Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem

Abstract

In Pseudomonas aeruginosa, resistance to imipenem is mainly related to a lack of protein OprD and resistance to fluoroquinolones is mainly related to alterations in DNA gyrase. However, strains cross resistant to fluoroquinolones and imipenem have been selected in vitro and in vivo with fluoroquinolones. We investigated the mechanisms of resistance to fluoroquinolones in 30 clinical strains of P. aeruginosa resistant to ciprofloxacin (mean MIC, >8 g/ml), 20 of which were also resistant to imipenem (mean MIC, >16 g/ml). By immunoblotting, OprD levels were markedly decreased in all of the imipenem-resistant strains. Plasmids carrying the wild-type gyrA gene (pPAW207) or gyrB gene (pPBW801) of Escherichia coli were introduced into each strain by transformation. MICs of imipenem did not change after transformation, whereas those of ciprofloxacin and sparfloxacin dramatically decreased (25-to 70-fold) for all of the strains. For 28 of them (8 susceptible and 20 resistant to imipenem), complementation was obtained with pPAW207 but not with pPBW801. After complementation, the geometric mean MICs of ciprofloxacin and sparfloxacin (MICs of 0.3 g/ml and 0.5 g/ml, respectively) were as low as those for wild-type strains. Complementation was obtained only with pPBW801 for one strain and with pPAW207 and pPBW801 for one strain highly resistant to fluoroquinolones. These results demonstrate that in clinical practice, gyrA mutations are the major mechanism of resistance to fluoroquinolones even in the strains of P. aeruginosa resistant to imipenem and lacking OprD, concomitant resistance to these drugs being the result of the addition of at least two independent mechanisms. Fluoroquinolones and carbapenems represent major progress in antimicrobial therapy for Pseudomonas aeruginosa infections. Because of good antibacterial activity, tissue diffusion, and oral bioavailability, fluoroquinolones have been widely used. However, the intensive use of fluoroquinolones has led to the selection of quinolone-resistant strains of P. aeruginosa (30,39). Imipenem, a carbapenem, has also been widely used in hospitals, and imipenem-resistant strains of P. aeruginosa have been readily selected (23, 32). Since there is generally no crossresistance between fluoroquinolones and other antibiotics, fluoroquinolones have been used for the treatment of infections caused by strains of P. aeruginosa already resistant to ␤-lactams, leading to the emergence of strains with associated resistance to quinolones and ␤-lactams, including imipenem. As an example, 22% of clinical strains of P. aeruginosa isolated in 1991 in French intensive care units were resistant to imipenem and half of them were also resistant to ciprofloxacin (19).Three mechanisms of resistance are known to cause quinolone resistance in P. aeruginosa (3, 39): alteration in DNA gyrase by mutations in gyrA or gyrB genes, decreased drug accumulation by decreased permeability of the cell wall, and enhanced efflux (22). gyrA mutations appear to be the most prominent cause of resistance in clin...

Quantitative Contributions of Target Alteration and Decreased Drug Accumulation to Pseudomonas aeruginosa Fluoroquinolone Resistance

“…1A. In accordance with the findings of several previous studies (25,26,27,28,29,30,31), the most frequently observed mutation, T83I, was encoded in the QRDR of gyrA, whereas mutations in gyrB were less frequent (30,32,33). Here the majority of mutations were found at amino acid positions 466 to 468; however, we also found two isolates with an I529V mutation, which, to our knowledge, has not been described previously.…”

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

“…Moreover, four of nine isolates of the former pair and 10 of 12 isolates of the latter pair also produced a CF166 A2 NM 16 4 T83I R82L A1 NM 2 1 R82L A3 M 2 1 R82L CF222 B1 NM 8 2 T83I R82L B3 NM 16 8 T83I R82L wk wk B2 M 2 2 R82L wk wk B4 M 2 0.5 wk CF86 C1 NM 4 2 D87N R82L C3 NM 16 8 T83I R82L wk detectable level of OprN or OprJ proteins related to the genes mexT (nfxC) and nfxB, respectively. Mutations in the target enzymes, DNA gyrase (gyrA) and topoisomerase IV (parC), and reduced intracellular accumulation of the drug either by decreased uptake and/or by increased efflux have been reported to be the main mechanisms of resistance to FQs in several bacterial species (1,7,10). A high level of resistance is commonly associated with gyrA and parC or two or more point mutations in the same gene or mutations involving more than one gene.…”

Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa

“…Part of the reason for the selection of gyrA mutations is probably the greater reduction in susceptibility that they afford. In P. aeruginosa, a 16-70-fold increase in MIC can be attributed to the T83I mutation (Cambau et al, 1995;Diver et al, 1991;Kureishi et al, 1994). By contrast, the transposable element mutations described by Breidenstein et al (2008) result in much more modest (2-46) increases in MIC.…”