The Influence of Growth Efficiency on the Inoculum Effect

Antibiotic resistance is a significant public health concern. Bacteria have evolved resistance to nearly every currently available antibiotic. Accordingly, there is a dire need to understand the common mechanisms by which bacterial populations can tolerate antibiotics towards extending their effective shelf-life. One mechanism that bacteria can use to tolerate antibiotics is the inoculum effect (IE); for a given antibiotic concentration, the initial bacterial density determines whether a population survives or goes extinct. If the initial density of the population is sufficiently high, the antibiotic is ineffective. IE has been reported for nearly all bactericidal antibiotics and bacteria. Moreover, evidence from the clinic suggests that IE can increase mortality rate of infected hosts. Despite its ubiquity, a mechanism to explain IE across diverse antibiotic classes has yet to be identified. Here, we report that IE can be largely explained through interactions between growth and metabolic rate, which we call growth efficiency. We observed that increasing growth efficiency decreased the strength of the IE. If growth efficiency is sufficiently high, IE is essentially abolished. Next, using flux balance analysis coupled with whole genome modeling and experimentation, we found growth efficiency, as determined by carbon source, can impact IE in Escherichia coli. This finding is consistent amongst several conditions including different clinically relevant ESKAPE pathogens, bactericidal antibiotics classes, and by decoupling growth and metabolism. Overall, our work is the first to experimentally demonstrate the effect of metabolism and growth on the IE, and it may serve to establish novel treatment approaches to reduce the occurrence of IE in the clinic.