Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci
- Source:
- Journal of applied microbiology 2006 v.101 no.5 pp. 977-985
- ISSN:
- 1364-5072
- Subject:
- Enterococcus, Escherichia coli, bacteria, beta-glucosidase, beta-glucuronidase, enzyme substrates, esters, fluorescence, glucose, glucosides, hydrolysis, indicator species, microbial growth
- Abstract:
- 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.
- Agid:
- 268750
- https://doi.org/10.1111/j.1365-2672.2006.03018.x