Biochemical analysis of the ceftazidime-hydrolysing extended-spectrum β-lactamse CTX-M-15 and of its structurally related β-lactamase CTX-M-3

Abstract

The extended-spectrum β-lactamase CTX-M-15 confers resistance to ceftazidime, unlike the majority of CTX-M-type enzymes. Kinetic parameters were determined from purified CTX-M-15 and CTX-M-3, which differ by the single amino acid substitution Asp-240 to Gly, according to the Ambler numbering of class A β-lactamases. Relative molecular masses of CTX-M-15 and CTX-M-3 were ∼29 kDa and pl values were 8.9 and 8.4, respectively. CTX-M-15 had higher affinities for β-lactams (lower Km values) than those of CTX-M-3 but catalytic efficiency (kcat/Km values) was variable depending on the β-lactam substrate. Only CTX-M-15 showed a measurable catalytic efficiency for ceftazidime. Clavulanic acid and tazobactam were good inhibitors of both enzymes. MICs of β-lactams for Escherichia coli reference strains expressing cloned β-lactamase genes in the same genetic background were similar except for ceftazidime. This work underlines the fact that some CTX-M enzymes may hydrolyse ceftazidime and thus confer resistance to this expanded-spectrum cephalosporin in Enterobacteriaceae.