Impact of the double deletion ΔG242-T243 in KPC-2 in the effectiveness of ceftazidime-avibactam and imipenem-relebactam

Combinations of β-lactam-diazabicyclooctane inhibitors (DBOs) like ceftazidime-avibactam (CZA) and imipenem-relebactam (IMR) have shown efficacy in treating KPC-2-producing <i>Klebsiella pneumoniae</i>. However, CZA-resistant <i>K. pneumoniae</i> strains have been identified, often linked to substitutions and/or insertions/deletions in three different loops of KPC: (i) the Ω-loop region (amino acids 164-179), (ii) the 237-243 loop; and (iii) the 266-275 loop. This study investigates the impact of the double deletion ΔG242-T243 present in KPC-14. Our results demonstrate that the lower effectiveness of CZA against KPC-14 can be explained by both increased hydrolysis of ceftazidime and a lower affinity and acylation rate by avibactam. In contrast, the IMR combination was efficient in restoring susceptibility to the KPC-14 producing-clone. Although we also observed a lower affinity and acylation rate for relebactam in KPC-14, this reduction in affinity was accompanied by a loss in the carbapenemase activity, finally resulting in an IMR susceptibility phenotype for KPC-14. Expansion of the substrate profile of KPC-14 toward ceftazidime is associated with a trade-off for carbapenems, other penicillins, and cephalosporins, as well as a higher inhibition by clavulanic acid compared to KPC-2. This study provides a better understanding of how deletions in the 237-243 loop affect the effectiveness of novel DBO-combinations and supports the hypothesis that these mutations result in CZA resistance by other different biochemical mechanisms than mutations in the Ω-loop.