Differential Selection of Multidrug Efflux Mutants by Trovafloxacin and Ciprofloxacin in an Experimental Model of Pseudomonas aeruginosa Acute Pneumonia in Rats

Abstract

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.

Mutations in PA2491 ( mexS ) Promote MexT-Dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas aeruginosa

“…In any case, these data suggest that P. aeruginosa may well encounter circumstances in vivo where PA2491 and MexEF-OprN are needed. Also, the demonstration that mutants overexpressing MexEF-OprN were readily recovered from an experimental model of rat pneumonia in the absence of antibiotic selection (28) indicates that there is some advantage to MexEF-OprN expression in vivo, independent of antimicrobial export. The recent observation that mutational loss of the VsqR quorum-sensing and virulence regulator compromises mexEF-oprN expression in cells under oxidative stress (i.e., stress due to H 2 O 2 ) suggests that this system may normally be induced in response to this stress (29).…”

Emergence of the P2 Phenotype in Pseudomonas aeruginosa PAO1 Strains Involves Various Mutations in mexT or mexF

“…Although the MexEF-OprN efflux pump has been extensively characterized in the context of antibiotics, it has been suggested that the main purpose of the pump may not actually be antimicrobial extrusion (33) but may rather be metabolic function (34). For example, some isolates of P. aeruginosa from a mouse model of pneumonia express the MexEF-OprN pump without any exposure to antimicrobials (35). There is increasing evidence that the MexEF-OprN pump func- Collagenase activity measured by degradation of fluorescein labeled gelatin.…”

Pseudomonas aeruginosaVirulence Analyzed in aDictyostelium discoideumHost System

“…The model of acute P. aeruginosa pneumonia used in this study is based on the model by Cash et al (4) and has been modified as described previously (16). Briefly, bacteria (10 6 CFU) were injected in agarenmeshed beads into anesthetized male Sprague-Dawley rats (Charles River, Saint Aubin les Elbeufs, France) via the transtracheal route.…”

“…The strains PT149 (NfxC) and PT637 (NfxC, mexE) were described recently in detail (16). Briefly, the mexT transcriptional activator gene (24) is interrupted by an 8-bp insertion in our P. aeruginosa wild-type strain, PT5.…”

“…To establish the correlation between Dictyostelium and a mammalian host system, the three efflux pump mutants and the wild-type strain PT5 were tested in the well-established model of acute pneumonia in rats (4,16). Bacteria were injected into rat trachea, and virulence of strains was determined by assessing mortality and time to death.…”