title: Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci.
abstract: 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.
authors.affiliation: Department of Microbiology, Freeman Hospital, Newcastle upon Tyne, UK.
authors.forename: J D
authors.initials: JD
authors.lastname: Perry
authors.forename: A L
authors.initials: AL
authors.lastname: James
authors.forename: K A
authors.initials: KA
authors.lastname: Morris
authors.forename: M
authors.initials: M
authors.lastname: Oliver
authors.forename: K F
authors.initials: KF
authors.lastname: Chilvers
authors.forename: R H
authors.initials: RH
authors.lastname: Reed
authors.forename: F K
authors.initials: FK
authors.lastname: Gould
mesh_terms: 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
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D014468:Umbelliferones; D044602:Cellulases; D005966:Glucuronidase