Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis

Abstract

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- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >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 an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k₂/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling 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 subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum β-lactamases.

Keywords: Asp240Gly; CTX-M-96; OmpF; avibactam; cefotaxime; ceftazidimase; ceftazidime; ceftazidime-avibactam.

FIG 1

(A and B) Far-UV (A) and near-UV (B) CD spectra for CTX-M^Asp (solid line) and CTX-M^Gly (dashed line) variants. (C and D) Thermally induced unfolding transitions. CD measurements were recorded for CTX-M^Asp (closed symbols) and CTX-M^Gly (open symbols) as temperature increased. (C) The transitions were monitored by the evolution of the molar ellipticity at 222 nm. (D) The value of the dynode voltage under each condition is shown.

FIG 2

Population analysis profile of E. coli K-12 strains expressing CTX-M-12 and CTX-M-96 recovered under CAZ pressure selection.

FIG 3

(Left) Two-dimensional representation of CTX-M-96 (Gly240) in association with AVI (created with LigPlot+); green dotted lines represent hydrogen bonds (HB), and red semicircles denote putative hydrophobic interactions. (Right) Three-dimensional in silico modeling showing the detailed interaction between AVI and CTX-M-96 residues; black dotted lines represent favored HB (see the text for further discussion).