Evolution of KPC-K pneumoniae that persist in patients on prolonged antibiotics

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 DESCRIPTION (provided by applicant): Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) have emerged as major pathogens, causing extensively drug resistant infections with crude mortality rates that can exceed 50%. Since 2008, we have maintained a biorepository of >700 KPC-Kp strains from our center, linked to clinical and pharmacy databases. We have shown that outcomes among patients with KPC-Kp bacteremia and intra- abdominal infections (IAI) range from acute death due to septic shock, to rapid cure, to recurrent or persistent infections that have lasted for more than a year. As is true at most U.S centers, KPC-Kp sequence type 258 (ST258) strains predominate among our patients. Our preliminary data demonstrate that ST258, KPC-Kp strains that are clonal by conventional molecular epidemiologic criteria exhibit significant genetic heterogeneity, including in antimicrobial resistance repertoire, porin function and within the chromosomal hypervariable region. Longitudinal strains from individual patients with persistent infections become increasingly resistant to antimicrobial agents, and have mutations in genes involved in lipopolysaccharide and capsule biosynthesis, iron transport, stress responses and other processes. Our major goal in this project is to identify the genetic bases by which KPC-Kp strains cause persistent infections. We hypothesize that persistence stems, at least in part, from the development of further antimicrobial resistance and attenuation of virulence by strains in response to treatment regimens and the stresses of in vivo environments. Our objectives are to comprehensively define genetic differences among ST258, KPC-Kp strains causing persistent infections, characterize the genetic evolution of strains during persistent infections, and correlate these results with antimicrobial resistance, virulence and gene expression in vivo. To achieve these objectives, we will pursue two specific aims. In aim 1, we will test longitudinally-collected ST258, KPC-Kp strains from patients with persistent infections for the emergence of resistance to the combination of doripenem and colistin (our front- line treatment regimen) during time-kill assays in vitro, and relative virulence during intra-abdominal infections of mice. Based on preliminary data, we anticipate that last longitudinal strains will be more resistant to doripenem-colistin and less acutely virulent than first longitudinal strains. In aim 2, we will perform whole genome and plasmid sequencing on ST258, KPC-Kp strains, use RNA-Seq to define transcriptomes in mice during IAIs, and create mutant strains to verify that genes identified by sequencing and RNA-Seq contribute to resistance and virulence. We anticipate that our results will afford insights into mechanisms by which KPC-Kp strains adapt to treatment and environmental pressures during infections. This project will generate new hypotheses about resistance and pathogenic mechanisms, and identify targets for the development of novel diagnostics, therapeutics, vaccines, and predictive markers for treatment responses.