Population Density Modulates Drug Inhibition and Gives Rise to Potential Bistability of Treatment Outcomes for Bacterial Infections

Abstract

The inoculum effect (IE) is an increase in the minimum inhibitory concentration (MIC) of an antibiotic as a function of the initial size of a microbial population. The IE has been observed in a wide range of bacteria, implying that antibiotic efficacy may depend on population density. Such density dependence could have dramatic effects on bacterial population dynamics and potential treatment strategies, but explicit measures of per capita growth as a function of density are generally not available. Instead, the IE measures MIC as a function of initial population size, and population density changes by many orders of magnitude on the timescale of the experiment. Therefore, the functional relationship between population density and antibiotic inhibition is generally not known, leaving many questions about the impact of the IE on different treatment strategies unanswered. To address these questions, here we directly measured real-time per capita growth of Enterococcus faecalis populations exposed to antibiotic at fixed population densities using multiplexed computer-automated culture devices. We show that density-dependent growth inhibition is pervasive for commonly used antibiotics, with some drugs showing increased inhibition and others decreased inhibition at high densities. For several drugs, the density dependence is mediated by changes in extracellular pH, a community-level phenomenon not previously linked with the IE. Using a simple mathematical model, we demonstrate how this density dependence can modulate population dynamics in constant drug environments. Then, we illustrate how time-dependent dosing strategies can mitigate the negative effects of density-dependence. Finally, we show that these density effects lead to bistable treatment outcomes for a wide range of antibiotic concentrations in a pharmacological model of antibiotic treatment. As a result, infections exceeding a critical density often survive otherwise effective treatments.

Delayed antibiotic exposure induces population collapse in enterococcal communities with drug-resistant subpopulations

“…To introduce non-constant antibiotic concentrations, we used customized, computer controlled bioreactors capable of precise control of inflow (e.g. drug and media) and outflow in each growth chamber ( Figure 2A; see also (63,64,11)). Cell density is monitored with light scattering (OD), and each chamber received fresh media and drug at a rate µ ≈ 0.12 hr −1 , which is approximately an order of magnitude slower than the per capita growth rate of sensitive cells in drug-free media.…”

“…For example, drug degradation by a sub-population of enzyme-producing cells can lead to cooperative resistance that allows sensitive (non-producing) cells to survive at otherwise inhibitory drug concentrations (5,6,7). Additional examples of collective resistance include density-dependent drug efficacy (8,9,10,11), indole-mediated altruism (12), and increased resistance in dense surface-associated biofilms (13). The growing evidence for collective resistance underscores the need to understand not just the molecular underpinnings of resistance, but also the ways in which these molecularlevel events shape population dynamics at the level of the bacterial community.…”

“…Increasing population density typically leads to decreased growth inhibition by antibiotics, consistent with the classical inoculum effect (IE) (8). However, β-lactams can also exhibit a surprising "reverse" inoculum effect (rIE) characterized by increased growth of the population at lower densities (11,58). In E. faecalis , the reverse IE arises from density-dependent changes in local pH (11), which are associated with increased activity of ampicillin and related drugs (59).…”

“…However, β-lactams can also exhibit a surprising "reverse" inoculum effect (rIE) characterized by increased growth of the population at lower densities (11,58). In E. faecalis , the reverse IE arises from density-dependent changes in local pH (11), which are associated with increased activity of ampicillin and related drugs (59). Similar growth-driven changes in pH (in antibioticfree environments) have been shown to modulate intercellular interactions (60) and even promote ecological suicide in other microbial species (61).…”

Interplay between antibiotic efficacy and drug-induced lysis underlies enhanced biofilm formation at subinhibitory drug concentrations

“…A quantitative understanding of biofilm formation may also inspire new optimized dosing protocols, similar to those used in, for example, references [48][49][50], and the current model could be easily extended to investigate the effects of clinically realistic antibiotic dosing regimens. In the long run, these results may lay the groundwork for improved systematic design of biofilm-specific therapies (51,52).…”