C. Lascols

Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli

OBJECTIVES Besifloxacin is a new fluoroquinolone in development for ocular use. We investigated its mode of action and resistance in two major ocular pathogens, Streptococcus pneumoniae and Staphylococcus aureus, and in the reference species Escherichia coli. METHODS Primary and secondary targets of besifloxacin were evaluated by: (i) mutant selection experiments; (ii) MIC testing of defined topoisomerase mutants; and (iii) inhibition and cleavable complex assays with purified S. pneumoniae and E. coli DNA gyrase and topoisomerase IV enzymes. RESULTS Enzyme assays showed similar besifloxacin activity against S. pneumoniae gyrase and topoisomerase IV, with IC(50) and CC(25) of 2.5 and 1 microM, respectively. In contrast to ciprofloxacin and moxifloxacin, besifloxacin was equally potent against both S. pneumoniae and E. coli gyrases. DNA gyrase was the primary target in all three species, with substitutions observed at positions 81, 83 and 87 in GyrA and 426 and 466 in GyrB (E. coli numbering). Topoisomerase IV was the secondary target. Notably, resistant mutants were not recovered at 4-fold besifloxacin MICs for S. aureus and S. pneumoniae, and S. aureus topoisomerase mutants were only obtained after serial passage in liquid medium. Besifloxacin MICs were similarly affected by parC or gyrA mutations in S. aureus and S. pneumoniae and remained below 1 mg/L in gyrA-parC double mutants. CONCLUSIONS Although mutant selection experiments indicated that gyrase is a primary target, further biochemical and genetic studies showed that besifloxacin has potent, relatively balanced activity against both essential DNA gyrase and topoisomerase IV targets in S. aureus and S. pneumoniae.

Low selection of topoisomerase mutants from strains of Escherichia coli harbouring plasmid-borne qnr genes

OBJECTIVES To investigate mutations in the type II topoisomerase genes in quinolone-resistant mutants selected from bacteria harbouring plasmid-borne qnr genes. METHODS Mutants were selected by nalidixic acid, ciprofloxacin and moxifloxacin from two Escherichia coli reference strains and corresponding transconjugants harbouring qnrA1, qnrA3, qnrB2 or qnrS1 genes. RESULTS The proportion of resistant mutants selected by the three quinolones was, respectively, in the same range for qnr-positive transconjugants and reference strains. Only 20% (65/329) of the mutants selected from the transconjugants showed a gyrase mutation, whereas 79% (94/119) of those from the reference strains without a qnr gene did (P < 0.0001). At four times the MIC of the selector quinolone, gyrA mutants represented 49% and 95% of the mutants selected with nalidixic acid, 4% and 94% with ciprofloxacin and 0% and 54% with moxifloxacin for qnr-positive transconjugants and reference strains, respectively. Mutations within gyrA were distributed at codon 87 (D87G, H, N or Y) and at codon 83 (S83L) with three novel mutations (gyrA Ser83stop, gyrA Asp82Asn and gyrB insertion of Glu at 465) and three rare mutations (gyrA Gly81Asp, gyrA Asp82Gly and gyrA Ser431Pro), mainly obtained from reference strains after moxifloxacin selection. Strikingly, none of the mutants selected by moxifloxacin from qnr-positive transconjugants harboured a mutation in the topoisomerase genes. CONCLUSIONS Topoisomerase mutants are rarely selected by ciprofloxacin and moxifloxacin from strains harbouring qnr. This suggests that the quinolone resistance-determining region domains are protected from quinolones by the Qnr protein and consequently other mechanisms are developed to acquire a further step of fluoroquinolone resistance.

Clarithromycin resistance of Helicobacter pylori has a major impact on the efficacy of the omeprazole-amoxicillin-clarithromycin therapy

Clarithromycin resistance of Helicobacter pylori is relatively frequent in France and is assumed to be the main cause of failure of the proton pump inhibitor-amoxicillin-clarithromycin (PPI-AC) therapy, which is the first-line regimen in our country. We determined the respective effects of clarithromycin primary and secondary resistances on efficacy of the PPI-AC regimen and examined whether failures were associated with persistence of the same strain or with emergence of a new one. Hundred and twenty three H. pylori-infected patients were treated for seven days with omeprazole 20 mg b.d., amoxicillin 1 g b.d., and clarithromycin 500 mg b.d. Eradication was assessed by breath test in 102 patients. MICs of clarithromycin were determined by E-test. Strain genotyping was performed by random amplified polymorphic DNA. The pre-treatment and post-treatment prevalences of clarithromycin resistance were 18.7% (23/123) and 69.2% (9/13), respectively. The rates of eradication were 67.6% (69/102), 78.8% (67/85), and 11.8% (2/17) for all, susceptible and resistant strains, respectively. The post-treatment isolate was available for six patients with a susceptible pre-treatment isolate and a persistent infection; resistance emerged in two patients and was associated with persistence of the pre-treatment strain in one and with selection of a new strain in the other. In conclusion, in our hospital, failures of the PPI-AC therapy are related to both clarithromycin primary and secondary resistances but emergence of secondary resistance does not explain all failures in the initial clarithromycin-susceptible group. In that group a new strain can emerge after failure.

First detection of plasmid-mediated quinolone resistance in the community setting and in hospitalized patients in Switzerland

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