Within-host diversity of Gram negative bacteria from bloodstream infections

Project Summary Klebsiella pneumoniae (KP) are major human pathogens, which have a propensity to develop antimicrobial resistance (AMR). Bloodstream (BSIs) and other serious KP infections are usually caused by strains that colonize the gastrointestinal (GI) tract. Emerging whole genome sequence (WGS) data show that the GI tract is colonized by clonal bacterial populations, in which genetic diversity develops. It is unknown how often BSIs are caused by clonal but genetically diverse strains of a given bacterium, since clinical labs generally characterize a single colony from positive cultures. In preliminary studies, we performed WGSing on strains isolated from 10 independent colonies from carbapenem-resistant KP (CRKP)-positive blood cultures of 6 patients. In all patients, blood cultures contained genetically distinct sequence type-258 CRKP strains, as evident by core genome single nucleotide polymorphisms, gene deletions and other mutations, and plasmid or plasmid-borne gene loss. Genetically distinct CRKP strains exhibited significant differences in antibiotic tolerance and AMR, capsular polysaccharide content, and other virulence-associated phenotypes. Genetic variant strains from 2 patients displayed significant differences in virulence during BSIs of mice. It is unclear whether our results were unique to CRKP BSIs, which typically occur in patients who face significant selection pressures from long-term antibiotic use and other stressors, or if the findings carry clinical significance. We hypothesize that: 1) baseline BCs from patients diagnosed with antibiotic susceptible (S)-KP BSIs contain genotypically and phenotypically diverse strains that are not identified by the clinical microbiology lab; 2) antibiotic treatment failures are associated with emergence of KP strains with pre-existing antibiotic tolerance, AMR or virulence attributes; and 3) specific gene variants in KP strains associated with treatment failure contribute to antibiotic tolerance, AMR, and/or virulence. In aim 1, we will perform WGSing and phenotypic assays on 10 strains from positive baseline BCs from each of 10 patients with S-KP BSIs who were treated successfully with antibiotics, and from 10 patients who failed treatment. We will determine diversity within each patient, and compare baseline diversity in patients who responded to antibiotics and in those who failed. In aim 2, we will perform WGSing and phenotypic assays on 10 KP strains from antibiotic treatment failure blood cultures in each of the 10 patients from aim 1. We will compare diversity at time of treatment failure with that at baseline. We will also validate certain genes and mutations associated with strains from treatment failures for contributions to antibiotic responses and virulence. Demonstrating within-host genotypic and phenotypic diversity of KP strains in this study would suggest that this phenomenon is not limited to the most highly resistant BSIs, but rather a more generalized phenomenon. Finding unrecognized antibiotic tolerance, AMR or virulence among KP in patients who fail treatment would indicate that diversity is clinically relevant. Our project has the potential to impact medical and clinical lab practices, and to afford new insights into determinants and mechanisms of KP antibiotic responses and pathogenesis.