We are losing the battle against antibiotic-resistant Gram-negative bacterial pathogens in our hospitals and clinical care facilities. The CDC estimates that over 2 million people are infected annually with 17 species of antibiotic-resistant bacterial pathogens, killing 23,000 people per year. Over half of the species are Gram-negative pathogens including carbapenem-resistant Enterobacteriaceae (CRE) and the non-fermenting opportunistic pathogen Acinetobacter baumannii. Although the CDC described carbapenem-resistant (CR) bacteria as “nightmare bacteria”, the factors required for virulence of these pathogens or their antibiotic-susceptible counterparts during bloodstream infections are largely unknown. Thus, there is an urgent need to identify unique (species-specific) and common (required by most of the six Gram-negative species) fitness and virulence factors required by these species for bacteremia, and to map key metabolic pathways and essential gene sets used by these pathogens in vivo. Our goal is to delineate the mechanisms of pathogenesis in Gram-negative bacteria that cause hospital-acquired infections. Our objective is to conduct RNA-seq and measure in vivo growth rates in representative isolates of E. coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter cloacae as well as A. baumannii, in the murine model of bacteremia in which Tn-seq has been largely completed for these isolates. Based on the relatedness of CR species at the family (Enterobacteriaceae) and class (A. baumannii) levels, these pathogens may require a combination of orthologous core functions and species-specific fitness factors to acquire nutrients and evade host responses during bacteremia. We are indentifying unique and common genes critical for bloodstream infection by antibiotic-susceptible and CR bacteria and measuring their in vivo gene expression and in vivo growth rates. Unique and common virulence determinants are being investigated to infer mechanisms of pathogenesis. We are defining the active metabolic pathways and resultant growth kinetics across six Gram-negative pathogens during bacteremia. Our aim is to identify shared and unique pathways required for bacteremia by six Gram-negative pathogens. Here we will measure growth kinetics of each pathogen during bacteremia, identify preferred and required pathways at equivalent growth phases in vivo, and construct maps of these pathways annotated with genes that are pathogen-specific or shared across multiple pathogens.