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

1991 ◽  
Vol 37 (12) ◽  
pp. 908-911 ◽  
Author(s):  
Lois C. Shadix ◽  
Eugene W. Rice
Keyword(s):  
Escherichia Coli ◽  
Accurate Method ◽  
Fecal Coliforms ◽  
Fluorogenic Substrate ◽  
Environmental Waters ◽  
E Coli ◽  
Glucuronidase Activity ◽  
Gas Fermentation ◽  
Fermentation Method ◽  
Glucuronidase Assay

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 β-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl β-D-glucuronide (MUG). This study was conducted to determine whether β-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. β-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 β-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters. Key words: Escherichia coli, β-glucuronidase, 4-methylumbelliferyl β-D-glucuronide, water.

An Improved Membrane Filtration Method for Enumeration of Faecal Coliforms and E. Coli by a Fluorogenic β-Glucuronidase Assay

1993 ◽  
Vol 27 (3-4) ◽  
pp. 267-270 ◽  
Author(s):  
M. T. Augoustinos ◽  
N. A. Grabow ◽  
B. Genthe ◽  
R. Kfir
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Membrane Filtration ◽  
Faecal Coliforms ◽  
Environmental Waters ◽  
Filtration Method ◽  
E Coli ◽  
Wide Range ◽  
Pure Cultures ◽  
Glucuronidase Assay

A fluorogenic β-glucuronidase assay comprising membrane filtration followed by selective enumeration on m-FC agar at 44.5°C and further confirmation using tlie 4-metliylumbelliferyl-β-D-glucuronide (MUG) containing medium was evaluated for the detection of Escherichia coli in water. A total of 200 typical blue and non-typical blue colonies were isolated from sea and fresh water samples using initial selective enumeration on m-FC agar. Pure cultures of the selected colonies were further tested using the MUG assay and identified using the API 20E method. Of the colonies tested which were shown to be positive using the MUG assay 99.4% were Escherichia coli. The results of this study indicate the combination of the m-FC method followed by the MUG assay to be highly efficient for the selection and confirmation of E. coli from a wide range of environmental waters.

scholarly journals Clonal Populations of ThermotolerantEnterobacteriaceae in Recreational Water and Their Potential Interference with Fecal Escherichia coli Counts

2001 ◽  
Vol 67 (10) ◽  
pp. 4934-4938 ◽  
Author(s):  
Sandra L. McLellan ◽  
Annette D. Daniels ◽  
Alissa K. Salmore
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Environmental Protection Agency ◽  
16S Rrna Gene Sequencing ◽  
Fecal Coliforms ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Diverse Range ◽  
E Coli ◽  
Glucuronidase Activity

ABSTRACT Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of β-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. β-Glucuronidase activity was critical in distinguishingE. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.

Comparison of culturable fecal coliforms and Escherichia coli enumeration in freshwaters

2007 ◽  
Vol 53 (6) ◽  
pp. 798-801 ◽  
Author(s):  
Tamara Garcia-Armisen ◽  
Josué Prats ◽  
Pierre Servais
Keyword(s):  
Escherichia Coli ◽  
Microbiological Quality ◽  
Fecal Coliforms ◽  
Quality Data ◽  
Plate Count ◽  
Most Probable Number ◽  
Tetrazolium Chloride ◽  
Probable Number ◽  
E Coli ◽  
Glucuronidase Activity

Fecal coliforms (FC) counts were compared with Escherichia coli counts in differently contaminated freshwater samples (n = 166). FC were enumerated by plate count on triphenyl 2,3,5-tetrazolium chloride Tergitol medium. Escherichia coli were enumerated by the most probable number microplate method based on the detection of glucuronidase activity. FC and E. coli counts were highly correlated; an average E. coli/FC ratio equal to 0.77 was found, meaning that on average, 77% of FC were E. coli. Knowing the E. coli/FC ratio allows us to convert the historical microbiological quality data expressed in FC counts into E. coli abundance and thus to compare with present and future monitoring data that are (or will be) based on E. coli enumeration.

Evaluation of the β-Glucuronidase Substrate 5-Bromo-4-Chloro-3-Indolyl-β-D-Glucuronide (X-GLUC) in a 24-Hour Direct Plating Method for Escherichia coli

1988 ◽  
Vol 51 (5) ◽  
pp. 402-404 ◽  
Author(s):  
ELON W. FRAMPTON ◽  
LAWRENCE RESTAINO ◽  
NANCY BLASZKO
Keyword(s):  
Escherichia Coli ◽  
Agar Medium ◽  
Plate Count ◽  
Light Illumination ◽  
Fluorogenic Substrate ◽  
Direct Plating ◽  
E Coli ◽  
Glucuronidase Activity ◽  
Plate Count Agar ◽  
Plating Method

A 24-h direct plating method for Escherichia coli using Peptone-Tergitol agar was used to compare the effectiveness of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC) with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) for β-glucuronidase activity. Values obtained for enumeration of two strains of E. coli recovered from artificially inoculated raw minced chicken (i.e., plating efficiencies on the inoculum, cells per g, and recovery percentages elated to those on Plate Count Agar) indicate that X-GLUC at 50 μg/ml was as effective as MUG in an agar medium. Unlike MUG, X-GLUC does not require ultraviolet light illumination, and the color reaction produced remains localized in the positive colonies.

scholarly journals Comparison of Fecal Coliform Agar and Violet Red Bile Lactose Agar for Fecal Coliform Enumeration in Foods

2002 ◽  
Vol 68 (4) ◽  
pp. 1631-1638 ◽  
Author(s):  
A. Leclercq ◽  
C. Wanegue ◽  
P. Baylac
Keyword(s):  
Escherichia Coli ◽  
Fecal Coliform ◽  
Food Samples ◽  
Fecal Coliforms ◽  
Predictive Values ◽  
Direct Plating ◽  
Hafnia Alvei ◽  
E Coli ◽  
Plating Method ◽  
Mashed Potatoes

ABSTRACT A 24-h direct plating method for fecal coliform enumeration with a resuscitation step (preincubation for 2 h at 37 ± 1°C and transfer to 44 ± 1°C for 22 h) using fecal coliform agar (FCA) was compared with the 24-h standardized violet red bile lactose agar (VRBL) method. FCA and VRBL have equivalent specificities and sensitivities, except for lactose-positive non-fecal coliforms such as Hafnia alvei, which could form typical colonies on FCA and VRBL. Recovery of cold-stressed Escherichia coli in mashed potatoes on FCA was about 1 log unit lower than that with VRBL. When the FCA method was compared with standard VRBL for enumeration of fecal coliforms, based on counting carried out on 170 different food samples, results were not significantly different (P > 0.05). Based on 203 typical identified colonies selected as found on VRBL and FCA, the latter medium appears to allow the enumeration of more true fecal coliforms and has higher performance in certain ways (specificity, sensitivity, and negative and positive predictive values) than VRBL. Most colonies clearly identified on both media were E. coli and H. alvei, a non-fecal coliform. Therefore, the replacement of fecal coliform enumeration by E. coli enumeration to estimate food sanitary quality should be recommended.

Distribution of Coliphages in Various Foods

1986 ◽  
Vol 49 (12) ◽  
pp. 944-951 ◽  
Author(s):  
J. E. KENNEDY ◽  
C. I. WEI ◽  
J. L. OBLINGER
Keyword(s):  
Escherichia Coli ◽  
Food Samples ◽  
Fecal Coliforms ◽  
Bacterial Indicators ◽  
Processed Meats ◽  
E Coli ◽  
Plate Counts ◽  
The Relationship

The distribution of coliphages in various foods and the relationship between the incidences of coliphages and bacterial indicators were investigated. A total of 120 food samples comprising twelve products and including fresh meats, shellfish, vegetables and processed meats, were analyzed for indigenous coliphages using Escherichia coli hosts C, C-3000 and B. Bacterial analyses included enumeration of E. coli, fecal coliforms and coliforms, as well as aerobic plate counts and Salmonella analyses. Coliphages were detected (≥10 PFU/100 g) in 56% of samples and eleven of twelve products. Coliphages, E. coli, fecal coliforms and coliforms were recovered at a level of at least 30 organisms per 100 g in 43, 43, 68 and 81% of samples, with overall mean recoveries of 13, 19, 93 and 4300 organisms/100 g, respectively. Highest and lowest recoveries of coliphages and E. coli were from fresh meats and vacuum-packaged processed meats, respectively. Significant nonparametric correlations between coliphages, E. coli, fecal coliforms and coliforms were found among all food samples.

Comparison of the β-Glucuronidase Assay and the Conventional Method for Identification of Escherichia coli on Eosin-Methylene Blue Agar

1997 ◽  
Vol 60 (1) ◽  
pp. 6-9 ◽  
Author(s):  
SHIU W. HUANG ◽  
CHUNG H. CHANG ◽  
TUNG F. TAI ◽  
TSUNG C. CHANG
Keyword(s):  
Escherichia Coli ◽  
Methylene Blue ◽  
Conventional Method ◽  
Meat Products ◽  
Mixed Cultures ◽  
Effective Alternative ◽  
Methyl Red ◽  
Raw Meat ◽  
E Coli ◽  
Glucuronidase Assay

The purpose of this study was to compare the IMViC (indole, methyl red, Voges-Proskauer and citrate utilization) tests with the β-glucuronidase (GUD) assay for the identification of suspect Escherichia coli on Levine's eosin-methylene blue (EMB) agar. After testing 258 suspect E. coli colonies from raw meat and meat products, 163 and 44 were found to be E. coli and non-E. coli, respectively, by both methods. Nine isolates were IMViC positive (i.e., + + − − or − + − −) but GUD negative; among these isolates, six were confirmed to be E. coli by API 20E (bioMérieux, Marcy-I'Etoile, France) with the remaining three being non-E. coli. There were 42 isolates that were IMViC negative but GUD positive; among these isolates, seven were pure E. coli cultures, 33 were mixed cultures containing E. coli, and the remaining two were Proteus spp. The sensitivities for the identification of E. coli on EMB were 80.9% (169/209) and 97.1% (203/209), respectively, by the IMViC tests and GUD assay; whereas the specificities were 93.9% (46/49) and 95.9% (47/49), respectively, by the IMViC tests and GUD assay. It is proposed that the GUD assay can be an effective alternative to the conventional IMViC tests for the identification of suspect E. coli on EMB.

Field Evaluation of the MUG Assay for Enumerating Escherichia coli in Seawater and Oysters from Southeastern United States

1991 ◽  
Vol 54 (4) ◽  
pp. 246-248 ◽  
Author(s):  
MILES L. MOTES ◽  
JAMES T. PEELER
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Southeastern United States ◽  
Field Evaluation ◽  
Fecal Coliforms ◽  
American Public Health Association ◽  
Suitable Alternative ◽  
E Coli ◽  
Significant Difference ◽  
Health Association

Oysters and seawater collected from the southeastern United States were examined for fecal coliforms and Escherichia coli, using the current procedure of the American Public Health Association (APHA) and the fluorogenic 4-methylumbelliferyl-β-D-glucuronide (MUG) modified APHA procedure. After the presence of E. coli in both methods was confirmed by conventional IMViC procedures, there was no significant difference between method means at the α = 0.05 level. In oysters, low confirmation rates of 67 and 77% were observed by the APHA and the MUG methods, respectively. Seawater had the greatest confirmation rates (95%) by the MUG method. The MUG method may be a suitable alternative to the current APHA method for the microbiological evaluation of oysters and seawater.

scholarly journals Phenotypic and Genotypic Characterization of Escherichia coli Isolated from Untreated Surface Waters

2013 ◽  
Vol 7 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Kristopher J Janezic ◽  
Blake Ferry ◽  
Eric W Hendricks ◽  
Brian A Janiga ◽  
Tiffany Johnson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Waters ◽  
Antimicrobial Agents ◽  
Genotypic Variation ◽  
Virulence Gene ◽  
Genotypic Diversity ◽  
Housekeeping Gene ◽  
Polymyxin B ◽  
Fecal Coliforms ◽  
E Coli

A common member of the intestinal microbiota in humans and animals isEscherichia coli. Based on the presence of virulence factors,E. colican be potentially pathogenic. The focus of this study was to isolateE. colifrom untreated surface waters (37 sites) in Illinois and Missouri and determine phenotypic and genotypic diversity among isolates. Water samples positive for fecal coliforms based on the Colisure®test were streaked directly onto Eosin Methylene Blue (EMB) agar (37°C) or transferred to EC broth (44.5°C). EC broth cultures producing gas were then streaked onto EMB agar. Forty-five isolates were identified asE. coliusing API 20E and Enterotube II identification systems, and some phenotypic variation was observed in metabolism and fermentation. Antibiotic susceptibility of each isolate was also determined using the Kirby-Bauer Method. Differential responses to 10 antimicrobial agents were seen with 7, 16, 2, and 9 of the isolates resistant to ampicillin, cephalothin, tetracycline, and triple sulfonamide, respectively. All of the isolates were susceptible or intermediate to amoxicillin, ciprofloxacin, polymyxin B, gentamicin, imipenem, and nalidixic acid. Genotypic variation was assessed through multiplex Polymerase Chain Reaction for four virulence genes (stx1andstx2[shiga toxin],eaeA[intimin]; andhlyA[enterohemolysin]) and one housekeeping gene (uidA[β-D-glucuronidase]). Genotypic variation was observed with two of the isolates possessing the virulence gene (eaeA) for intimin. These findings increase our understanding of the diversity ofE. coliin the environment which will ultimately help in the assessment of this organism and its role in public health.

scholarly journals Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments

2010 ◽  
Vol 76 (17) ◽  
pp. 5659-5668 ◽  
Author(s):  
G. M. Luna ◽  
C. Vignaroli ◽  
C. Rinaldi ◽  
A. Pusceddu ◽  
L. Nicoletti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Marine Sediments ◽  
Large Scale ◽  
Microbiological Quality ◽  
Genotypic Diversity ◽  
Coastal Sediments ◽  
Fecal Coliforms ◽  
Biochemical Tests ◽  
Term Survival ◽  
E Coli

ABSTRACT Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx 1, stx 2, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.

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