Detection of Escherichia coli O157 in Foods by a Novel Polymyxin-Based Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) system was developed using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Extracts from cell suspensions were reacted with polymyxin-coated microwells followed by immunoenzymatic detection of captured LPS antigens using a commercially available anti–E. coli O157 antibody–peroxidase conjugate and a 3,3′,5,5′-tetramethylbenzidine substrate. The polymyxin ELISA was highly sensitive and specific for E. coli strains bearing the O157 antigen. When this ELISA was combined with enrichment, results were in complete agreement with those of standard culture techniques for the detection of this pathogen in a variety of artificially inoculated and naturally contaminated foods. The polymyxin ELISA is a simple and inexpensive assay for E. coli O157 with a 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

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Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin

Journal of Clinical Microbiology ◽

10.1128/jcm.6.5.439-444.1977 ◽

1977 ◽

Vol 6 (5) ◽

pp. 439-444 ◽

Cited By ~ 2

Author(s):

R H Yolken ◽

H B Greenberg ◽

M H Merson ◽

R B Sack ◽

A Z Kapikian

Keyword(s):

Escherichia Coli ◽

Immunosorbent Assay ◽

Radioactive Isotope ◽

Enzyme Linked Immunosorbent Assay ◽

Adrenal Cell ◽

Elisa System ◽

Culture Techniques ◽

E Coli ◽

Heat Labile ◽

Tissue Culture Techniques

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.

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Use of a Shiga Toxin (Stx)-Enzyme-Linked Immunosorbent Assay and Immunoblot for Detection and Isolation of Stx-Producing Escherichia coli from Naturally Contaminated Beef

Journal of Food Protection ◽

10.4315/0362-028x-63.9.1167 ◽

2000 ◽

Vol 63 (9) ◽

pp. 1167-1172 ◽

Cited By ~ 22

Author(s):

HEBA NASHED ATALLA ◽

ROGER JOHNSON ◽

SCOTT MCEWEN ◽

R. W. USBORNE ◽

C. L. GYLES

Keyword(s):

Escherichia Coli ◽

Vero Cell ◽

Immunosorbent Assay ◽

Shiga Toxin ◽

Total Coliform ◽

Complete Agreement ◽

Enzyme Linked Immunosorbent Assay ◽

Spearman Correlation ◽

Southern Ontario ◽

E Coli

The purpose of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA) and an immunoblot procedure for detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from beef, and to correlate the presence of STEC in beef with E. coli and total coliform counts. A total of 120 samples of boneless beef supplied to a meat processor in southern Ontario were tested for the presence of STEC, E. coli, and total coliforms. Following enrichment in modified tryptic soy broth, samples were screened for Shiga toxin (Stx) by a Stx-ELISA and a Vero cell assay (VCA). Samples that were positive in the Stx-ELISA were subjected to the Stx-immunoblot for STEC isolation. Overall, 33.3% of samples were positive in the VCA, and 34.2% were positive in the Stx-ELISA. There was almost complete agreement between the Stx-ELISA and the VCA results (kappa = 0.98). The sensitivity and specificity of the Stx-ELISA with respect to the VCA were 100% and 98.75%, respectively. STEC were isolated by the Stx-immunoblot from 87.8% of the samples that were positive in the Stx-ELISA. The STEC isolates belonged to 19 serotypes, with serotype O113:H21 accounting for 10 of 41 isolates. No STEC of serotype O157:H7 were isolated. There was a significant correlation between E. coli counts and total coliform counts (Spearman correlation coefficient = 0.68, P < 0.01). The E. coli count was positively correlated with detection of STEC by both the Stx-ELISA and the VCA (P < 0.01).

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Evaluation of home-mode ELISA system for thedetection of antibodies against Escherichia coli O157:H7 using purified lipopolysaccharide

Baghdad Science Journal ◽

10.21123/bsj.5.4.563-568 ◽

2008 ◽

Vol 5 (4) ◽

pp. 563-568

Author(s):

Baghdad Science Journal

Keyword(s):

Escherichia Coli ◽

Immunosorbent Assay ◽

Gel Filtration ◽

Laboratory Animals ◽

Partial Purification ◽

Nucleic Acid Content ◽

Enzyme Linked Immunosorbent Assay ◽

Biochemical Basis ◽

Elisa System ◽

E Coli

An enzyme linked immunosorbent assay (ELISA) for the detection and quantitation of human immunoglobulin G (IgG) antibodies against vero- cytotoxine (VT) producing Escherichia coli serogroup O157:H7 was produced. E. coli O157: H7 lipopolysaccharide was extracted from locally isolated strains by using hot phenol- water method, followed by partial purification using gel filtration chromatography by sepharose- 4B. The purity of the lipopolysaccharide was checked by measuring the protein and nucleic acid content and then used as antigen. Four isolates of vero- cytotoxin producing E. coli serogroup O157:H7 was obtained by culturing 350 stool samples from children suffering from bloody diarrhea. These isolates were identified on bacteriological, serological and biochemical basis. Toxin production was confirmed on laboratory animals as well as by cytopathic effect on tissue culture. The possibility of using E. coli O157:H7 lipopolysaccharide in an enzyme- linked immunosorbent assay for the routine diagnostic testing of serum from patients for evidence of O157:H7 infection is discussed.

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Comparison of Rapid Enzyme-Linked Immunosorbent Assay and Immunomagnetic Separation Methods for Detection of Escherichia coli O157 in Fecal, Hide, Carcass, and Ground Beef Samples

Journal of Food Protection ◽

10.4315/0362-028x-70.10.2230 ◽

2007 ◽

Vol 70 (10) ◽

pp. 2230-2234 ◽

Cited By ~ 4

Author(s):

T. W. THOMPSON ◽

T. P. STEPHENS ◽

G. H. LONERAGAN ◽

M. F. MILLER ◽

M. M. BRASHEARS

Keyword(s):

Escherichia Coli ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Separation Method ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

Perfect Agreement ◽

Fecal Samples ◽

E Coli ◽

Beef Products

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coli O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

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Evaluation of Three Commercially Available Enzyme-Linked Immunosorbent Assay Kits for Detection of Shiga Toxin

Journal of Food Protection ◽

10.4315/0362-028x-72.4.741 ◽

2009 ◽

Vol 72 (4) ◽

pp. 741-747 ◽

Cited By ~ 34

Author(s):

JOHN WILLFORD ◽

KENNETH MILLS ◽

LAWRENCE D. GOODRIDGE

Keyword(s):

Escherichia Coli ◽

Immunosorbent Assay ◽

Shiga Toxin ◽

Screening Method ◽

Enzyme Linked Immunosorbent Assay ◽

Microplate Assay ◽

E Coli ◽

Elisa Kit ◽

Pure Cultures ◽

Order Of Magnitude

Three commercially available Shiga toxin (Stx) enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their ability to detect Stx in pure cultures of Stx-producing Escherichia coli (specificity). The detection limits (sensitivity) of each ELISA kit were also evaluated. Seventy-eight Stx-producing E. coli (STEC) isolates that produced Stx1, Stx2, or Stx1 and Stx2 variants were examined in this study. The specificities of the tests were comparable, and the sensitivities of two of the tests (Premier EHEC and rBiopharm Ridascreen Verotoxin Enzyme Immunoassay) were within the same order of magnitude. The ProSpecT Shiga Toxin E. coli Microplate Assay was approximately 10-fold less sensitive. The inability of all three tests to detect the Stx2d and Stx2e variants indicated that some STEC strains may not be detected by Stx ELISA. The ability of the Premier EHEC ELISA to detect toxin in artificially inoculated bovine fecal samples (following enrichment) indicated that this kit may be used to screen cattle for the presence of Stx as an indicator of the presence of STEC. In particular, such a screening method could be useful during the summer, when the number of STEC-positive animals and the number of STEC that they shed increase.

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Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of IgM and IgG Antibodies to Lipopolysaccharide of Escherichia coli O157

Kansenshogaku zasshi ◽

10.11150/kansenshogakuzasshi1970.73.593 ◽

1999 ◽

Vol 73 (6) ◽

pp. 593-599 ◽

Cited By ~ 1

Author(s):

Naoto SUGAWARA ◽

Takeshi SAIKA ◽

Miyuki HASEGAWA ◽

Intetsu KOBAYASHI ◽

Fumihiko KURIMOTO ◽

...

Keyword(s):

Escherichia Coli ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

Igg Antibodies

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Comparison of Shiga-Like Toxin II Expression between Two Genetically Diverse Lineages of Escherichia coli O157:H7

Journal of Food Protection ◽

10.4315/0362-028x-71.8.1673 ◽

2008 ◽

Vol 71 (8) ◽

pp. 1673-1678 ◽

Cited By ~ 18

Author(s):

SCOT E. DOWD ◽

JASON B. WILLIAMS

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

Pathogenic Potential ◽

Genomic Changes ◽

E Coli ◽

Gene Expression Analyses ◽

Shiga Toxin 2 ◽

The Difference

The existence of two separate lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that one of these lineages (lineage I) might be more pathogenic toward human hosts. We postulated that the lineage more pathogenic expresses higher levels of Shiga toxin 2 (Stx2) than do the nonpathogenic lineage II. A comprehensive set of methodologies were used to investigate the difference in Stx2 protein and mRNA expression between the two lineages. An initial Stx2-specific enzyme-linked immunosorbent assay was conducted, and lineage I overall demonstrated significantly more toxin proteins expressed (P < 0.01). Gene expression analyses all showed significantly higher stx2 gene expression in lineage I (P = 0.02). PCR mapping revealed a possible explanation for decreased amounts of stx2 transcripts in the potentially nonpathogenic lineage II isolates, suggesting that genomic changes have modified the toxin-encoding region of the phage. This study provides additional data to support the existence of two diverse lineages of E. coli O157:H7, one of which may have lower pathogenic potential in relation to human hosts. The PCR described also provides a possible screening tool for E. coli O157 populations to differentiate these lineages. This study provides useful information on the ecology of E. coli O157, with broad implications within the clinical, scientific, and livestock industries.

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Estimation of Listeria monocytogenes and Escherichia coli O157:H7 Prevalence and Levels in Naturally Contaminated Rocket and Cucumber Samples by Deterministic and Stochastic Approaches

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-261 ◽

2015 ◽

Vol 78 (2) ◽

pp. 311-322 ◽

Cited By ~ 4

Author(s):

AGNI HADJILOUKA ◽

KYRIAKI-SOFIA MANTZOURANI ◽

ANASTASIA KATSAROU ◽

MARINA CAVAIUOLO ◽

ANTONIO FERRANTE ◽

...

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Culture Media ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

E Coli ◽

Molecular Tests ◽

The Media ◽

High Level

The aims of the present study were to determine the prevalence and levels of Listeria monocytogenes and Escherichia coli O157:H7 in rocket and cucumber samples by deterministic (estimation of a single value) and stochastic (estimation of a range of values) approaches. In parallel, the chromogenic media commonly used for the recovery of these microorganisms were evaluated and compared, and the efficiency of an enzyme-linked immunosorbent assay (ELISA)-based protocol was validated. L. monocytogenes and E. coli O157:H7 were detected and enumerated using agar Listeria according to Ottaviani and Agosti plus RAPID'L.mono medium and Fluorocult plus sorbitol MacConkey medium with cefixime and tellurite in parallel, respectively. Identity was confirmed with biochemical and molecular tests and the ELISA. Performance indices of the media and the prevalence of both pathogens were estimated using Bayesian inference. In rocket, prevalence of both L. monocytogenes and E. coli O157:H7 was estimated at 7% (7 of 100 samples). In cucumber, prevalence was 6% (6 of 100 samples) and 3% (3 of 100 samples) for L. monocytogenes and E. coli O157:H7, respectively. The levels derived from the presence-absence data using Bayesian modeling were estimated at 0.12 CFU/25 g (0.06 to 0.20) and 0.09 CFU/25 g (0.04 to 0.170) for L. monocytogenes in rocket and cucumber samples, respectively. The corresponding values for E. coli O157:H7 were 0.59 CFU/25 g (0.43 to 0.78) and 1.78 CFU/25 g (1.38 to 2.24), respectively. The sensitivity and specificity of the culture media differed for rocket and cucumber samples. The ELISA technique had a high level of cross-reactivity. Parallel testing with at least two culture media was required to achieve a reliable result for L. monocytogenes or E. coli O157:H7 prevalence in rocket and cucumber samples.

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Evaluation of an Enzyme-Linked Immunosorbent Assay, Direct Immunofluorescent Filter Technique, and Multiplex Polymerase Chain Reaction for Detection of Escherichia coli O157:H7 Seeded in Beef Carcass Wash Water†

Journal of Food Protection ◽

10.4315/0362-028x-61.8.934 ◽

1998 ◽

Vol 61 (8) ◽

pp. 934-938 ◽

Cited By ~ 11

Author(s):

PINA M. FRATAMICO ◽

TERENCE P. STROBAUGH

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Multiplex Pcr ◽

Multiplex Polymerase Chain Reaction ◽

Wash Water ◽

Enzyme Linked Immunosorbent Assay ◽

Escherichia Coli O157 ◽

Chain Reaction ◽

E Coli ◽

Polymerase Chain

In commercial beef processing, carcasses are customarily washed with water to remove physical and microbial contamination. Assaying the water that is shed from the carcasses after washing is a convenient method to determine whether the carcass is contaminated with Escherichia coli O157:H7 or other bacterial pathogens. E. coli O157:H7 was inoculated into carcass wash water at various levels and the bacteria were then concentrated by filtration. After collection of bacteria in the filter units, the nylon membranes were cut out and placed in tubes containing growth medium, and the tubes were mixed vigorously to dislodge the bacteria from the membranes. Prior to enrichment, samples were removed for testing by a multiplex polymerase chain reaction (PCR) and a direct immunofluorescent filter technique (DIFT). The remaining samples were subjected to 4-h enrichment culturing at 37°C, after which aliquots were removed for testing by multiplex PCR, DIFT, and an enzyme-linked immunosorbent assay (ELISA). Following 4-h enrichment culturing, E. coli O157:H7 was detected in wash water samples initially inoculated with ca. 100, 0.1, and 1 CFU/ml by ELISA, DIFT, and multiplex PCR, respectively. Testing of the wash water using the ELISA and the DIFT can be accomplished in less than 8 h. On the basis of these results, assaying carcass wash water by ELISA, DIFT, or multiplex PCR can be useful for detection of E. coli O157:H7 beef carcass contamination and can potentially be employed to identify carcasses for further processing to inactivate the organism.

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