GyrA Subunit Of DNA Gyrase Research Articles

Prevalence and mechanisms of ciprofloxacin resistance in Escherichia coli isolated from hospitalized patients, healthy carriers, and wastewaters in Iran

BackgroundThis study was aimed to evaluate the prevalence and molecular characteristics of ciprofloxacin resistance among 346 Escherichia coli isolates collected from clinical specimens (n = 82), healthy children (n = 176), municipal wastewater (n = 34), hospital wastewater (n = 33), poultry slaughterhouse wastewater (n = 12) and livestock (n = 9) slaughterhouse wastewater in Iran.MethodsCiprofloxacin minimum inhibitory concentration (MIC) was determined by agar dilution assay. Phylogroups and plasmid-mediated quinolone resistance (PMQR) genes were identified using PCR. Mutations in gyrA, gyrB, parC, and parE genes and amino acid alterations were screened through sequencing assay. The effect of efflux pump inhibitor (PAβN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method.ResultsIn total, 28.03% of E. coli isolates were phenotypically resistant to ciprofloxacin. Based on sources of isolation, 64.63%, 51.51%, 33.33%, 14.70%, 10.22% and 8.33% of isolates from clinical specimens, hospital wastewater, livestock wastewater, municipal wastewater, healthy children and poultry wastewater were ciprofloxacin-resistant, respectively. Eighty-one point eighty-one percent (Ser-83 → Leu + Asp-87 → Asn; 78.78% and Ser-83 → Leu only; 3.03% (of ciprofloxacin-resistant E. coli isolates showed missense mutation in GyrA subunit of DNA gyrase, while no amino-acid substitution was noted in the GyrB subunit. DNA sequence analyses of the ParC and ParE subunits of topoisomerase IV exhibited amino-acid changes in 30.30% (Ser-80 → Ile + Glu-84 → Val; 18.18%, Ser-80 → Ile only; 9.10% and Glu-84 → Val only; 3.03%0 (and 15.38% (Ser-458 → Ala) of ciprofloxacin-resistant E. coli isolates, respectively. The PMQR genes, aac(6')-Ib-cr, qnrS, qnrB, oqxA, oqxB, and qepA were detected in 43.29%, 74.22%, 9.27%, 14.43%, 30.92% and 1.03% of ciprofloxacin-resistant isolates, respectively. No isolate was found to be positive for qnrA and qnrD genes. In isolates harboring the OqxA/B efflux pump, the MIC of ciprofloxacin was reduced twofold in the presence of PAβN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates.ConclusionsThis study proved the high incidence of ciprofloxacin-resistant E. coli isolates in both clinical and non-clinical settings in Iran. Chromosomal gene mutations and PMQR genes were identified in ciprofloxacin resistance among E. coli population.

The Relationship between Ciprofloxacin Resistance and Genotypic Changes in S. aureus Ocular Isolates.

Staphylococcus aureus (S. aureus) is a frequent cause of eye infections with some isolates exhibiting increased antimicrobial resistance to commonly prescribed antibiotics. The increasing resistance of ocular S. aureus to ciprofloxacin is a serious concern as it is a commonly used as a first line antibiotic to treat S. aureus keratitis. This study aimed to analyse genetic mutations in the genomes of 25 S. aureus isolates from infections or non-infectious ocular conditions from the USA and Australia and their relationship to ciprofloxacin resistance. Overall, 14/25 isolates were phenotypically resistant to ciprofloxacin. All isolates were analyzed for mutations in their quinolone resistance-determining regions (QRDRs) and efflux pump genes. Of the fourteen resistant isolates, 9/14 had ciprofloxacin resistance mutations within their QRDRs, at codons 80 or 84 within the parC subunit and codon 84 within the gyrA subunit of DNA gyrase. The highest resistance (MIC = 2560 μg/mL) was associated with two SNPs in both gyrA and parC. Other resistant isolates (3/14) had mutations within norB. Mutations in genes of other efflux pumps and their regulator (norA, norC, mepA, mdeA, sepA, sdrM, mepR, arlR, and arlS) or the DNA mismatch repair (MMR) system (mutL and mutS) were not associated with increased resistance to ciprofloxacin. The functional mutations associated with ciprofloxacin resistance in QRDRs (gyrA and parC) and norB suggests that these are the most common reasons for ciprofloxacin resistance in ocular isolates. Novel SNPs of gyrA Glu-88-Leu, Asn-860-Thr and Thr-845-Ala and IIe-855-Met, identified in this study, need further gene knock out/in studies to better understand their effect on ciprofloxacin resistance.

Novel Amino Acid Derivatives of Quinolines as Potential Antibacterial and Fluorophore Agents

A new series of amino acid derivatives of quinolines was synthesized through the hydrolysis of amino acid methyl esters of quinoline carboxamides with alkali hydroxide. The compounds were purified on silica gel by column chromatography and further characterized by TLC, NMR and ESI-TOF mass spectrometry. All compounds were screened for in vitro antimicrobial activity against different bacterial strains using the microdilution method. Most of the synthesized amino acid-quinolines show more potent or equipotent inhibitory action against the tested bacteria than their correspond esters. In addition, many of them exhibit fluorescent properties and could possibly be utilized as fluorophores. Molecular docking and simulation studies of the compounds at putative bacterial target enzymes suggest that the antimicrobial potency of these synthesized analogues could be due to enzyme inhibition via their favorable binding at the fluoroquinolone binding site at the GyrA subunit of DNA gyrase and/or the ParC subunit of topoisomerase-IV.

Inhibition of indole production increases the activity of quinolone antibiotics against E. coli persisters

Persisters are a sub-population of genetically sensitive bacteria that survive antibiotic treatment by entering a dormant state. The emergence of persisters from dormancy after antibiotic withdrawal leads to recurrent infection. Indole is an aromatic molecule with diverse signalling roles, including a role in persister formation. Here we demonstrate that indole stimulates the formation of Escherichia coli persisters against quinolone antibiotics which target the GyrA subunit of DNA gyrase. However, indole has no effect on the formation of E. coli persisters against an aminocoumarin, novobiocin, which targets the GyrB subunit of DNA gyrase. Two modes of indole signalling have been described: persistent and pulse. The latter refers to the brief but intense elevation of intracellular indole during stationary phase entry. We show that the stimulation of quinolone persisters is due to indole pulse, rather than persistent, signalling. In silico docking of indole on DNA gyrase predicts that indole docks perfectly to the ATP binding site of the GyrB subunit. We propose that the inhibition of indole production offers a potential route to enhance the activity of quinolones against E. coli persisters.

Identification of new DNA gyrase inhibitors based on bioactive compounds from streptomyces: structure-based virtual screening and molecular dynamics simulations approaches

DNA gyrase enzyme has vital role in bacterial survival and can be considered as a potential drug target. Owing to the appearance of resistance to gyrase-targeted drugs, especially fluoroquinolone, screening new compounds which bind more efficiently to the mutant binding pocket is essential. Hence, in this work, using Smina Autodock and through structure-based virtual screening of StreptomeDB, several natural products were discovered based on the SimocyclinoneD8 (SD8) binding pocket of GyrA subunit of DNA gyrase. After evaluation of binding affinity, binding modes, critical interactions and physicochemical and pharmaceutical properties, three lead compounds were selected for further analysis. Afterward 60 ns molecular dynamics simulations were performed and binding free energies were calculated by the molecular mechanics/Poisson–Boltzmann surface area method. Also, interaction of the selected lead compounds with the mutated GyrA protein was evaluated. Results indicated that all of the selected compounds could bind to the both wild-type and mutated GyrA with the binding affinities remarkably higher than SimocyclinoneD8. Interestingly, we noticed that the selected compounds comprised angucycline moiety in their structure which could sufficiently interact with GyrA and block the DNA binding pocket of DNA gyrase, in silico. In conclusion, three DNA gyrase inhibitors were identified successfully which were highly capable of impeding DNA gyrase and can be considered as potential drug candidates for treatment of fluoroquinolone-resistant strains.Communicated by Ramaswamy H. Sarma

Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria.

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC90 values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10-8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC50], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1.

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6')-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6')-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.

Structural signature of Ser83Leu and Asp87Asn mutations in DNA gyrase from enterotoxigenic Escherichia coli and impact on quinolone resistance

Enterotoxigenic Escherichia coli (ETEC) is among the most frequent microorganisms causing traveler's diarrhea (TD). Quinolones are potent antimicrobial agents used for the treatment of TD. Resistance to quinolones is typically caused by substitutions in QRDR region of gyrA subunit of DNA gyrase. The aim of this study was to seek insights into the effect of these substitutions at structural level and their association with observed quinolone resistance.Majority of the ETEC strains have gyrA mutations at amino acid position 83 and 87. To understand the quinolone resistance mechanism at molecular level, we have studied the interaction of wild type and mutant forms of ETEC gyrA with nalidixic acid and ciprofloxacin by molecular modeling using Discovery Studio and LeadIt.All the mutants had reduced affinity towards both ciprofloxacin and nalidixic acid relative to the wild type due to the mutations introduced in gyrA. Besides Ser83 and Asp87, for nalidixic acid binding Arg91 and His45 residues were observed to be critical while in ciprofloxacin binding Lys42 and Arg91 residues played a significant role.Amino acid substitutions contribute to the emergence of drug resistance in sensitive strains by causing structural alterations leading to reduced affinity of the drug towards receptor. Analysis of the effect of amino acid substitutions at structural level is of utmost importance to establish possible associations between mutations and the diseases. These studies accelerate the identification of pharmaceutical targets for relevant treatments and could also be helpful in guiding the design of further experimental research.

Polymorphic mutation frequencies in clinical isolates ofStaphylococcus aureus: the role of weak mutators in the development of fluoroquinolone resistance

The polymorphic mutation frequencies for 154 Staphylococcus aureus isolates from Chinese bovine clinical mastitis cases were investigated. We found that nearly 29% of the isolates presented as weak mutators, while only two (1.3%) strong mutators were detected. Of the 15 weak mutators that exhibited ciprofloxacin resistance phenotypes, only one isolate was found to be mutS deficient. All of the ciprofloxacin-resistant isolates had the classic ciprofloxacin resistance mutations at codon 80 within the ParC subunit of topoisomerase IV and codon 84/88 within the GyrA subunit of DNA gyrase. The proportion of ciprofloxacin-resistant isolates among the weak mutators (34.1%) was significantly higher than that found in the normomutators (11.4%) and hypomutators (0%) (P < 0.001, Fisher's exact test), suggesting a positive correlation between weak mutators and ciprofloxacin resistance.

Multiplex Bead Suspension Array for Screening Neisseria gonorrhoeae Antibiotic Resistance Genetic Determinants in Noncultured Clinical Samples

The increasing threat of antibiotic-resistant Neisseria gonorrhoeae highlights the need for new diagnostic options. A high-throughput multiplex bead suspension array assay was developed for profiling 29 N. gonorrhoeae genomic mutations and 2 plasmid genes conferring resistance to 6 antimicrobial agents: penicillin, ciprofloxacin, cefixime, tetracycline, azithromycin, and spectinomycin. The three steps of this assay include amplification of 12 N. gonorrhoeae chromosomal and plasmid loci, multiplex allele-specific primer extension reaction, and multiplex bead suspension array detection. Antibiotic resistance genetic determinants were identified successfully in 239 cervicovaginal N. gonorrhoeae-positive noncultured swab samples. This molecular assay can be used for detection of gonococci in clinical specimens, molecular typing, mutation profiling, and predictive assessment of N. gonorrhoeae susceptibility to antibiotics without the need for culture.

Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

Fluoroquinolone (FQ) resistance is emerging in Mycobacterium tuberculosis. The main mechanism of FQ resistance is amino acid substitution within the quinolone resistance-determining region (QRDR) of the GyrA subunit of DNA gyrase, the sole FQ target in M. tuberculosis. However, substitutions in GyrB whose implication in FQ resistance is unknown are increasingly being reported. The present study clarified the role of four GyrB substitutions identified in M. tuberculosis clinical strains, two located in the QRDR (D500A and N538T) and two outside the QRDR (T539P and E540V), in FQ resistance. We measured FQ MICs and also DNA gyrase inhibition by FQs in order to unequivocally clarify the role of these mutations in FQ resistance. Wild-type GyrA, wild-type GyrB, and mutant GyrB subunits produced from engineered gyrB alleles by mutagenesis were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. MICs and DNA gyrase inhibition were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. All these substitutions are clearly implicated in FQ resistance, underlining the presence of a hot spot region housing most of the GyrB substitutions implicated in FQ resistance (residues NTE, 538 to 540). These findings help us to refine the definition of GyrB QRDR, which is extended to positions 500 to 540.

Study of Organic Solvent Tolerance and Increased Antibiotic Resistance Properties in E. coli gyrA Mutants.

Ciprofloxacin is one of the most widely used antibiotics for the treatment of several infections caused by Gram-negative bacteria, like E. coli. Changes in gyrA, encoding GyrA subunit of DNA gyrase, cause the resistance to ciprofloxacin. Some ciprofloxacin resistant gyrA mutants acquired constitutive expression of marRAB operon due to the gaining mutations in marR, a repressor of this operon. This leads to the expression of a multidrug resistance phenotype and high organic solvent tolerance. Thus, this study was aimed to provide more information on extra mechanisms of resistance in gyrA mutants with different ciprofloxacin MICs. For this purpose, the tolerance of organic solvent, resistance to tetracycline and presence of possible mutation in marOR were investigated in 10 gyrA mutants. Results showed that most of gyrA mutants behaved like MG1655, control strain, but 3 out of 10 were slightly more resistant to tetracycline than MG1655 and had better growth on hexane. Among three mutants, two possess a mutation in marOR. In conclusion, the generation of mutation in marOR is not enough by itself to produce the multidrug resistance phenotype and complete activation of AcrAB-TolC.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal domain of the GyrA subunit of DNA gyrase fromStaphylococcus aureusstrain Mu50

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological form of bacterial DNA. In this study, the C-terminal domain of the GyrA subunit of DNA gyrase from Staphylococcus aureus Mu50 strain was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.80 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P2(1), with unit-cell parameters a = 37.28, b = 80.19, c = 50.22 Å, β = 110.64°. The asymmetric unit contained one molecule, with a corresponding V(M) of 2.02 Å(3) Da(-1) and a solvent content of 39.2%.

Preliminary X-ray crystallographic analysis of the breakage-reunion domain of the GyrA subunit of DNA gyrase from Colwellia psychrerythraea strain 34H.

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal breakage-reunion domain of the GyrA subunit of DNA gyrase from Colwellia psychrerythraea 34H was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.60 A resolution using a synchrotron-radiation source. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 98.98, b = 101.56, c = 141.83 A. The asymmetric unit contained two molecules, with a corresponding V(M) of 3.18 A(3) Da(-1) and a solvent content of 59.9%.

Emerging fluoroquinolone resistance in Neisseria meningitidis in India: cause for concern

Sir, Despite our improved understanding of the epidemiological features, pathogenesis, risk factors and advances in diagnosis and treatment of meningococcal disease, the disease remains a leading cause of meningitis and sepsis. In India, meningococcal cases are reported sporadically from all parts of the country and epidemic waves occur at regular intervals affecting children and young adults. Persons in close contact with patients who have meningococcal disease are at elevated risk of contracting the disease and it has been seen that institution of chemoprophylaxis during the first few days after onset of disease in the index patient reduces the risk of secondary disease in close contacts. The high rates of morbidity and mortality associated with meningococcal disease make antimicrobial chemoprophylaxis a primary means of preventing the spread of meningococcal disease and curtailing an impending outbreak. Rifampicin, ciprofloxacin and ceftriaxone are recommended antimicrobials for chemoprophylaxis against meningococcal disease. Although rifampicin is commonly used as chemoprophylaxis to eradicate meningococcal carriage, it has been suggested to replace rifampicin with ciprofloxacin in adults, because of high rates of nasopharyngeal clearance seen with use of a single dose of ciprofloxacin compared with twice daily administration of rifampicin for 2 days. Although meningococcal resistance to the fluoroquinolones may not be a major problem, reports of emerging ciprofloxacin resistance make it essential to re-examine the use of this drug for chemoprophylaxis. First documented resistance to ciprofloxacin in Neisseria meningitidis was reported in 1992 from Greece where a single isolate with an MIC of 1 mg/L was recovered. In 1999 Kaczmarski et al. and Casin et al. from England and Wales reported meningococci with raised MICs of ciprofloxacin, but their MIC ranges were not reported. After that low-level resistance to ciprofloxacin with MICs 0.12–0.25 mg/L was reported on four more occasions from France (1999), Australia (2000), Spain (2004) and Argentina (2005). In India, as yet ciprofloxacin-resistant meningococci have not been documented but personal communications and a recent review article on meningococcal infection mention an increase in resistance to ciprofloxacin. During a recent epidemic (April 2005–April 2006) in New Delhi, 13 strains of N. meningitidis serogroup A were isolated from 249 patients clinically suspected of meningococcal infection and referred to our laboratory, which is affiliated to two teaching hospitals in Central Delhi. Bacteriological culture, isolation, identification, serogrouping and antimicrobial susceptibility testing was performed as per standard guidelines. MICs were determined for penicillin, azithromycin, ceftriaxone, chloramphenicol, ampicillin and ciprofloxacin by Etest strips using breakpoints recently recommended by NCCLS/CLSI on Mueller-Hinton agar supplemented with defibrinated sheep blood. Antimicrobial susceptibility revealed 100% susceptibility to penicillin (MIC 0.06 mg/L), azithromycin (MIC 2 mg/L), ceftriaxone (MIC 0.12 mg/L), chloramphenicol ( 2 mg/L) and ampicillin ( 0.12 mg/L). Only four isolates were found to be resistant to ciprofloxacin with MICs of 0.25 mg/L (resistance: MIC 0.12 mg/L). Two major mechanisms of fluoroquinolone resistance have been identified in meningococci. One is chromosomal mutations in the quinolone resistance determining regions (QRDRs) of the target sites for fluoroquinolones, namely the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV and the other is overexpression of endogenous efflux pumps. It has been suggested that resistance in meningococci may arise in the same manner and reach similar levels as seen in quinolone-resistant Neisseria gonorrhoeae. Oropharyngeal or nasopharyngeal cultures are not really helpful in determining the need for chemoprophylaxis and may unnecessarily delay the use of chemoprophylactic antibiotics. Thus chemoprophylaxis needs to be given empirically and knowledge of antibiotic susceptibility patterns during the previous epidemic and the current situation may help in the selection of antibiotics. Scanty data is available from India regarding antimicrobial susceptibility patterns of N. meningitidis, only sulfadiazine resistance has been previously documented. The recent encounter with meningococcal strains showing decreased susceptibility to ciprofloxacin is a cause for real concern and will have implications in chemoprophylaxis since ciprofloxacin is used to eradicate meningococci from nasopharyngeal carriers. Indiscriminate and inadequate use of this easily available (over the counter) and orally effective antibiotic in the community for a variety of infections has probably resulted in increased resistance to this valuable molecule. Continuous surveillance is necessary to monitor the emergence and spread of resistance to ciprofloxacin and other quinolones and to guide appropriate public health interventions in preventing drug-resistant meningococci. It is also another case for regulating over the counter sale of antibiotics in this and other countries.

Linkage of ciprofloxacin resistance with a single genotypic cluster of Klebsiella pneumoniae

The objective of this study was to examine the epidemiology of ciprofloxacin-resistant, extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strains. Sixty-nine unique patient isolates of K. pneumoniae isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of The Royal Infirmary of Edinburgh and associated General Practices were identified and susceptibility testing was performed with the Vitek system. Strains flagged as ESBL-positive by the Vitek system were subjected to isoelectric focusing. The results suggested that all 69 isolates harboured at least one ESBL, which was later confirmed by polymerase chain reaction (PCR) with bla TEM and/or bla SHV primers. The purified PCR product was subjected to automated sequencing and the results were compared with the BLAST online search engine. Of the 69 isolates, 32 (46.4%) were found to be resistant to ciprofloxacin, 11 (15.9%) were intermediate and 26 (37.7%) were sensitive. To investigate the epidemiological relationship between the ciprofloxacin-resistant ESBL-positive strains, pulsed-field gel electrophoresis (PFGE) was performed. Rapidest software was used to calculate the genetic distance by the Nei distance method. PFGE analysis indicated that the clinical isolates belonged to four distinct genotype clusters (Groups A, B, C and D); each group or cluster was homogeneous or compact with respect to certain characteristics. Group A consisted of 25 isolates, group B of 3 isolates and Groups C and D of 2 isolates each. These results indicate that the spread of resistance is largely as a result of the dissemination of a single clonal strain. PCR was used to amplify the gyrA and parC genes from genomic DNA of the ciprofloxacin-resistant isolates. The amplified product was sent for analysis by automated DNA sequencing and the resulting DNA sequences were compared with the gyrA gene of K. pneumoniae. The sequencing results demonstrated that alteration of the GyrA subunit of DNA gyrase at amino acid 83 and/or amino acid 87 plays a central role in conferring high-level quinolone resistance in K. pneumoniae possessing ESBLs.

Alteration in the GyrA Subunit of DNA Gyrase and the ParC Subunit of Topoisomerase IV in Quinolone-Resistant Shigella dysenteriae Serotype 1 Clinical Isolates from Kolkata, India

Alteration in the GyrA Subunit of DNA Gyrase and the ParC Subunit of Topoisomerase IV in Quinolone-Resistant <i>Shigella dysenteriae</i> Serotype 1 Clinical Isolates from Kolkata, India

MSP017 Emergence of antibiotic resistance among S. pneumoniae and S. pyogenes strains isolated from the respiratory tract infections of children in Germany, 2002

Enterotoxigenic Escherichia coli (ETEC) is among the most frequent microorganisms causing traveler's diarrhea (TD). Quinolones are potent antimicrobial agents used for the treatment of TD. Resistance to quinolones is typically caused by substitutions in QRDR region of gyrA subunit of DNA gyrase. The aim of this study was to seek insights into the effect of these substitutions at structural level and their association with observed quinolone resistance.Majority of the ETEC strains have gyrA mutations at amino acid position 83 and 87. To understand the quinolone resistance mechanism at molecular level, we have studied the interaction of wild type and mutant forms of ETEC gyrA with nalidixic acid and ciprofloxacin by molecular modeling using Discovery Studio and LeadIt.All the mutants had reduced affinity towards both ciprofloxacin and nalidixic acid relative to the wild type due to the mutations introduced in gyrA. Besides Ser83 and Asp87, for nalidixic acid binding Arg91 and His45 residues were observed to be critical while in ciprofloxacin binding Lys42 and Arg91 residues played a significant role.Amino acid substitutions contribute to the emergence of drug resistance in sensitive strains by causing structural alterations leading to reduced affinity of the drug towards receptor. Analysis of the effect of amino acid substitutions at structural level is of utmost importance to establish possible associations between mutations and the diseases. These studies accelerate the identification of pharmaceutical targets for relevant treatments and could also be helpful in guiding the design of further experimental research.

Frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae in Tokyo in 2002.

The frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae obtained in Tokyo in 2002 was studied. The frequencies of GyrA and ParC mutations in these 19 isolates were 100% (19 of 19) and 84.2% (16 of 19), respectively, and these results were 1.48-fold (100%/67.6%) and 3.58-fold (84.2%/23.5%) higher, respectively, than the frequencies reported in 1998 in 68 isolates obtained in Fukuoka during the period from 1992 to 1996. Isolates with increasing numbers of mutations were more resistant not only to levofloxacin but also to other antibiotics. The 50% and 90% minimum inhibitory concentrations (MICs) to levofloxacin during the period from 1995 to 1996 were 0.063 and 1 micro g/ml, and they increased to 4 and 8 micro g/ml, respectively, in the present study. All 19 cases of gonoccocal urethritis in the present study were cured with a single intramuscular injection of 2 g spectinomycin.

Trends in antimicrobial susceptibility among isolates of Campylobacter species in Ireland and the emergence of resistance to ciprofloxacin

Measurements were made of the susceptibility to six commonly prescribed antibiotics, including erythromycin, tetracycline and ciprofloxacin, of 130 isolates of Campylobacter jejuni and 15 isolates of Campylobacter coli cultured from...