An electrochemiluminescent method for the detection of beta-lactamases in microorganisms.

GRACIELA PRIANO; SANDRA RUZAL; FERNANDO BATTAGLINI

ANALYTICAL BIOCHEMISTRY

Elsevier

Lugar: Amsterdam; Año: 2001 vol. 290 p. 379 - 382

0003-2697

The most important resistance mechanism toward b-lactam antibiotics is the manifestation of b-lactamases. These enzymes catalyze the opening of the cyclic amide, shown in Fig. 1. The assays for the resistance evaluation involve commercial kits (antibiograms or the Kirky-Bauer method). The limitations of this method are the ability of the microorganism to grow in the selected medium, the presence of antibiotic inhibitors in this medium, the diffusion in agar, and the incubation for at least 16 h to obtain results. These assays are rationalized using tables which establish levels of resistance for the microorganism to them. Electrochemically generated tris(2,29-bipyridyl) ruthenium(III), Ru(bpy)3 oxidizes some organic molecules producing luminescence. This process called electrochemiluminescence (ECL) was studied by several authors, and a comprehensive review on the analytical applications can be found elsewhere. Liang et al. described the use of electrochemiluminescence to detect b-lactam antibiotics and their hydrolysis products. They observed that the hydrolyzed antibiotic shows a dramatic increase in the light emitted compared with the nonhydrolyzed antibiotic. They show its potential use in the determination of b-lactamases in microorganisms or in the detection of b-lactam antibiotics in a sample. In their work, they do not discuss which antibiotics are convenient for detecting b-lactamases in an unknown microorganism and the ECL responses for different antibiotics are presented as a ratio between hydrolyzed and nonhydrolyzed antibiotic, which does not provide information on the sensitivity of the method for each antibiotic. In this work we present a detailed description of three different antibiotics that could be used to assess the presence of b-lactamases in microorganism using a simple wall-jet FIA system. The required care in the treatment of the microorganism samples is also discussed.b-lactam antibiotics is the manifestation of b-lactamases. These enzymes catalyze the opening of the cyclic amide, shown in Fig. 1. The assays for the resistance evaluation involve commercial kits (antibiograms or the Kirky-Bauer method). The limitations of this method are the ability of the microorganism to grow in the selected medium, the presence of antibiotic inhibitors in this medium, the diffusion in agar, and the incubation for at least 16 h to obtain results. These assays are rationalized using tables which establish levels of resistance for the microorganism to them. Electrochemically generated tris(2,29-bipyridyl) ruthenium(III), Ru(bpy)3 oxidizes some organic molecules producing luminescence. This process called electrochemiluminescence (ECL) was studied by several authors, and a comprehensive review on the analytical applications can be found elsewhere. Liang et al. described the use of electrochemiluminescence to detect b-lactam antibiotics and their hydrolysis products. They observed that the hydrolyzed antibiotic shows a dramatic increase in the light emitted compared with the nonhydrolyzed antibiotic. They show its potential use in the determination of b-lactamases in microorganisms or in the detection of b-lactam antibiotics in a sample. In their work, they do not discuss which antibiotics are convenient for detecting b-lactamases in an unknown microorganism and the ECL responses for different antibiotics are presented as a ratio between hydrolyzed and nonhydrolyzed antibiotic, which does not provide information on the sensitivity of the method for each antibiotic. In this work we present a detailed description of three different antibiotics that could be used to assess the presence of b-lactamases in microorganism using a simple wall-jet FIA system. The required care in the treatment of the microorganism samples is also discussed.