Resistance to pefloxacin in Pseudomonas aeruginosa

Abstract

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 ...

Comparative in-vitro activity of new quinolones against clinical isolates and resistant mutants

“…We also tested a strain of P. aeruginosa with altered permeability due to reduced expression of Omp D2 combined with increased amount of lipopolysaccharides in the outer membrane. These alterations more markedly reduced the activity of quinolones than did deficiency of OmpF, which confirms our recent observation that both Omp D2 and lipo-polysaccharides are involved in the diffusion of quinolones through the outer membrane (Michea-Hamzehpour et al, 1991). Interestingly, we observed that in a P. aeruginosa strain with only a mutated DNA gyrase, similar changes in the MICs of quinolones were seen as observed for E. cloacae.…”

Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa

“…This indicates the importance of utilizing truly isogenic strains obtained without direct antibiotic selection. Other authors have also suggested a role for OprD in uptake of quinolones (16,17) or other antibiotics (24) in P. aeruginosa. These conclusions are based in part on poorly defined clinical or experimental animal isolates or on in vitro model liposome swelling experiments that have been criticized on other grounds (3,31,33).…”

“…Some, but not all, fluoroquinolone-resistant mutants are cross resistant to imipenem and lack OprD. For example, Michea-Hamzehpour et al demonstrated that decreased fluoroquinolone susceptibility was associated with a decrease or loss of OprD and proposed that OprD can catalyze the facilitated diffusion of fluoroquinolone as it does for imipenem (16,17).…”

A pleiotropic, posttherapy, enoxacin-resistant mutant of Pseudomonas aeruginosa

“…While several strains with decreased susceptibilities to several agents have been isolated, which suggests a mutation in nalB, there are biochemical differences that are commensurate with different mutations encoding multiple resistance. Some quinolone-resistant P. aeruginosa strains contain a new 54-kDa outer membrane protein (14, 18) or lack a 31.5-kDa outer membrane protein (9, 17), possibly OprF, or have decreased levels of proteins D2 and Hi or Gl (5,20,23). Several workers have also reported changes in lipopolysaccharide (LPS) (6,18,34).…”

“…Many laboratory mutants contain alleles of nalB and require nalidixic acid MICs of >500 ,ug/ml and are cross resistant to carbenicillin, ureidopenicillins, chloramphenicol, and novobiocin (33, 34). Resistant strains have arisen during experimental P. aeruginosa infections, with several classes of mutants isolated with mutations resembling naL4 and nfxC (10,22,23). Quinolone-resistant P. aeruginosa isolated from patients may also have the nal4 or nalB phenotype (43) or a nalB-like phenotype lacking OprF.…”