DISCRIMINATION OF EXTENDED-SPECTRUM BETA-LACTAMASES BY A NOVEL NITROCEFIN COMPETITION ASSAY

We describe a nitrocefin competition assay for determining inhibition profiles as a useful adjunct to existing biochemical methods for the discrimination of β-lactamases. The hydrolysis rate of nitrocefin was measured with a plate photometer as the change in A480 over 45 min in the presence of 17 inhibitors. Fourteen well-established β-lactamases and 13 extended-spectrum β-lactamases were tested. Correlations with data from isoelectric focusing and amino acid sequencing suggested that the inhibition profile reflects alterations in the active-site configuration of β-lactamases. The method was especially useful in measuring the relative affinities of β-lactamases against poorly hydrolyzed substrates and in screening large numbers of isolates for the detection of new β-lactamase types.