Antimicrobial Combination Effects at Subinhibitory Doses do not Reliably Predict Effects at Inhibitory Concentrations

Abstract

Assessing whether drug combinations synergise or antagonise is difficult for several reasons: (i) measuring bacterial death rates at clinically relevant inhibitory drug concentrations is methodologically challenging, (ii) there is no unifying definition of what constitutes synergy or antagonism, and (iii) both synergy and antagonism may be concentration- and mixing-ratio-dependent. To assess how well sub-inhibitory measurements predict inhibitory behaviour, we quantified drug interactions for 15 pairwise drug combinations on a concentration checkerboard covering a wide range of inhibitory and sub-inhibitory concentrations. To this end, we tracked the population dynamics of 8640 bioluminescent E. coli cultures by recording their light-intensity trajectories. To handle time-varying treatment effects and allow fair comparisons between drugs with distinct killing dynamics, we used a time-weighted net growth rate {psi} to summarise each trajectory and assigned interaction labels (synergistic/independent/antagonistic) based on Bliss independence and Loewe additivity. We found that the interaction label depends on both the concentration and the mixing ratio, frequently changing between the sub-inhibitory and inhibitory regimes. Characterising drug combinations at a single sub-inhibitory concentration is therefore not sufficient. Instead, their combined effects should be assessed at the conditions of their intended use.