Antibiotic resistance among hypermutator carbapenem resistant Klebsiella pneumoniae

Project Summary Carbapenem resistant Enterobacteriaceae (CRE) are major public health threats. CR-Klebsiella pneumoniae (CRKP) are the most common CRE globally. Treatment of CRKP and other CRE infections with new antibiotics like ceftazidime-avibactam (CZA) and meropenem-vaborbactam (MVB) has improved survival, but recurrent infections are common and emergent resistance is problematic. CRE infections are usually due to strains that colonize the GI tract. Among other bacteria, it is now apparent that GI colonization is caused by a population of closely related, but genetically distinct strains. These strains can exhibit a range of antibiotic resistance and biologic attributes, which are often not appreciated by studying single colonies from microbiologic cultures. In studies of natural bacterial populations, ~1 to 5% of isolates exhibit high spontaneous mutation rates, which may confer selective advantages under environmental stress and increase diversity within the population. Such hypermutation (HM) strains are most prevalent in humans during chronic colonization and recurrent infections, and they most often stem from mutations to DNA mismatch repair (MMR) genes like mutS, mutL or mutH. There are few studies of HM among CRKP or CRE. We believe that HM is under-recognized in these bacteria because studies have not assessed long-term GI colonization, recurrent infections, or populations of strains from clinical samples. In this project, we hypothesize that 1) HM CRKP can be recovered from patients with chronic GI colonization and persistent/recurrent infections; 2) MMR and other gene mutations promote HM in vitro, and CZA and MVB resistance in vitro and within infected organs; and 3) these mutations promote transmission and receipt of antibiotic resistance gene (ARG)-bearing plasmids by CRKP in vitro and during GI colonization. In pilot screening studies, we recovered HM CRKP from ~30% of patients with chronic GI colonization, or persistent or recurrent infections. MMR gene mutations were identified in most HM CRKP strains. One of these mutations (MutH V76G) was proven to contribute to HM, MVB and CZA resistance, and enhanced transfer and acceptance of plasmids containing ARGs in vitro, and to CRKP tissue burdens and emergence of MVB resistance within infected organs of intravenously (IV)-infected mice. In aim 1 of this proposal, we will continue to screen clinical CRKP isolates for HM phenotype. We will perform whole genome sequencing on HM isolates, and create isogenic mutant strains to determine if certain mutations contribute to HM. In aim 2, we will evaluate the role of HM mutations in emergence of CZA, MVB and colistin resistance in vitro and within mouse organs following IV infection. Then, we will determine impact of HM mutations on transfer of ARG-bearing plasmids in vitro and during mouse GI colonization. This project will mark the first systematic investigations of CRE clinical isolates for HM phenotypes. If successful, experiments will provide new understanding of HM and its crucial roles in regulating CRKP antibiotic resistance. Isogenic HM strains created here will also be powerful tools for assessing resistance barriers for novel antibiotics and new targets of antibiotic discovery.