Molecular Mechanisms of Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Abstract

Twenty P. aeruginosa isolates were collected from six cystic fibrosis (CF) patients, aged 27 to 33, in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulse-field gel electrophoresis (PFGE) or ribotyping. Five of the patients had isolates with the same PFGE or ribotyping patterns in 1997 as in 1994, and ciprofloxacin had a two-to fourfold higher MIC for the isolates collected in 1997 than those from 1994. Genomic DNA was amplified for gyrA, parC, mexR, and nfxB by PCR and sequenced. Eleven isolates had mutations in gyrA, seven isolates had mutations at codon 83 (Thr to Ile), and four isolates had mutations at codon 87 (Asp to Asn or Tyr). Sixteen isolates had mutations in nfxB at codon 82 (Arg to Leu). Increased amounts of OprN were found in six isolates and OprJ in eight isolates as determined by immunoblotting. No isolates had mutations in parC or mexR. Six isolates had mutations in efflux pumps without gyrA mutations. The average number of mutations was higher in isolates from 1997 than in those from 1994. The results also suggested that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-CF patients, in which mutations in gyrA and parC dominate (S. Jalal and B. Wretlind, Microb. Drug Resist. 4:257-261, 1998).Fluoroquinolones (FQ) are the only available antibiotics for oral treatment of Pseudomonas aeruginosa infections in most countries. However, P. aeruginosa easily becomes resistant to these drugs, which severely limits their usefulness. The main mechanisms of resistance are mutations in the target genes, those encoding DNA gyrase (gyrA) and topoisomerase IV (parC) (3, 9), and mutations in regulatory genes for drug efflux pumps. Three different multidrug efflux-pump systems have been identified, MexAB-OprM, MexCD-OprJ, and MexEFOprN, which are regulated by mexR (nalB), nfxB, and mexT (nfxC), respectively (4,8,17).In this study, 20 quinolone-resistant P. aeruginosa isolates from cystic fibrosis (CF) lungs have been analyzed for gyrA, parC, nfxB, and mexR genes and the expression of membrane proteins mexT (nfxC)-related OprN and nfxB-related OprJ associated with FQ resistance. Our aim was to determine molecular mechanisms for quinolone resistance of repeated isolates of P. aeruginosa from CF patients. MATERIALS AND METHODSBacterial strains. Twenty P. aeruginosa isolates were collected from six CF patients (ages, 27 to 33) with chronic P. aeruginosa infections (duration, 18 to 26 years) in 1994 (9 isolates) and 1997 (11 isolates) at the CF Center, Copenhagen, Denmark, and were typed by pulsed-field gel electrophoresis (PFGE) ( Table 1). However, four isolates (B1, B2, B3, and D1) could not be typed by PFGE, probably because of high DNase activities, and were subjected to ribotyping using Riboprinter (Qualicon, Wilmington, Del.). All patients had been exposed to repeated treatment periods with ciprofloxacin since 1986, and all of them had received at least 15 treatment courses (14 days/course)...

Overexpression of MexCD-OprJ Reduces Pseudomonas aeruginosa Virulence by Increasing Its Susceptibility to Complement-Mediated Killing

“…This notion is supported by the low number of natural nfxB mutant strains isolated in clinical settings (21)(22)(23)(24) and is in agreement with the results of the experiments performed by Hirakata et al (10), who showed that an nfxB mutant was less virulent that the parent strain in a mouse model of bacteremia. In contrast, nfxB mutants are highly prevalent among P. aeruginosa isolates from cystic fibrosis patients with chronic respiratory infections (25). Indeed, mutation of nfxB is involved in the early adaptation to the chronic setting (26), perhaps by promoting biofilm growth.…”

Real-Time Monitoring of nfxB Mutant Occurrence and Dynamics in Pseudomonas aeruginosa Biofilm Exposed to Subinhibitory Concentrations of Ciprofloxacin

“…A variety of substrates can be accommodated by MexCD-OprJ, including antibiotics used in clinics, such as fluoroquinolones, macrolides, and glycylcyclines (9), as well as biocides like triclosan and chlorhexidine, dyes, detergents, and organic solvents (10). Efflux-mediated resistance due to the overexpression of MexCD-OprJ is commonly found in resistant P. aeruginosa isolates from chronic lung infection in cystic fibrosis (CF) patients and occasionally in non-CF patients (11)(12)(13).…”

Amino Acid Substitutions Account for Most MexS Alterations in Clinical nfxC Mutants of Pseudomonas aeruginosa

“…This phenotype was attributed to (i) reduced intracellular levels of the Pseudomonas quinolone signal (PQS), caused by a shortage of a metabolic precursor (kynurenine or 4-hydroxy-2-heptylquinoline [HHQ]) exported by the pump (17,19), and (ii) MexT acting as a global regulator and indirectly impairing the T3SS in an MexEF-OprN-independent way (18). Information about the rates and traits of nfxC mutants in cystic fibrosis (CF) patients (20,21) and non-CF patients (12,(22)(23)(24) remains scarce. As a plausible explanation, the low virulence of these mutants would be detrimental to their survival in the host or in the hospital setting and would account for their infrequent isolation from clinical samples.…”