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

journal contribution
posted on 06.12.2017, 00:00 by J Perry, A James, K Morris, M Oliver, K Chilvers, Robert Reed, F Gould
Aims: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of b-glucuronidase and b-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 b-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-b-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 b-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-b-dglucoside(MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised b-glucoside derivatives of umbelliferone-3-carboxylicacid 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-b-d-glucoside was inferior. However, the variability in detectable b-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 enumerationof these faecal indicator bacteria. Conclusions: The b-glucuronidase substrate CMUG appears to be a more promising detection system than the various b-glucosidase substrates tested. Significance and Impact of the Study: The novel substrate CMUG showed enhanced sensitivity for the detection of b-glucuronidase-producing bacteriasuch 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.

History

Volume

101

Start Page

977

End Page

985

Number of Pages

9

ISSN

1364-5072

Location

United Kingdom

Publisher

Wiley-Blackwell Publishing Ltd

Language

en-aus

Peer Reviewed

Yes

Open Access

No

Era Eligible

Yes

Journal

Journal of applied microbiology.

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