Mutations In Gyrase Research Articles

Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli

Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis.

Structure based in silico analysis of quinolone resistance in clinical isolates of Salmonella Typhi from India.

Enteric fever is a major cause of morbidity in several parts of the Indian subcontinent. The treatment for typhoid fever majorly includes the fluoroquinolone group of antibiotics. Excessive and indiscriminate use of these antibiotics has led to development of acquired resistance in the causative organism Salmonella Typhi. The resistance towards fluoroquinolones is associated with mutations in the target gene of DNA Gyrase. We have estimated the Minimum Inhibitory Concentration (MIC) of commonly used fluoroquinolone representatives from three generations, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, for 100 clinical isolates of Salmonella Typhi from patients in the Indian subcontinent. The MICs have been found to be in the range of 0.032 to 8 μg/ml. The gene encoding DNA Gyrase was subsequently sequenced and point mutations were observed in DNA Gyrase in the quinolone resistance determining region comprising Ser83Phe/Tyr and Asp87Tyr/Gly. The binding ability of these four fluoroquinolones in the quinolone binding pocket of wild type as well as mutant DNA Gyrase was computationally analyzed by molecular docking to assess their differential binding behaviour. This study has revealed that mutations in DNA Gyrase alter the characteristics of the binding pocket resulting in the loss of crucial molecular interactions and consequently decrease the binding affinity of fluoroquinolones with the target protein. The present study assists in understanding the underlying molecular and structural mechanism for decreased fluoroquinolone susceptibility in clinical isolates as a consequence of mutations in DNA Gyrase.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥ 0.25 µg/ml), SFM2 (≥ 4 µg/ml) and SFM3 (≥ 32 µg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥ 0.25 µg/ml) and SDM2 (≥ 4 µg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Mutations were observed in gyrA Ser [83]→Leu, Asp [87]→Asn/Gly, Val [196]→Ala and in parC Phe [93]→Val, Ser [80]→Ile, Asp [101]→Glu and Asp [110]→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance ( p0 <0.05); while tolC and acrR expression levels did not. Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val [196]→Ala in gyrA in clinical isolates and Phe [93]→Val, Asp [101]→Glu, Asp [110]→Glu and in parC in majority of laboratory-grown mutants.

Molecular diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis.

As a consequence of the use of fluoroquinolones (FQ), resistance to FQ has emerged, leading to cases of nearly untreatable and extensively drug-resistant tuberculosis. Mutations in DNA gyrase represent the main mechanism of FQ resistance. A full understanding of the pattern of mutations found in FQ-resistant (FQ(r)) clinical isolates, and of their proportions, is crucial for improving molecular methods for the detection of FQ resistance in Mycobacterium tuberculosis. In this study, we reviewed the detection of FQ resistance in isolates addressed to the French National Reference Center for Mycobacteria from 2007 to 2012, with the aim of evaluating the performance of PCR sequencing in a real-life context. gyrA and gyrB sequencing, performed prospectively on M. tuberculosis clinical isolates, was compared for FQ susceptibility to 2 mg/liter ofloxacin by the reference proportion method. A total of 605 isolates, of which 50% were multidrug resistant, were analyzed. The increase in FQ(r) strains among multidrug-resistant (MDR) strains during the time of the study was alarming (8% to 30%). The majority (78%) of the isolates with gyrA mutations were FQ(r), whereas only 36% of those with gyrB mutations were FQ(r). Only 12% of the FQ(r) isolates had a single mutation in gyrB. Combined gyrA and gyrB sequencing led to >93% sensitivity for detecting resistance. The analysis of the four false-positive and the five false-negative results of gyrA and gyrB sequencing illustrated the actual limitations of the reference proportion method. Our data emphasize the need for combined gyrA and gyrB sequencing in the investigation of FQ susceptibility in M. tuberculosis and challenge the validity of the current phenotype-based approach as the diagnostic gold standard for determining FQ resistance.

Bacillus anthracis GrlAV96A topoisomerase IV, a quinolone resistance mutation that does not affect the water-metal ion bridge.

The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an ∼ 3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.

Conjugation between quinolone-susceptible bacteria can generate mutations in the quinolone resistance-determining region, inducing quinolone resistance

Quinolones are an important group of antibacterial agents that can inhibit DNA gyrase and topoisomerase IV activity. DNA gyrase is responsible for maintaining bacteria in a negatively supercoiled state, being composed of subunits A and B. Topoisomerase IV is a homologue of DNA gyrase and consists of two subunits codified by the parC and parE genes. Mutations in gyrA and gyrB of DNA gyrase may confer resistance to quinolones, and the majority of resistant strains show mutations between positions 67 and 106 of gyrA, a region denoted the quinolone resistance-determining region (QRDR). The most frequent substitutions occur at positions 83 and 87, but little is known about the mechanisms promoting appearance of mutations in the QRDR. The present study proposes that some mutations in the QRDR could be generated as a result of the natural mechanism of conjugation between bacteria in their natural habitat. This event was observed following conjugation in vitro of two different isolates of quinolone-susceptible Pseudomonas aeruginosa, which transferred plasmids of different molecular weights to a recipient strain of Escherichia coli (HB101), also quinolone-susceptible, generating two different transconjugants that presented mutations in DNA gyrase and acquisition of resistance to all quinolones tested.

The Molecular Genetics of Fluoroquinolone Resistance in Mycobacterium tuberculosis.

The fluoroquinolones (FQs) are synthetic antibiotics effectively used for curing patients with multidrug-resistant tuberculosis (TB). When a multidrug-resistant strain develops resistance to the FQs, as in extensively drug-resistant strains, obtaining a cure is much more difficult, and molecular methods can help by rapidly identifying resistance-causing mutations. The only mutations proven to confer FQ resistance in M. tuberculosis occur in the FQ target, the DNA gyrase, at critical amino acids from both the gyrase A and B subunits that form the FQ binding pocket. GyrA substitutions are much more common and generally confer higher levels of resistance than those in GyrB. Molecular techniques to detect resistance mutations have suboptimal sensitivity because gyrase mutations are not detected in a variable percentage of phenotypically resistant strains. The inability to find gyrase mutations may be explained by heteroresistance: bacilli with a resistance-conferring mutation are present only in a minority of the bacterial population (>1%) and are therefore detected by the proportion method, but not in a sufficient percentage to be reliably detected by molecular techniques. Alternative FQ resistance mechanisms in other bacteria--efflux pumps, pentapeptide proteins, or enzymes that inactivate the FQs--have not yet been demonstrated in FQ-resistant M. tuberculosis but may contribute to intrinsic levels of resistance to the FQs or induced tolerance leading to more frequent gyrase mutations. Moxifloxacin is currently the best anti-TB FQ and is being tested for use with other new drugs in shorter first-line regimens to cure drug-susceptible TB.

Plasmid-related quinolone resistance determinants in epidemic Vibrio parahaemolyticus, uropathogenic Escherichia coli, and marine bacteria from an aquaculture area in Chile.

Marine bacteria from aquaculture areas with industrial use of quinolones have the potential to pass quinolone resistance genes to animal and human pathogens. The VPA0095 gene, related to the quinolone resistance determinant qnrA, from clinical isolates of epidemic Vibrio parahaemolyticus conferred reduced susceptibility to quinolone after cloning into Escherichia coli K-12 either when acting alone or synergistically with DNA gyrase mutations. In addition, a plasmid-mediated quinolone resistance gene from marine bacteria, aac(6')-Ib-cr, was identical to aac(6')-Ib-cr from urinary tract isolates of E. coli, suggesting a recent flow of this gene between these bacteria isolated from different environments. aac(6')-Ib-cr from E. coli also conferred reduced susceptibility to quinolone and kanamycin when cloned into E. coli K-12.

Mechanisms of fluoroquinolone monoresistance in Mycobacterium tuberculosis.

The gyrase mutations and efflux pumps confer fluoroquinolones (FQ) resistance in Mycobacterium tuberculosis. However, the contribution of two mechanisms in FQ mono-resistant M. tuberculosis is still unclear. Here, we investigated the contribution of gyrase mutations and efflux pumps to FQ resistance among 17 clinical FQ mono-resistant strains. Our data showed that gyrase mutations in gyrA QRDR were only responsible for four FQ mono-resistant strains. Mutations located in Ala90 and Asp94 of GyrA confer high-level LFX resistance, which can be explained by 3D modeling affinity change between GyrA and LFX. In addition, we found that a high level of efflux pump pstB transcripts may confer FQ resistance in two high-level FQ-resistant isolates (MIC ≥ 4 μg mL−1). The recombinant Escherichia coli with pstB revealed greatly increased MIC level from < 0.125 μg mL−1 to 2 μg mL−1. For the two isolates harboring high-level pstB transcripts, the presence of CCCP reduced LFX resistance to 1.0 μg mL−1. The transcriptional levels of pstB showed no significant difference among 10 clinical M. tuberculosis isolates with different drug susceptibility profiles. In conclusion, our findings demonstrate that both QRDR mutation and efflux pump mechanisms are responsible for monoresistance to FQ. PstB may serve as FQ-related efflux pumps in M. tuberculosis.

Mechanism of Quinolone Action and Resistance

Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone–enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

Molecular characterization and PCR-based replicon typing of multidrug resistant Shigella sonnei isolates from an outbreak in Thimphu, Bhutan

BackgroundShigella species are an important cause of diarrhea in developing countries. These bacteria normally acquire their antibiotic resistance via several different mobile genetic elements including plasmids, transposons, and integrons involving gene cassettes. During a diarrhea surveillance study in Thimphu, Bhutan in June and July, 2011, Shigella sonnei were isolated more frequently than expected. This study describes the antibiotic resistance of these S. sonnei isolates.MethodsA total of 29 S. sonnei isolates from Thimphu, Bhutan was characterized for antimicrobial susceptibility by disc diffusion assay and minimum inhibitory concentration (MIC) assay. All isolates were tested by pulsed-field gel electrophoresis (PFGE) with restriction enzyme XbaI and were tested for plasmid. The plasmid patterns and the PFGE patterns were analyzed by Bionumerics software. DNA sequencing was performed on amplified products for gyraseA gene and class 1 and class 2 integrons. S. sonnei isolates were classified for incompatibility of plasmids by PCR-based replicon typing (PBRT).ResultsThese S. sonnei were resistant to multiple drugs like ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, streptomycin, and tetracycline but susceptible to azithromycin. All isolates had class 2 integrons dfrA1, sat1 and aadA1 genes. Two point mutations in Gyrase A subunit at position Ser83Leu and Asp87Gly were detected in these quinolone resistant isolates. The plasmid and PFGE patterns of S. sonnei isolates suggested a clonal relationship of the isolates. All isolates carried common ColE plasmid. ColE plasmid co-resided with B/O plasmid (nine isolates) or I1 plasmid (one isolate).ConclusionsThe characteristics of 29 S. sonnei isolates from Thimphu, Bhutan in June and July, 2011 are identical in PFGE, plasmid and resistance pattern. This study suggests that these recent S. sonnei isolates are clonally related and multidrug-resistant.

A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain

Of all known Salmonella enterica serovars, S. Infantis is one of the most commonly isolated and has been recently emerging worldwide. To understand the recent emergence of S. Infantis in Israel, we performed extensive comparative analyses between pre-emergent and the clonal emergent S. Infantis populations. We demonstrate the fixation of adaptive mutations in the DNA gyrase (gyrA) and nitroreductase (nfsA) genes, conferring resistance to quinolones and nitrofurans, respectively, and the carriage of an emergent-specific plasmid, designated pESI. This self-transferred episome is a mosaic megaplasmid (∼280 kb), which increases bacterial tolerance to environmental mercury (mer operon) and oxidative stress, and provides further resistance to tetracycline, sulfamethoxazole and trimethoprim, most likely due to the presence of tetRA, sulI and dfrA genes respectively. Moreover, pESI carries the yersiniabactin siderophore system and two novel chaperone-usher fimbriae. In vitro studies established that pESI conjugation into a plasmidless S. Infantis strain results in superior biofilm formation, adhesion and invasion into avian and mammalian host cells. In vivo mouse infections demonstrated higher pathogenicity and increased intestinal inflammation caused by an S. Infantis strain harboring pESI compared with the plasmidless parental strain. Our results indicate that the presence of pESI that was found only in the emergent population of S. Infantis in Israel contributes significantly to antimicrobials tolerance and pathogenicity of its carrier. It is highly likely that pESI plays a key role in the successful spread of the emergent clone that replaced the local S. Infantis community in the short time of only 2-3 years.

Molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis clinical isolates.

To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.

INHIBITORY ACTIVITY OF POTENTOX AGAINST DNA GYRASE OF ENTEROBACTERIACEAE

DNA gyrase, a type II topoisomerase catalyzes the supercoiling of relaxed closed circular DNA. In this report we evaluated the in vitro inhibitory effect of non antibiotic adjuvant compounds disodium edetate and sodium citrate against supercoiling activity of bacterial DNA gyrase. Further, we assessed the effect of different drugs alone and in combination with one or more adjuvants in overcoming FQ resistance. A total of twelve DNA gyrase mutant strains three of each Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae were used in the study. MICs of drugs were determined by the agar dilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Potentox was the most active against all the selected mutants of with MIC values ranging 16-32 µg mL-1. DNA gyrase showed optimum supercoiling activity at 60 mM Mg2+. DNA gyrase inhibition was observed with low concentrations of disodium edetate and 50 and 100% inhibition on DNA gyrase activity was noted at approximately 4.2 and 10 mM disodium edetate. On the other hand, 50 and 100% inhibition of DNA gyrase was found at 65 and 140 mM sodium citrate. Furthermore, results revealed that Potentox inhibited the supercoiling activity of DNA gyrase at half of MIC of drug whereas other comparator drugs except gatifloxacin did not inhibit significantly the supercoiling activity of mutants as well as positive control. Potentox and gatifloxacin inhibited DNA gyrase and supercoiling at lower concentration and were the most effective. Although DNA gyrase from mutants was highly resistant to quinolones but sensitive to Potentox. Based on these results, it can be concluded that Potentox is an effective solution for the treatment of infections caused by fluoroquinolone resistant organism having DNA gyrase mutation.

MECANISMOS MOLECULARES DE RESISTENCIA A QUINOLONAS EN CEPAS DE Escherichia coli UROPATÓGENAS

El empleo de quinolonas para el tratamiento de infecciones del tracto urinario (ITU) se ha incrementado notablemente, hecho que ha conllevado a la aparición de cepas resistentes. El objetivo del estudio fue determinar los perfiles y mecanismos moleculares por los que las cepas de E. coli uropatógenas resistentes a quinolonas sobreviven a dichos antibióticos. Para ello, se utilizaron 3 cepas de E. coli uropatógenas sensibles y 58 cepas resistentes. En cuanto a la sensibilidad antimicrobiana, se utilizó el método de difusión en disco para ácido nalidixico (W), ciprofloxacino (Cip), levofloxacino (Lvx), norfloxacino (Nfx) y moxifloxacino (Mxf). Las pruebas moleculares consistieron en la reacción en cadena de la polimerasa (PCR), polimorfismo en longitud de los fragmentos de restricción (RFLP) y secuenciación del gen gyr A. Para el análisis de datos se utilizaron los programas Excel, Blastn y ClustalX. Las cepas de E. coli uropatógenas presentaron dos perfiles mayoritarios: resistencia a W 25,90% y cepas resistentes a WCipNorLvxMxf 43,10%. Mediante RFLP-PCR del gen gyr A, se determinó que 50 (86,20%) cepas presentaron una mutación puntual en el codón que cambió el aminoácido Ser 83 por Leu en la ADN girasa A; mientras que cuatro cepas no presentaron esta mutación. Con el análisis de las secuencias nucleotídicas del gen gyr A de 26 cepas, se confirmaron los perfiles de restricción; además, se detectó un cambio en el codón de Asp 87 a Asn en 22 de las cepas analizadas. Las cepas de E. coli uropatógenas resistentes a WCipNorLvxMxf representaron el principal perfil de resistencia a quinolonas. Asimismo, la mayoría de cepas contienen una mutación en la ADN girasa A que cambia de Ser 83 a Leu.

Resistance of M. leprae to Quinolones: A Question of Relativity?

Multidrug resistant leprosy, defined as resistance to rifampin, dapsone and fluoroquinolones (FQ), has been described in Mycobacterium leprae. However, the in vivo impact of fluoroquinolone resistance, mainly mediated by mutations in DNA gyrase (GyrA2GyrB2), has not been precisely assessed. Our objective was to measure the impact of a DNA gyrase mutation whose implication in fluoroquinolone resistance has been previously demonstrated through biochemical studies, on the in vivo activity of 3 fluoroquinolones: ofloxacin, moxifloxacin and garenoxacin.Methodology/Principal FindingsWe used the proportional bactericidal method. 210 four-week-old immunodeficient female Nude mice (NMRI-Foxn1nu/Foxn1nu) were inoculated in the left hind footpad with 0.03 ml of bacterial suspension containing 5×103, 5×102, 5×101, and 5×100 M. leprae AFB organisms of strain Hoshizuka-4 which is a multidrug resistant strain harboring a GyrA A91V substitution. An additional subgroup of 10 mice was inoculated with 5×10−1 bacilli in the untreated control group. The day after inoculation, subgroups of mice were treated with a single dose of ofloxacin, moxifloxacin, garenoxacin or clarithromycin at 150 mg/kg dosing. 12 months later mice were sacrificed and M. leprae bacilli were numbered in the footpad. The results from the untreated control group indicated that the infective inoculum contained 23% of viable M. leprae. The results from the moxifloxacin and garenoxacin groups indicated that a single dose of these drugs reduced the percentage of viable M. leprae by 90%, similarly to the reduction observed after a single dose of the positive control drug clarithromycin. Conversely, ofloxacin was less active than clarithromycin.Conclusion/SignificanceDNA gyrase mutation is not always synonymous of lack of in vivo fluoroquinolone activity in M. leprae. As for M. tuberculosis, in vivo studies allow to measure residual antibiotic activity in case of target mutations in M. leprae.

Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi

Purpose: There are increasing reports on failure of clinical response to ciprofloxacin in typhoid fever despite the strain being sensitive to drug in in-vitro using standard guidelines and showing mutations in DNA gyrase. But this increased MIC and clinical failures with ciprofloxacin are not always co-related with mutations presently identified in gyrA and parC genes. This shows that there may be other mechanisms such as an active drug efflux pump responsible as has been shown in other Enterobacteriaceae. This study was carried out to determine the role of efflux pump in Salmonella Typhi isolates. Materials and Methods: Total 25 already characterized nalidixic acid sensitive and nalidixic acid resistant S. Typhi strains with different range of ciprofloxacin MIC were included to study the role of efflux pump in the presence of CCCP (efflux pump inhibitor). For genotypic characterization, the entire acrR gene was sequenced to confirm the presence of any mutation in the gene. Results: The MIC of ciprofloxacin remained same in the presence and absence of CCCP in the studied strains and no significant mutations were found in the acrR gene in any of the isolates studied. Conclusions: No role of efflux pump in ciprofloxacin resistance was found in strains studied. There is a need to explore further mechanism of ciprofloxacin resistance in Salmonella Typhi.

Risk factors and clinical characteristics of patients with qnr-positive Klebsiella pneumoniae bacteraemia

Plasmid-mediated quinolone resistance (PMQR) caused by qnr genes has been known for 15 years. Information about global distribution and prevalence of qnr genes is abundant, but clinical information concerning infections produced by these isolates and risk factors for their acquisition is limited. Klebsiella pneumoniae blood isolates (n = 227) from a 1 year prospective cohort of patients in Taiwan were studied. MICs of quinolones were determined for all isolates, and multiplex PCR for the presence of PMQR genes and DNA gyrase mutations was applied to all 24 isolates with ciprofloxacin MICs ≥ 0.12 mg/L and a control group of 72 isolates with MICs ≤ 0.06 mg/L. All qnr isolates were in the group with ciprofloxacin MICs ≥ 0.12 mg/L, constituting 9.4% of tested isolates and 3.9% (qnrB 2.6% and qnrS 1.3%) of total isolates. aac(6')-Ib-cr and qepA were not found. Risk factors for qnr included nosocomial infection, bedridden status, surgery within 3 months, non-K1/K2 serotypes and prior antimicrobial use. Ciprofloxacin MIC ≥ 0.12 mg/L was associated with prior quinolone use; in contrast, prior cephalosporin use was more closely linked to the presence of qnr. Fourteen-day mortality was similar in patients infected with qnr-positive versus qnr-negative isolates, but there was a trend for increased in-hospital mortality in patients infected with qnr-positive isolates. In K. pneumoniae blood isolates collected at a hospital in Taiwan, the overall prevalence of qnr genes was 3.9%. Prior quinolone use was linked to increased ciprofloxacin MIC, but not with the prevalence of qnr, which was most strongly linked to exposure to other antimicrobials, especially cephalosporins.

Accumulation of mutations in DNA gyrase and topoisomerase IV genes contributes to fluoroquinolone resistance in Vibrio cholerae O139 strains

High resistance rates to nalidixic acid (NAL) in Vibrio cholerae serogroup O139 strains have been found, and ciprofloxacin (CIP) resistance is also observed. In this study, mutations within the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV from NAL-resistant O139 strains were analysed. The predominant mutation profile was S83I in GyrA in combination with S85L in ParC. In addition, the combination substitutions of D87N in GyrA and D420N in ParE in combination with S83I in GyrA and S85L in ParC as well as D87N in GyrA and P439S in ParE in combination with S83I in GyrA and S85L in ParC were found in the CIP-resistant strains. A series of site-directed mutants comprising D87 in GyrA, D420 in ParE and P439 in ParE were constructed from a wild-type V. cholerae O139 strain carrying the common mutations S83I in GyrA and S85L in ParC. Introduction of the mutation D87N in GyrA increased the CIP minimum inhibitory concentration (MIC) of the mutant strain by nearly 4-fold compared with the initial strain. The second introduction of D420N in ParE further significantly increased the CIP MIC to ca. 23-fold compared with the initial strain. A second introduction of P439S in ParE also increased the CIP MIC by 17-fold. Therefore, it is concluded that the emergence of D87N in GyrA and D420N or P439S in ParE dramatically induces resistance to fluoroquinolones in V. cholerae O139, and the accumulation of multiple mutations in the QRDRs confers significant resistance to fluoroquinolones in V. cholerae.

Consistent mutations in Type II Topoismerases of Vibrio cholerae O1 and global reduction in fluoroquinolone susceptibility

Vibrio cholerae O1 the epidemic strains causing devastating cholera harbors two related type II topoisomerases, gyrase and topoisomerase IV which alter DNA topology through a DNA cleavage complex. Fluoroquinolones stabilizing the DNA breakage‐reunion complex are effective WHO recommended antibiotics used in the treatment of severe cholera cases. A total of 200 Vibrio cholerae O1 strains (clinical and environmental) collected from different parts of India were subjected for MIC calculations for ciprofloxacin according to the CLSI, 2010 guidelines. The strains showed reduced susceptibility to ciprofloxacin (MIC 0.1 to 1 μg/ml). Two mutation in quinolone resistance determining region, 1 mutation in gyrA (83S&gt;;I) and 1 mutation in parC (85S&gt;;L) were found consistent in all strains. These amino acid substitutions are previously known to be associated with quinolone resistance in V. cholerae. Molecular modelling studies of mutant gyrase and topoisomerase IV revealed change in binding pocket for ciprofloxacin and would lead to alter holding over DNA cleavage complex. Such type of strains experiences the inhibitory dose of ciprofloxacin as sub‐inhibitory concentration, and could be the reason of longer duration of diarrhoea. The reduced susceptibility to ciprofloxacin was also noticed in the hybrid V. cholerae O1 strains from recent large cholera epidemics in Western Africa and Haiti. The study suggested that the circulating epidemic strains are consistently spreading globally. It hampers the outbreak control policies which can be a serious concern in future. The use of alternate chemotherapeutic agents stabilizing the transition complex of typeII topoisomerases with DNA breakage‐reunion should be of pressing importance.