Use of the ecf1 Gene To Detect Shiga Toxin–Producing Escherichia coli in Beef Samples

2015 ◽  
Vol 78 (4) ◽  
pp. 675-684 ◽  
Author(s):  
KRISTIN W. LIVEZEY ◽  
BETTINA GROSCHEL ◽  
MICHAEL M. BECKER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
False Positive ◽  
Screening Method ◽  
Severe Illness ◽  
Specific Gene ◽  
E Coli ◽  
Detection Assay ◽  
Meat Samples ◽  
Inspection Service

Escherichia coli O157:H7 and six serovars (O26, O103, O121, O111, O145, and O45) are frequently implicated in severe clinical illness worldwide. Standard testing methods using stx, eae, and O serogroup–specific gene sequences for detecting the top six non-O157 STEC bear the disadvantage that these genes may reside, independently, in different nonpathogenic organisms, leading to false-positive results. The ecf operon has previously been identified in the large enterohemolysin-encoding plasmid of eae-positive Shiga toxin–producing E. coli (STEC). Here, we explored the utility of the ecf operon as a single marker to detect eae-positive STEC from pure broth and primary meat enrichments. Analysis of 501 E. coli isolates demonstrated a strong correlation (99.6%) between the presence of the ecf1 gene and the combined presence of stx, eae, and ehxA genes. Two large studies were carried out to determine the utility of an ecf1 detection assay to detect non-O157 STEC strains in enriched meat samples in comparison to the results using the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) method that detects stx and eae genes. In ground beef samples (n = 1,065), the top six non-O157 STEC were detected in 4.0% of samples by an ecf1 detection assay and in 5.0% of samples by the stx- and eae-based method. In contrast, in beef samples composed largely of trim (n = 1,097), the top six non-O157 STEC were detected at 1.1% by both methods. Estimation of false-positive rates among the top six non-O157 STEC revealed a lower rate using the ecf1 detection method (0.5%) than using the eae and stx screening method (1.1%). Additionally, the ecf1 detection assay detected STEC strains associated with severe illness that are not included in the FSIS regulatory definition of adulterant STEC.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals Detex for Detection of Escherichia coli O157 in Raw Ground Beef and Raw Ground Poultry

2001 ◽  
Vol 84 (3) ◽  
pp. 752-760 ◽  
Author(s):  
Yvette M Henry ◽  
Nandini Natrajan ◽  
Wendy F Lauer
Keyword(s):  
Escherichia Coli ◽  
False Positive Rate ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Department Of Agriculture ◽  
E Coli ◽  
Metal Plating ◽  
Positive Rate ◽  
Meat Samples ◽  
Inspection Service

Abstract A method for detection of Escherichia coli O157 in beef and poultry is presented. The method is antibody-based and uses a patented antibody-specific metal-plating procedure for the detection of E. coli O157 in enriched meat samples. Both raw ground beef and raw ground poultry were tested as matrixes for the organism. The sensitivity and specificity of the assay were 98 and 90%, respectively. The accuracy of the assay was 96%. Overall, the method agreement between the E. coli O157 Detex assay and the U.S. Department of Agriculture/Food Safety Inspection Service method was 96%. Sample temperature upon loading of the apparatus was critical to the observed false-positive rate of the system.

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

2009 ◽  
Vol 72 (4) ◽  
pp. 741-747 ◽  
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.

Isolation and Characterization of Shiga Toxin–Producing Escherichia coli Serogroups O26, O45, O103, O111, O113, O121, O145, and O157 Shed from Range and Feedlot Cattle from Postweaning to Slaughter

2014 ◽  
Vol 77 (7) ◽  
pp. 1052-1061 ◽  
Author(s):  
ABEL B. EKIRI ◽  
DOUGLAS LANDBLOM ◽  
DAWN DOETKOTT ◽  
SUSAN OLET ◽  
WEILIN L. SHELVER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Beef Cows ◽  
Laboratory Culture ◽  
Feedlot Cattle ◽  
Fecal Shedding ◽  
E Coli ◽  
Isolation And Characterization ◽  
Inspection Service

Cattle are the main reservoirs for Shiga toxin–producing Escherichia coli (STEC) strains. E. coli O26, O45, O103, O111, O121, O145, and O157 are among the STEC serogroups that cause severe foodborne illness and have been declared as adulterants by the U.S. Department of Agriculture, Food Safety and Inspection Service. The objectives of this study were (i) to estimate the prevalence of non-O157 STEC and E. coli O157 in naturally infected beef cows and in steer calves at postweaning, during finishing, and at slaughter and (ii) to test non-O157 STEC isolates for the presence of virulence genes stx1, stx2, eaeA, and ehlyA. Samples were collected from study animals during multiple sampling periods and included fecal grabs, rectal swabs, and midline sponge samples. Laboratory culture, PCR, and multiplex PCR were performed to recover and identify E. coli and the virulence genes. The prevalence of non-O157 STEC (serogroups O26, O45, O103, O111, O121, O113, and O145) fecal shedding ranged from 8% (4 of 48 samples) to 39% (15 of 38 samples) in cows and 2% (1 of 47 samples) to 38% (9 of 24 samples) in steer calves. The prevalence of E. coli O157 fecal shedding ranged from 0% (0 of 38 samples) to 52% (25 of 48 samples) in cows and 2% (1 of 47 samples) to 31% (15 of 48 samples) in steer calves. In steer calves, the prevalence of non-O157 STEC and E. coli O157 was highest at postweaning, at 16% (15 of 96 samples) and 23% (22 of 96 samples), respectively. Among the 208 non-O157 STEC isolates, 79% (164 isolates) had stx1, 79% (165 isolates) had stx2, and 58% (121 isolates) had both stx1 and stx2 genes. The percentage of non-O157 STEC isolates encoding the eaeA gene was low; of the 165 isolates tested, 8 (5%) were positive for eaeA and 135 (82%) were positive for ehlyA. Findings from this study provide further evidence of non-O157 STEC shedding in beef cows and steer calves particularly at the stage of postweaning and before entry into the feedlot.

scholarly journals Is Shiga Toxin-Producing Escherichia coli O45 No Longer a Food Safety Threat? The Danger is Still Out There

2020 ◽  
Vol 8 (5) ◽  
pp. 782 ◽  
Author(s):  
Yujie Zhang ◽  
Yen-Te Liao ◽  
Xiaohong Sun ◽  
Vivian C.H. Wu
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Shiga Toxin ◽  
Evolutionary Relationship ◽  
Severe Illness ◽  
Next Generation Sequencing Technology ◽  
E Coli ◽  
Related Information ◽  
Foodborne Outbreaks ◽  
Enterocyte Effacement

Many Shiga toxin-producing Escherichia coli (STEC) strains, including the serogroups of O157 and most of the top six non-O157 serotypes, are frequently associated with foodborne outbreaks. Therefore, they have been extensively studied using next-generation sequencing technology. However, related information regarding STEC O45 strains is scarce. In this study, three environmental E. coli O45:H16 strains (RM11911, RM13745, and RM13752) and one clinical E. coli O45:H2 strain (SJ7) were sequenced and used to characterize virulence factors using two reference E. coli O45:H2 strains of clinical origin. Subsequently, whole-genome-based phylogenetic analysis was conducted for the six STEC O45 strains and nine other reference STEC genomes, in order to evaluate their evolutionary relationship. The results show that one locus of enterocyte effacement pathogenicity island was found in all three STEC O45:H2 strains, but not in the STEC O45:H16 strains. Additionally, E. coli O45:H2 strains were evolutionarily close to E. coli O103:H2 strains, sharing high homology in terms of virulence factors, such as Stx prophages, but were distinct from E. coli O45:H16 strains. The findings show that E. coli O45:H2 may be as virulent as E. coli O103:H2, which is frequently associated with severe illness and can provide genomic evidence to facilitate STEC surveillance.

scholarly journals Identification of Human-Pathogenic Strains of Shiga Toxin-Producing Escherichia coli from Food by a Combination of Serotyping and Molecular Typing of Shiga Toxin Genes

2007 ◽  
Vol 73 (15) ◽  
pp. 4769-4775 ◽  
Author(s):  
Lothar Beutin ◽  
Angelika Miko ◽  
Gladys Krause ◽  
Karin Pries ◽  
Sabine Haby ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Severe Illness ◽  
Shiga Toxins ◽  
E Coli ◽  
Food Borne ◽  
Length Polymorphisms ◽  
Beef Products ◽  
Pcr Products ◽  
Shiga Toxin Genes

ABSTRACT We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx 1 or variant genes were detected in 88 (40.2%) strains and stx 2 and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx 1, stx 1c, and stx 1d) and the Stx2 (stx 2, stx 2d, stx 2-O118, stx 2e, and stx 2g) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx 2 and/or mucus-activatable stx 2d genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx 2 and stx 2d STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx 2e), lamb, and wildlife meat (stx 1c). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

scholarly journals Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers

2022 ◽  
Vol 11 ◽  
Author(s):  
Xiaomei Zhang ◽  
Michael Payne ◽  
Sandeep Kaur ◽  
Ruiting Lan
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
In Silico ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Specific Gene ◽  
Metagenomic Sequencing ◽  
Accurate Identification ◽  
Gene Markers ◽  
E Coli

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

scholarly journals Prevalence of Shiga toxin-producing Escherichia coli isolated from chicken meat in west of Iran

2019 ◽  
Author(s):  
omid zarei ◽  
Leili Shokoohizadeh ◽  
Hadi Hossainpour ◽  
Mohammad Yousef Alikhani
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Culture Media ◽  
Chicken Meat ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Meat Samples ◽  
High Level

Abstract Objective: Shiga toxin producing Escherichia coli (STEC) has known as a crucial zoonotic food borne pathogen. A total of 257 row chicken meat samples were collected from different markets in Hamadan city from January 2016 to May 2017. Samples were cultured on selective and differential culture media, and the virulence genes of E. coli isolates were analyzed by PCR assay. The antibiotic resistance patterns of E. coli isolates were determined by disk diffusion method. The genetic relatedness of STEC isolates were analyzed by ERIC-PCR. Results: Totally, 93(36%) of isolates were identified as E. coli in this current study. According serological and microbiological tests, 5(5.3%), 31(33.3%) and 7(7.5%) of E. coli isolates, characterized as Enterohemorrhagic E. coli (EHEC), STEC and attaching and effacing E. coli (AEEC) strains, respectively. High level resistance to tetracycline (89.8), ampicillin (82.8%) and sulfametoxazole-trimotoprime (71%) were detected among E. coli isolates. Analysis of ERIC-PCR results showed five different ERIC types among EHEC isolates. Based on our findings, chicken meat identified as a sources of STEC strains, therefore, the controlling and checkup the chicken meats for the resistance and virulent strains of E. coli should be consider as a crucial issues in public health.

scholarly journals NeoSeekTM STEC: A Multiplex Molecular Method for Detection and Identification of Select Shiga Toxin–Producing Escherichia coli in Beef

2020 ◽  
Vol 103 (2) ◽  
pp. 523-532
Author(s):  
Edan Hosking ◽  
Brooke Roman ◽  
Susan Alles ◽  
Mark Mozola ◽  
Susanne Hinkley ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Probability Of Detection ◽  
Molecular Method ◽  
Single Strain ◽  
E Coli ◽  
Enrichment Cultures ◽  
Reference Methods ◽  
Detection And Identification ◽  
Inspection Service

Abstract Background: NeoSeekTM STEC is a single-source, service-based method for detection and identification of select Shiga toxin–producing Escherichia coli (STEC), including E. coli O157:H7 and STEC of somatic groups O26, O45, O103, O111, O121, and O145. The method is a multiplex molecular method utilizing more than 80 genetic targets to identify STEC in complex matrices such as food enrichment cultures. Objective: A study was conducted to validate the NeoSeek method for detection of select STEC in raw beef trim. Methods: Performance of the NeoSeek STEC method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods for E. coli O157:H7 and non-O157 STEC for detection of E. coli O157:H7 and E. coli O26:H11 in raw beef trim. Additionally, inclusivity/exclusivity testing and method robustness testing were performed. Results: Results of raw beef trim testing showed no statistically significant differences in performance between the NeoSeek and reference methods in the ability to detect either E. coli O157:H7 or E.coli O26:H11, as determined by probability of detection analysis. Results of inclusivity and exclusivity testing showed 100% expected results with target and nontarget bacteria, with the exception of a single strain of E. coli O157:H7, which was subsequently verified to be stx-negative by PCR. Conclusions and Highlights: NeoSeek STEC is an accurate, reliable method for rapid detection and identification of select STEC in complex populations such as beef trim enrichment cultures.

A Novel Selective Medium for Simultaneous Enrichment of Shiga Toxin–Producing Escherichia coli and Salmonella in Ground Beef

2018 ◽  
Vol 81 (8) ◽  
pp. 1252-1257 ◽  
Author(s):  
JOSEPH EGGERS ◽  
JOELLEN M. FEIRTAG ◽  
ALAN D. OLSTEIN ◽  
JOSEPH M. BOSILEVAC
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Ground Beef ◽  
Performance Standards ◽  
Selective Medium ◽  
Microbiological Analysis ◽  
Department Of Agriculture ◽  
Selective Enrichment ◽  
E Coli ◽  
Inspection Service

ABSTRACT Microbiological analysis of ground beef for contamination by both Salmonella and Shiga toxin–producing Escherichia coli (STEC) is performed by the U.S. Department of Agriculture, Food Safety Inspection Service (FSIS), as part of its Performance Standards Verification Testing program. FSIS has established a zero tolerance for STEC serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 because they are regarded as adulterants. The detection and isolation of these specific serogroups presents a technical challenge necessitating time-consuming and costly laboratory procedures that often exceed the technical capabilities of many small internal and reference laboratories. We describe here a method using a novel STEC and Salmonella selective (SSS) broth that allows for simultaneous selective enrichment of STEC and Salmonella sp., providing isolation and detection from the same broth. The method only involves direct plating from beef enrichments to detect suspect isolates that can be easily confirmed by using immunoassays or PCR, rendering the isolation simpler and less costly than the current described methods. In a side-by-side comparison with modified tryptic soy broth (mTSB), the use of SSS broth resulted in primarily isolating STEC and Salmonella sp., while substantially suppressing the growth of other gram-negative Enterobacteriacae by 90%. Significantly more (χ2 < 3.84) samples containing E. coli O157:H7 and STEC O26, O111, O121, and O145 and a nondifferent (χ2 > 3.84) number of samples containing STEC O103 and O45 were identified when enriching in SSS broth. Coenrichment using six different Salmonella serovars showed numerically greater but not significant (χ2 < 3.84) positive samples by using SSS broth compared with mTSB for a majority of serotypes.

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