Genome Analysis of Carbapenemase-Negative,Extensively Drug-Resistant Klebsiella pneumoniae Clinical Isolates

Abstract

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) poses a major global health threat due to limited treatment options and high mortality. While the genomes of carbapenemase-positive XDR-KP strains are well-documented, there is limited data on carbapenemase-negative XDR-KP. This study characterized the genomes of two such clinical isolates, obtained from abdominal fluid and urine, that were resistant to nearly all tested antibiotics. Antimicrobial susceptibility testing was performed at the Hamilton Regional Microbiology Laboratory, ON. Carbapenemase production and the presence of carbapenemase genes were assessed using the NG-Test^®CARBA-5 and a multiplex carbapenemase PCR assay, respectively. Whole-genome sequencing via Oxford Nanopore technology revealed that both strains lacked carbapenemase genes but harbored multiple other resistance genes (e.g., bla_CTX-M-15, qnrB1/S1, sul1/2, dfrA17/A14, fosA) and acrR mutations. Notably, both carried ompK37 mutations, an outer membrane porin previously linked to carbapenem resistance. A number of antibiotic resistance genes were found on conjugative plasmids (IncFIB/IncFII, IncFIA, IncFIA/IncFII). Multilocus sequence typing identified different sequence types (ST307, ST45) and virulence profiling showed overlapping and distinct features, including variability in capsular and siderophore genes. These findings suggest a significant role for porin loss and efflux pump regulation in conferring resistance to broad-spectrum and last-resort antibiotics in clinical XDR-KP isolates that lack carbapenemases and other related antibiotic resistance genes.