scholarly journals Recovery Rate of Cells of the Seven Regulated Serogroups of Shiga Toxin-Producing Escherichia coli from Raw Veal Cutlets, Ground Veal, and Ground Beef from Retail Stores in the Mid-Atlantic Region of the United States

2020 ◽  
Author(s):  
YangJin Jung ◽  
Anna C. S. Porto-Fett ◽  
Salina Parveen ◽  
Joan Meredith ◽  
Bradley A Shoyer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Specific Gene ◽  
Atlantic Region ◽  
Recovery Rates ◽  
Related Data ◽  
The U.S

A total of 482 veal cutlet, 555 ground veal, and 540 ground beef samples were purchased from retail establishments in the Mid-Atlantic region of the U.S. over a non-contiguous, two-year period between 2014 and 2017. Samples (325 g each) were individually enriched and screened via real-time PCR for all seven regulated serogroups of Shiga toxin-producing Escherichia coli (STEC). Presumptive STEC positive samples were subjected to serogroup-specific immunomagnetic separation and plated onto selective media. Up to five isolates typical for STEC from each sample were analyzed via multiplex PCR for both the virulence genes (i.e., eae , stx 1 and/or stx 2 , and ehxA ) and serogroup-specific gene(s) for the seven regulated STEC serogroups. The recovery rates of non-O157 STEC from veal cutlets (3.94%, 19 of 482 samples) and ground veal (7.03%, 39 of 555 samples) were significantly higher (P < 0.05) than that from ground beef (0.93%, 5 of 540 samples). In contrast, only a single isolate of STEC O157:H7 was recovered; this isolate originated from one (0.18%) of 555 samples of ground veal. Recovery rates for STEC were not associated with state, season, packaging type, or store type (P > 0.05), but were associated with brand and fat content (P < 0.05). Pulsed-field subtyping of the 270 viable/confirmed STEC isolates from the 64 total samples testing positive revealed 78 pulsotypes (50 to 80% similarity) belonging to 39 pulsogroups, with ≥90% similarity among pulsotypes within pulsogroups. Also, multiple isolates from the same sample displayed an indistinguishable pulsotype for 43 of 64 (67.7%) samples testing positive.  These findings support related data from regulatory sampling exercises over the past decade and confirm that recovery rates for the regulated STEC serogroups are appreciably higher for raw veal compared to raw beef samples as was also observed herein for meat purchased at food retailers in the Mid-Atlantic region of the U.S.

Isolation of Shiga Toxin–Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145 from Ground Beef Using Modified Rainbow Agar and Post–Immunomagnetic Separation Acid Treatment†

2012 ◽  
Vol 75 (9) ◽  
pp. 1548-1554 ◽  
Author(s):  
GLENN E. TILLMAN ◽  
JAMIE L. WASILENKO ◽  
MUSTAFA SIMMONS ◽  
TODD A. LAUZE ◽  
JOSEPH MINICOZZI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Acid Treatment ◽  
Agar Medium ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Treatment Target ◽  
Potassium Tellurite ◽  
Treatment Step

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin–producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post–immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

Detection and Isolation of the “Top Seven” Shiga Toxin–Producing Escherichia coli in Ground Beef: Comparison of RapidFinder Kits to the U.S. Department of Agriculture Microbiology Laboratory Guidebook Method

2017 ◽  
Vol 80 (5) ◽  
pp. 829-836 ◽  
Author(s):  
Pina M. Fratamico ◽  
Lori K. Bagi ◽  
Aisha Abdul-Wakeel
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Microbiology Laboratory ◽  
Department Of Agriculture ◽  
E Coli ◽  
The U.S

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the “top seven” STEC, and these have been declared to be adulterants in beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems RapidFinder STEC real-time PCR method to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157–specific targets was determined with 132 top seven STEC strains and 283 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g, were also detected. For RapidFinder analysis, 375-g ground beef samples spiked with ≥4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h at 42 ± 1°C, and for the MLG method, 325-g samples were similarly spiked and enriched in 975 mL of modified TSB for 15 h at 42 ± 1°C. Following DNA extraction, real-time PCR was performed using RapidFinder Express software, and for the MLG method, the BAX Real-Time PCR STEC Suite and the BAX Real-Time E. coli O157:H7 assay were used with the BAX System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder and BAX assays were similar; all samples were positive after 10 and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder system can be used for routine and rapid detection of the top seven STEC in beef.

Performance of Chromogenic Agar Media for Isolation of Shiga Toxin–Producing Escherichia coli from Ground Beef

2020 ◽  
Vol 83 (7) ◽  
pp. 1149-1154
Author(s):  
GENTRY L. LEWIS ◽  
NATALIA CERNICCHIARO ◽  
RODNEY A. MOXLEY
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Culture Media ◽  
Geometric Mean ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Broth Culture ◽  
True Negative ◽  
Chromogenic Agar ◽  
Agar Media

ABSTRACT The performance of three chromogenic agar media for detection of the “top seven” Shiga toxin–producing Escherichia coli (STEC) in beef was compared. Samples of retail ground beef were inoculated with STEC O26, O45, O103, O111, O121, O145, or O157 at geometric mean (±standard error of the mean) levels of 0, 48 (±1), 420 (±1), 4,100 (±1), or 45,000 (±1) CFU/10 g and enriched 1:10 (90 mL) in EC broth (40°C for 6 h). Following enrichment, aliquots of broth culture were treated by immunomagnetic separation with one of three pools of beads against the seven STEC serogroups: pool I, O26, O45, and O121; pool II, O103, O111, and O145; and pool III, O157. After immunomagnetic separation, 50 μL of washed bead suspensions in buffered peptone water were spiral plated onto modified Rainbow Agar O157 (mRBA), CHROMagar STEC (CS), or modified Possé differential medium (mPossé2) and incubated at 37°C for 18 h. Up to six isolated colonies were picked from each spiral plate based on expected colony phenotypes for STEC on the respective media, and isolate identity was confirmed with an 11-plex PCR assay targeting the O serogroups and virulence genes. Overall, mRBA had the highest sensitivity (99.2%), correctly detecting a significantly higher proportion of STEC serogroups than either CS (79.4%; P < 0.05) or mPossé2 (91.7%; P < 0.05). mRBA also had the highest negative predictive value (90.0%), correctly identifying a significantly higher proportion of true-negative samples compared with CS (25.7%; P < 0.05) and mPossé2 (46.2%; P < 0.05). However, mRBA also had the lowest analytical specificity of 83.2% (P < 0.05), yielding the lowest proportion of colonies tested that were STEC positive (3,548 of 4,263) compared with 97.7% (3,607 of 3,693) for mPossé2 and 98.0% (2,875 of 2,935) for CS. Reduced specificity results in more work and higher expense due to the increased number of colonies that must be tested. Further improvements in agar culture media for non-O157 STEC isolation are needed. HIGHLIGHTS

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

2015 ◽  
Vol 78 (1) ◽  
pp. 196-202 ◽  
Author(s):  
SARITA RAENGPRADUB WHEELER ◽  
PRECIAUS HEARD ◽  
CHRISTOPHE DUFOUR ◽  
DELPHINE THEVENOT-SERGENTET ◽  
ESTELLE LOUKIADIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Detection Methods ◽  
Primary Concern ◽  
The European Union ◽  
Commercial Method

Although serotype O157:H7 remains the pathogenic Shiga toxin–producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin–Producing Escherichia coli

2019 ◽  
Vol 82 (11) ◽  
pp. 1844-1850
Author(s):  
YANGJIN JUNG ◽  
ANNA C. S. PORTO-FETT ◽  
BRADLEY A. SHOYER ◽  
LAURA E. SHANE ◽  
ELIZABETH HENRY ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
The United States ◽  
Retail Stores ◽  
Pcr Assay ◽  
Atlantic Region ◽  
O Antigens ◽  
Pork Samples

ABSTRACT A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin–producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.

scholarly journals Application of real-time PCR for detection of antibiotic resistant pathogens and Shiga-toxin producing Escherichia coli

2015 ◽  
Author(s):  
◽  
Prashant Singh
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Ground Beef ◽  
The United States ◽  
Multiple Drug ◽  
United States Department ◽  
Antibiotic Resistant ◽  
Food Pathogens

Salmonella and Shiga toxin producing Escherichia coli (STEC) are among the most important food pathogens. Increasing use of antibiotics for treatment and as a therapeutic agent on food animals has been proposed as a reason for the emergence of multiple drug resistant (MDR) strains of food pathogens. In this study real-time PCR methods were developed for the detection antibiotic resistant strains of Salmonella, extended-spectrum [beta]-lactam (ESBL) and carbapenem resistant pathogens. The United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) declared seven STEC serogroups O157, O26, O45, O103, O111, O121 and O145 as adulterants in ground beef and beef trims. Multiplex real-time PCR melt curve assays with IAC were standardized for the detection of seven STEC serogroups with their virulence genes and Salmonella. The assay was able to detect all STEC strains in 325 g of ground beef and beef trims spiked with 10 CFU.

Establishment of non-O157 Shiga Toxin-Producing Escherichia coli (STEC) baseline of retail ground beef in the United States

Meat Science ◽  
2014 ◽  
Vol 96 (1) ◽  
pp. 484
Author(s):  
Y.T. Liao⁎ ◽  
G.H. Loneragan ◽  
J.C. Brooks ◽  
A. Echeverry ◽  
M.F. Miller ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Ground Beef ◽  
The United States

Characterization and Virulence Potential of Serogroup O113 Shiga Toxin–Producing Escherichia coli Strains Isolated from Beef and Cattle in the United States

2017 ◽  
Vol 80 (3) ◽  
pp. 383-391 ◽  
Author(s):  
Peter Feng ◽  
Sabine Delannoy ◽  
David W. Lacher ◽  
Joseph M. Bosilevac ◽  
Patrick Fach
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Severe Disease ◽  
Ground Beef ◽  
The United States ◽  
Intestinal Epithelial ◽  
Virulence Potential ◽  
Beef Products ◽  
Genetic Profiles

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) of serotype O113:H21 have caused severe diseases but are unusual in that they do not produce the intimin protein required for adherence to intestinal epithelial cells. Strains of serogroup O113 are one of the most common STEC found in ground beef and beef products in the United States, but their virulence potential is unknown. We used a microarray to characterize 65 O113 strains isolated in the United States from ground beef, beef trim, cattle feces, and fresh spinach. Most were O113:H21 strains, but there were also nine strains of O113:H4 serotype. Although strains within the same serotype had similar profiles for the genes that were tested on the array, the profiles were distinct between the two serotypes, and the strains belonged to different clonal groups. Analysis by clustered regularly interspaced short palindromic repeat analysis showed that O113:H4 strains are conserved genetically, but the O113:H21 strains showed considerable polymorphism and genetic diversity. In comparison to the O113:H21 strains from Australia that were implicated in severe disease, the U.S. isolates showed similar genetic profiles to the known pathogens from Australia, suggesting that these may also have the potential to cause infections.

Non-O157 Shiga Toxin–Producing Escherichia coli in U.S. Retail Ground Beef

2014 ◽  
Vol 77 (7) ◽  
pp. 1188-1192 ◽  
Author(s):  
YEN-TE LIAO ◽  
MARKUS F. MILLER ◽  
GUY H. LONERAGAN ◽  
J. CHANCE BROOKS ◽  
ALEJANDRO ECHEVERRY ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Ground Beef ◽  
The United States ◽  
Latex Agglutination ◽  
Retail Stores ◽  
Convenience Sample ◽  
Two Samples ◽  
Beef Products

Shiga toxin–producing Escherichia coli (STEC) serotype O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are the leading cause of STEC-associated infections in humans in the United States. In the United States, these organisms are considered adulterants in raw nonintact beef products and in intact beef destined to be made into or used in nonintact raw beef products. The objective of this study was to provide an estimate of the burden of the six serogroups of non-O157 STEC in ground beef obtained from retail stores across the United States. A convenience sample of commercial ground beef products (n = 1,129) were purchased from retail stores in 24 states from October 2011 to May 2012. The samples had various lean/fat proportions, muscle group of origin (chuck, round, sirloin, or not specified), and packaging types. For each ground beef sample, 25 g was inoculated in 225 ml of modified tryptic soy broth, stomached for 1 min, and then incubated at 41°C for 18 ± 2 h. These enrichment cultures were then screened for stx, eae, and O group genes using a commercially available, closed-platform PCR-based method. The potential positive samples were subjected to immunomagnetic separation and plated on modified Rainbow agar. Morphologically typical colonies were subjected to latex agglutination and PCR determination of stx and eae genes. Nine (0.8%) of the ground beef samples were potentially positive for at least one STEC serogroup after PCR screening. The serogroups detected by PCR assay were O26 (four samples), O103 (four samples), O145 (three samples), O45 (two samples), and O121 (one sample). No STEC isolates belonging to these serogroups were recovered from the sample cultures. The current research provides updated surveillance data for non-O157 STEC isolates among commercial ground beef products and information regarding the potential sources of contamination from different parts of beef trims destined for ground beef production.

National Survey of Shiga Toxin–Producing Escherichia coli Serotypes O26, O45, O103, O111, O121, O145, and O157 in Australian Beef Cattle Feces

2016 ◽  
Vol 79 (11) ◽  
pp. 1868-1874 ◽  
Author(s):  
GLEN E. MELLOR ◽  
NARELLE FEGAN ◽  
LESLEY L. DUFFY ◽  
KATE E. McMILLAN ◽  
DAVID JORDAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Beef Cattle ◽  
Shiga Toxin ◽  
Immunomagnetic Separation ◽  
The United States ◽  
Pcr Analysis ◽  
Fecal Samples ◽  
E Coli ◽  
Beef Products ◽  
Low Prevalence

ABSTRACT Escherichia coli O157 and six non-O157 Shiga toxin–producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, and O145, colloquially referred to as the “big 6”) have been classified as adulterants of raw nonintact beef products in the United States. While beef cattle are a known reservoir for the prototype STEC serotype, E. coli O157, less is known about the dissemination of non-O157 STEC serotypes in Australian cattle. In the present study, 1,500 fecal samples were collected at slaughter from adult (n =628) and young (n =286) beef cattle, adult (n =128) and young (n =143) dairy cattle, and veal calves (n = 315) across 31 Australian export-registered processing establishments. Fecal samples were enriched and tested for E. coli O157 and the big 6 STEC serotypes using BAX System PCR and immunomagnetic separation methods. Pathogenic STEC (pSTEC; isolates that possess stx, eae, and an O antigen marker for O157 or a big 6 serotype) were isolated from 115 samples (7.7%), of which 100 (6.7%) contained E. coli O157 and 19 (1.3%) contained a big 6 serotype. Four of the 115 samples contained multiple pSTEC serotypes. Among samples confirmed for big 6 pSTEC, 15 (1%) contained E. coli O26 and 4 (0.3%) contained E. coli O111. pSTEC of serotypes O45, O103, O121, and O145 were not isolated from any sample, even though genes indicative of E. coli belonging to these serotypes were detected by PCR. Analysis of animal classes revealed a higher pSTEC prevalence in younger animals, including veal (12.7%), young beef (9.8%), and young dairy (7.0%), than in adult animals, including adult beef (5.1%) and adult dairy (3.9%). This study is the largest of its kind undertaken in Australia. In contrast to E. coli O157 and consistent with previous findings, this study reports a relatively low prevalence of big 6 pSTEC serotypes in Australian cattle populations.

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