title: Molecular characterization and phylogenetic analysis of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE gene loci in viridans group streptococci isolated from adult patients with cystic fibrosis.
abstract: OBJECTIVE
  Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs).
  
  
  METHODS
  The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis.
  
  
  RESULTS
  Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%).
  
  
  CONCLUSIONS
  Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.
authors.affiliation: Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Lisburn Road, Belfast BT9 7AD, Northern Ireland, UK.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Maeda
authors.forename: Mayumi
authors.initials: M
authors.lastname: Murayama
authors.forename: Colin E
authors.initials: CE
authors.lastname: Goldsmith
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Coulter
authors.forename: Charlene
authors.initials: C
authors.lastname: Mason
authors.forename: B Cherie
authors.initials: BC
authors.lastname: Millar
authors.forename: James S G
authors.initials: JS
authors.lastname: Dooley
authors.forename: Colm J
authors.initials: CJ
authors.lastname: Lowery
authors.forename: Motoo
authors.initials: M
authors.lastname: Matsuda
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Rendall
authors.forename: J Stuart
authors.initials: JS
authors.lastname: Elborn
authors.forename: John E
authors.initials: JE
authors.lastname: Moore
mesh_terms: D000328:Adult; D000900:Anti-Bacterial Agents / Q000009:adverse effects / Q000494:pharmacology* / Q000627:therapeutic use; D002939:Ciprofloxacin / Q000009:adverse effects / Q000494:pharmacology / Q000627:therapeutic use; D003550:Cystic Fibrosis / Q000150:complications; D027081:DNA Gyrase / Q000235:genetics*; D027101:DNA Topoisomerase IV / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004363:Drug Utilization; D005260:Female; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D015363:Quinolones / Q000009:adverse effects / Q000494:pharmacology* / Q000627:therapeutic use; D017422:Sequence Analysis, DNA; D013290:Streptococcal Infections / Q000382:microbiology; D034363:Viridans Streptococci / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D015363:Quinolones; D002939:Ciprofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase