Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin

Mechanisms of resistance to pefloxacin were investigated in four isogenic Pseudomonas aeruginosa strains: S (parent isolate; MIC, 2 ,ug/ml), PT1 and PT2 (posttherapy isolates obtained in animals; MICs, 32 and 128 ,ug/ml, respectively), and PT2-r (posttherapy isolate obtained after six in vitro subpassages of PT2; MIC, 32 ,ug/ml). [2-3H]adenine incorporation (indirect evidence of DNA gyrase activity) in EDTA-permeabilized cells was less affected by pefloxacin in PT2 and PT2-r (50% inhibitory concentration, 0.27 and 0.26 ,ug/ml, respectively) than it was in S and PT1 (50% inhibitory concentration, 0.04 and 0.05 ,ug/ml, respectively). Reduced [14C]pefloxacin labeling of intact cells in strains PT1 and PT2 correlated with more susceptibility to EDTA and the presence of more calcium (P < 0.05) and phosphorus in the outer membrane fractions. Outer membrane protein analysis showed reduced expression of protein D2 (47 kDa) in strains PT1 and PT2. Other proteins were apparently similar in all strains. The addition of calcium chloride (2 mM) to the sodium dodecyl sulfate-solubilized samples of outer membrane proteins, before heating and Western blotting, probed with monoclonal antibody anti-OmpF showed electrophoretic mobility changes of OmpF in strains PT1 and PT2 which were not seen in strain S. Calcium-induced changes were reversed with ethyleneglycoltetraacetate.Decreased [14C]pefloxacin labeling was further correlated with an altered lipopolysaccharide pattern and increased 3-deoxy-D-mannooctulosonic acid concentration (P < 0.01). These findings suggested that resistance to pefloxacin is associated with altered DNA gyrase in strain PT2-r, with altered permeability in PT1, and with both mechanisms in PT2. The decreased expression of protein D2 and the higher calcium and lipopolysaccharide contents of the outer membrane could be responsible for the permeability deficiency in P. aeruginosa.The activities of fluoroquinolones against Pseudomonas aeruginosa have been fully documented (45), but bacteria can develop resistance to these agents (12,18,19,36,38). This resistance results from reduced permeability of the outer membrane (OM), altered DNA gyrase, or a combination of the two processes (7,8,18,36,38). However, mechanisms of resistance at the molecular level are not yet entirely elucidated (45).In a pseudomonal peritonitis model, we showed previously that resistance emerges rapidly when mice are treated with pefloxacin or ciprofloxacin (26). In mice infected with a quinolone-susceptible P. aeruginosa isolate, posttherapy variants (strain PT1) emerged after a single dose of pefloxacin, showing an 8-to 16-fold decrease in susceptibility to quinolones. Subinoculation of a PT1 strain, followed by therapeutic exposure to ciprofloxacin, produced a highly resistant posttherapy variant (strain PT2) for which the MIC was increased 64-fold (26).We report here results of further studies on the mechanism of resistance of these strains. As expected, we observed involvements of DNA gyrase and permeability, but we were surprised by ...

Section: Discussionmentioning

confidence: 99%

Resistance to pefloxacin in Pseudomonas aeruginosa

Michéa-Hamzehpour

Lucain

Péchère

1991

“…Such systems have been described in E. coli (10,14) and P. aeruginosa (7,13,20). In the former, mutations in the regulatory marRAB region result in decreased expression of OmpF and alterations of unidentified OM proteins (9,32), while in the latter antibiotic efflux is linked to the overexpression of OprK (29) or OprM (22), two OM proteins of about 50 to 54 kDa.…”

Section: Discussionmentioning

confidence: 99%

Evidence for an efflux pump in multidrug-resistant Campylobacter jejuni

Charvalos

Tselentis

Hamzehpour

et al. 1995

Mechanisms of drug resistance in Campylobacter jejuni were investigated. Mutant strains 34PEF r , which was resistant to pefloxacin (128-fold increase in the MIC), and 34CTX r , which was resistant to cefotaxime (32-fold increase in the MIC) and which was derived from the susceptible parent 34 s , were obtained by serial passages on pefloxacin and cefotaxime gradient plates, respectively. Both mutants showed cross-resistance to erythromycin, chloramphenicol, tetracycline, ␤-lactams, and quinolones. While the quinolone resistance of strain PEF r could be explained by a mutation at codon 86 of the gyrA gene, the multidrug resistance phenotype of both strains was further investigated. Accumulation of pefloxacin, ciprofloxacin, and minocycline was measured by fluorometry and was found to be lower in the mutant strains than in the parent strain. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone, however, completely abolished this difference. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations from both mutant strains showed overexpression of two proteins of 55 and 39 kDa which were absent from the outer membranes of the wild-type strain. These results indicate that in C. jejuni 34PEF r and 34CTX r , multidrug resistance is associated with an efflux system with a broad specificity.Campylobacter jejuni is recognized as a leading cause of diarrhea in the world (3, 5). Typically, C. jejuni diarrhea is a self-limiting disease lasting up to 5 days, but it may persist longer and may develop into severe forms, especially in children and immunocompromised patients (2, 3).Fluoroquinolones are powerful antimicrobial agents against many gram-negative enteric bacteria including C. jejuni (4,35,37). Besides their potent in vitro activity against susceptible organisms (37), quinolones can also achieve high concentrations in the gastrointestinal tract (11). Nevertheless, the resistance of C. jejuni to fluoroquinolones during therapy has been reported (1,12,15,30,31).In gram-negative bacteria three mechanisms of quinolone resistance have been described, including alterations in the DNA gyrase structure (17,26,38), decreased outer membrane permeability (10), and export through an active efflux system (10,18,21).Quinolone-resistant mutants of C. jejuni selected in vitro by single-step quinolone exposure have been shown to be due to point mutations in the gyrA gene, the molecular target of these agents (16,36). However, little is known about another possible resistance mechanism, namely, elimination of antibiotics through an active efflux system. This mechanism was investigated by stepwise in vitro selection of C. jejuni mutants resistant to the quinolone pefloxacin or to the ␤-lactam cefotaxime. Study of the cross-resistance of the generated mutants provided evidence for an active antibiotic efflux system in C. jejuni. MATERIALS AND METHODSBacterial strains and selection of resistant mutants. The pefloxacin-susceptible strain 34 s was isolated from a stool specimen...

“…In contrast, Hirai et al (8) and Legakis et al (13) have observed the appearance of an additional OMP of 54 kDa in P. aeruginosa for mutants which exhibited cross-resistance to quinolones, tetracycline, and chloramphenicol. It was suggested that the new protein would act as a permeability barrier (8).…”

mentioning

confidence: 89%

Susceptibility of Xanthomonas maltophilia to six quinolones and study of outer membrane proteins in resistant mutants selected in vitro

Lecsö-Bornet

Pierre

Sarkis-Karam

et al. 1992

The in vitro susceptibilities of 75 clinical isolates of Xanthomonas maltophilia to nalidixic acid, five fluoroquinolones, latamoxef, and doxycycline were determined. Spontaneous mutants were selected, at a frequency of about 10'-to 10-from four strains by culturing the strains in the presence of each quinolone.Selection in the presence of nalidixic acid provided mutants that were either resistant only to that compound or that exhibited cross-resistance to all the fluoroquinolones tested. Cross-resistance was always observed for mutants selected on any of the five fluoroquinolones. It was always associated with chloramphenicol resistance and, frequently, with doxycycline resistance. The electrophoretic alterations of the outer membrane proteins of the mutants suggest that different mechanisms may be involved in quinolone resistance in X. maltophilia.Xanthomonas maltophilia has recently been isolated with increasing frequency and is mainly responsible for nosocomial infections in compromised hosts (17). Its natural resistance restricts the choice of antibiotics for use in the treatment of infected patients (1, 2, 11, 16), and quinolones have been considered as a possible option for therapy (17). The agar diffusion method has revealed a particular phenotype of quinolone susceptibility for this species (12). In the current study, we determined the MICs of nalidixic acid, pefloxacin, ciprofloxacin, ofloxacin, temafloxacin, sparfloxacin, latamoxef, and doxycycline for 75 clinical isolates ofX. maltophilia. Since the emergence of mutants resistant to quinolones and unrelated drugs has been described in several species of gram-negative bacilli (5-7, 14, 18, 20), especially Pseudomonas aeruginosa (8,15), from 4 of the 75 strains of X. maltophilia, we selected mutants that were resistant to six quinolones. The resistance patterns of the mutants were studied, and the electrophoretic profiles of the outer membrane proteins (OMPs) were analyzed for each type of mutant.MICs were determined by a twofold serial agar dilution method on Mueller-Hinton agar by using a replicating spot device, with an inoculum of about 104 CFU per spot.Spontaneous mutants were selected from four susceptible strains by spreading approximately 2 x 108 CFU onto plates of Mueller-Hinton agar containing each of the six quinolones at concentrations of 4, 8, and 16 times the respective MICs for the strains. After 48 h at 37°C, the frequencies of mutation were evaluated and the MICs of the six quinolones, doxycycline, latamoxef, and chloramphenicol were determined. The mutants were classified according to their patterns of resistance to these nine antibiotics. From each type of mutant, OMP fractions were prepared as described previously (6) and were submitted to sodium dodecyl sulfatepolyacrylamide gel electrophoresis for separation (13.5% acrylamide, 0.36% bisacrylamide).