In vitro and in vivo activity of ciprofloxacin/fosfomycin combination therapy against ciprofloxacin-resistant Shigella flexneri isolates

Objective: Ciprofloxacin resistance (CIP(R)) for Shigella isolates is becoming more prevalent. This study systematically investigated the antibacterial activity of ciprofloxacin (CIP)/fosfomycin (FOS) combination in vitro and in vivo against CIP(R) S. flexneri isolates. Method: Eighty CIP(R) S. flexneri isolates were selected for synergy studies by the microtiter plate checkerboard assay. Two S. flexneri isolates (GN120471, CIP(R)FOS(R); GN120454, CIP(R)FOS(S)) were used to investigate the efficacy of the CIP/FOS combination by the time-kill methodology. Clinically relevant concentrations (CIP, 0.5, 1, or 2.5 μg/mL; FOS, 30, 150, or 300 μg/mL) were combined, and the colony counts were conducted at 3, 5, 8, and 24 hours. The in vivo activity of the CIP/FOS combination was assessed using a Galleria mellonella larvae model. Results: In checkerboard assays, 31 strains (38.75%) showed synergy for the CIP/FOS combination. For the isolate GN120471, monotherapy with CIP or FOS at all concentrations produced little or no bacterial killing, while the CIP/FOS combination produced enhanced bacterial killing with FOS concentrations of 150 and 300 μg/mL, especially when combined with CIP at 2.5 μg/mL. For the isolate GN120454, the CIP/FOS combination at all concentrations produced more rapid and extensive killing (up to 5log(10) colony forming units (CFU)/mL with many combinations) than with either antibiotic alone. Mortality at 96 hours was around 80% at approximately 10(4) CFU/larva for GN120471 and GN120454. When CIP at 2.5 μg/mL was combined with FOS at 150 μg/mL for the bactericidal activity in vivo, the survival rates for CIP/FOS combination against GN120471-infected and GN120454-infected larvae were significantly higher than that of CIP (68.75% vs 25%, P=0.013; 81.25% vs 37.5%, P=0.012, respectively). Conclusion: Against CIP(R) S. flexneri isolates, the CIP/FOS combination induced synergy, and increased bacterial killing in vitro and in a simple invertebrate model of infection.