Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and β-D- glucuronidase

Abstract

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and β-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the α-decarboxylation of L- glutamic acid to yield γ-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4- methylumbelliferyl-β-D-glucuronide to the fluorogenic compound 4- methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65°C but not at 68°C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than tines the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.