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Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

Perry, J.D., James, A.L., Morris, K.A., Oliver, M., Chilvers, K.F., Reed, R.H., Gould, F.K.
Journal of applied microbiology 2006 v.101 no.5 pp. 977-985
Enterococcus, Escherichia coli, bacteria, beta-glucosidase, beta-glucuronidase, enzyme substrates, esters, fluorescence, glucose, glucosides, hydrolysis, indicator species, microbial growth
Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of β-glucuronidase and β-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. A novel β-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-β- [smallcapital d]-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four β-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-β- [smallcapital d]-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised β-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-β- [smallcapital d]-glucoside was inferior. However, the variability in detectable β-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. The β-glucuronidase substrate CMUG appears to be a more promising detection system than the various β-glucosidase substrates tested. The novel substrate CMUG showed enhanced sensitivity for the detection of β-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.