Drug resistance enablers and their role in antibiotic treatment failure

PROJECT 1: SUMMARY Antimicrobial resistance is one of the greatest worldwide health challenges lacking a clear path toward a solution. Chief among the concerns are nosocomial diseases recalcitrant to antimicrobial treatment, turning treatable illnesses unrelated to infectious diseases into end-of-life events. The source of this antibiotic treatment failure (ATF) varies, but failure itself is remarkably common, with healthcare-associated pneumonia resulting ATF in up to 70% of the cases. Although ATF is often tied to the spread of antimicrobial resistant bacteria, a large proportion of cases cannot be so easily explained. Furthermore, the acquisition of antimicrobial resistance (AMR) itself appears to be more complex than originally envisioned, as precursor mutations appear to exist in microbial populations that may be required as stepping stones for the development of AMR. These precursor mutations are enablers, resulting in increasing drug tolerance, persistence, or heteroresistance, with the consequence that a subpopulation can remain viable in the presence of antimicrobials allowing the outgrowth of resistant populations as antibiotic concentrations ebb and flow. The central hypothesis of this work is twofold: these enabler mutations increase the evolvability of the microbe to acquire antimicrobial resistance, and these strains having enabler mutations can be demonstrated to cause ATF even in the absence of acquisition of clinically-defined AMR. To test this hypothesis, experiments are proposed to analyze the Gram-negative bacterium Acinetobacter baumannii, a causative agent of nosocomial pneumonia that has become increasingly difficult to treat due to the acquisition of multidrug resistance. To study the connection between A. baumannii enabler variants, ATF and the outgrowth of drug resistant mutants, a series of experiments are proposed that systematically identifies a large spectrum of enabler mutations, including partial function lesions in essential genes. The enablers will be characterized to determine the relative size of subpopulations that provide precursor pools for the development of AMR, in work involving collaboration with Project 3 and the Scientific Core. This will allow an evaluation of the likelihood that a variant will eventually give rise to AMR. Once the pool of enablers is identified, they will be tested under multiple growth conditions for their ability to cause ATF in culture conditions as well as their ability to generate resistant mutants. Similarly, the clinical development of ATF and AMR will be modeled in a murine pneumonia model. This will be accomplished by first determining if acquisition of an enabler mutation, in the absence of known determinants of AMR, can result in ATF, and then determining if enabler mutations increase the evolvability to drug resistance during the course of disease.