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.     

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.     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

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.     

The use of lauryl tryptose broth containing 4-methylumbelliferyl-β-D-glucuronide (MUG) to enumerate Escherichia coli from freshwater sediment

Most-Probable-Number tests for Escherichia coli were performed on 45 sediment samples using Lauryl Tryptose broth (LTB) containing 4-methylumbelliferyl-β-D-glucuronide (MUG). Eighty-three percent of 493 LTB-MUG reactions agreed with results obtained by conventional faecal coliform analysis. Although false-negative reactions are suspected in about 9% of the LTB-MUG tubes, anaerogenic E. coli , that would not be enumerated by conventional faecal coliform tests were recovered using LTB-MUG. The high percentage of agreement between the two methods suggests that the MPN method employing LTB-MUG is adequate to enumerate E. coli from some freshwater sediments.     

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 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.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Dipstick immunoassay to detect enterohemorrhagic Escherichia coli O157:H7 in retail ground beef.

A sensitive and easy-to-perform dipstick immunoassay to detect Escherichia coli O157:H7 in retail ground beef was developed by using a sandwich-type assay (with a polyclonal antibody to E. coli O157 as the capture antibody and a monoclonal antibody to E. coli O157:H7 as the detection antibody) on a hydrophobic polyvinylidine difluoride-based membrane. E. coli O157:H7 in ground beef could be detected within 16 h, including incubation for 12 h in enrichment broth and the immunoassay, which takes 4 h. Pure culture cell suspensions of 10(5) or 10(6) E. coli O157:H7 organisms per ml produced intense color reactions in the immunoassay, whereas faint but detectable reactions occurred with 10(3) CFU/ml. The sensitivity of the combined enrichment-immunoassay procedure as determined by using ground beef inoculated with E. coli O157:H7 was 0.1 to 1.3 cells per g, with a false-positive rate of 2.0%. A survey of retail ground beef using this procedure revealed that 1 of 76 samples was contaminated by E. coli O157:H7.     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

Evaluation of test media for routine monitoring of Escherichia coli in nonpotable waters.

Six test media, m-TEC, m-TEC with 4-methylumbelliferyl-beta-D-glucuronide (MUG), lauryl tryptose agar (LTA) with MUG, LTA with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Glue), EC medium with MUG, and lauryl tryptose broth with MUG, were evaluated for their usefulness in enumerating Escherichia coli in nonpotable waters on a routine basis. The media were chosen for their case of interpretation of target colonies, ability to allow enumeration at low and high concentrations, and ability to inhibit nontarget microorganisms. The recoveries on the test media were compared with those on three reference media, R2A, m-FC, and m-Endo, by analysis of spiked samples of filter-sterilized waters. The test media were then further tested for their ability to differentiate nontarget but closely related microorganisms. Statistical analysis indicated that the best recoveries were obtained with lauryl tryptose agar with added MUG and X-Gluc. The media were then tested with surface waters that could be expected to have high levels of total and fecal coliforms along with Escherichia coli.     

使用MUG培养基定量检测植物药中大肠杆菌时荧光猝灭现象的发生与克服方法

实验中观察到,用ＭＵＧ培养基对植物药中的大肠杆菌定量时多发生荧光猝灭现象,影响检测结果。本文对此现象产生的原因与克服方法进行了系统的考察,发现以一种简便的转接方法可排除植物药介质对菌检的干扰。该方法由两组检验系列构成,当怀疑正常稀释系列（第一系列）４０ｈ培养液的荧光结果可能因猝灭现象呈假阴性时,立即分别将该系列的１—３号管培养液以０．５ｍｌ的接种量转接入新鲜的ＭＵＧ培养基（第二系列）,重新培养２４ｈ,荧光猝灭现象即可克服。综合两系列的荧光、产气和吲哚三项生化特征得出检品中大肠杆菌含量。实际应用表明,此法能显著提高使用该培养基时菌检结果的可靠性。     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Detection of Escherichia coli in potable water using direct impedance technology

Direct impedance measurement utilizing a medium previously described as being specific for Escherichia coli and which contains trimethylamine N -oxide (TMAO) and glucuronic acid was used to detect E. coli in water samples. The system was compared with the Colilert® presence/absence test and the United Kingdom standard membrane nitration technique using membrane lauryl sulphate broth. The impedance method correlated well with both the traditional membrane method (93%) and the Colilert® method (93.95%) for a number of different water types. No interference from Citrobacter spp. (as reported in previous studies) was detected in this study although some Salmonella spp. did give false-positive results. The data presented here suggest that the use of direct impedance may offer an alternative to conventional methods for the detection of E. coli in water.     

Comparison of LST + MUG broth technique and conventional method for the enumeration of Escherichia coli in foods

AIMS: To reduce the analysis time needed for the enumeration of Escherichia coli, a rapid fluorogenic method (MUG) which takes only 48 h was compared with the standard most probable number (MPN) method which takes 6 days as described in the International Standards Organization (ISO). This study provides reliability data for the fluorogenic method applied to certain foods. METHODS AND RESULTS: Both methods were applied to 500 food samples which were analysed for E. coli enumeration. Agreement between the two methods was found in 409 (81 x 8%) samples; 81 (16 x 2%) samples gave higher values by the fluorogenic method, and only 10 (2 x 0%) samples were more effectively assayed by the ISO method. According to statistical analysis, the reliability between the methods was r = 0 x 9706, r(2) = 0 x 9421 and Cronbach's alpha = 0 x 9851. While all three values showed a high degree of correlation (P < 0 x 0001) between the two methods, McNemar's test demonstrated a significant difference between them, indicating that the MUG method was more reliable than the ISO method. CONCLUSIONS: The data suggest that the fluorogenic method is more reliable and shorter to perform than the standard ISO method. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of the two methods may provide a rapid and more reliable alternative for the enumeration of E. coli in food samples.     

Ability of the Colilert method to recover oxidant-stressed Escherichia coli

Methods for the microbiological analysis of drinking water must be able to detect Escherichia coli that may be injured by treatment. The Colilert method, which simulaneously detects total coliforms and E. coli in water samples by the observation of direct colour changes produced by defined substrates in the media, was found to be equivalent to the reference EC MUG method in its ability to recover low numbers (< 4/100 ml) of oxidant-stressed E. coli.     

An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli.

A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1- and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 10(2) and 2 x 10(3) 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29% more E. coli O157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including O157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains.     

Membrane filtration differentiation of E. coli from coliforms in the examination of water

A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed. Membranes containing coli-form colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-β-d-glucuronide (MUG) and incubated at 35°C for 4 h. The MUG is hydrolyzed by the glucuronidase of E. coli and the fluorogenic product is visualized. The method recovered 98% of E. coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.     

Membrane filtration differentiation of E. coli from coliforms in the examination of water

A membrane filter-Endo agar method for enumerating Escherichia coli as distinct from other coliforms in drinking water was developed. Membranes containing coliform colonies are transferred to nutrient agar containing 4-methyl umbelliferyl-beta-D-glucuronide (MUG) and incubated at 35 degrees C for 4 h. The MUG is hydrolyzed by the glucuronidase of E. coli and the fluorogenic product is visualized. The method recovered 98% of E. coli without false positives and is proposed as an additional test in routine water examination for the detection of pollution.