A rapid fluorogenic method for the detection of Escherichia coli by the production of β-glucuronidase

A medium containing the fluorogenic substrate 4-methylumbelliferyl-β-d-glucuronide was developed for the isolation and identification of Escherichia coli within 7·5 h and was based on the detection of β-glucuronidase. Optimum conditions for the rapid development of fluorescent colonies were determined. The optimum temperature was 41·5°C. Development of fluorescence was delayed when carbohydrates were incorporated into the medium. Water samples were used to evaluate the medium by surface plating and membrane filtration. The frequency of false-negative results was 6·1% and false-positives were 3·7% for freshwater samples. The false-positive organisms were identified as Klebsiella spp. and Shigella sonnei. The potential applications of the medium are discussed.     

A method of enhancing verocytotoxin production by Escherichia coli

The number of verocytotoxin producing Escherichia coli (VTEC) present in the faeces during an infection may be very low, making their detection difficult. We report a method for enhancing toxin production by VTEC using mitomycin C as an inducing agent with the aim of improving the detection of VTEC. In pure culture, mitomycin C enhanced toxin production up to 100-fold. When applied to mixed faecal culture, toxin could be detected in mitomycin C treated samples when standard cultures were negative and when substantially fewer verocytotoxin-producing bacteria were present. Use of this method may aid in the detection of VTEC and is appropriate for use in the routine diagnostic laboratory.     

Evaluation of a fluorogenic assay for detection of Escherichia coli in foods

A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive.     

Evaluation of a commercial beta-glucuronidase test for the rapid and economical identification of Escherichia coli

A commercial beta-glucuronidase (beta-GUR) test for the rapid and economical identification of Escherichia coli was evaluated. A total of 762 clinical strains and 228 environmental isolates were studied. More than 95% of the E. coli strains were found to be beta-GUR positive. Thirty-one clinical isolates of Shigella sonnei, 10 of Enterobacter cloacae, eight of Enterobacter aerogenes, nine of Citrobacter freundii and one of Salmonella enteritidis also gave positive results. The enzyme beta-GUR was also detected in two environmental strains of E. cloacae and one C. freundii. A comparative study between the beta-GUR test and the conventional identification system was carried out in 233 consecutive isolates of lactose positive enterobacteria. Agreement was observed in 223 cases and 190 E. coli strains were correctly identified using this test. Discrepancies were found in 10 cases: nine E. coli were beta-GUR negative and one C. freundii was beta-GUR positive. Escherichia coli was the only species positive for both beta-GUR and indole tests. This procedure permits a rapid, easy, precise and inexpensive identification of E. coli. beta-GUR positive Enterobacter strains have not previously been described.     

Evaluation of beta-glucuronidase assay for the detection of Escherichia coli from environmental waters.

The new United States Drinking Water Regulations state that water systems must analyze for Escherichia coli or fecal coliforms on any routine or repeat sample that is positive for total coliforms. The proposed methods for the detection of E. coli are based on beta-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide (MUG). This study was conducted to determine whether beta-glucuronidase negative E. coli were present in significant numbers in environmental waters. Two hundred and forty E. coli cultures were isolated from 12 water samples collected from different environmental sources. beta-glucuronidase activity was determined using lauryl tryptose broth with MUG, EC broth with MUG, and the Autoanalysis Colilert (AC) procedure. The isolates were also evaluated by the standard EC broth gas fermentation method for fecal coliforms. The results confirm that assaying for the enzyme beta-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters.     

Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity

Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.     

Evaluation of a fluorogenic assay for detection of Escherichia coli in foods.

A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods. Fluorescence is indicative of the presence of E. coli; extensive biochemical confirmation is unnecessary with this assay. The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E. coli on 270 samples of raw ingredients and powdered food products. Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives. We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive.     

A new rapid fluorogenic method for measuring bacteriocin activity

We describe a novel bacteriocin screening assay based on fluorescence emitted by berberine following its influx into compromised cells. This technique showed agreement with the conventional well-diffusion method, and results can be obtained within one hour. This assay could facilitate the rapid identification of bacteriocinogenic bacterial isolates.     

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.     

Implications of alpha-amylase production and beta-glucuronidase expression in Escherichia coli strains.

Two Escherichia coli strains in which alpha-amylase production differed were used to study in depth some characteristics related to beta-glucuronidase induction by starch. The beta-glucuronidase background activity in Luria broth medium was comparable for the two isolates, but only amylase positive S1 was able to grow on starch molecules supplied as the sole carbon source. In this case growth resulted at higher beta-glucuronidase levels (p < 0.01) with respect to basal activity and the induced expression was maximal (6.1-fold) when cultures reached the stationary phase. Growth in the presence of a protein synthesis inhibitor (chloramphenicol) was associated with a marked reduction of activity. The beta-glucuronidase activity of amylase negative M94 remained unchanged during starvation on starch medium, but an induced response was observed with methylumbelliferyl-glucuronide. These results further support the hypothesis that starch metabolism is involved in the complex beta-glucuronidase regulation of E. coli strains. This is relevant not only for basic research but also to investigating gut microbial enzymology.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.     

Enzyme-capture assay for rapid detection of Escherichia coli in oysters

Enzyme-capture assays (ECAs) for Escherichia coli beta-D-glucuronidase (GUD) were performed directly from 24-h gas-positive lauryl tryptose broth (LTB) fermentation tubes that had been inoculated with oyster homogenate seeded with E. coli. The LTB-ECA method yielded results in 1 day that were equivalent to those obtained in 2 days by an LTB and EC-4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) method. Overall, 62 of 64 (97%) positive EC-MUG broths from which E. coli was isolated were correctly identified by ECA. Of 61 LTB tubes identified as GUD negative by ECA, 59 were confirmed to be free of E. coli by using EC-MUG; thus, the false-negative rate was approximately 3%. Polyclonal antibodies prepared against E. coli GUD reacted only with GUDs of E. coli, Escherichia vulneris, and Shigella sonnei. The antibodies did not react with GUDs from Flavobacterium spp., Staphylococcus spp., Yersinia enterocolitica, shellfish, or bovine liver. The GUD ECA test, when used in conjunction with the most-probable-number technique, was a rapid method for E. coli enumeration in oysters.     

Fluorogenic assay for rapid detection of Escherichia coli in food

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.     

Efficacy of beta-glucuronidase assay for identification of Escherichia coli by the defined-substrate technology

In 1976, Kilian and Bulow described the association of beta-glucuronidase with the genus Escherichia (97% positive) and suggested that a beta-glucuronidase assay would be a useful identification test. Since that report, papers about the sensitivity and specificity of this enzyme for the identification of Escherichia coli from clinical sources, food, seawater, potable-water supplies, and various environmental sources have appeared. A study was undertaken to determine the efficacy and specificity of the defined-substrate technology beta-glucuronidase (Colilert) assay for the identification of this species from fecal samples. A total of 460 human, 105 cow, and 55 horse E. coli isolates were tested. Results showed 95.5% beta-glucuronidase-positive isolates in 24 h and 99.5% positive after 28 h of incubation. Only one E. coli isolate was negative. There were no significant differences in the percentage of beta-glucuronidase-positive isolates among the human or animal isolates. There were no non-E. coli isolates that were positive. All subjects carried beta-glucuronidase-positive E. coli.     

A fluorogenic technique for the confirmation and enumeration of Escherichia coli in beansprouts

The use of the 4-methylumbelliferyl glucuronide (MUG) to differentiate between Escherichia coli and biotypes of Klebsiella pneumoniae isolated from beansprouts is described. The incorporation of MUG into the selective media ensured that the presence of Klebsiella spp. on the beansprouts was not confused with E. coli.     

An agar-overlay method for detection of toxins produced by Escherichia coli

Abstract An agar overlay method, with Vero and Hela cells, was used for detection of heat-labile enterotoxin and verotoxin from Escherichia coli . The method is more sensitive than the conventional cell culture assay, is rapid, easy to perform, and is suitable for epidemiological studies.     

One step purification of Escherichia coli beta-glucuronidase

beta-glucuronidase was purified by affinity chromatography on thiophenyl-glucuronide coupled to Sepharose. The enzyme was more than 95% pure. This enzyme is a tetramer composed of identical 74 kDa monomers. The amino-terminal sequence determined was: NH2-Met-Leu-Arg-Pro-Val.     

Expression and purification of Escherichia coli beta-glucuronidase

A strong and constitutive expression vector of Escherichia coli beta-glucuronidase with the isocitrate dehydrogenase promoter has been developed for producing a large amount of recombinant protein. More than 95% pure enzyme was obtained by a four step purification procedure-ammonium sulfate precipitation, DEAE ion-exchange chromatography, Superose 12 gel filtration, and hydroxyapatite steric ion-exchange chromatography. The overexpressed gene can produce 23 mg of pure enzyme from one liter of bacterial culture.     

Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method

Here we report a loop-mediated isothermal amplification (LAMP) method for detecting Shigella and enteroinvasive Escherichia coli. The target for this LAMP method is the ipaH gene which is carried by both of the pathogens. The LAMP method efficiently detected the gene within 2 h at a minimal amount of bacteria (8 CFU) per reaction.     

A fluorescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing Escherichia coli

A hybridization assay using fluorescence polarization was combined with the asymmetric polymerase chain reaction (PCR) in a method for the detection of the verotoxin type 2 gene of verotoxin-producing Escherichia coli. Six oligonucleotide probes labeled with FITC were designed and evaluated. One of these gave a detection limit of 10(3) colony forming units per assay, and assay results could be obtained within 5 min after PCR. It appears that the detection limit was restricted mainly by the extent and fidelity of PCR amplification, rather than by the sensitivity of the fluorescence polarization technique, indicating that good probe design facilitates the rapid detection of the PCR product. The fluorescence polarization assay, in conjunction with DNA amplification by PCR, is a powerful and widely applicable method for the rapid and sensitive detection of oligonucleotide sequences.