Rapid, inexpensive method for specific detection of microbial β-lactamases by detection of fluorescent end products

Abstract

A rapid method was developed for specific detection of microbial β-lactamases which uses ampicillin and cephalexin as substrates. The end products (open β-lactam ring forms) generated after separately incubating either substrate with β-lactamase-producing organisms initially were separated from the unhydrolyzed substrates by high-voltage electrophoresis at pH 2.1. The end products of both antibiotics were highly fluorescent and could be analyzed visually and semiquantitatively under a long-wave UV lamp. Application of 5 μl of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120° C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between β-lactamase and acylase activities, since the end products to acylase [the common side chain, D(-)-α-aminophenylacetic acid, and the intact β-lactam nuclei, 6-aminopenicillanic acid and 7-aminodeacetoxycephalosporanic acid] are not fluorescent. This method was relatively rapid, inexpensive, and more sensitive than the chromogenic cephalosporin (nitrocefin) method when 21 strains of 7 gram-positive species and 77 strains of 29 gram negative species of bacteria were tested.