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

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.

Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial beta-galactosidase.

AIMS: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-beta-D-galactoside (4-methylumbelliferone-beta-D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the beta-galactosidase activity of coliform bacteria in water analysis. The intense fluorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. METHODS AND RESULTS: Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-carboxylate (EHC) giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a beta-galactoside derivative, EHC-GAL generated a more rapid fluorescence than any other tested substrate. CONCLUSIONS: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC-GAL around 1 h earlier than with 4-MU-GAL. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that EHC-GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased fluorescence; (iii) pH stability of fluorescence; and (iv) rapid detection.

Evaluation of p-naphtholbenzein-beta-D-galactoside as a substrate for bacterial beta-galactosidase.

We describe the synthesis of a new substrate for the detection of beta-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) and cyclohexenoesculetinbeta-D-galactoside (CHE-Gal). Of 206 Enterobacteriaceae strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed p-naphtholbenzein-beta-D-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of beta-galactosidase.

Phototoxicity of rose bengal in mycological media--implications for laboratory practice.

The effects of Rose Bengal (RB) on plate counts of the bacteria Escherichia coli and Pseudomonas aeruginosa, and the yeast Saccharomyces cerevisiae, were studied under natural sunlight and artificial fluorescent lighting. While RB was not inherently toxic in darkness at concentrations found in mycological media, the illumination of media containing RB caused a decrease in colony counts in all cases, and especially for surface spread plates. A negative synergy was observed between chloramphenicol, RB and illumination using a spring water sample containing substantial numbers of Gram-negative bacteria. Exposure of media containing RB to moderate amounts of light during standard laboratory procedures may inhibit microbial growth, with positive benefits in relation to the suppression of contaminant bacteria, or negative implications where fungi are inhibited.