Rapid, inexpensive method for specific detection of microbial beta-lactamases by detection of fluorescent end products.
A rapid method was developed for specific detection of microbial beta-lactamases which uses ampicillin and cephalexin as substrates. The end products (open beta-lactam ring forms) generated after separately incubating either substrate with beta-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 microliters of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120 degrees C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between beta-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-alpha-aminophenylacetic acid, and the intact beta-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.
D000581:Amidohydrolases / Q000378:metabolism; D000667:Ampicillin / Q000378:metabolism; D001431:Bacteriological Techniques; D002506:Cephalexin / Q000378:metabolism; D002510:Cephalosporinase / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism; D005453:Fluorescence; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D007202:Indicators and Reagents; D010405:Penicillinase / Q000378:metabolism*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D007202:Indicators and Reagents; D000667:Ampicillin; D000581:Amidohydrolases; C019665:amidase; D002510:Cephalosporinase; D010405:Penicillinase; D001618:beta-Lactamases; C021720:nitrocefin; D002506:Cephalexin