Emergence and characterization of IncFII/IncR plasmids with multiple 5,692 bp- blaKPC−2-bearing tandem repeats in ceftazidime/avibactam non-susceptible Klebsiella pneumoniae strains

Ceftazidime/avibactam (CAZ/AVI) is widely recognized as an effective treatment for infections caused by KPC-producing <i>Klebsiella pneumoniae</i> (KPC-Kp). However, the prevalence of CAZ/AVI resistance among KPC-Kp isolates has increased rapidly in recent years. In this study, high-level carbapenem resistance and enhanced CAZ/AVI resistance were observed in two hypervirulent carbapenem-resistant <i>K. pneumoniae</i> isolates, KP1878 and KP3034, following prolonged carbapenem use. Virulence phenotypes were confirmed using the string test and a <i>Galleria mellonella</i> larvae infection model. Real-time quantitative PCR revealed that the relative expression of <i>bla</i> _KPC-2 in KP1878 and KP3034 was 2.4-fold and 11.6-fold higher, respectively, than that in the CAZ/AVI-susceptible KPC-Kp strain KP1880. Whole-genome sequencing showed that the <i>bla</i> _KPC-2 gene resided within an identical 5,692-bp Δ<i>klcA</i>-<i>korC</i>-ΔIS<i>Kpn6</i>-<i>bla</i> _KPC-2-IS<i>Kpn8</i>-Δ<i>tnpR-</i>IS<i>26</i> tandem repeat, which was replicated twice and four times in plasmids pKPC1878 and pKPC3034, respectively. Compared with KP1880, the β-lactamase hydrolysis activities of crude cell lysates derived from KP1878 and KP3034 were significantly higher in their ability to hydrolyze meropenem, ceftazidime, and nitrocefin. S1-nuclease-digested pulsed-field gel electrophoresis, along with Southern blot and restriction fragment length polymorphism fingerprinting, identified plasmid profiles but revealed one or more 5.6-kilobase variations in the regions hybridized with the KPC-specific probe. Further comparative genomic analysis suggested that a potential homologous recombination event occurred between the <i>bla</i> _KPC-2-carrying plasmid and the pLVPK-like virulence plasmid of KP3034, leading to the generation of a cointegrated plasmid that combined both virulence and CAZ/AVI resistance.