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


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.


CONCLUSIONS
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.
Perry
J D
JD
Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, UK.
James
A L
AL
Morris
K A
KA
Oliver
M
M
Chilvers
K F
KF
Reed
R H
RH
Gould
F K
FK
D044602:Cellulases / Q000096:biosynthesis; D003470:Culture Media / Q000737:chemistry; D016983:Enterococcus / Q000201:enzymology / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D005966:Glucuronidase / Q000096:biosynthesis; D006801:Humans; D014468:Umbelliferones; D014871:Water Microbiology
D003470:Culture Media; D005456:Fluorescent Dyes; D014468:Umbelliferones; D044602:Cellulases; D005966:Glucuronidase