Role of lon, an ATP-dependent protease homolog, in resistance of Pseudomonas aeruginosa to ciprofloxacin

Abstract

With few novel antimicrobials in the pharmaceutical pipeline, resistance to the current selection of antibiotics represents a significant therapeutic challenge. Microbial persistence in subinhibitory antibiotic environments has been proposed to contribute to the development of resistance. Pseudomonas aeruginosa cultures pretreated with subinhibitory concentrations of ciprofloxacin were found to exhibit an adaptive resistance phenotype when cultures were subsequently exposed to suprainhibitory ciprofloxacin concentrations. Microarray experiments revealed candidate genes involved in such adaptive resistance. Screening of 10,000 Tn5-luxCDABE mutants identified several mutants with increased or decreased ciprofloxacin susceptibilities, including mutants in PA1803, a close homolog of the ATP-dependent lon protease, which were found to exhibit > or = 4-fold-increased susceptibilities to ciprofloxacin and other fluoroquinolones, but not to gentamicin or imipenem, as well as a characteristic elongated morphology. Complementation of the lon mutant restored wild-type antibiotic susceptibility and cell morphology. Expression of the lon mutant, as monitored through a luciferase reporter fusion, was found to increase over time in the presence of subinhibitory ciprofloxacin concentrations. The data are consistent with the hypothesis that the induction of Lon by ciprofloxacin is involved in adaptive resistance.

FIG. 1.

Survival ability of P. aeruginosa in ciprofloxacin at 2× MIC following growth in subinhibitory concentrations of ciprofloxacin. Cultures of P. aeruginosa strain H103 were grown in the presence (□, 0.01 μg/ml; ▵, 0.03 μg/ml) or absence (⧫) of subinhibitory concentrations of ciprofloxacin to mid-logarithmic phase; then they were washed and exposed to 0.2 μg/ml ciprofloxacin. CFU were counted every 15 min for 2 h. Results from a representative data set of four independent experiments are shown.

FIG. 2.

Percentage of genes in each of the gene functional categories exhibiting expression changes after growth of P. aeruginosa in subinhibitory and inhibitory concentrations of ciprofloxacin. A.A., amino acid; FA, fatty acid.

FIG. 3.

Induction of the lon::luxCDABE fusion (H1105) with 0.3× MIC ciprofloxacin (shaded bars) over expression in untreated H1105 cultures (open bars). Results are modes from four independent experiments.

FIG. 4.

Gram-stained P. aeruginosa strains grown for 3 h in the presence or absence of subinhibitory ciprofloxacin concentrations. As evident from the Gram stain images of cells grown in the absence or presence of 0.3× MIC ciprofloxacin [0.03 μg/ml for H103 and H1105/lon⁺; 0.0075 μg/ml for H1105 (lon::lux)], cell division of P. aeruginosa mutants with alterations in the Lon protease homolog [H1105 (lon::lux)] was inhibited relative to that of wild-type H103 cells. Complementation of the lon mutant (H1105/lon⁺) restored cell division. All micrographs are at ×1,000 magnification.