Molecular determinants underlying enhanced ceftazidime resistance mediated by the β-lactamase CTX-M-55 compared with CTX-M-14

Abstract

Salmonella strains carrying the bla_CTX-M-55 gene encoding the β-lactamase CTX-M-55 present markedly greater resistance to ceftazidime (CAZ) than those carrying the bla_CTX-M-14 gene encoding CTX-M-14, but the structural basis remains unclear. In this study, we combined susceptibility testing, binding affinity measurement, molecular dynamics simulation, and site-directed point mutation to examine how CTX-M-55 enhances CAZ hydrolysis. Compared with CTX-M-14, CTX-M-55 conferred an eightfold higher minimum inhibitory concentration for CAZ and exhibited more than tenfold stronger CAZ binding affinity. Structural and simulation analyses revealed that CTX-M-55 possesses a larger and more hydrophobic active pocket that supports more rapid interactions with CAZ and forms a more stable binding environment, driven mainly by favorable van der Waals and solvation energies. Mutation analysis further revealed two functionally distinct classes of residues contributing to CAZ resistance. Asp-131 and Asn-132 represent functional determinants of CAZ hydrolysis in CTX-M-55, as their substitution disrupts the structure of the binding pocket and hinders the effective binding of the substrate. In contrast, Ser-272 acts as an optimized residue that strengthens the hydrolytic capacity of CTX-M-55 compared with that of CTX-M-14 by modulating CAZ accommodation within a stable pocket. Together, these results indicate that the stronger CAZ resistance of CTX-M-55 results from both a favorable pocket structure and specific residue effects, with Asp-131 and Asn-132 providing the basic hydrolysis capacity and Ser-272 playing an optimized role for CAZ. This work helps clarify the differences in CAZ resistance among the CTX-M family and points to pocket features that may be useful targets for inhibitor design.

Keywords

CTX-M β-lactamase / Ceftazidime / Resistance / Substrate-binding pocket / Molecular dynamics simulation / Bacteria

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Funding

National Key Research and Development Program of China(2022YFD1800402)

RIGHTS & PERMISSIONS

The Author(s)