Ceftazidime-Avibactam Resistance Associated with Increased bla _KPC-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 blaKPC 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.

Reduced Ceftazidime-Avibactam Susceptibility in KPC-Producing Klebsiella pneumoniae From Patients Without Ceftazidime-Avibactam Use History – A Multicenter Study in China

“…The results were consistent with some previously published studies (Shen et al, 2017;Zhang et al, 2020). The higher copy numbers of bla KPC−2 may potentially result from the high copy numbers of the plasmids carrying bla KPC (Roth et al, 2011) and/or a duplication of mobile genetic elements associated with bla KPC (Coppi et al, 2020). Since the examination of the bla KPC−2 promoter regions failed to identify any mutations, the higher bla KPC gene expression may potentially be affected by the higher copy numbers of bla KPC or other gene regulatory mechanisms.…”

Distribution of β-Lactamase Genes and Genetic Context of blaKPC-2 in Clinical Carbapenemase-Producing Klebsiella pneumoniae Isolates

“…Intriguingly, in addition to the high level of carbapenem resistance with a meropenem MIC of 1,024 mg/L, strain KP3034 exhibited a higher MIC level for CAZ/AVI (16/4 mg/L) than the other strains detected. In contrast to the alteration recently reported by Coppi et al 26 two-amino-acid duplications (Gly136-Asp137) within transmembrane β-strand loop 3 (L3) of OmpK36 were observed in KP3034, which was previously associated with reduced susceptibility to carbapenems. 27 Since there is no history of previous antimicrobial exposure to CAZ/AVI treatment or specific mutations were observed in the bla KPC-2 gene, hyperexpression of the bla KPC-2 gene associated with alteration in the outer membrane or other unknown mechanisms might account for this low level of CAZ/AVI resistance.…”

Emergence and rising of ceftazidime-avibactam resistant KPC-producing Pseudomonas aeruginosa in China: a molecular epidemiology study

“…It was reasonable to assume that there was a correlation between the bla KPC-2 copy number and CAZ-AVI resistance. Previous studies had demonstrated β-lactamase gene amplification correlated with the susceptibility to β-lactam/β-lactamase inhibitor combination agents [54-56]. Our results supported these conclusions.…”