Project Abstract Candida species are fourth leading cause of bloodstream infections (BSIs) in United States hospitals. BSIs due to C. albicans and C. glabrata, the most common Candida species to cause invasive infections, are 20%-40% despite treatment with echinocandins, the frontline antifungal agents. Echinocandin resistance is increasingly described among C. albicans and C. glabrata clinical isolates. Nevertheless, most echinocandin treatment failures are not associated with emergence of resistance. Echinocandin tolerance, in which Candida growth is inhibited but cells remain viable, may predispose to subsequent development of resistance, but clinical relevance of this phenotype is unclear. The longstanding paradigm is that almost all candidemia and other BSIs stem from a single, clonal organism. In preliminary studies, however, we showed by whole genome sequencing (WGS) and phylogenetic analyses that C. albicans and C. glabrata BSIs are caused by mixed populations of genetically diverse strains, which are not typically recognized by the clinical microbiology laboratory. Upon deeper analysis of C. albicans WGS data, we found that gene variants identified in multiple patients with BSIs were enriched for biological processes that are known to be important in echinocandin responses and virulence. Our objectives in this project are to characterize in greater detail the genetic and phenotypic diversity of bloodstream C. albicans and C. glabrata strains, with particular attention to strains associated with persistent or recurrent infections despite echinocandin treatment, and to implicate specific Candida genes and gene variants in echinocandin tolerance, resistance and virulence. We hypothesize that by studying strains from longitudinal BCs and extra- blood sites of patients with persistent or recurrent C. albicans or C. glabrata bloodstream infections despite echinocandin treatment, we will identify novel genes or gene variants that are responsible for echinocandin tolerance/resistance and virulence. In our first aim, we will complete WGS and analyses of C. albicans and C. glabrata from baseline BCs (10 patients each). Then, we will perform WGS and analyses of strains from positive longitudinal blood and extra-blood cultures collected during or after echinocandin treatment. Finally, we will determine phenotypes of genetically diverse strains from BCs, including echinocandin tolerance and resistance. In our second aim, we will construct isogenic mutant and complemented C. albicans and C. glabrata strains for genes and gene variants that are identified and prioritized in aim 1. We will test strains for phenotypes in vitro and for echinocandin treatment responses and virulence using mouse models of hematogenously disseminated infections. Results will affirm the extent and type of C. albicans and C. glabrata genetic diversity in BCs, afford new insights into Candida responses to echinocandins during persistent or recurrent BSIs, and identify novel gen...