title: Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy.
abstract: Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.
authors.affiliation: Department of Microbiology and Biomedical Sciences, Polytechnic University of Marche Medical School, 60131 Ancona, Italy.
authors.forename: Maria Pia
authors.initials: MP
authors.lastname: Montanari
authors.forename: Emily
authors.initials: E
authors.lastname: Tili
authors.forename: Ileana
authors.initials: I
authors.lastname: Cochetti
authors.forename: Marina
authors.initials: M
authors.lastname: Mingoia
authors.forename: Aldo
authors.initials: A
authors.lastname: Manzin
authors.forename: Pietro Emanuele
authors.initials: PE
authors.lastname: Varaldo
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001692:Biological Transport; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase / Q000235:genetics; D027101:DNA Topoisomerase IV / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D007558:Italy; D008826:Microbial Sensitivity Tests; D017354:Point Mutation; D012703:Serotyping; D013296:Streptococcus pneumoniae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D000900:Anti-Bacterial Agents; D024841:Fluoroquinolones; D002939:Ciprofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase