Dissecting the host-plasmid interplay to unravel the spread of ESBL-producing K. pneumoniae

ABSTRACT Multidrug-resistant Klebsiella pneumoniae (Kp) infections, especially carbapenem resistant (CR-Kp) and extended spectrum β-lactamase (ESBL-Kp) producing strains, have become successful global pathogens that are recalcitrant to most effective antibiotics and are associated with high rates of morbidity and mortality. Extensive molecular epidemiological studies over the last 20 years described the successful global spread of CR-Kp ST258, but improved diagnostics and treatment have calmed this epidemic. In this background, we have investigated the increase and national spread of ESBL-Kp isolates, especially in the community, by partnering with Quest Diagnostics who captured 1,338 ceftriaxone resistant from 10 regional sites for whole genome sequencing. The collected population, primarily from community patients (>85%), were cultured from elderly (mean age 70) women (68%) with urinary tract infections (92%). These isolates showed 53% resistance to cefepime, 33% resistance to piperacillin/tazobactam and of clinical significance, 89% were resistant to fluroquinolones, 85% were resistant to trimethoprim/sulfamethoxazole, and 86% were resistant to nitrofurantoin; oral agents commonly used to treat UTIs. In contrast to the CR-Kp ST258 epidemic, 1,338 ESBL-Kp isolates revealed remarkable genetic diversity, 199 sequence types (STs) and 104 capsule polysaccharides (CPS) were identified, and 81% harbored multidrug resistant IncF transferable plasmids. Most significantly, genomic and plasmid dissection of the major STs and CPS types identified transmissible clusters with highly conserved genomes (<21 SNPs), harboring common plasmid(s) and spreading across the US. The distribution of the IncF plasmids is evidence that there are positive and negative relationships between plasmids and hosts and in this grant, we hypothesize that transmissible clusters are examples of positive interplay with increased "fitness" and successful spread. In this proposal we will build on our collaboration with Quest Diagnostics and repeat our snapshot collection over years 2 and 4 and create a longitudinal dataset to build on our phylogenetic structure. Aim 1 will use both Illumina and Nanopore sequencing to discern the trajectory of transmissible clusters, CPS prevalence, and IncF plasmid microevolution. The data generated will further enable us to develop and refine a predictive model for the molecular epidemiology of ESBL-Kp, advancing our ability to assess and track resistance dynamics. These results will guide our studies in Aim 2 where both the genetic backgrounds from transmissible clusters and unique strain types will be experimentally tested by creating plasmid-less strains, using our novel CRISPR-Cas "curing" method. In comparison to our molecular epidemiology findings, we will evaluate plasmid- host interactions regarding plasmid acquisition, stability, antibiotic resistance gene expression, fitness, and host response. We will also address the challenges of drug resistant UTIs by building on our expertise in CPS-targeted antibody development and in Aim 3, we will evaluate the preventive and therapeutic efficacy of these antibodies against the most prevalent ESBL-Kp serotypes using murine models of septicemia and ascending UTI.