PprA, a pleiotropic protein for radioresistance, works through DNA gyrase and shows cellular dynamics during postirradiation recovery in Deinococcus radiodurans.

PprA, a pleiotropic protein involved in radioresistance of Deinococcus radiodurans was detected in multiprotein DNA processing complex identified from this bacterium. pprA mutant expressing GFP-PprA could restore its wild type resistance of γ radiation. Under normal conditions, GFP-PprA expressing cells showed PprA localization on both septum trapped nucleoids (STN) and nucleoids located elsewhere (MCN). Cell exposed to 4 kGy γ radiation showed nearly 2 h growth lag and during this growth arrest phase, the majority of the cells had GFP-PprA located on MCN. While in late phase (~120 min) PIR cells, when cells are nearly out of growth arrest, PprA was maximally found with STN. These cells when treated with nalidixic acid showed diffused localization of PprA across the septum. gyrA disruption mutant of D. radiodurans showed growth inhibition, which increased further in gyrA pprA mutant. Interestingly, gyrA mutant showed ~20-fold less resistance to γ radiation as compared to wild type, which did increase further in gyrA pprA mutant. These results suggested that PprA localization undergoes a dynamic change during PIR, and its localization on nucleoid near septum and functional interaction with gyrase A might suggest a mechanism that could explain PprA role in genome segregation possibly through topoisomerase II.

Transferable quinolone resistance in Vibrio cholerae.

Ciprofloxacin was introduced for treatment of patients with cholera in Bangladesh because of resistance to other agents, but its utility has been compromised by the decreasing ciprofloxacin susceptibility of Vibrio cholerae over time. We correlated levels of susceptibility and temporal patterns with the occurrence of mutation in gyrA, which encodes a subunit of DNA gyrase, followed by mutation in parC, which encodes a subunit of DNA topoisomerase IV. We found that ciprofloxacin activity was more recently further compromised in strains containing qnrVC3, which encodes a pentapeptide repeat protein of the Qnr subfamily, members of which protect topoisomerases from quinolone action. We show that qnrVC3 confers transferable low-level quinolone resistance and is present within a member of the SXT integrating conjugative element family found commonly on the chromosomes of multidrug-resistant strains of V. cholerae and on the chromosomes of Escherichia coli transconjugants constructed in the laboratory. Thus, progressive increases in quinolone resistance in V. cholerae are linked to cumulative mutations in quinolone targets and most recently to a qnr gene on a mobile multidrug resistance element, resulting in further challenges for the antimicrobial therapy of cholera.

Impact of low-level resistance to fluoroquinolones due to qnrA1 and qnrS1 genes or a gyrA mutation on ciprofloxacin bactericidal activity in a murine model of Escherichia coli urinary tract infection.

We investigated the impact of low-level resistance to fluoroquinolones on the bactericidal activity of ciprofloxacin in a murine model of urinary tract infection. The susceptible Escherichia coli strain CFT073 (ciprofloxacin MIC [CIP MIC] of 0.008 microg/ml) was compared to its transconjugants harboring qnrA1 or qnrS1 and to an S83L gyrA mutant. The three derivatives showed similar low-level resistance to fluoroquinolones (CIP MICs, 0.25 to 0.5 microg/ml). Bactericidal activity measured in vitro after 1, 3, and 6 h of exposure to 0.5 microg/ml of ciprofloxacin was significantly lower for the derivative strains (P < 0.01). In the murine model of urinary tract infection (at least 45 mice inoculated per strain), mice were treated with a ciprofloxacin regimen of 2.5 mg/kg, given subcutaneously twice daily for 2 days. In mice infected with the susceptible strain, ciprofloxacin significantly decreased viable bacterial counts (log10 CFU/g of tissue) in the bladder (4.2 +/- 0.5 versus 5.5 +/- 1.3; P = 0.001) and in the kidney (3.6 +/- 0.8 versus 5.0 +/- 1.1; P = 0.003) compared with those of untreated mice. In contrast, no significant decrease in viable bacterial counts was observed with any of the three derivative strains. The area under the concentration-time curve from 0 to 24 h/MIC and the maximum concentration of drug in serum/MIC ratios measured in plasma were indeed equal to 827 and 147, respectively, for the parental strain, and only 12.4 to 24.8 and 2.2 to 4.4, respectively, for the derivative strains. In conclusion, low-level resistance to fluoroquinolones conferred by a qnr gene is associated with decreased bactericidal activity of ciprofloxacin, similar to that obtained with a gyrA mutation.

In vitro activity of garenoxacin against Streptococcus pneumoniae mutants with characterized resistance mechanisms.

We evaluated the potency of garenoxacin in selecting resistant Streptococcus pneumoniae mutants by determining its mutant prevention concentration, using strains with and without topoisomerase gene mutations, and compared its potency to that of other quinolones. Garenoxacin had a significantly greater potency against pneumococci, including strains containing topoisomerase mutations. Genetic analysis of the S. pneumoniae mutants created by garenoxacin revealed that the gyrA gene was a primary target of garenoxacin.

Nuclear gyrB encodes a functional subunit of the Plasmodium falciparum gyrase that is involved in apicoplast DNA replication.

The DNA replication machinery of the Plasmodium falciparum apicoplast is a validated drug target. Nuclear-encoded gyrase subunits are predicted to play a critical role in maintaining DNA topology during the D-loop/bi-directional ori replication process of the parasite. We show the presence of P. falciparum gyrase subunits in parasite lysates by using antibodies generated against recombinant gyrase A and B. The ATPase activity of PfGyrB was inhibited by novobiocin that also caused parasite death in culture. Reduction of apicoplast/nuclear DNA ratio in the presence of novobiocin indicated that the drug targets apicoplast DNA replication. Molecular modeling of gyrase A and B subunits revealed extensive fold conservation with the Escherichia coli counterparts as well as the presence of a long disordered loop adjacent to the ATPase domain of PfGyrB. Our results have implications for development of PfGyrB as a drug target against malaria.

Substitutions of amino acids in alpha-helix-4 of gyrase A confer fluoroquinolone resistance on Clostridium perfringens.

DNA gyrase, an essential enzyme that regulates DNA topology in bacteria, is the target of fluoroquinolones. Three fluoroquinolone-resistant mutants derived from one strain of Clostridium perfringens had amino acid substitutions of glycine 81 to cysteine, aspartic acid 87 to tyrosine, or both, in alpha-helix-4 of gyrase A. The gyrase mutations affected the growth kinetics of mutants differently when the mutants were exposed to increasing concentrations of gatifloxacin and ciprofloxacin. Fluoroquinolone concentration-dependent effects observed during growth in the exponential and stationary phases depended on the presence of particular gyrA mutations. Introduction of a wild-type gyrA gene into the mutants enhanced their susceptibility to fluoroquinolones and decreased their growth rates proportional to increases in fluoroquinolone concentrations. Amino acid substitutions in alpha-helix-4 of gyrase A protected C. perfringens from fluoroquinolones, and a strain with two substitutions was the most resistant.

Analysis of pleiotropic transcriptional profiles: a case study of DNA gyrase inhibition.

Genetic and environmental perturbations often result in complex transcriptional responses involving multiple genes and regulons. In order to understand the nature of a response, one has to account for the contribution of the downstream effects to the formation of a response. Such analysis can be carried out within a statistical framework in which the individual effects are independently collected and then combined within a linear model. Here, we modeled the contribution of DNA replication, supercoiling, and repair to the transcriptional response of inhibition of the Escherichia coli gyrase. By representing the gyrase inhibition as a true pleiotropic phenomenon, we were able to demonstrate that: (1) DNA replication is required for the formation of spatial transcriptional domains; (2) the transcriptional response to the gyrase inhibition is coordinated between at least two modules involved in DNA maintenance, relaxation and damage response; (3) the genes whose transcriptional response to the gyrase inhibition does not depend on the main relaxation activity of the cell can be classified on the basis of a GC excess in their upstream and coding sequences; and (4) relaxation by topoisomerase I dominates the transcriptional response, followed by the effects of replication and RecA. We functionally tested the effect of the interaction between relaxation and repair activities, and found support for the model derived from the microarray data. We conclude that modeling compound transcriptional profiles as a combination of downstream transcriptional effects allows for a more realistic, accurate, and meaningful representation of the transcriptional activity of a genome.

[Decrease in bactericidal activity of ciprofloxacin in Salmonella mutants generated following repeated exposure to various fluorquinolones]. / Disminución de la actividad bactericida del ciprofloxacino en mutantes de Salmonella generados tras exposición repetida a diversas fluoroquinolonas.

Salmonella mutants with reduced fluoroquinolone susceptibility were generated in vitro following repeated exposure to subinhibitory concentrations of the drugs and the alterations in the gyrA gene were characterized. Afterwards, the change in bactericidal activity exhibited by ciprofloxacin against the resulting mutants was determined. A decrease in the bactericidal activity of ciprofloxacin was found in all the mutants, but was more pronounced in mutants of nalidixic acid-resistant strains. This may help to explain the therapeutic failures sometimes described when fluoroquinolones are used in the treatment of these strains. In addition, our model evaluates the first interactions produced between the microorganism and the antibiotics, and demonstrates that the loss of bactericidal activity of ciprofloxacin occurs following exposure to all of the fluoroquinolones tested.

Enhanced in vivo fitness of fluoroquinolone-resistant Campylobacter jejuni in the absence of antibiotic selection pressure.

Campylobacter jejuni, a major foodborne human pathogen, has become increasingly resistant to fluoroquinolone (FQ) antimicrobials. By using clonally related isolates and genetically defined mutants, we determined the fitness of FQ-resistant Campylobacter in chicken (a natural host and a major reservoir for C. jejuni) in the absence of antibiotic selection pressure. When monoinoculated into the host, FQ-resistant and FQ-susceptible Campylobacter displayed similar levels of colonization and persistence in the absence of FQ antimicrobials. The prolonged colonization in chickens did not result in loss of the FQ resistance and the resistance-conferring point mutation (C257 --> T) in the gyrA gene. Strikingly, when coinoculated into chickens, the FQ-resistant Campylobacter isolates outcompeted the majority of the FQ-susceptible strains, indicating that the resistant Campylobacter was biologically fit in the chicken host. The fitness advantage was not due to compensatory mutations in the genes targeted by FQ and was linked directly to the single point mutation in gyrA, which confers on Campylobacter a high-level resistance to FQ antimicrobials. In certain genetic backgrounds, the same point mutation entailed a biological cost on Campylobacter, as evidenced by its inability to compete with the FQ-susceptible Campylobacter. These findings provide a previously undescribed demonstration of the profound effect of a resistance-conferring point mutation in gyrA on the fitness of a major foodborne pathogen in its natural host and suggest that the rapid emergence of FQ-resistant Campylobacter on a worldwide scale may be attributable partly to the enhanced fitness of the FQ-resistant isolates.

Illegitimate recombination mediated by double-strand break and end-joining in Escherichia coli.

The frequency of illegitimate recombination has been measured by a lambda bio transducing phage assay during the induction of the E. coli lambda cI857 lysogen. Illegitimate recombination falls into two classes, short homology-independent and short homology-dependent illegitimate recombination. The former involves sequences with virtually no homology, and is mediated by DNA topoisomerases and controlled by the DNA binding protein HU. The latter is induced by UV irradiation or other DNA damaging agents and requires short regions of homology, usually contain 4 to 13 base pairs, at sites involved in recombination. It has been shown that the RecJ exonuclease promotes short homology-dependent illegitimate recombination, but that the RecQ helicase suppresses it. In addition, we have shown that the overexpression of RecE and RecT enhances the frequencies of spontaneous and UV-induced illegitimate recombination and that the RecJ, RecF, RecO, and RecR functions are required for this RecE-mediated illegitimate recombination. Moreover, we have also indicated that RecQ plays a role in the suppression of RecE-mediated illegitimate recombination, with the participation of DnaB, Fis, ExoI, and H-NS. Models have been proposed for these modes of recombination: the DNA gyrase subunit exchange model for short homology-independent illegitimate recombination and the "double-strand break and join" model for short homology-dependent illegitimate recombination. Many features of these models remain to be tested in future studies.

Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica.

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.

Mutations in the gyrA gene in Salmonella enterica clinical isolates with decreased ciprofloxacin susceptibility.

The incidence of resistance to nalidixic acid has increased in the University General Hospital of Elche, Spain, and this paper describes the investigation of this phenomenon. This increase was mainly due to an increase of nalidixic-resistant Salmonella enterica serotype Enteritidis. Resistance to nalidixic acid is an indicator of decreased ciprofloxacin susceptibility (sensitivity 100%, specificity 96.7%). Strains that were resistant to nalidixic acid and exhibited decreased susceptibility to ciprofloxacin had a single mutation in QRDR of gyrA: Asp87-Asn, Asp87-Tyr or Ser83-Phe. The sensitivity of S. enterica strains to nalidixic acid should be tested and the breakpoint of ciprofloxacin established by MENSURA applied, instead of that of the NCCLS for these strains.

Quinolone resistance mechanisms in pneumococci.

Quinolones are widely used in the treatment of respiratory infections, in large part because of their activity against Streptococcus pneumoniae and other commonly encountered respiratory tract pathogens. Pneumococcal isolates that are resistant to these "respiratory quinolones" have now begun to emerge. Resistance is attributable to mutations affecting the intracellular targets of these drugs, topoisomerase IV and DNA gyrase; drug efflux contributes to quinolone resistance in some isolates. Most commonly, strains fully resistant to the newer quinolones have one or more mutations affecting DNA gyrase and topoisomerase IV. Although various agents of this class exhibit selectivity in primarily targeting one or the other of these enzymes, the passage of isolates in the presence of any agent can result in selection of mutations affecting both enzymes. Quinolone resistance in S. pneumoniae has arisen in heterogeneous genetic backgrounds but, ominously, has now appeared in strains that are well adapted for regional and global transmission.

[Resistance to quinolones and beta-lactams in Salmonella enterica due to mutations in topoisomerase-encoding genes, altered cell permeability and expression of an active efflux system]. / Resistencia a quinolonas y betalactámicos en Salmonella enterica, y su relación con mutaciones en las topoisomerasas, alteraciones en la permeabilidad celular y expresión de un mecanismo de expulsión activa.

BACKGROUND: The mechanisms of resistance to fluoroquinolones and beta-lactams were studied in isolates of Salmonella enterica resistant to both antimicrobial groups, isolated over time from two patients treated with fluoroquinolones. METHODS: The clonal relationships among the various strains was established by serotyping and pulsed-field gel electrophoresis. MICs for beta-lactams, quinolones, chloramphenicol and tetracycline were determined. Presence of beta-lactamases was ruled out by a colorimetric assay. Quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes were sequenced, and the relevance of the mutations in these regions was evaluated by complementation assays. Outer membrane protein profiles, the effect of phenylalanyl-arginyl-naphthylamide (PAN, 20 mg/l) on the MICs of several quinolones, and norfloxacin accumulation in the absence and in the presence of a metabolic inhibitor were also determined. RESULTS: The following mutations were found: gyrA (Asp87 --> Gly; Ser83 --> Phe; Asp87 --> Lys), gyrB (Ser463 --> Phe) and parC (Glu84 --> Gly). Altered outer membrane protein profiles, including decreased expression of a porin equivalent to OmpF from Escherichia coli was observed. Active efflux of norfloxacin was proved in both a clinical isolate and a mutant obtained in vitro. In the presence of PAN, nalidixic acid MICs decreased 4-32 times (except in one strain), pefloxacin MICs decreased 4-16 times for 5 out of 9 evaluated strains, and MICs of both norfloxacin and ciprofloxacin did not change or changed within a single dilution step. CONCLUSIONS: Quinolone-resistance is the consequence of a combination of mutations in topoisomerase-encoding genes, altered permeability and active efflux. Altered permeability and active efflux would also contribute to decreased susceptibility to beta-lactams.

PriA is essential for viability of the Escherichia coli topoisomerase IV parE10(Ts) mutant.

The parE10(Ts) mutation, which renders Escherichia coli thermosensitive for growth by inactivation of the essential E. coli topoisomerase topo IV, is lethal at all temperatures when PriA, the main replication restart protein, is absent. This lethality is suppressed by the activation of a PriA-independent replication restart pathway (dnaC809 mutation). This result suggests that topo IV acts prior to full-chromosome replication completion.

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin.

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.

In vitro fluoroquinolone-resistant mutants of Salmonella enterica serotype Enteritidis: analysis of mechanisms involved in resistance.

This study analysed the mechanisms involved in the acquisition of resistance to quinolones in mutants obtained in vitro of Salmonella enterica serotype Enteritidis. Two nalidixic acid-resistant (minimal inhibitory concentrations, MIC>256 mg/l), ciprofloxacin-susceptible (MIC 0.5 mg/l) clinical isolates of Salmonella Enteritidis with a mutation at amino acid codon Ser-83 of the gyrA gene were grown on plates containing increasing concentrations of ciprofloxacin. The increase in MIC to ciprofloxacin, sparfloxacin and trovafloxacin was totally or partially associated with over-expression of an AcrAB-like efflux pump. In addition, unidentified mechanism(s) may have been involved in the increased MIC to these antimicrobials. This study demonstrated that AcrAB-like efflux pumps appear to play a relevant role in the increase in MIC to some quinolones although, other, as yet undefined, mechanisms may be involved.

Molecular evaluation of antibiotic susceptibility: Tropheryma whipplei paradigm.

Tropheryma whipplei, the agent of Whipple's disease, grows fastidiously only in cell cultures without plaque production, and only three strains have been passaged. The formation of bacterial clumps in the supernatant precludes enumeration of viable bacteria and MIC determination. We evaluated the bacteriostatic effects of fluoroquinolones against two T. whipplei isolates by measuring the inhibition of the DNA copy number increase by real-time quantitative PCR. The analysis of the T. whipplei genome database allowed the identification not only of the gyrA gene but also the parC gene encoding the alpha subunit of the natural fluoroquinolone targets DNA gyrase (GyrA) and topoisomerase IV (ParC), respectively. The parC gene was detected in actinobacteria for the first time. High ciprofloxacin MICs (4 and 8 micro g/ml) were correlated with the presence in T. whipplei GyrA and ParC sequences with an alanine residue at positions 83 and 80 (Escherichia coli numbering), respectively. Alanines at these positions have previously been associated with increased fluoroquinolone resistance in E. coli and mycobacteria. However, the MIC of levofloxacin was low (0.25 micro g/ml). The same T. whipplei GyrA and ParC sequences were found in two other cultured strains and in nine uncultured tissue samples from Whipple's disease patients, allowing one to speculate that T. whipplei is naturally relatively resistant to fluoroquinolones.