Ceftazidime-Avibactam Resistance Associated with Increased blaKPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae

Abstract

This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the bla_KPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.

Keywords: CAZ-AVI; KPC; Tn4401; avibactam; carbapenem-resistant Enterobacterales; carbapenemase; double copy; porin alterations.

FIG 1

Linear map of pIT-14519 and pIT-8788, two novel KPC-encoding pKpQIL derivative plasmids. A comparison with most similar elements (i.e., pKpQIL-UK and pIT-FIPP-1) is also shown, together with the linear map of ColE-14159 plasmid. The straight lines or the checkered pattern between plasmids indicate a sequence identity ≥99% in the same or in the opposite orientation, respectively. Genes are represented as rectangles with a color-based legend according to their function. Elements belonging to the Tn4401a transposon are grouped with a light yellow rectangle.