(686e) Investigating How Antibiotic Target Network Components Impact the Transience of Heteroresistance

Heteroresistance describes a phenomenon where a seemingly isogenic bacterial population contains minority subpopulations displaying elevated resistance (higher minimum inhibitory concentrations [MICs]) relative to the susceptible majority. Once treatment concludes, the resistance of these subpopulations can either be stable (their progeny maintains higher MICs) or unstable/transient (their progeny’s MICs revert back to that of the original population), either of which can adversely impact treatment outcomes. However, instances of transient heteroresistance present clinical conundrums, because after antibiotic failure, those isolates’ MICs are identical to that of the initial population. Although heteroresistance has been observed in various clinical isolates, few studies have delved into quantitatively evaluating heteroresistance stability and the genetic factors that influence this phenotype. To address this knowledge gap, we screened E. coli K-12 substr. MG1655 for heteroresistance to various, mechanistically distinct antibiotics with population analysis profiling and developed an assay and metric to quantify the degree to which the resulting subpopulations exhibited stable or transient resistance after treatment. We found that MG1655 exhibits heteroresistance to several of the drugs assayed, including D-cycloserine (DCS), a cell wall synthesis inhibitor that targets multiple enzymes and enters the cytoplasm via a transporter. Using DCS as a model antibiotic, we probed how genetic alterations to its target network impacted heteroresistance and its stability. Interestingly, only a subset of the network components altered DCS heteroresistance, and their impacts on stability were counter-intuitive due to what we propose is a non-monotonic dependency of heteroresistant stability on antibiotic concentration. Collectively, these findings demonstrate the utility of MG1655 as a tool for understanding genetic drivers of heteroresistance, which could facilitate the identification of therapeutic targets that mitigate treatment complications from heteroresistant strains.