AAC Accepts, published online ahead of print on 10 September 2007

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00391-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In Vitro Infection Model Characterizing the Effect of Efflux Pump Inhibition on Prevention of Resistance to Levofloxacin and Ciprofloxacin in Streptococcus pneumoniae

Arnold Louie^*, David L. Brown, Weiguo Liu, Robert W. Kulawy, Mark R. Deziel, and George L. Drusano

Emerging Infections and Pharmacodynamics Laboratory, Ordway Research Institute, Albany, NY 12208

^* To whom correspondence should be addressed. Email: alouie{at}ordwayresearch.org.

Background: The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae (Spn) is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by Spn may facilitate emergence of fluoroquinolone resistance.

Methods: Using an in vitro pharmacodynamic infection system, a wild-type Spn (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to simulated human serum concentration-time profiles for ciprofloxacin 750 mg po Q 12 hr and levofloxacin 500 and 750 mg po QD (± continuous infusions of 20 µg/ml of reserpine).

Results: The MICs of Spn-058 to ciprofloxacin and levofloxacin were both 1 µg/ml when susceptibility testing was conducted for each antibiotic alone and in the presence of reserpine. For Spn-RC2, the MICs for levofloxacin alone and with reserpine were also 1 µg/ml; the MICs for ciprofloxacin were 2 and 1 µg/ml, respectively, when determined alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For the treatment of Spn-058, simulated regimens for ciprofloxacin 750 Q 12 hr or levofloxacin 500 mg QD were associated with emergence of fluoroquinolone resistance. However, the use of ciprofloxacin 750 mg q 12 hr and levofloxacin 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented emergence of resistance. For Spn-RC2, levofloxacin 500 mg QD was associated with emergence of resistance that was again prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin 750 mg q 12 h also rapidly selected for ciprofloxacin-resistant mutants from Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the time till emergence of resistance. Levofloxacin 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at baseline, Spn-RC2 had greater efflux pump expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of efflux pump expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in efflux pump expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged with suboptimal antibiotic regimens had a stable increase in efflux pump expression.

Conclusion: Levofloxacin 500 mg QD in combination with reserpine, an efflux pump inhibitor, or the use of 750 mg QD of levofloxacin alone, killed wild-type Spn and Spn that overexpressed reserpine-inhibitable efflux pumps and was highly effective in preventing emergence of fluoroquinolone resistance in Spn during therapy. Ciprofloxacin 750 mg Q 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented emergence of resistance for Spn-058 but not for Spn-RC2, the efflux pump over expressing strain.

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