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

2008 ◽  
Vol 71 (8) ◽  
pp. 1673-1678 ◽  
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.

scholarly journals Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

2009 ◽  
Vol 76 (2) ◽  
pp. 474-482 ◽  
Author(s):  
Yongxiang Zhang ◽  
Chad Laing ◽  
Zhengzhong Zhang ◽  
Jennyka Hallewell ◽  
Chunping You ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Rt Pcr ◽  
Genetic Lineages ◽  
E Coli ◽  
Shiga Toxin 2 ◽  
Flanking Region ◽  
Q Gene

ABSTRACT Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated “mobile genetic elements” such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx 2 flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx 2) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx 2, whereas all LII strains carried variant stx 2c and 4 of 14 LI/II strains had copies of both stx 2 and variant stx 2c. Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx 2 mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

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

2007 ◽  
Vol 70 (10) ◽  
pp. 2230-2234 ◽  
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.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

scholarly journals Outbreaks of Escherichia coli O157:H7 Infections Linked to Romaine Lettuce in Canada from 2008 to 2018: An Analysis of Food Safety Context

2020 ◽  
Vol 83 (8) ◽  
pp. 1444-1462 ◽  
Author(s):  
GENEVIÈVE COULOMBE ◽  
ANGELA CATFORD ◽  
AMALIA MARTINEZ-PEREZ ◽  
ENRICO BUENAVENTURA
Keyword(s):  
Escherichia Coli ◽  
The United States ◽  
Escherichia Coli O157 ◽  
Romaine Lettuce ◽  
E Coli ◽  
Leafy Greens ◽  
Iceberg Lettuce ◽  
Canadian Population ◽  
The Difference ◽  
Commercial Distribution

ABSTRACT Foodborne diseases are a major cause of illness in Canada. One of the main pathogens causing cases and outbreaks of foodborne illness in Canada is Escherichia coli O157:H7. From 2008 to 2018, 11 outbreaks of E. coli O157:H7 infection in Canada were linked to leafy greens, including 7 (63.6%) linked to romaine lettuce, 2 (18.2%) linked to iceberg lettuce, and 2 (18.2%) linked to other or unspecified types of leafy greens. The consumption of lettuce in Canada, the behavior of E. coli O157:H7 on lettuce leaves, and the production practices used for romaine and iceberg lettuce do not seem to explain why a higher number of outbreaks of E. coli O157:H7 infection were linked to romaine than to iceberg lettuce. However, the difference in the shape of iceberg and romaine lettuce heads could be an important factor. Among the seven outbreaks linked to romaine lettuce in Canada between 2008 and 2018, an eastern distribution of cases was observed. Cases from western provinces were reported only twice. The consumption of romaine and iceberg lettuce by the Canadian population does not seem to explain the eastern distribution of cases observed, but the commercial distribution, travel distances, and the storage practices used for lettuce may be important factors. In the past 10 years, the majority of the outbreaks of E. coli O157:H7 infection linked to romaine lettuce occurred during the spring (March to June) and fall (September to December). The timing of these outbreaks may be explained by the availability of lettuce in Canada, the growing region transition periods in the United States, and the seasonality in the prevalence of E. coli O157:H7. The consumption of romaine lettuce by the Canadian population does not explain the timing of the outbreaks observed. HIGHLIGHTS

Glucose Decreases Virulence Gene Expression of Escherichia coli O157:H7

2012 ◽  
Vol 75 (4) ◽  
pp. 748-752 ◽  
Author(s):  
V. DELCENSERIE ◽  
G. LaPOINTE ◽  
T. CHARASLERTRANGSI ◽  
A. RABALSKI ◽  
M. W. GRIFFITHS
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Quorum Sensing ◽  
Shiga Toxin ◽  
Virulence Gene ◽  
Escherichia Coli O157 ◽  
Virulence Gene Expression ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Enterocyte Effacement

Escherichia coli O157:H7 is responsible for a human toxico-infection that can lead to severe complications such as hemolytic uremic syndrome. Inside the intestine, E. coli O157:H7 forms typical attaching-effacing lesions and produces Shiga toxins. The genes that are responsible for these lesions are located in a pathogenicity island called the locus of enterocyte effacement (LEE). LEE gene expression is influenced by quorum sensing through the luxS system. In this study, the effect of glucose on the expression of several genes from LEE, on the expression of Shiga toxin genes, and on the expression of luxS was assessed with real-time, reverse transcription PCR. All concentrations of glucose (from 0.1 to 1%) were able to down-regulate genes from the LEE operon. A slight down-regulation of genes implicated in Shiga toxin expression was also observed but was significant for low doses of glucose (0.1 to 0.5%) only. A slight but significant increase in luxS expression was observed with 1% glucose. This confirms that in addition to quorum sensing, the presence or absence of nutrients such as glucose has an impact on the down- or upregulation of LEE-encoded virulence genes by the bacterium. The influence of glucose on the virulence of E. coli O157:H7 has received little attention, and these results suggest that glucose can have an important effect on the virulence of E. coli O157:H7.

Seasonal Prevalence of Escherichia coli O157:H7 in Beef Cattle Feces†

2006 ◽  
Vol 69 (12) ◽  
pp. 3018-3020 ◽  
Author(s):  
M. J. ALAM ◽  
L. ZUREK
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Shiga Toxin ◽  
Bacterial Colonization ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Human Pathogen ◽  
E Coli ◽  
Cattle Feces ◽  
Shiga Toxin 2

Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.

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

2015 ◽  
Vol 78 (2) ◽  
pp. 311-322 ◽  
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.

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†

1998 ◽  
Vol 61 (8) ◽  
pp. 934-938 ◽  
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.

Development and Validation of a Most-Probable-Number–Immunomagnetic Separation Methodology of Enumerating Escherichia coli O157 in Cattle Feces

2007 ◽  
Vol 70 (5) ◽  
pp. 1072-1075 ◽  
Author(s):  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
W. E. CHANEY ◽  
L. A. BRANHAM ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Immunomagnetic Separation ◽  
Feedlot Cattle ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
Direct Plating ◽  
Fecal Samples ◽  
E Coli ◽  
The Difference

A method to validate enumeration of Escherichia coli O157 in fecal samples from feedlot cattle was developed in these studies. Due to background flora, bovine fecal sample enumeration cannot be performed by simple direct plating techniques. Known quantities of E. coli O157:H7 were inoculated into feces, and populations were determined by direct plating of the cocktail (studies 1, 2, and 3) and manure and cocktail (studies 4 and 5) mixtures and compared with a most-probable-number (MPN)–immunomagnetic separation (IMS) method. The three-tube MPN combined preenrichment in gram-negative broth with confirmation using IMS. Five separate enumeration studies (study 1, sterile feces inoculated with 102 E. coli O157:H7 per g; study 2, nonsterile feces inoculated with 103 E. coli O157:H7 per g; study 3, nonsterile feces inoculated with 101 E. coli O157:H7 per g; study 4, sterile feces inoculated with 104 streptomycin-resistant E. coli O157:H7 per g; and study 5, sterile feces inoculated with 102 streptomycin-resistant E. coli O157:H7 per g) were conducted. These studies were performed to determine the precision, accuracy, and specificity at low and high levels of pathogen contamination in feces, using direct plating compared with the MPN-IMS methodology tested. There was an overall difference (P < 0.01) between direct plating and MPN-IMS methodologies, but this difference was biologically negligible due to the difference in least-squares means (0.29 ± 0.10) being so low. The direct plating and MPN-IMS methods were correlated (r = 0.93). These results suggest that using the MPN-IMS procedures is an effective method of estimating E. coli O157 populations in naturally infected bovine fecal samples.

scholarly journals Escherichia coli O157:H7 Strain Origin, Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

2009 ◽  
Vol 75 (15) ◽  
pp. 5074-5081 ◽  
Author(s):  
Ross M. S. Lowe ◽  
Danica Baines ◽  
L. Brent Selinger ◽  
James E. Thomas ◽  
Tim A. McAllister ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Escherichia Coli O157 ◽  
Human Origin ◽  
E Coli ◽  
Unknown Factor ◽  
Shiga Toxin 2 ◽  
The Impact ◽  
Dose Dependent

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 103 to 105 CFU and maximum colonization at 107 CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx 1) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx 2). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx 2 expression in modulating the amount of E. coli O157:H7 colonization of cattle.

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