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

Abstract

A rapid method was developed for specific detection of microbial 1-lactamases which uses ampicillin and cephalexin as substrates. The end products (open 3-lactam ring forms) generated after separately incubating either substrate with 3-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 ,ul 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 3-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-a-aminophenylacetic acid, and the intact 3-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 gramnegative species of bacteria were tested. P-lactamases which hydrolyze the amide bonds of the ,Blactam ring of sensitive penicillins and cephalosporins (Fig. 1 and 2) are widely distributed among microorganisms (14) and play ah important role in microbial resistance to f-lactam antibiotics. Chemical methods used for detecting microbial * Corresponding author.

Ascorbate as an induction inhibitor of β-lactamase in a strain of Enterobacter cloacae

“…In that respect, Ent. cloacae ATCC 13047 was reported to have an inducible fl-lactamase with predominant cephalosporinase specificity (Chen et al 1984). With such specificity, lower Plactamase content would hydrolyse cephalosporins so efficiently that it affords protection to the cell against these cephalosporins but not penicillins.…”

Pattern recognition analysis of in vivo enzyme-substrate fluorescence velocities in microorganism detection and identification

“…The P-lactamases (4) penicillinase and cephalosporinase are two other extracellular enzymes that could be considered in the pattern recognition set, because an in vivo reaction with the nonfluorescent penicillin and cephalosporin substrates produces fluorescent products. Their investigation is further desirable in that the products are generated within 5 to 15 min at room temperature, producing a bluegreen fluorescence with 300to 350-nm excitation.…”

“…These methods include pyrolysis mass spectrometry (7,11,12,21), microbial phospholipid fatty acid extracts (15), countercurrent chromatographic bacterial separation (10), excitation-emission matrices with a video fluorometer of whole-cell supernatants (19) and differential dye-cell wall binding (20), time-resolved fluorescence with wavelengthdependent lifetimes (3), resonance Raman spectra of UVexcited organisms (9), staining organisms in blood cultures (18), and the enzyme-linked lectinosorbent assay (5,8) for detecting and differentiating Bacillus anthracis from closely related bacilli. Recent methods developed to probe in vivo enzymes include the chromogenic ax-glucosidase substrate probe in distinguishing B. anthracis from other bacilli (17), the fluorogenic 4-methylumbelliferone-3-D-glucuronide assay of p-glucuronidase in Escherichia coli (16), and the fluorometric assay of the P-lactamases (4) with penicillin and cephalosporin as substrates for a variety of gram-positive and gram-negative organisms. These methods span various degrees of complexity of design, sensitivity, specificity, convenience, and time required to generate sufficient data.…”

Two-dimensional thin-layer chromatography for the specific detection of hippurate hydrolysis by microorganisms

“…isolated from vaginal washings on human blood bilayer agar media with Tween 80 or without Tween 80 (20) in 36°C candle extinction jars and identified by typical microscopic and colonial morphology (20). The growth conditions and sources of gram-negative bacteria (enteric and nonenteric) and other gram-positive bacteria listed in Table 1 were described previously (6). Hippurate hydrolysis and dansyiation.…”