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International Research Journal of Applied and Basic Sciences 2013 Available online at www.irjabs.com ISSN 2251-838X / Vol, 4 (10): 3063-3067 Science Explorer Publications Generation of clones with higher resistance to ciprofluoxacin resistant Escherichia coli mutants Razieh Pourahmad Jaktaji1 and Rayhaneh Ebadi2 1. Assistant professor in Genetics at Department of Genetics, Faculty of Science, University of Shahrekord, 2. M. Sc student in Genetics at University of Shahrekord, Shahrekord, Iran Corresponding Author email: [email protected] ABSTRACT: Resistance to ciprofloxacin is mostly associated with mutations in gyrA or marR or a combination of both in Escherichia coli. Mutation in marR reduces the accumulation of ciprofloxacin due to activation of AcrAB-TolC, a multidrug efflux pump. The activation of this pump causes low resistance to other antibiotics, such as tetracycline and chloramphenicol and even organic solvents, such as hexane and cyclohexane. However, this low resistance can be amplified by stepwise exposure to increasing amounts of tetracycline in both clinical and laboratory isolates of Escherichia coli. The aim of this study was to produce clones with higher resistance to tetracycline and chloramphenicol from Escherichia coli gyrA mutants with or without mutation in marR useing above procedure. Resulted clones had intermediate levels of resistance to tetracycline and chloramphenicol and better growth on hexane in comparison with original mutants. However, they still could not grow on cyclohexane. It is concluded that to produce clones with high levels of resistance to antibiotics the above procedure should be continued with higher amounts of tetracycline. Key words: AcrAB-TolC pump; gyrA mutants; marR mutation; multiple antibiotic resistance; organic solvent tolerance Fluoroquinolones, such as ciprofloxacin (Cip) are a class of synthetic antibiotics which have greater activity against gram negative bacteria especially E. coli than have older quinolones like nalidixic acid (Lindgren et al., 2003; Pena et al., 1995). However, spontaneous Cip resistant mutants can be isolated by cultivation of Cip sensitive E. coli strains (both clinical and laboratory strains) on medium plus Cip (Cohen et al., 1989; Cirz et al., 2005). Most of these Cip resistant mutants has mutation in gyrA gene encoding the A subunit of DNA gyrase (Maneewannakul and Levy, 1996). Some of them are low multiple antibiotic resistant (Mar) mutants which are resistant to low levels of other antibiotics, including tetracycline (Tc) and chloramphenicol (Cm) (Cohen et al., 1989). Thus, they are not a clinical threat, but they can be used for the development of increased clinically relevant resistances against antibiotics and organic solvents as well (Lindgren et al., 2003; Asako et al., 1997). There are examples of multidrug resistance in clinical isolates of Enterobacteriaceae, such as E. coli (Mazzariol et al., 2000; Swick et al., 2011; Yasufuku et al., 2011). mar mutations locate mostly either in marR or acrR gene (Grkovic et al., 2002). marR is a member of marRAB operon. marR encodes the repressor of this operon called MarR (Maneewannakul and Levy, 1996). This repressor, like AcrR repressor possesses DNA binding and drug binding sites (Perera & Grove, 2010). The N-terminal site of this protein has DNA binding domains and the C-terminal site has dimerization and drug binding domains (Perera & Grove, 2010). On the other hands, marA, the other member of marRAB operon encodes not only the activator of this operon but also other genes involve in generation of low multiple antibiotic resistance phenotype, like acrAB operon and tolC gene which collectively encode AcrAB-TolC efflux pump (Mazzariol et al., 2000; Rhee et al., 1998). Mar phenotype was linked to alteration of outer membrane properties which causes decreased intracellular drug concentration. This is due to decrease in OmpF, a membrane porin protein and increase in AcrAB-TolC membrane pump activity (Martin et al., 2008). Both of these activities happen following the overexpression of marA (Martin et al., 2008; Viveiros et al., 2007). It was demonstrated that it is possible to induce high levels of Intl. Res. J. Appl. Basic. Sci. Vol., 4 (10), 3063-3067, 2013 resistance to Tc and Cm in susceptible E. coli k12 strain by a gradual step wise increase in amount of Tc in the medium (Viveiros et al., 2007; George and Levy, 1983). Tc is one of ligands that can bind to drug binding site of MarR and promote the dissociation of this repressor from operator site of marRAB operon and thereby leading to activation of this operon via MarA (Perera and Grove, 2010). Tc can also help to dissociate AcrR repressor (encoded by acrR) from operator site of acrAB operon and again to activate the operon via MarA (Su et al., 2007; Ma et al., 1996). Previous study described the isolation of Cip resistant mutants harboring a mutation in gyrA gene (Pourahmad and Mohiti, 2010). These gyrA mutants were with and without mutation in marR (Pourahmad et al., 2011). The aim of this study was to use gyrA mutants with and without a marR mutation to generate clones with higher resistance to antibiotics by using increasing amount of Tc. Antimicrobial agent and Chemicals Tetracycline hydrochloride (Tc) and chloramphenicol were obtained from Sigma and their stock solution were 4 mg/ml and 10 mg/ml, repectively. Organic solvents used for this study were n-hexane (Merck) and cyclohexane (Merck). Bacterial strain and mutants MG1655 was parent strain. gyrA mutants isolated in previous work (Pourahmad and Mohiti, 2010) are listed in Table 1. These mutants were used to generate clones with higher resistance to tetracycline and choloramphenicol. Media LB broth and agar (Merck) were used for cultivation of bacteria and antibiotic susceptibility test. LBGMg agar medium, containing 0.1% glucose, 10 mM MgSO4 and 1.5% agar other than LB was used for organic solvent tolerance assay (Asako et al., 1997). Generation of Clones For generation of clones with higher resistance to Tc and Cm from gyrA mutants with low resistance to these antibiotics (have MICs slightly higher than MG1655, see Table 1) the increasing amounts of Tc were used according to previously described method with modification (Viveiros et al., 2007; George and Levy, 1983). Briefly, gyrA mutants cultivated on LB agar containing 5 µg/ml Tc (higher than their Tc MICs) at 37º C for 24-48 h (three plates for each mutants). Then one colony from each plate (totally 9 colonies) was picked and purified and on same condition cultivated separately on LB agar containing 10 µg/ml Tc. Then the procedure was repeated for cultivation on LB agar containing 20 µg/ml Tc. Finally, one clone from each plate (totally 9 clones) was isolated and used for antibiotic susceptibility test. Antibiotic susceptibility test As described in previous study (Pourahmad and Mohiti, 2010), MICs of antibiotics for control strain, MG1655, and gyrA mutants were determined using broth dilution method. Different concentrations of Tc and Cm ranging from 1 µg/ml to 50 µg/ml, were used. MICs for control strain and clones were determined in three independent experiments. Organic solvent tolerance assay Serial dilutions were prepared from fresh cultures of control strain and clones in 0.9% NaCl and 5 µl of each dilution spotted on a solid LBGMg medium as described previously (Asako et al., 1997). The surface of the medium was overlaid with an organic solvents (pure hexane or a mixture of hexane-cyclohexane in a ratio of 3:1, 1:1 and 1:3) and incubated at 37° C for 24 h. Then the number of colonies per spot was counted on each plate. PCR amplification and DNA sequencing A single colony from each clone grown on LB agar was used as a template for PCR reaction as described previously (Pourahmad et al., 2011). Primers used for PCR amplification and DNA sequencing were, forward primer 5`-GGTGGTTGTTATCCTGTGTA-3` and reverse primer 5`-CGGCAGGACTTTCTTAAGC-3`. PCR products (700 bp in size) which contained a part of marO and the entire marR gene were sequenced. Intl. Res. J. Appl. Basic. Sci. Vol., 4 (10), 3063-3067, 2013 Clones with higher resistance to antibiotics Resistance to tetracycline and chloramphenicol can be divided to three levels, including low levels of resistance (MIC: 1 to 10 µg/ml), intermediate levels of resistance (MIC: 10 to 50 µg/ml) and high levels of resistance (MIC: >50 µg/ml (George and Levy, 1983). After isolation and purification of 9 clones from plate containing 20 µg/ml Tc, their MICs against Tc and Cm were assessed. This was conducted for wild type and original mutants as well. Table 1 shows MICs of wild type, mutants and clones. According to this table the maximum resistance for both antibiotics was 45 µg/ml. Tolerance of hexane and cyclohexane Purified clones were used for organic solvents tolerance assay. Table 2 indicates the results. Based on this results clones had better growth on pure hexane compared to their original gyrA mutants, but like their original mutants, still could not grow on any ratio of hexane-cyclohexane mixture. Presence of extra mutation in marR As clones were more resistance to Tc and Cm than original mutants, it was possible that they would gain extra mutation in marR gene. Fig. 1 shows the result of gel electrophoresis for control strain (MG1655) and some clones following PCR reaction. The results for other clones were the same as these results. However, DNA sequencing of marR gene in clones did not demonstrate extra mutation in this gene compared to sequence of marR in MG1655. Figure 1. Gel analysis of PCR product. First lane contains 1 kb DNA ladder and other lanes contain PCR products. Multidrug resistance (MDR) phenotype was found in all bacterial species, but has been studied greatly in members of Enterobacteriaceae, especially E. coli (Lindgren et al., 2003; Pena et al., 1995). MDR produces from unsuitable drug treatment with one antibiotic can cause cross-resistance to other structurally unrelated antibiotics (Lindgren et al., 2003; Pena et al., 1995). Multidrug resistance phenotype in E. coli is due to mar regulon (Mazzariol et al., 2000). In this regulatory system, MarA has a key role. MarA overactivity induces firstly, the synthesis of micF antisense RNA that downregulates the OmpF outer membrane porine synthesis and secondly, the over production of AcrAB-TolC drug efflux pump (Lindgren et al., 2003). Mutations located in marR leading to constitutive expression of the marRAB operon have been described in fluoroquinolone resistant isolates of E. coli (Cohen et al., 1989; Grkovic et al., 2002; Lindgren et al., 2003). In the previous study ciprofloxacin resistant mutants of E. coli having mutation in gyrA were isolated (Pourahmad and Mohiti, 2010). Among these mutants two had mutation in marR and one did not have any mutation in marR (Pourahmad et al., 2011). They also had low levels of resistance to tetracycline and choloramphenicol and low level of organic solvent tolerance (Pourahmad et al., 2011). It was described that long exposure to tetracycline produces clones with higher resistance to tetracycline and other antibiotics, such as chloramphenicol (Viveiros et al., 2007; George and Levy, 1983). The aim of this study was to produce clones with higher levels of resistance to tetracycline and chloramphenicol from gyrA mutants with low levels of resistance to these antibiotics. Intl. Res. J. Appl. Basic. Sci. Vol., 4 (10), 3063-3067, 2013 It was found that increasing amounts of tetracycline in stepwise manner up to 20 µg/ml can produce clones with intermediate levels of resistance to tetracycline and choloramphenicol. These clones can be used for quantification of mRNA of genes, such as marA, acrA involved in production of multiple antibiotic resistance phenotype. As these clones did not gain one or more mutation in marR, it may imply that acquiring intermediate levels of resistance to Tc and Cm is not solely dependent on possession of defect in MarR, but is dependent on exposure to increasing amounts of tetracycline. This is consistent with previous studies that showed the inducibility of marRAB operon (Martin and Rosner, 1995). Thus, for production of high levels of resistance this procedure should be continued with higher amounts of tetracycline (more than 20 µg/ml). Meanwhile this does not mean that after long exposure to increasing amounts of Tc, resulting clones would not acquire extra mutations in marR or other genes involved in production of Mar phenotype. Moreover, It was shown that high tolerance of organic solvents happens following over activity of AcrAB-TolC pump (White et al., 1997; Aono et al., 1998). The finding that resulted clones were still low organic solvent tolerant indirectly reveals that AcrAB-TolC did not over activated in these clones. Further study on quantification of acrA expression in these mutants revealed that this gene was not over expressed in these clones (submitted for publication). However, another study on quantification of marA expression shown that two clones had higher expression of marA in comparison to wild type strain, but probably the levels of over expression were not enough to overexpress acrAB operon (submitted for publication). This work was financially supported by the University of Shahrekord. We thank Prof. R. G. Lloyd for kind gift of MG1655. Cip, Tc and Cm are abbreviations for ciprofloxacin, tetracycline and chloramphenicol, respectively. Table 2. Organic solvent tolerance of control strain, gyrA mutants and their derivative clones Intl. Res. J. Appl. Basic. Sci. Vol., 4 (10), 3063-3067, 2013 Aono R, Tsukagoshi N, Yamamoto M. 1998. Involvement of outer membrane protein TolC a possible member of the mar-sox regulon in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12. J. Bacteriol. 180: 938-944. Asako H, Nakajima H, Kobayashi K, Kobayashi M, Aono R. 1997. Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli. Appl Environ Microbiol. 63: 1428-1433. Cirz RT, Chin JK, Andes DR, Crecy-Lagard V, Craig WA, Romesberg FE. 2005. Inhibition of mutation and combating the evolution of antibiotic resistance. PLOS Biology. 3: 1024-1033. Cohen SP, McMurry LM, Hooper DC, Wolfson JS, Levy SB. 1989. Cross resistance to fluoroquinolones in multiple antibiotic resistant (Mar) Escherichia coli selected by tetracycline or chloramphenicol: Decreased drug accumulation associated with membrane changes in addition to OmpF reduction. Antimicrobial Agents Chemother. 33: 1318-1325. George AM, Levy SB. 1983. Amplifiable resistance to tetracycline, chloramphenicol and other antibiotics in Escherichia coli: Involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol. 155: 531-540. Grkovic S, Brown MH, Skurray RA. 2002. Regulation of bacterial drug export systems. Microbiol and Mol Biol Rev. 66: 671-701. Lindgren PK, Karlsson A, Hughes D. 2003. Mutation rate and evolution of fluoroquinolone resistance in Escherichia coli isolates from patients with urinary tract infections. Antimicrobial Agents Chemother. 47: 3222-3232. Ma D, Alberti M, Lynch C, Nikaido H, Hearst J. 1996. The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol. 19: 101-112. Maneewannakul K, Levy SB. 1996. Identification of mar mutants among quinolone resistant clinical isolates of Escherichia coli. Antimicrobial Agents Chemother. 40: 1695-1698. Martin RG, Bartlett ES, Rosner JL, Wall ME. 2008. Activation of the E. coli marA/soxS/rob regulon in response to transcriptional activator Martin RG, Rosner JL. 1995. Binding of purified multiple antibiotic resistance repressor protein (MarR) to mar operator sequences. Proc Mazzariol A, Tokue Y, Kanegawa TM, Cornaglia G, Nikaido H. 2000. High level fluoroquinolone resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrobial Agents Chemother. 44: 3441-3443. Pena C, Albareda JM, Pallares R, Pujol M, Tubau F, Javier A. 1995. Relationship between quinolone use and emergence of ciprofloxacin- resistant Escherichia coli in bloodstream infections. Antimicrobial Agents Chemother. 39: 520-524. Perera IC, Grove A. 2010. Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators. Pourahmad Jaktaji R, Ebadi R, Karimi M. 2011. Study of organic solvent tolerance and increased antibiotic resistance properties in E. coli Pourahmad Jaktaji R, Mohiti E. 2010. Study of mutations in the DNA gyrase gyrA gene of Escherichia coli. IJPR. 9: 43-45. Rhee S, Martin RG, Rosner JL, Davies DR. 1998. A novel DNA binding motif in MarA: The first structure for an AraC family transcriptional activator. Proc Natl Acad Sci USA. 95: 10413-10418. Su CC, Rutherford DJ, Yu EW. 2007. Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem Biophys Res Swick MC, Morgan-Linnell SK, Carlson KM, Zechiedrich L. 2011. Expression of multidrug efflux pump genes acrAB-tolC, mdfA and norE in Escherichia coli clinical isolates as a function of fluoroquinolone and multidrug resistance. Antimicrobial Agents Chemother. 55: 921-924. Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pages JM, Amaral L. 2007. Antibiotic stress, genetic response and altered permeability of E. coli. PLoS. 4: e365. White DG, Goldman JD, Demple B, Levy SB. 1997. Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS or robA in Escherichia coli. J Bacteriol. 179: 6122-6126. Yasufuku T, Shigemura K, Shirakawa T, Matsumoto M, Nakano Y, Tanaka K, Arakawa S, Kinoshita S, Kawabata M, Fujisawa M. 2011. Correlation of overexpression of efflux pump genes with antibiotic resistance in Escherichia coli strains clinically isolated from urinary tract infection patients. J Clinic Microbiol. 49: 189-194.

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