Project Summary S. epidermidis is the most common cause of hospital-acquired blood stream infections and new strategies for prevention and acute, point of care therapeutics are critically needed. However, a lack of identified virulence determinants, conflicting evidence regarding the source of infectious strains, and the limitations of older technologies to resolve strain origin or function present barriers to developing such strategies. Thus, we aim to 1) advance prevention of S. epidermidis BSIs through a better understanding of the origin of infectious strains and 2) advance treatment of S. epidermidis BSIs through discovery of the molecular mechanisms of infection, providing potential therapeutic targets. In Aim 1, we will collect blood samples and skin swab samples from patients with a diagnosed S. epidermidis catheter-related blood stream infection (CRBSI). Using shotgun sequencing and high-resolution computational analyses, we will determine co-occurrence of skin and infection strains within an individual to better understand the origin of infectious strains. Ancillary analyses will identify genes enriched in infection and multi-species composition of catheter biofilms. In Aim 2, we complement genomic analyses of Aim 1 to address the hypothesis that regulatory, not genic differences between skin and infection-associated strains may underlie virulence transition. We will perform metatranscriptomic sequencing on the samples from Aim 1 to identify genes correlated with infection. Finally, in Aim 3, we investigate the molecular mechanisms underlying S. epidermidis infections. Candidate infection-associated genes identified from the literature and genes identified in Aims 1 and 2 will be knocked down using a modified CRISPR/Cas system and subjected to in vitro and in vivo phenotypic profiling in a variety of growth assays that parallel conditions in which S. epidermidis thrives as pathogen. Secondary analyses (including gene over-expression, multi-gene knockdowns, network analyses) will be conducted for genes identified as contributors to infection. Successful completion of this aim would represent the first large-scale use of the CRISPR/Cas system in staphylococci. Overall, our proposed aims are designed to reveal the genetic determinants of S. epidermidis’ survival as an opportunistic pathogen and the contribution of strain variation to this role.