequently meet sub-MICs of various anti- antibiotic resistance by sub-MIC of ciprofloxacin. Plate biotics. Therefore, careful evaluation of sub-MIC effects cultures were grown on LB agar with or without on bacterial physiology is needed prior to therapeutic use antimicrobial according to their needs. Incubation temper- of sub-MICs and animal growth promotion. atures were 37°C throughout, except for strain Sal94, The mechanisms leading to the development and which was grown at 26°C. transmission of antibiotic resistance by sub-MICs of antibiotics are not fully understood. Recent researchers Susceptibility testing indicated that sub-MICs of various antibiotics changed the expression of many proteins in bacteria, especially Minimal inhibitory concentrations (MICs) of antimicro- proteins related to SOS response (Cirz et al. 2007; bial agents were determined by means of the Agar Nandurietal. 2006, 2008). But the relationship of Dilution Method according to a standard procedure antibiotic resistance mutations and SOS response induced described by the Clinical and Laboratory Standards by sub-MICs of antibiotics, and/or whether sub-MICs of Institute/NCCLS (2005). The antimicrobial agents were antibiotics exerting natural selection for resistance on incorporated into Mueller-Hinton Agar (Oxoid, UK) in these organisms is still not very clear. In this work, we serial twofold concentrations from 0.06 to 128 μg/ml. used Salmonella typhimurium strains (Sal94) containing Quality control strains were E. coli ATCC 25922 and E. plasmid-borne fusions of Vibrio fischeri lux to the recA coli ATCC 35218withevery batchofclinical strains to promoter (Mingli et al. 2009), directly examined the SOS ensure accurate and comparable performance of assays. induction factor induced by 1/2 and 1/4 MIC of cipro- The plates were incubated in ambient air at 35°C for 18 h. floxacin in bacteria, and compared the MF of ciproflox- The inoculating concentration of bacteria was approximate acin resistance at different induction times to evaluate the 1.5×10 CFU/ml, equivalent to a 0.5 McFarland standard. relationship of SOS response and mutations of resistance An inoculum of 10 CFU per spot was delivered with a by sub-MICs of antibiotics. multipoint inoculator (AQS, UK). Ann Microbiol (2010) 60:511–517 513 Table 1 Strains used in this work (all are Escherichia coli K-12 strain except Sal94) Strain/plasmid Genotype Source/reference AB1157 thr-1 araC14 leuB6(Am)Δ (gpt-proA)62 lacY1 tsx-33 supE44(AS) galK2(Oc) hisG4 Our laboratory/(Bachmann 1972) (Oc) rfbD1 mgl-51 rpoS396(Am) rpsL31(Str ) kdgK51 xylA5 mtl-1 argE3(Oc) thi-1 IC400 AB1157 recA430 E. Botello/(Grompone et al. 2003) C R R Sal94 S. typhimurium strain with pRecA::LuxCDABE tolC Cm Amp S. Belkin/(Davidov et al. 2000) Influence of the bacteria growth by sub-MIC of ciprofloxacin The mutant frequencies were obtained by the mean of the mutant colonies divided by the mean of the survival Overnight cultured cells were diluted 100-fold with fresh colonies. LB medium, incubated with shaking with or without sub- MIC of ciprofloxacin. The cell density at 600 nm was Determination of transformation frequency examined sequentially with a Beckman DU640 spectro- photometer every 20 min. IC400 and AB1157 strain were used as a recipient for pMD18-T Vector (lacZ ori Amp ) (Takara, China). Cells of The SOS response induced by sub-MIC of ciprofloxacin IC400 and AB1157 were cultured to logarithmic phase in Mueller-Hinton medium. Then 1/2 and 1/4 MICs of Mid-log Sal94 cells were supplemented with sub-MIC ciprofloxacin were added to the mixture, respectively, and of ciprofloxacin, and cultured to the desired times. incubated for 1–2 h with surveillance. After the calcium The induced samples were diluted 100-fold with fresh chloride treatment, pMD18-T Vector was introduced into LB medium and continually cultured for 1.5 h for the the mixture, and incubation continued at 30°C for 30 min SOS expressing. and then at 37°C for a further 2 h for transformation. Cells Luminescence assay: 1 ml of expression cells of were plated on Mueller-Hinton agar supplemented with induction or control were directly transferred to a 1.5-ml ampicillin (100 μg /ml) overnight for screening the Eppendorf tube. The emitted luminescence (arbitrary transformer. The frequency of ampicillin-resistant trans- relative light units, RLUs) was monitored using a lumin- formants was determined by colonies on plate with and ometer (GLOMAX 20/20 Luminometer) and the cell without ampicillin (100 μg /ml). density was measured using a Beckman DU640 spectro- photometer at 600 nm. The SOS induction factor of control sample (no Results induction at 0 time) was defined as 1.0. The SOS induction factor of induced samples was calculated by dividing the Antimicrobial susceptibility mean luminescence (RLUs/OD ) of an induced sample displayed at the different times by that of the non-induced The MICs of bacteria strains in this work are shown in sample at 0 time. Table 2. AB1157 and IC400 are E.coli strains but different in recA gene (wild-type in AB1157 and deficient in IC400). Induction of antibiotic resistance by sub-MIC of ciprofloxacin The MIC of the IC400 strain is lower than that of the AB1157 strain. Overnight culture cells of AB1157 and IC400 were diluted 100-fold with fresh LB medium and grown to mid-log with Growth kinetics 8 8 shaking to a density of about 1×10 to 2×10 bacteria per ml, then supplemented with ciprofloxacin at 1/4 or 1/2 MIC The growth of strains Sal94, IC400, AB1157 affected by 1/4 of these strains, and incubated continually with shaking. and 1/2 MIC of ciprofloxacin are shown in Figs. 1, 2 and 3, Samples at desired times were diluted to appropriate concentration with sterile water, then 0.1 ml was spread Table 2 The ciprofloxacin sus- −1 Strain MIC ( μgml ) ceptibility of bacteria on the screen plate containing ciprofloxacin of 5-fold MIC and on the LB agar plate without ciprofloxacin for AB1157 0.125 overnight culture. The colonies on the screen plate were IC400 0.06 ciprofloxacin-resistant mutants, and the colonies on the LB Sal94 0.5 plate without antibiotic represented the total survival cells. 514 Ann Microbiol (2010) 60:511–517 Fig. 1 The growth of S. typhimurium strain Sal94 affected by Fig. 3 The growth of AB1157 strain affected by subinhibitory subinhibitory concentrations of ciprofloxacin. Sub-minimum inhibito- concentrations of ciprofloxacin. Sub-minimum inhibitory concentra- ry concentration doses used are based on MIC values in Table 2. tion doses used are based on MIC values in Table 2. Symbols: ■ Symbols: ■ Control, ● 1/4 MIC, ▲ 1/2 MIC Control, ● 1/4 MIC, ▲ 1/2 MIC respectively. When compared with the control, the growth of Induction factor induced by sub-MIC of ciprofloxacin all strains was influenced by 1/4 and 1/2 MICs of ciprofloxacin treatments over 320 min. The data also showed Bacteria Sal94 with plasmid pRecALux3 was induced by a dose-dependent effect (the influence of 1/2 MIC of sub-MIC of ciprofloxacin, and the luminescence and cell ciprofloxacin on the growth of all these strains was density (arbitrary relative light units, RLUs) at different obviously more than that of 1/4 MIC of ciprofloxacin). times were assayed. RLUs/OD and the SOS induction Meanwhile, when the three strains were compared, the factor was shown in Fig. 4. The data indicated that the SOS growth of strain IC400 with mutant recA gene was more response gene recA could be induced rapidly by sub-MIC impacted by sub-MIC of ciprofloxacin than that of strain of ciprofloxacin in 10 min, but slowly came down to a Sal94 and AB1157 with wild-type recA gene. definite level thereafter. The SOS response induced by 1/2 MIC of ciprofloxacin was relatively higher than that of 1/4 MIC of ciprofloxacin and displayed a dose-dependency. Antibiotic resistance induced by sub-MIC of ciprofloxacin Mutation frequencies (MF) of ciprofloxacin resistance in AB1157 strain induced by sub-MIC of ciprofloxacin were showninFig. 5. The MF induced by sub-MIC of ciprofloxacin was higher than that of spontaneous muta- tions, and its increase was time- and dose-dependent in this work (the MF increased with induced time, and 1/2 MIC of ciprofloxacin resulted in higher mutations than that of the 1/ 4 MIC of ciprofloxacin). Figure 6 compares the spontaneous mutation frequen- cy of anti-ciprofloxacin resistance with that induced by sub-MIC of ciprofloxacin in strains AB1157 and IC400. The anti-ciprofloxacin resistance mutations produced by 1/4 and 1/2 MICs of ciprofloxacin treatment were Fig. 2 The growth of IC400 strain affected by subinhibitory remarkably increased in the AB1157 strain (p<0.05), concentrations of ciprofloxacin. Sub-minimum inhibitory concentra- but in the IC400 strain these changes were not very tion doses used are based on MIC values in Table 2. Symbols: ■ Control, ● 1/4 MIC, ▲ 1/2 MIC distinct (p>0.05). Ann Microbiol (2010) 60:511–517 515 Fig. 4 The expression (right) and induction factor (left) of the recA in as mean values of the experiments. Sub-minimum inhibitory concen- S. typhimurium strain Sal94 induced by subinhibitory concentrations tration doses used are based on MIC values in Table 2. Symbols: ■1/2 of ciprofloxacin. RLUs/OD are expressed as the mean ± SD of at MIC, ● 1/4MIC least three repeated experiments, and induction factors are expressed Transformation frequency induced by sub-MIC Discussion ciprofloxacin Bacteria often encounter sub-MICs of antibiotics in wild The pMD18-T Vector with ampicillin resistance gene niches and therapeutic conditions, and many aspects of the was transformed into AB1157 and IC400 strains which bacteria response to these conditions have been investigated were induced by sub-MIC of ciprofloxacin. Treatment of in recent years (Davies et al. 2006; Fajardo and Martínez sub-MIC of ciprofloxacin significantly increased the 2008; Nanduri et al. 2006; Henderson-Begg et al. 2006). transformation frequency in the AB1157 strain, but The data of growth kinetics in this work showed that sub- barely changed the MF of transformation in the IC400 MIC of ciprofloxacin impaired the bacteria growth in all strain (Fig. 7). Fig. 6 Camparison of the mutation frequency of the AB1157 and IC400 strains induced by subinhibitory concentrations of ciprofloxacin. Mid-log Fig. 5 Mutation frequencies of ciprofloxacin resistance induced by cells of AB1157 and IC400 were incubated with shaking without (C) or subinhibitory concentrations of ciprofloxacin in AB1157. Mid-log with 1/4 and 1/2 MICs of ciprofloxacin for 2 h, anti-ciprofloxacin cells of AB1157 were induced by 1/4 and 1/2 MIC of ciprofloxacin, mutants were selected on 5-fold MIC ciprofloxacin plates. Data were at anti-ciprofloxacin mutants at desired times were selected on 5-fold least three independent experiments. Sub-minimum inhibitory concen- MIC of ciprofloxacin plates. Data were at least three independent tration doses used are based on MIC values in Table 2. Symbols: insert experiments. Sub-minimum inhibitory concentration doses used are IC400, in the two square AB1157. *Statistically significant based on MIC values in Table 2. Symbols: ■ 1/2 MIC, ● 1/4 MIC difference from those of spontaneous mutations (p<0.05) 516 Ann Microbiol (2010) 60:511–517 But this induction was relatively weak because of a lower concentration, and some of the subsequent damage might be rehabilitated by the bacteria repair system stimulated by the response. The mutations of ciprofloxacin resistance of the AB1157 strain in this work were dose-dependent (Fig. 5); the MF of 1/2 MIC induced was comparatively higher than that of 1/4 MIC. Previous studies have shown antibiotics with different concentrations have different effects on bacteria: high concentrations of antibiotics (equal or above inhibitory concentrations) mainly deal with cell growth inhibition (or death), while low concentrations may act as signaling molecules modulating expression of specific genes (Fajardo and Martínez 2008). The data of this work reflected that the different intensity or a different kind of effect also existed Fig. 7 Camparison of the transformation frequency of the AB1157 and IC400 strains induced by subinhibitory concentrations of cipro- below inhibitory concentrations. Combining the data of the floxacin. Data were at least three independent experiments. C Control induction factor resulting from the 1/2 and 1/4 MICs of groups, and sub-minimum inhibitory concentration doses are based on ciprofloxacin (Fig. 4), a positive relationship between the MIC values in Table 2. Symbols: IC400, AB1157. SOS response and the mutations of anti-ciprofloxacin *Statistically significant difference from those of spontaneous muta- tions (p <0.05) resistance was found with co-culture of sub-MIC of ciprofloxacin in the AB1157 strain. Further evidence for this positive relationship was obtained by comparing the strains AB1157, IC400, and Sal94 compared to the control, MF of anti-ciprofloxacin resistance induced by sub-MIC of and the influence level was positively correlated with the ciprofloxacin in the recA mutant strain IC400 (deficient concentration (Figs. 1, 2 and 3). These were consistent with strain in SOS response) with the recA wild strain AB1157 the results obtained in bacteria by sub-MICs of antibiotics (SOS inducible strain) (Fig. 6); the MF of sub-MIC (Lorian 1975; Reeks et al. 2005). The growth of strain ciprofloxacin induction was significantly higher than the IC400 with mutant recA gene was markedly reduced spontaneous mutations in the AB1157 strain (p<0.05), but compared with those of AB1157 and Sal94 strains with only a slight higher in the IC400 strain (p>0.05). The SOS wild-type recA gene by sub-MIC of ciprofloxacin. The response which stimulated the bacteria error prone repair possible reason is that the IC400 cells cannot repair the system has been considered the main reason for the damage induced by sub-MICs of antibiotics because of mutations of antibiotic resistance induced by antibiotics, the deficient recA gene, which results in more cell death especially the type of beta-lactams and quinolones (Lewin than those with the wild-type recA gene. et al. 1989; Miller et al. 2004). The evidence of this work For a growing number of bacteria, the SOS response has indicated that SOS response was one of the main reasons been recognized as a critical component of the response to for mutations of antibiotic resistance induced by sub-MIC environmental stress, in particular to antibiotics such as of ciprofloxacin. ciprofloxacin (Cirz et al. 2007; Miller et al. 2004; Power The mutations were time dependent with the supplement and Phillips 1992). Ciprofloxacin induces double-stranded of sub-MIC of ciprofloxacin in AB1157 (Fig. 5). It is DNA breaks and stalled replication forks, both of which are essential that the high concentrations of antibiotics have processed to single-stranded DNA. RecA forms filaments selective effects for mutations of antibiotic resistance, on the single-stranded DNA, and these nucleoprotein because only the mutants can grow in this condition. The filaments facilitate recombination repair as well as bind data of Fig. 3 showed that cells of AB1157 could be the SOS gene repressor LexA, stimulating its auto- slightly inhibited when sub-MIC of ciprofloxacin was proteolysis. This cleavage inactivates the LexA repressor supplemented. However, it was uncertain the increases of and results in the induction of the SOS genes. In this work, mutations were resulting from the different growth with and sub-MIC of ciprofloxacin markedly induced SOS response without sub-MIC of ciprofloxacin supplement. in Sal94 strain in 10 min (Fig. 4), but slowly came down Gene transfer is an important way for the acquirement of thereafter. The induction factor resulting from 1/2 MIC was antibiotic resistance (Beaber et al. 2004; Hastings et al. comparatively higher than that of the 1/4 MIC. These 2004; Lewin et al. 1989). Generally, antibiotic resistance results indicated that even sub-MIC of ciprofloxacin could genes exist in plasmid, transposon, integron, and other rapidly induce double-stranded DNA breaks and stall transposable elements. In this work, the impact of anti- replication forks, resulting in SOS response in bacteria. biotics on transformation frequency was investigated for the Ann Microbiol (2010) 60:511–517 517 AB1157 and IC400 strains. The transformation frequency Grompone G, Ehrlich SD, Michel B (2003) Replication restart in gyrB Escherichia coli mutants. Mol Microbiol 48:845–854 of pMD18-T Vector was significantly increased in the Hastings PJ, Rosenberg SM, Slack A (2004) Antibiotic-induced lateral presence of subinhibitory concentrations of ciprofloxacin in transfer of antibiotic resistance. Trends Microbiol 12:401–404 the AB1157 strain, but there was almost no change in the Henderson-Begg SK, Livermore DM, Lucinda MCH (2006) Effect of subinhibitory concentrations of antibiotics on mutation frequency IC400 strain (Fig. 7). These results were similar to the in Streptococcus pneumoniae. J Antimicro Chemother 57:849– mutations induced by sub-MIC of ciprofloxacin in these strains (Fig. 6), and suggested that the recA gene played an Hersh MN, Ponder RG, Hastings PJ, Rosenberg SM (2004) Adaptive important role for the transformation of plasmid in bacteria mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress. Res Microbiol 155:352–359 impacted by the stress of antibiotics. Lewin CS, Howard MA, Ratcliffe NT, Smith JT (1989) 4-Quinolones and the SOS response. J Med Microbiol 29:139–144 Acknowledgements We would like to thank Dr Emilia Botello Lipsitch M, Samore MH (2002) Antimicrobial use and antimicrobial (University of Extremadura department of Biochemistry and Molec- resistance: a population perspective. Emerg Infect Dis 8:347–354 ular Biology and Genetics. 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Annals of MicrobiologySpringer Journals

Published: Jun 16, 2010

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