Collateral Consequences of Enabler Genotypes in Antibiotic Treatment Failure.

Project 2: Collateral Consequences of Enabler Genotypes in Antibiotic Treatment Failure Antibiotic treatment failure (ATF) is of critical medical importance as delays in effective antibiotic therapy promotes prolonged morbidity and increase mortality leading to overall poorer patient outcomes. While antibiotic resistance is one primary mechanism for ATF, the roles for transient cell-states conferring antibiotic tolerance, persistence, and hetero-resistance, herein referred to as “enablers” are increasingly recognized as a major mechanism for bacterial evasion of antibiotic therapy. These enabler bacterial populations are of considerable scientific and medical importance as typically precede the subsequent evolution or acquisition of high-level resistance mechanisms, making these populations a “canary in the coal mine” for understanding how resistance and treatment failures can emerge. Enabler mutants identified thus far in central cellular pathways indicate commonalities for developing an enabler state suggest these findings will be broadly applicable in a cross-species manner. Our underlying hypothesis is that the collateral phenotypes of enabler genotypes can be identified and rationally targeted to optimize the treatment outcomes and prevent subsequent acquisition of high-level resistance. We plan on using a multifaceted approach to understand the genetic and mechanistic basis of enabler mutations. We will experimentally select for enabler mutations using continuously variable antibiotic exposures in both sensitive strains and those with defined enabler mutations. These antibiotic sensitive but tolerant populations will be used to model the impact of different enabler genotypes on the probability and genetic pathways to subsequent evolution of high-level antibiotic resistance, the role of these genotypes on horizontal transfer of resistance, and how enabler mutants impact antibiotic treatment failure in vivo. These enabler strains will be used to model how the respective genotypes, upon antibiotic exposure, affect key pathways such as bacterial stringent response, transcriptional signaling, and translational fidelity, and how this in turn leads to the production of enabler phenotypes. These lines of investigation will provide insight into whether different enabler genotypes have common mechanistic underpinnings. We also plan to examine if rational targeting of enablers could provide therapeutic benefit to either stop the spread of resistance or improve antibiotic efficacy when enabler mutants are present. Enabler mutations are often associated with collateral consequences that confer sensitivity or resistance to metabolic of chemical stresses. We will determine the possibility of exploiting these cross-sensitivities by both targeted and unbiased chemical screening approaches. These synergies will then be investigated for their capacity to prevent emergence of enabler mutants as well as their capacity to more effectively treat infections caused by enabler strains recalcitrant to antibiotic therapy. The successful completion of these aims will provide a genetic basis and mechanistic insight into transient cell-states conferring enabler phenotypes. We anticipate these findings will be applicable across multiple bacterial species given the likelihood of conserved mechanisms underlying enablers and may provide additional strategies to prevent antibiotic treatment failure during bacterial infection.