Mechanisms of high-level resistance to quinolones in urinary tract isolates of Pseudomonas aeruginosa

Abstract

Twenty-eight strains of Pseudomonas aeruginosa with various degrees of norfloxacin resistance were isolated from patients with urinary tract infections. P. aeruginosa strains (norfloxacin MICs, 3.13 to 200 ,ug/ml) were transformed by either pPAW207 or pNF111 plasmid DNA, which included either the gyrA or nfxB gene, respectively. For transformants with pPAW207, norfloxacin MICs decreased 8-to 128-fold. It was suggested that moderate and high degrees of resistance to norfloxacin were expressed as a result of alterations in gyrA. No strain manifesting only an alteration in nfxB permeability was observed. The MICs of norfloxacin (200 ,ug/ml) for two P. aeruginosa strains, GN17605 and GN17434-2, were decreased following transformation not only by pPAW207 but also by pNF111. Analysis of outer membrane proteins disclosed the presence of a 54,000-Da protein in these parent strains that was not expressed in the pNF111 transformants. The level of accumulation of norfloxacin by the pNF111 transformant of GN17605 was higher than that by the parent strain. The norfloxacin susceptibility of DNA gyrase subunit A purified from GN17605 was only 1/35th that of the gyrase containing a subunit A from P. aeruginosa PAO1. These findings suggest that GN17605 is a gyrA-nfjxB double mutant and that strain GN17434-2 possesses double mutations in both nfxB and some unknown gene.Quinolones have a broad spectrum of antibacterial activity against gram-positive and -negative bacteria and have strong bactericidal activities (2,11,16,17 The mechanisms of resistance to quinolones in P. aeruginosa include reductions in the susceptibilities of intracellular targets to the drugs, DNA gyrase, and alterations in outer membrane permeability (6, 7). Many findings relating an alteration in an outer membrane protein and its effect on resistance to quinolones have been presented by Hirai et al. (6) . In addition, mechanisms of resistance, including alterations in both DNA gyrase and outer membrane permeability, have been reported recently in studies of clinical isolates of P. aeruginosa (8,19).In the study described here, P. aeruginosa strains isolated from patients with urinary tract infections were analyzed by transformation with plasmids carrying either a gyrA or a nfxB gene in order to determine the mechanisms of resistance to quinolones. MATERIALS AND METHODSBacterial strains. Twenty-eight strains of norfloxacin-resistant (norfloxacin MIC, >1.56 ,ug/ml) P. aeruginosa originating from patients with urinary tract infections were used in the study. These strains were isolated between 1988 and 1991 from various hospitals in Japan and were maintained in glycerol at

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus

“…Sequence analysis of the gyrA and grlA regions homologous to the E. coli gyrA QRDR showed that only grlA mutations occurred in first-step mutants, supporting the previous finding that in S. aureus DNA topoisomerase IV is a primary target of fluoroquinolones (6). This may in part explain why some firststep mutants reported in the literature (19,21,39) have no mutation in the QRDR of gyrA. In contrast, no mutation has been described in ParC for E. coli, even though it has been demonstrated that topoisomerase IV activity was inhibited by the coumarin and quinolone drugs, requiring concentrations 3-to 30-fold higher than those required to inhibit E. coli DNA gyrase (12,29).…”

“…The notion that another locus is involved in resistance to fluoroquinolones has been recently alluded to by groups studying resistance mechanisms in other bacteria (20,39). A third locus proposed to be responsible for resistance in S. aureus, termed flqA (36), was shown to be distinct from either the structural genes encoding DNA gyrase or NorA.…”

Resistance and Virulence of Pseudomonas aeruginosa Clinical Strains Overproducing the MexCD-OprJ Efflux Pump

“…Many studies have highlighted the fact that fluoroquinolones readily select for nfxB mutants in vitro (24,25,32,46) as well as in infected animals (29,31,43). Although very few clinical nfxB strains have been characterized to date (24,25,62), the circumstances of their selection in vivo have rarely been documented (56). Review of the treatments administered to the patients with strains 3308, 2439, 2126, and 1956 clearly showed that these nfxB mutants emerged under long-term single therapy (as cefpodoxime and ceftriaxone are quite inefficient against P. aeruginosa) with fluoroquinolones (mostly ciprofloxacin), as in the case described by Reinhardt et al (56).…”

“…Such changes in the NfxB phenotype have been attributed to variations in the levels of MexCD-OprJ production (45). More recently, the increased susceptibility of in vitro-selected nfxB mutants to ␤-lactams (except imipenem) was attributed to the decreased expression of system MexAB-OprM and/or deficient drug induction of intrinsic ␤-lactamase AmpC (13, 47).Since the first description of these mutants by Hirai et al (15) 2 decades ago, very few nfxB strains have been identified in the clinical setting (24,25,26,56,62), thus suggesting that these mutants are either underrecognized because of their variable drug resistance phenotype or poorly pathogenic for humans. In support of the latter proposal, Linares et al and Sanchez et al (38,57) recently reported that MexCD-OprJ overproduction is associated with reduced fitness, virulence, and type 3 secretion system (T3SS)-dependent cytotoxicity in laboratory nfxB mutants.…”

“…Since the first description of these mutants by Hirai et al (15) 2 decades ago, very few nfxB strains have been identified in the clinical setting (24,25,26,56,62), thus suggesting that these mutants are either underrecognized because of their variable drug resistance phenotype or poorly pathogenic for humans. In support of the latter proposal, Linares et al and Sanchez et al (38,57) recently reported that MexCD-OprJ overproduction is associated with reduced fitness, virulence, and type 3 secretion system (T3SS)-dependent cytotoxicity in laboratory nfxB mutants.…”