Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum beta-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.