Synergistic effect of antibiotic combinations on Staphylococcus aureus biofilms and their persister cell populations

Treatment of staphylococcal infections is difficult due to multidrug resistance with their persister forms posing an added threat of recalcitrant infections. Antibiotic combinations are widely studied as an alternative strategy to combat them; therefore, they merit further investigation into their effect on the number of persister cells. In the present study, the fractional inhibitory concentrations of antibiotic combinations ciprofloxacin-daptomycin, ciprofloxacin-vancomycin, daptomycin-tobramycin, and tobramycin-vancomycin (checkerboard assay) were determined against two previously studied clinical (S48 and J6) and one standard (NCIM 5021) isolate of Staphylococcus aureus. They showed synergistic effects with a 2 to 256-fold reduction in MICs. All combinations also resulted in inhibition and disruption of biofilms in a concentration-dependent manner. All antibiotic combinations, except ciprofloxacin-daptomycin, showed total biofilm inhibition at 100X MICs. Similarly, antibiotic combination at 100X MIC showed 77-97% disruption of preformed biofilms. Time-kill assays performed at a 100X MIC combination against stationary-phase cells showed a two to six log10 reduction in CFU followed by a plateau indicating the presence of persisters. Significant differences were observed in persister cell fraction remaining after treatment with antibiotic combinations compared to monotherapies (p < 0.05) and therefore merit further investigation in clinical use for treatment against persisters.