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First variants of the Klebsiella pneumoniae carbapenemase (KPC), KPC-2 and KPC-3, have encountered a worldwide success, particularly in K. pneumoniae isolates. These beta-lactamases conferred resistance to most beta-lactams including carbapenems but remained susceptible to new beta-lactam/beta-lactamase inhibitors, such as ceftazidime-avibactam. After the marketing of ceftazidime-avibactam, numerous variants of KPC resistant to this association have been described among isolates recovered from clinical samples or derived from experimental studies. In KPC variants resistant to ceftazidime-avibactam, point mutations, insertions and/or deletions have been described in various hot spots. Deciphering the impact of these mutations is crucial, not only from a therapeutic point of view, but also to follow the evolution in time and space of KPC variants resistant to ceftazidime-avibactam. In this review, we describe the mutational landscape of the KPC beta-lactamase toward ceftazidime-avibactam resistance based on a multidisciplinary approach including epidemiology, microbiology, enzymology, and thermodynamics. We show that resistance is associated with three hot spots, with a high representation of insertions and deletions compared with other class A beta-lactamases. Moreover, extension of resistance to ceftazidime-avibactam is associated with a trade-off in the resistance to other beta-lactams and a decrease in enzyme stability. Nevertheless, the high natural stability of KPC could underlay the propensity of this enzyme to acquire in vivo mutations conferring resistance to ceftazidime-avibactam (CAZavi), particularly via insertions and deletions.

Objectives: Ceftazidime-avibactam (CZA) and cefiderocol are recently commercialized molecules active against highly drug-resistant bacteria, including carbapenem-resistant members of the Enterobacteriaceae. Mutants resistant to CZA have been described, notably in Klebsiella pneumoniae carbapenemase (KPC) producers. Considering the structural similarities between ceftazidime and cefiderocol, we hypothesized that resistance to CZA in KPC-producing members of the Enterobacterales may lead to cross-resistance to cefiderocol., Methods: CZA-resistant mutants from three clinical isolates of the Enterobacterales carrying either bla KPC-2 or bla KPC-3 were selected in vitro. Mutants with increased MIC to CZA compared to the ancestral allele were cloned in a pBR322 plasmid and expressed in Escherichia coli TOP10. We evaluated the impact of these mutations on cefiderocol MICs and minimal bactericidal concentrations (MBCs), and we assessed the impact of bacterial inoculum size on cefiderocol MICs., Results: We used 37 KPC mutants with increased CZA MICs. Of these, six have been described previously in clinical isolates. Compared to the wild-type alleles, increases in the cefiderocol MICs of 4- to 32-fold were observed for 75.6% of tested mutants (28/37), MICs reaching up to 4 mg/L in E. coli TOP10 for KPC-31 (D179Y-H274Y mutations). MBCs and MICs of cefiderocol were similar, confirming the bactericidal activity of this drug. Finally, when using higher inocula (10 7  CFU/mL), a large increase in cefiderocol MIC was observed, and all isolates were categorized as resistant., Conclusion: We observed that most of the CZA-resistant KPC variants have a possible impact on cefiderocol by increasing the cefiderocol MICs. In addition, cefiderocol is greatly impacted by the inoculum effect, suggesting that precautions should be taken when treating infections with a suspected high inoculum., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To explore the mutational possibilities of insertions and deletions (indels) in the Klebsiella pneumoniae carbapenemase (KPC) beta-lactamase, we selected for ceftazidime-avibactam-resistant mutants. Of 96 screened mutants, we obtained 19 indels (2 to 15 amino acids), all located in the loops surrounding the active site. Three antibiotic susceptibility phenotypes emerged: an extended-spectrum-beta-lactamase-like phenotype, an activity restricted to ceftazidime, and a carbapenem-susceptible KPC-like phenotype. Tolerance for indels reflects the evolvability of KPC beta-lactamase, which could challenge the therapeutic management of patients., (Copyright © 2020 American Society for Microbiology.)