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

Lookup NU author(s): Dr John Perry, Professor Kate Gould

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Abstract

Aims: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. Methods and Results: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-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 beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-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-beta-D-glucoside was inferior. However, the variability in detectable beta-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. Conclusions: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. Significance and Impact of the Study: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-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.

Publication metadata

Author(s): Perry JD, James AL, Morris KA, Oliver M, Chilvers KF, Reed RH, Gould FK

Publication type: Article

Publication status: Published

Journal: Journal of Applied Microbiology

Year: 2006

Volume: 101

Issue: 5

Pages: 977-985

ISSN (print): 1364-5072

ISSN (electronic): 1365-2672

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1111/j.1365-2672.2006.03018.x

DOI: 10.1111/j.1365-2672.2006.03018.x

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