Down the Resistance Road: An Exploration of the Evolution and Emergence of Antibiotic Resistance in Enterococcus faecalis Communities

Abstract

Antibiotic resistance poses a growing and global threat to public health. The rapid resistance evolution of microbes challenges our ability to control infections and leads to increased mortality. In this work, we explore the dynamics of antibiotic resistance evolution and the emergence of cooperative resistance---a phenomenon where antibiotic susceptible cells survive in drug environments due to cooperative interactions with antibiotic resistant cells---through a combination of experimental assays and mathematical modeling. To examine these dynamics, we use Enterococcus faecalis, a common pathogen in hospital-acquired infections that is notorious for its capacity to acquire resistance and to remain viable in biofilms.

In the first study, we use a continuous culture bioreactor to subject an isogenic bacterial population to a combination of linezolid and levofloxacin to explore the effects of antagonistic antibiotics (i.e., ones that work less effectively in combination as opposed to synergistic combinations, where the efficacy of the drug is enhanced by the presence of the other) on the rate of resistance evolution. Our results show how this pair can inhibit evolutionary resistance, challenging conventional therapies favoring synergistic combinations. In the second study, we present a fluorescent-reporter library tailored to E. faecalis that facilitates following the single-cell level dynamics of populations in spatially fixed systems and in bulk assays. Lastly, we apply this reporter library to study mixed biofilms of differently labeled Ampicillin susceptible and Ampicillin resistant Enterococci. We combine experimental and in silico methods to show that biofilm structure can shape resistance, an effect driven by cooperative interactions among resistant and sensitive subpopulations.

Our results indicate that we can actively modulate and potentially disrupt cooperative resistance behaviors by adjusting the population composition and the spatial arrangement of individual cells. In this way, we expand our understanding of the key parameters that play a role in the emergence of antibiotic resistance in spatially fixed bacterial communities. We open new directions for antimicrobial management that had not been appreciated before that focus on disrupting interactions between individuals in a mixed bacterial community instead of mechanisms of resistance that are intrinsic to a single cell.