Resistance mechanisms selected after in vitro exposure to 12 quinolones were analyzed for Pseudomonas aeruginosa. Efflux-type mutants were predominant. Quinolones differed in their ability to select a particular efflux system. While the newer fluoroquinolones favored the MexCD-OprJ system, the older quinolones selected exclusively the MexEF-OprN or MexAB-OprM systems. A protonable C-7 substituent in combination with a C-6 fluorine atom is a structural determinant of quinolones involved in efflux pump substrate specificity.

The ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo was studied in a model of acute Pseudomonas aeruginosa pneumonia in rats. Twelve hours after intratracheal inoculation of 10 6 CFU of P. aeruginosa strain PAO1 enmeshed in agar beads, two groups of 12 rats were treated by three intraperitoneal injections of each antibiotic given every 5 h. Dosing regimens were chosen to obtain a comparable area under the concentration-time curve from 0 to infinity/MIC ratio of 27.9 min for trovafloxacin (75 mg/kg of body weight) and of 32.6 min for ciprofloxacin (12.5 mg/kg). Twelve rats were left untreated and served as controls. Rats were sacrificed 12 h after the last injection (34 h after infection) for lung bacteriological studies. Selection of resistant bacteria was determined by plating lung homogenates on Trypticase soy agar plates containing antibiotic. In untreated animals, the frequency of resistant colonies was 10-fold higher than in agar beads. Compared to controls, both treatment regimens resulted in a 2-log reduction of lung bacterial load. The frequency of resistant colonies was 10-fold less with trovafloxacin than with ciprofloxacin at twice the MIC (7.4 ؋ 10 ؊5 versus 8.4 ؋ 10 ؊4 , respectively) (P < 0.05) and at four times the MIC (6.2 ؋ 10 ؊4 versus 5.0 ؋ 10 ؊5 , respectively) (P < 0.05). A multidrug resistance phenotype typical of efflux mutants was observed in all 41 randomly tested colonies obtained from treated and untreated rats. In agreement with in vitro results, trovafloxacin and ciprofloxacin preferentially selected MexCD-OprJ and MexEF-OprN overproducers, respectively. These results demonstrate the differential ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo and highlight the rapid emergence of those mutants, even without treatment.

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 ...

The ability of six antibiotics to produce resistance by stepwise selection on agar medium was assessed in 24 gram-negative rods. Escherichia coli was the strain least prone to selection of resistance, whereas Pseudomonas aeruginosa frequently developed resistance to all antibiotics. When used alone, ciprofloxacin, pefloxacin, amikacin, ceftazidime and cefpirome were associated with a comparable risk of acquired resistance (in 14 to 17 out of 24 strains); imipenem selected resistant strains in 10/24 isolates (5/18 in non-pseudomonas strains). The number of strains exhibiting cross resistance with structurally unrelated antibiotics was 11 after pefloxacin treatment, eight after exposure to ciprofloxacin, six after ceftazidime, and one after imipenem or cefpirome. The combination of ciprofloxacin with amikacin was less efficient in reducing acquisition of resistance than the combination of ciprofloxacin with a beta-lactam: ciprofloxacin plus cefpirome was especially potent in this respect.

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46 degrees C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25 degrees C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.