Role In Fluoroquinolone Resistance Research Articles

Fluoroquinolone-specific resistance trajectories in E. coli and their dependence on the SOS-response

BackgroundFluoroquinolones are indispensable antibiotics used in treating bacterial infections in both human and veterinary medicine. However, resistance to these drugs presents a growing challenge. The SOS response, a DNA repair pathway activated by DNA damage, is known to influence resistance development, yet its role in fluoroquinolone resistance is not fully understood. This study aims to unfold the mechanisms of fluoroquinolone resistance by investigating the impact of the SOS response on bacterial adaptation.ResultsWe exposed Escherichia coli to four fluoroquinolones—ciprofloxacin, enrofloxacin, levofloxacin, and moxifloxacin. Using a recA knockout mutant, deficient in the SOS response, as a control, we assessed how the presence or absence of this pathway affects resistance development. Our findings demonstrated that the rate of resistance evolution varied between the different fluoroquinolones. Ciprofloxacin, enrofloxacin, and moxifloxacin exposures led to the most evident reliance on the SOS response for resistance, whereas levofloxacin exposed cultures showed less dependency. Whole genome analysis indicated distinct genetic changes associated with each fluoroquinolone, highlighting potential different pathways and mechanisms involved in resistance.ConclusionsThis study shows that the SOS response plays a crucial role in resistance development to certain fluoroquinolones, with varying dependencies per drug. The characteristic impact of fluoroquinolones on resistance mechanisms emphasizes the need to consider the unique properties of each antibiotic in resistance studies and treatment strategies. These findings are essential for improving antibiotic stewardship and developing more effective, tailored interventions to combat resistance.

The prevalence and functional characteristics of CrpP-like in Pseudomonas aeruginosa isolates from China.

Modifying enzyme-CrpP and its variants reduced the MICs of fluoroquinolones in Pseudomonas aeruginosa. This study investigated the dissemination and functional characteristics of CrpP-like in P. aeruginosa from China. The positive rate of crpP-like genes in 228 P. aeruginosa was 25.4% (58/228), and 6 new crpP-like genes were determined. Transformation experiments showed that CrpP-like had a low effect on CIP and LEV susceptibility. The genetic of crpP-positive was diverse. Furthermore, the mean expression level of crpP was no statistical difference between fluoroquinolone-susceptible and -resistant group (P > 0.05). CrpP-like may not play a significant role in fluoroquinolone resistance in P. aeruginosa.

Co-Incidence of Type II Topoisomerase Mutations and Efflux Expression in High Fluoroquinolone Resistant Enterococcus faecalis Isolated from Urinary Tract Infections.

IntroductionEnterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. This study aimed to elucidate the mechanisms involved in fluoroquinolone resistance in clinical E. faecalis isolates by analyzing mutations in quinolone- resistance-determining regions (QRDRs) of gyrA and parC and investigating the role of some efflux pumps.MethodsIn total, 70 clinical E. faecalis isolates collected from UTIs were identified by phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed and multidrug-resistant (including ciprofloxacin resistant) isolates were studied for minimum inhibitory concentrations to ciprofloxacin, levofloxacin, and ofloxacin. In the following, mutations in QRDRs of gyrA and parC and expression of EfrA, EfrB, and EmeA efflux pumps were investigated in 20 high-level ciprofloxacin resistant and two ciprofloxacin susceptible isolates.ResultsHigh-level resistance to ciprofloxacin was detected in 97.5% of isolates. Sequencing of QRDRs revealed that 65% and 75% of isolates carried mutations in gyrA and parC, respectively. The presence of efflux genes was detected in all studied isolates, but expression of efrA, emeA, and efrB was demonstrated in 50%, 40%, and 30% of resistant isolates, respectively. Neither QRDR mutation nor the expression of efflux genes showed any significant association with MIC.ConclusionCo-incidence of mutation and efflux gene expression in more than half of isolates (13/20) suggests that both mechanisms may play a role in fluoroquinolone resistance. The other unknown mechanisms including different efflux pumps and probably other QRDRs mutations may contribute to fluoroquinolone resistance in E. faecalis.

Efflux Pump Inhibitors, Alpha-Tocopherol and Aspirin: Role in Campylobacter jejuni and Campylobacter coli Fluoroquinolone Resistance.

This study aimed to investigate how efflux pump activity contributes to high fluoroquinolone (FQ) resistance in Campylobacter jejuni and Campylobacter coli isolates and to evaluate the modulatory effects of α-tocopherol and aspirin on FQ phenotypic resistance profiles. Minimum inhibitory concentration (MIC) values were obtained for different FQ agents following exposure to different efflux pump inhibitors (EPIs), including PaβN (50 μg/mL), which targets the cmeABC efflux system, and chlorpromazine (45 μg/mL) and verapamil (120 μg/mL), which target the MFS efflux system. The modulatory effects of aspirin (100 and 200 μg/mL) and α-tocopherol (4 and 10 μg/mL) on FQ resistance profiles were examined. PaβN had no effect on the MIC values of all FQ agents, while MFS efflux system inhibitors reduced the resistance level of different FQ agents and achieved an effect nearly comparable with that of α-tocopherol (10 μg/mL). Aspirin exerted a dose-dependent excitatory effect on phenotypic resistance profiles, and this may raise concerns about its usage in both veterinary and clinical settings. While an efflux system other than cmeABC may play a role in FQ resistance in Campylobacter species, lipophilic substances may represent a new approach for controlling efflux pump activities.

Transcriptional Response of AcrEF-TolC against Fluoroquinolone and Carbapenem in Escherichia coli of Clinical Origin

Introduction: Efflux pump systems constitute a major means of intrinsic resistance in Escherichia coli. AcrEF-TolC pump is known to exhibit higher expression level in quinolone resistant isolates. However, the transcriptional response of this pump is yet to be known when exposed to quinolone and other group of antibiotics. Objective: The present study analyses the transcriptional response of AcrEF-TolC in the presence of quinolones and carbapenems. Methodology: A total of 167 non-duplicate clinical isolates from Silchar medical college and Hospital, Silchar, India were included in this study. Of which 27 were devoid of any carbapenemase activity and among them 13 isolates showed overexpression of AcrE and AcrF gene. Transcriptional response of AcrE was directly proportional to increasing concentration of levofloxacin and ofloxacin. However, the response of AcrE and AcrF was inconsistent with carbapenems. Result: The study isolates showed susceptibility towards amikacin (68.4%), gentamicin (59.6%), cefepime (52.7%) and pipercillin/tazobactam (48.3%). The present investigation highlights that apart from qnr genes and mutational changes in gyr region, AcrEF-TolC plays a major role in fluoroquinolone resistance in this part of the world. Conclusion: Upregulation of AcrE in the presence of levofloxacin and ofloxacin warrants further investigation to establish their active role in efflux of this drug.

The Mutations of Topoisomerase Genes and Their Effect on Resistance to Fluoroquinolones in Extended-Spectrum β-Lactamase-Producing Escherichia coli

Background: Fluoroquinolones have been used for empirical treatment of urinary tract infections in recent years. This study aimed at identifying mutations in gyrA and parC genes and their correlation with fluoroquinolone minimal inhibitory concentration (MIC) among extended-spectrum β-lactamase-producing (ESBL) Escherichia coli. Methods: A total of 240 E. coli were isolated from urine of patients during 2014 and 2015 in the west of Iran. The isolates were screened for ESBL-producing phenotype using combined disc diffusion test. The susceptibility of ESBL isolates to ciprofloxacin and levofloxacin was determined by disk diffusion and microdilution assay. PCR, sequencing and bioinformatics analysis were performed to identify mutations in gyrA and parC genes. Results: Of 240 isolates, 66 (27.5%) were ESBL positive. Of them, 45 (68.1%) isolates were resistant to both ciprofloxacin and levofloxacin. Sequence analysis showed mutations in the quinolone resistance determining region (QRDR) in the 2 codons (Ser-83, Asp-87) of gyrA and 2 codons (Ser-80, Glu-84) of parC. One mutation at codon of Ala-192 was found outside the QRDR in parC. Conclusions: Isolates with mutations in QRDR of parC and gyrA had the higher MIC level compared to isolates with mutations only in gyrA. The highest level of resistance was detected in isolates with accumulation of mutations in gyrA and parC. A high frequency of fluoroquinolone resistance among ESBL producing E. coli isolates indicated the clustered transmission of resistance genes in this bacterium. A new mutation outside the QRDR of parC was detected, which may play a role in fluoroquinolone resistance.

Effect of Efflux Pump Inhibitor Carbonyl Cyanide 3-Chlorophenylhydrazone on the Minimum Inhibitory Concentration of Ciprofloxacin in Acinetobacter baumannii Clinical Isolates.

Background:Acinetobacter baumannii is an important human pathogen with increasing notoriety in the recent years, as a causative organism of drug resistant nosocomial infections, particularly in immunocompromised patients hospitalized in burn centers.Objectives:The aim of this study was to determinate the role of efflux pump(s) in ciprofloxacin resistance of A. baumannii strains isolated from burn patients.Materials and Methods:Sixty-five A. baumannii strains were isolated from the burn patients hospitalized in Motahari Burns and Reconstruction Center in Tehran, Iran. Susceptibility test to ciprofloxacin was carried out by disk agar diffusion and agar dilution methods, according to the CLSI guidelines. Activity of the efflux system was evaluated using efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP).Results:All Acinetobacter isolates were resistant to ciprofloxacin. The Minimum inhibitory concentration (MIC) range of ciprofloxacin in isolates was 4 to 128 µg/mL or greater. Moreover, susceptibility of strains to ciprofloxacin was highly increased in the presence of efflux pump inhibitor; So that, for 86.1% (56/65) of isolates, CCCP reduced the MIC by 2 to 64 folds.Conclusions:Our findings are suggestive that efflux-based system may play a role in fluoroquinolone resistance in A. baumannii isolates, affecting hospitalized patients. The ability of Acinetobacter to acquire resistance to these potent antimicrobials by the efflux pump mechanism is a concern. Therefore, new strategies are required in order to eliminate the efflux transport activity from the resistant bacteria causing nosocomial infections and provide more appropriate approaches for treatment and management of troubling infections.

P63 The second nationwide surveillance of bacterial urinary pathogens for complicated urinary tract infection conducted by JSC, JAID and JSCM

There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli.

Does mutation in gyrA and/or parC or efflux pump expression play the main role in fluoroquinolone resistance in Escherichia coli urinary tract infections?: A statistical analysis study

There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli.

Escherichia coli producing VIM-1 carbapenemase isolated on a pig farm

and the source from which the isolate was obtained. Further target gene mutations were detected in single isolates: (i) a mutation that resulted in the Glu471Asp exchange in GrlB was present in an avian ST5 MRSA; (ii) a mutation at codon 517 in the gyrB gene (resulting in an Arg to Lys exchange) was found in the ST1791 MRSA of turkey meat origin; and (iii) the ST2269 isolate had an additional grlA mutation at codon 84 that caused a Glu to Asp exchange, and a gyrA mutation that resulted in a Glu88Asp exchange. The GrlA alterations Ser80Leu and Glu84Asp and the GyrA exchange Glu88Asp have not been identified so far in S. aureus. The role in fluoroquinolone resistance of the Glu422Asp exchange in GrlB needs further investigation as the corresponding mutation was present along with other mutations in staphylococci that varied in their enrofloxacin MICs between 1 and 8 mg/L, but it was also the only mutation detected in two porcine MRSA isolates with an enrofloxacin MIC of 1 mg/L. All but one of the MRSA and MSSA isolates investigated in this study showed one of two types of point mutations (A and B in Figure S1, available as Supplementary data at JAC Online) in the norA promoter region; these, however, affected neither the 235 and 210 positions, nor the norA-associated ribosome binding site. For one avian MSSA isolate, no norA-specific PCR product could be obtained in repeated attempts. The results of this study show that increased MICs of enrofloxacin among MRSA and MSSA isolates from diseased foodproducing animals or food of animal origin is mainly mediated by grlA and/or gyrA mutations, which—aside from the four novel mutations detected during this study—correspond to those reported previously in S. aureus isolates of different origin. 2

First Molecular Characterization of Fluoroquinolone Resistance in Aerococcus spp

Aerococcus urinae and Aerococcus sanguinicola are emerging Gram-positive pathogens responsible for urinary tract infections, especially in elderly patients (2). Although they seem to be intrinsically susceptible to fluoroquinolones (3, 8, 10), acquired fluoroquinolone resistance has not been yet reported. Resistance to fluoroquinolones in Gram-positive bacteria is mainly due to point mutations in the quinolone-resistance determining regions (QRDRs) of the GyrA and GyrB subunits of the DNA gyrase and QRDRs of ParC and ParE subunits of the topoisomerase IV (4). Decreased accumulation of fluoroquinolones is a second resistance mechanism that is mediated by the overexpression of efflux pump systems (4). Since QRDR sequences of A. urinae and A. sanguinicola are not available, the aim of this study was to elucidate the mechanisms associated with the fluoroquinolone resistance. Nineteen A. urinae and 8 A. sanguinicola urinary isolates, previously identified by 16S rRNA sequencing, were studied (2). The MICs of ofloxacin, ciprofloxacin, levofloxacin, and moxifloxacin were established using the Etest method (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar supplemented by 5% horse blood. The MICs of ciprofloxacin were also determined in the presence of reserpin, an efflux pump inhibitor, incorporated in the medium (10 μg/ml) (11). Against A. urinae isolates, moxifloxacin (MIC50, 0.12 μg/ml) was 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 0.5 μg/ml) and ofloxacin (MIC50, 2 μg/ml), respectively (Table ​(Table1).1). Against A. sanguinicola isolates, moxifloxacin (MIC50, 0.25 μg/ml) was also 4- and 16-fold more active than ciprofloxacin/levofloxacin (MIC50, 1 μg/ml) and ofloxacin (MIC50, 4 μg/ml), respectively (Table ​(Table1).1). The potent activity of moxifloxacin against Aerococcus spp. is concordant with data previously reported (8). Finally, active efflux did not seem to play a major role in fluoroquinolone resistance in aerococci, since the MICs of ciprofloxacin were similar in the absence or presence of reserpin (Table ​(Table11). TABLE 1. Susceptibility to fluoroquinolones and mutations in QRDRs of gyrA, gyrB, parC, and parE genes of A. urinae and A. sanguinicola Following the use of degenerate primers, the DNA fragments corresponding to QRDRs of GyrA, GyrB, ParC, and ParE were amplified using standard PCR conditions with novel specific primers (Table ​(Table2).2). The sequences of GyrA, GyrB, ParC, and ParE of A. urinae were 100%, 98%, 88%, and 93% identical to those of A. sanguinicola, respectively. In A. urinae, a serine residue and a glutamate residue were found at positions 84 and 88 (corresponding to 83 and 87 in Escherichia coli numbering) in GyrA and also at positions 79 and 83 (corresponding to 80 and 84 in E. coli numbering) in ParC, as described in Enterococcus faecalis (9). The unique difference with A. sanguinicola was the presence of an aspartate residue at position 83 in ParC, as described in Streptococcus pneumoniae (9). Whereas all susceptible strains possessed no mutation, at least one mutation was found in gyrA and/or parC in all four resistant strains (Table ​(Table1).1). Except for S79T, similar amino acid changes in hot spot positions of ParC have been identified in other Gram-positive bacteria; these are S79R in E. faecalis and Enterococcus faecium, E83K in E. faecium and Staphylococcus aureus, and D78N and D83G in S. pneumoniae (1, 5, 7, 9). Concerning GyrA, an identical mutation (S84L) has also been identified in S. aureus, E. faecium, and Streptococcus agalactiae (6, 9). These findings suggest that topoisomerase IV seems to be the primary target of fluoroquinolones in Aerococcus spp., as previously described in other Gram-positive bacteria (4, 9). TABLE 2. Deoxynucleotide primers used in this study

Overexpression of patA and patB , Which Encode ABC Transporters, Is Associated with Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance.

Joint Effects of Topoisomerase Alterations and Plasmid-Mediated Quinolone-Resistant Determinants inSalmonella entericaTyphimurium

Alterations in topoisomerases and plasmid-mediated quinolone-resistant (PMQR) determinants have been identified as main quinolone-resistant mechanisms in Salmonella enterica Typhimurium. But the joint effects of these mechanisms have not been thoroughly characterized in homologous Salmonella Typhimurium strains. In this study, isogenic topoisomerase mutants were constructed using phage λ Red recombinase system and phage transduction. And the joint effects of topoisomerase mutations (gyrA [S83F], gyrA [S83F, D87N] and parC [S80I]) and PMQR determinants (including qnrB4, qnrS1, aac(6')-Ib-cr, and qepA) were studied in homologous genetic constitutions. Our data showed that mutations in gyrA played a dominant role in fluoroquinolone resistance in Salmonella Typhimurium and have a synergistic effect with other resistant mechanisms. The mutation (S80I) in parC would have no effect in quinolone resistance without gyrA mutations. The joint effect of aac(6')-Ib-cr and topoisomerase mutations were only observed for ciprofloxacin among tested quinolones. Different joint effects between topoisomerase mutations and qepA, qnrB4, or qnrS1 were observed for tested quinolones. Importantly, the acquirement of the PMQR determinants could improve 0- to 32-fold of the host mutant prevention concentration to ciprofloxacin. Our data showed that the acquirement of PMQR determinants could not only improve the host minimal inhibitory concentrations to quinolones but also accelerate the generation of high-level fluoroquinolone-resistant mutants.

Presence of qnr gene in Escherichia coli and Klebsiella pneumoniae resistant to ciprofloxacin isolated from pediatric patients in China

BackgroundQuinolone resistance in Enterobacteriaceae results mainly from mutations in type II DNA topoisomerase genes and/or changes in the expression of outer membrane and efflux pumps. Several recent studies have indicated that plasmid-mediated resistance mechanisms also play a significant role in fluoroquinolone resistance, and its prevalence is increasing worldwide. In China, the presence of the qnr gene in the clinical isolates of Enterobacteriaceae has been reported, but this transmissible quinolone resistance gene has not been detected in strains isolated singly from pediatric patients. Because quinolones associated with a variety of adverse side effects on children, they are not authorized for pediatric use. This study therefore aimed to investigate the presence of the qnr gene in clinical isolates of E. coli and K. pneumoniae from pediatric patients in China.MethodsA total 213 of non-repetitive clinical isolates resistant to ciprofloxacin from E. coli and K. pneumoniae were collected from hospitalized patients at five children's hospital in Beijing, Shanghai, Guangzhou, and Chongqing. The isolates were screened for the plasmid-mediated quinolone resistance genes of qnrA, qnrB, and qnrS by PCR. Transferability was examined by conjugation with the sodium azide-resistant E. coli J53. All qnr-positive were analyzed for clonality by enterobacterial repetitive intergenic consensus (ERIC)-PCR.ResultsThe study found that 19 ciprofloxacin-resistant clinical isolates of E. coli and K. pneumoniae were positive for the qnr gene, and most of the qnr positive strains were ESBL producers. Conjugation experiments showed that quinolone resitance could be transferred to recipients. Apart from this, different DNA banding patterns were obtained by ERIC-PCR from positive strains, which means that most of them were not clonally related.ConclusionThis report on transferable fluoroquinolone resistance due to the qnr gene among E. coli and K. pneumoniae strains indicated that plasmid-mediated quinolone resistance has emerged in pediatric patients in China.

Selection and characterization of fluoroquinolone-resistant mutants of Campylobacter jejuni using enrofloxacin.

Significant levels of fluoroquinolone resistance were obtained in Campylobacterjejuni isolates after an unique step of selection using enrofloxacin. An Asp90-to-Asn and a Thr86-to-Ile change in the gyrase subunit GyrA were found associated with a low (MIC < or = 8 /microg/ml) or a high (MIC > or = 16 microg/ml) level of resistance to ciprofloxacin, respectively. An association of both mutations conferred a higher level of resistance (MIC > or = 128 microg/ml). Further steps of selection increased the MICs of fluoroquinolones but did not result in a multiple antibiotic resistance phenotype. The Thr86-to-Ile change was found to confer different levels of resistance, pointing out other mechanisms of resistance. However, sequencing revealed no mutation in gyrB, and several attempts did not enable any amplification of the parC gene coding for topoisomerase IV, suggesting an absence of this secondary target in C. jejuni. In addition, no difference in the major outer membrane protein expression was found among the isolates. Furthermore, the use of the recently identified efflux pump inhibitor Phe-Arg-beta-naphthylamide did not result in a significant decrease of fluoroquinolone MICs or change in the frequency of isolation of enrofloxacin-resistant mutants, and thus appears ineffective against fluoroquinolone-resistant C. jejuni isolates. Results obtained during ciprofloxacin accumulation studies confirmed that efflux probably plays a minor role in fluoroquinolone resistance of C. jejuni.

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.