ABSTRACT Antibiotic resistance is one of the most serious medical challenges of our time. This crisis puts patients at risk of untreatable bacterial infections and threatens major advances of modern medicine that rely on antibiotics (transplants, chemotherapy, etc). There are at least 2.8 million antibiotic resistant infections each year in the US, leading to over 35,000 deaths [1]. Without significant action, worldwide annual mortality due to these infections is predicted to reach 10 million by 2050, surpassing that predicted for cancer [2]. Understanding resistance mechanisms is critical to designing novel approaches and therapeutics to combat resistant bacteria. Heteroresistance (HR) is an enigmatic form of antibiotic resistance in which a bacterial isolate harbors a resistant subpopulation that can rapidly replicate in the presence of an antibiotic, while a susceptible subpopulation is killed [3, 4]. We have observed HR to the antibiotic, fosfomycin, which is a member of its own drug class and has primarily been used in the US in an oral form to treat urinary tract infections (UTIs) [5]. The use of fosfomycin has recently increased as bacteria become resistant to other classes of drugs [6] and due to its strong safety profile. Due to its increased need and expected expanded approval for IV use, fosfomycin is expected to become a much more prominent part of the antibiotic arsenal in the US. Therefore, it is essential that we elucidate the biology of fosfomycin resistance to guide clinical use. Strikingly, our surveillance data revealed that the rate of fosfomycin HR among carbapenem-resistant Enterobacteriaceae (CRE; 72%) and Acinetobacter baumannii (CRAB; 89%) was higher than that of any other antibiotic tested, and that a large proportion was not detected by clinical diagnostics [7]. We recently demonstrated that HR to diverse antibiotics, including fosfomycin, can cause treatment failure in vivo [4]. Interestingly, and thus far unique among studied examples of HR, we found that fosfomycin HR is caused by two distinct, co-existing resistant subpopulations, both of which replicate in the presence of drug and are not persisters, but form resistant small (R-SM) or large (R-LG) colonies. Results from a transposon screen and metabolomic experiments revealed the underlying basis for the R-SM and R-LG cells to be metabolic heterogeneity, rather than unstable genetic changes such as gene amplification. We will dissect how metabolic signaling drives the expansion of the resistant R-SM subpopulation and the roles of glutamate and glutathione in this process. We will then study the prevalence of distinct fosfomycin resistant subpopulations among diverse clinical isolates. This work will have a sustained and powerful impact on our understanding of non- genetic mechanisms of HR and metabolic and phenotypic heterogeneity. This will complement Project 1 which focuses on unstable genetic mechanisms of HR. The new and fundamental insights gained will lay the foundation for the discovery of novel therapeutics and interventions targeting subpopulations to reduce human disease.

PROJECT NARRATIVE In accordance with RFA-AI-20-001, the project narrative should not be completed for the research project.