Comparison of in vitro static and dynamic assays to evaluate the efficacy of an antimicrobial drug combination against Staphylococcus aureus

Abstract

An easily implementable strategy to reduce treatment failures in severe bacterial infections is to combine already available antibiotics. However, most in vitro combination assays are performed by exposing standard bacterial inocula to constant concentrations of antibiotics over less than 24h, which can be poorly representative of clinical situations. The aim of this study was to assess the ability of static and dynamic in vitro Time-Kill Studies (TKS) to identify the potential benefits of an antibiotic combination (here, amikacin and vancomycin) on two different inoculum sizes of two S. aureus strains. In the static TKS (sTKS), performed by exposing both strains over 24h to constant antibiotic concentrations, the activity of the two drugs combined was not significantly different the better drug used alone. However, the dynamic TKS (dTKS) performed over 5 days by exposing one strain to fluctuating concentrations representative of those observed in patients showed that, with the large inoculum, the activities of the drugs, used alone or in combination, significantly differed over time. Vancomycin did not kill bacteria, amikacin led to bacterial regrowth whereas the combination progressively decreased the bacterial load. Thus, dTKS revealed an enhanced effect of the combination on a large inoculum not observed in sTKS. The discrepancy between the sTKS and dTKS results highlights that the assessment of the efficacy of a combination for severe infections associated with a high bacterial load could be demanding. These situations probably require the implementation of dynamic assays over the entire expected treatment duration rather than the sole static assays performed with steady drug concentrations over 24h.

Fig 1. Time-kill curves of the 2 strains subjected to constant concentrations of antibiotic.

Evolution of the bacterial population (log₁₀ CFU/mL) for the MSSA (A and B) and MRSA (C and D) strains after exposure to 70μg/mL amikacin (blue) or 18μg/mL vancomycin (green) or the combination of both (red) over 24hours in MHB (A and C) and in RPMI (B and D). The marks represent the mean ±SD of the bacterial counts for the different tested treatments (n = 2 for each treatment). Curves with open squares represent the bacterial counts on a large initial inoculum and curves with crosses represent the bacterial counts on a standard initial inoculum. (LOD = 2.5 log₁₀ CFU/mL). For the low inoculum of MSSA in MHB and RPMI (A and B), there was no difference between drugs alone or in combination. For the high inoculum of MSSA, there was no difference between drugs in MHB (A) but vancomycin led to significantly higher bacterial counts than amikacin and the combination in RPMI (B). For the low inoculum of MRSA, there was no difference between drugs alone or in combination in MHB (C) but amikacin led to significantly higher bacterial counts than vancomycin and combination in RPMI (D). For the high inoculum of MRSA, there was no difference between drugs in MHB (C) but amikacin led to significantly higher bacterial counts than the combination in RPMI (D).

Fig 2. Time-kill curves of the MSSA strain subjected to dynamic concentrations of antibiotic.

Evolution of the bacterial population (log₁₀ CFU/mL) after exposure to amikacin or vancomycin alone or the combination of both over 5 days in RPMI. The marks represent the mean ±SD of the bacterial counts for the different tested treatments (black: control without antibiotic, green: vancomycin twice a day, blue: amikacin once a day, red: amikacin once a day and vancomycin twice a day. Curves with open squares represent the bacterial counts on a large initial inoculum and curves with crosses represent the bacterial counts on a standard initial inoculum. (n = 2 for each treatment on the large inocula and n = 1 for each treatment on the standard inocula) and LOD = 2.5 log₁₀ CFU/mL). The bacterial counts from 0 to 24 h after exposure to vancomycin were significantly higher (p<0.01) than those obtained with amikacin or the combination. The bacterial counts from 0 to 104 h were significantly different for each treatment (p<0.01).