An investigation of gene dosage reveals that increased sensitivity to D-cycloserine divergently impacts the transience of heteroresistance in Escherichia coli

Heteroresistance describes the phenomenon where seemingly isogenic bacterial populations contain subpopulations with elevated resistance compared to the susceptible majority that are often missed in routine susceptibility testing. The enhanced resistance of those subpopulations can either be maintained (stable) or quickly lost (unstable/transient) after treatment, where transient cases return susceptibility results identical to those of original cultures. Recent work has implicated increased gene dosage of resistance determinants as a major cause of unstable heteroresistance in clinical isolates. Inspired by that work, we sought to systematically evaluate how gene dosage of an antibiotic’s target network components impacted heteroresistance and its stability. To accomplish that, we used Escherichia coli MG1655 as a model organism, and D-cycloserine (DCS), a cell-wall synthesis inhibitor that enters through a transporter (CycA) and inhibits multiple enzymes (DdlA, DdlB, DadX, and Alr), as a model antibiotic. To measure heteroresistance, we used population analysis profiling, and to quantify stability, we used a population-level model to define a heteroresistance stability index (HSIj∣t), which quantifies the proportion of heteroresistant subpopulation j that maintained elevated resistance over t generations. We found that increased sensitivity to DCS through gene dosage variation of cycA and ddlB divergently impacted heteroresistance stability, with ddlB enhancing stability and cycA fostering transience. These findings translated to uropathogenic E. coli (UTI89) and suggested that increasing the number of antibiotic targets and/or points of antibiotic entry could decrease the propensity of heteroresistance to yield stable resistance. This knowledge could impact the development of new antibiotics and improve understanding of antibiotic treatment failure. IMPORTANCE Heteroresistance is a concern because heteroresistant strains escape clinical detection and facilitate treatment failure. Heteroresistant cells can produce stably resistant or transiently resistant populations, and enhanced understanding of genetic factors that influence the level of heteroresistance and its stability has the potential to improve treatment strategies. Here, we introduce the heteroresistance stability index, which is a quantitative metric of heteroresistance stability, and use it to analyze heteroresistance of Escherichia coli to D-cycloserine. We investigated how gene dosage of antibiotic target network components (transporter, enzymatic targets) influences heteroresistance and its stability and found diverging outcomes on stability for comparable declines in heteroresistance. Specifically, these results suggest that designing antibiotics to enter through multiple transporters or target multiple enzymes would reduce the emergence of stable resistance.