Defining substrate specificity in the CTX-M family: the role of Asp240 in ceftazidime hydrolysis [PublishAheadOfPrint]

The natural diversification of CTX-M β-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime/avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the k_cat/K_m for CAZ was 5-15-fold higher for all Asp240Gly variants, but remained 200-725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ resistant clones yielded non-susceptible CTX-M-producers (MIC>16 μg/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96 with K_iapp comparable to SHV-2 and 1,000-fold greater than PER-2 and CMY-2; k₂/K for CTX-M-12 was 24- and 35-fold higher than CTX-M-96 and CTX-M-15, respectively. Molecular modelling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e. OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant sub-populations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA could be an effective preventive strategy to delay the development of resistance in this family of ESBLs.

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