Double Carbapenemases in Klebsiella pneumonia blood isolates in Athens, Greece
Acquisition of multiple carbapenemase genes by Klebsiella pneumoniae (Kp) is an emerging public health threat. Here, we aim to elucidate the population structure of Kp blood isolates carrying two different carbapenemase genes and identify the mechanism facilitating their dissemination.  The study was conducted in a tertiary healthcare center between 2014 and 2022. Twenty-four patients with bacteremia caused by Kp carrying two different carbapenemase genes were identified. All 24 blood isolates were analyzed by short-read genome sequences supplemented by long-reads in a selected number of isolates.  All isolates carried blaKPC (23 blaKPC-2, one blaKPC-3) and blaVIM-1 genes along with a variety of antimicrobial resistance determinants. The isolates were clustered in six clonal lineages (ST39, ST147, ST323, ST258, ST3035, ST340). Long-read genome sequences demonstrated that each carbapenemase gene was located on separate group of plasmids; the blaKPC-2 on a fusion of IncFIB(pQil) and IncFII(K) plasmids, the blaKPC-3 on IncX3, the blaVIM-1 on IncC or a fusion of the IncFIB(pNDM-Mar) and IncHI1B(pNDM-MAR) plasmids. Comparison of plasmid content of eight isolates carrying a single carbapenemase gene from a previous study with eight isolates carrying two carbapenemase genes from the present study, matched by clonal lineages, revealed that the second carbapenemase gene was acquired by addition of another plasmid. Identical plasmids were found within the same lineage and across lineages.  These findings suggest that dissemination of carbapenemase genes in our hospital setting follows the pattern of multiple plasmids/multiple lineages and is mainly driven by a small number of highly efficient clones.