Unravelling ceftazidime/avibactam resistance of KPC-28, a KPC-2 variant lacking carbapenemase activity

• Published in: Journal of antimicrobial chemotherapy Vol. 74; no. 8; pp. 2239 - 2246
• Main Authors: Oueslati, Saoussen, Iorga, Bogdan I, Tlili, Linda, Exilie, Cynthia, Zavala, Agustin, Dortet, Laurent, Jousset, Agnès B, Bernabeu, Sandrine, Bonnin, Rémy A, Naas, Thierry
• Format: Journal Article
• Language: English
• Published: England Oxford University Press (OUP) 01.08.2019
• Subjects: Bacteriology; Life Sciences; Microbiology and Parasitology; Pharmaceutical sciences; Pharmacology
• ISSN: 0305-7453; 1460-2091; 1460-2091
• DOI: 10.1093/jac/dkz209

KPC-like carbapenemases have spread worldwide with more than 30 variants identified that differ by single or double amino-acid substitutions. To describe the steady-state kinetic parameters of KPC-28, which differs from KPC-2 by a H274Y substitution and the deletion of two amino acids (Δ242-GT-243). The blaKPC-2, blaKPC-3, blaKPC-14 and blaKPC-28 genes were cloned into a pTOPO vector for susceptibility testing or into pET41b for overexpression, purification and subsequent kinetic parameter (Km, kcat) determination. Molecular docking experiments were performed to explore the role of the amino-acid changes in the carbapenemase activity. Susceptibility testing revealed that Escherichia coli producing KPC-28 displayed MICs that were lower for carbapenems and higher for ceftazidime and ceftazidime/avibactam as compared with KPC-2. The catalytic efficiencies of KPC-28 and KPC-14 for imipenem were 700-fold and 200-fold lower, respectively, than those of KPC-2, suggesting that Δ242-GT-243 in KPC-28 and KPC-14 is responsible for reduced carbapenem hydrolysis. Similarly, the H274Y substitution resulted in KPC-28 in a 50-fold increase in ceftazidime hydrolysis that was strongly reversed by clavulanate. We have shown that KPC-28 lacks carbapenemase activity, has increased ceftazidime hydrolytic activity and is strongly inhibited by clavulanate. KPC-28-producing E. coli isolates display an avibactam-resistant ESBL profile, which may be wrongly identified by molecular and immunochromatographic assays as the presence of a carbapenemase. Accordingly, confirmation of carbapenem hydrolysis will be mandatory with assays based solely on blaKPC gene or gene product detection.