Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Manafi, M.; Kneifel, W.; Bascomb, Shoshana

doi:10.1128/mmbr.55.3.335-348.1991

Cited by 173 publications

(110 citation statements)

References 97 publications

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“…The use of media containing substrates for the enzyme b-dglucuronidase for the detection of Escherichia coli in water has become widely adopted in many regions of the world (Cowburn et al 1994;Eckner 1998;Edberg et al 1988;Manafi et al 1991) and such methods have been shown to be more specific than methods based upon fermentation of lactose and production of indole from tryptophan at 44°C (Fricker et al 2008). However, it is clear that not all such media perform equally (Fricker and Fricker 1996;Niemela et al 2003;Fricker et al 2008;Olstadt et al 2007).…”

Section: Introductionmentioning

confidence: 99%

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

Fricker

DeSarno

Warden

et al. 2008

Letters in Applied Microbiology

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Aims: Testing for β‐d‐glucuronidase activity has become the basis of many methods for the detection of Escherichia coli in both food and water. Used in combination with tests for the presence of β‐d‐glucuronidase, these tests offer a simple method for simultaneously detecting coliforms and E. coli. The purpose of this study was to determine the effectiveness of several different procedures in detecting β‐d‐glucuronidase activity and hence in detecting E. coli. Methods and Results: The ability of membrane lactose glucuronide agar (MLGA), Colilert‐18®, MI agar, Colitag® and Chromocult agar to detect β‐d‐glucuronidase activity was tested with over 1000 isolates of E. coli recovered from naturally contaminated water samples. Four of the media gave very similar results but MLGA failed to detect glucuronidase activity in 15·6% of the cultures tested. Conclusions: MLGA had very poor sensitivity for the detection of β‐d‐glucuronidase activity in strains of E. coli isolated from naturally contaminated water. This is probably because of the fact that β‐d‐glucuronidase activity is pH‐sensitive and that acid is formed by E. coli during fermentation of lactose in MLGA. Significance and Impact of the Study: The detection of E. coli in drinking water is the primary test used to establish faecal contamination. The poor sensitivity of MLGA in detecting β‐d‐glucuronidase activity suggests that this medium and others containing high concentrations of fermentable carbohydrate should not be used for the detection of E. coli.

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Section: Introductionmentioning

confidence: 99%

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

Fricker

DeSarno

Warden

et al. 2008

Letters in Applied Microbiology

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“…Kramer and Liu 2002). However, the limitations of 4-MU are well-recognized (Manafi et al 1991) and several studies have described the synthesis of novel umbelliferones with lower pK a values (Goodwin and Kavanagh 1950;Sherman and Robins 1968;Koller and Wolfbeis 1985;Wolfbeis et al 1985). Substrates based on such fluorophores have been shown to be particularly advantageous for the assay of enzymes that have a nonalkaline pH optimum (Tsvetkova et al 1996;Gee et al 1999).…”

Section: Discussionmentioning

confidence: 99%

“…derivatives of 4-MU are widely used for detection of bacterial enzymes such as b-galactosidase, b-glucuronidase and b-glucosidase (Manafi et al 1991;Dealler 1993). Such assays have been widely applied in the water industry as these enzymes provide convenient markers for the detection of coliforms, Escherichia coli and enterococci respectively (Berg and Fiksdal 1988;Edberg et al 1988;Clarke et al 1991;Brenner et al 1993;George et al 2000;Caruso et al 2002;Kramer and Liu 2002;Pope et al 2003).…”

mentioning

confidence: 99%

“…A major limitation of 4-MU is its relatively high pK a of 7AE8, which leads to the partial dissociation of the molecule to its highly fluorescent anion at physiological pH values Wolfbeis et al 1985). This results in a sub-optimal fluorescent signal in microbiological assays unless alkali is added at the end of an assay to amplify the fluorescence (Manafi et al 1991;Gee et al 1999). Other fluorescent coumarins have been synthesized which have lower pK a values and consequently higher fluorescence intensities at pH 7 (e.g.…”

mentioning

confidence: 99%

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

et al. 2006

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Aims: 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. Methods and Results: A novel β‐glucuronide derivative based on 6‐chloro‐4‐methylumbelliferone (CMUG) was synthesized and compared with 4‐methylumbelliferyl‐β‐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‐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‐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. Conclusions: The β‐glucuronidase substrate CMUG appears to be a more promising detection system than the various β‐glucosidase substrates tested. Significance and Impact of the Study: 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.

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“…Substrates based on 4-methylumbelliferone (4-MU) have been used extensively for the detection of enzymes in diagnostic microbiology (Mana® et al 1991;Dealler 1993;James 1994). This is due to the ease of hydrolysis and intense¯uorescence generated on release of 4-MU from the substrate by speci®c enzymes (Berg and Fiksdal 1988;Shadix and Rice 1991;Brenner et al 1993).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2001

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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.

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Fluorogenic and chromogenic substrates used in bacterial diagnostics.

Cited by 173 publications

References 97 publications

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

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

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

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