Aims: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-b-D D-galactoside (4-methylumbelliferone-b-D D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the b-galactosidase activity of coliform bacteria in water analysis. The intense¯uorescence 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-caboxylate (EHC) giving a combination of greater uorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a b-galactoside derivative, EHC-GAL generated a more rapid¯uorescence 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. Signi®cance 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¯uorescence; (iii) pH stability of¯uorescence; and (iv) rapid detection.
Novel 7-N-(beta-alanyl)aminophenoxazin-3-one salts 27a-d have been synthesized and tested as chromogenic substrates for beta-alanyl aminopeptidase, which is present in Pseudomonas aeruginosa, the most common respiratory pathogen in patients with cystic fibrosis. The biological results show that 7-N-(beta-alanyl)amino-1-pentylphenoxazin-3-one trifluoroacetate salt 27a is a chromogenic substrate for this bacterium, with a low degree of diffusion in nutrient media for growing bacterial cultures and a bright red colour, making it easily distinguishable from the agar background.
We describe the synthesis of two new substrates for the detection of -galactosidase and evaluate their performance in comparison with that of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). Of 171 Enterobacteriaceae strains that were able to hydrolyze X-Gal, 166 (97.1%) also hydrolyzed cyclohexenoesculetin--D-galactoside whereas only 96 (56.1%) showed evidence of hydrolysis of 8-hydroxyquinoline--D-galactoside. No false-positive results were observed with either substrate.
Aims: To synthesize and evaluate¯uorogenic substrates for the detection of microbial nitroreductases. These substrates, all based on 7-nitrocoumarin, may be reduced to form uorescent aminocoumarins. Methods and Results: Thirty pathogenic microbial strains, including both bacteria and yeasts, were examined for nitroreductase activity in a whole-cell assay. All strains readily reduced each of the seven substrates to generate¯uorescence, suggesting the widespread presence of nitroreductase activity in pathogenic bacteria.Conclusions, Signi®cance and Impact of the Study: These novel substrates facilitate the direct detection of nitroreductase activity and have potential as sensitive indicators of microbial growth.
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