Multiplex PCR Detection of Shiga Toxin–Producing Escherichia coli Strains Belonging to Serogroups O157, O103, O91, O113, O145, O111, and O26 Experimentally Inoculated in Beef Carcass Swabs, Beef Trim, and Ground Beef

2011 ◽  
Vol 74 (2) ◽  
pp. 228-239 ◽  
Author(s):  
ANGELA M. VALADEZ ◽  
CHITRITA DEBROY ◽  
EDWARD DUDLEY ◽  
CATHERINE N. CUTTER
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shiga Toxin ◽  
Gastrointestinal Disease ◽  
Simultaneous Detection ◽  
Ground Beef ◽  
Multiplex Pcr Assay ◽  
Significant Difference ◽  
Beef Products ◽  
Beef Carcass

Numerous foodborne outbreaks are attributed to Shiga toxin–producing Escherichia coli (STEC) and have been recognized for causing gastrointestinal disease in humans. Beef products have been considered the principal source of STEC. A multiplex PCR assay enabling simultaneous detection of STEC O103, O91, O113, O145, O111, O157, and O26 was developed and evaluated in artificially contaminated beef carcass swabs, beef trim, and ground beef after overnight enrichment. Individual serogroups were experimentally inoculated at low (1 to 10 CFU/ml) and high (11 to 100 CFU/ml) levels, and with a cocktail of strains belonging to two, four, and six serogroups. There was no significant difference in detecting single STEC strains under the different conditions. Only when strains were combined were there significant differences in detection of all cocktail isolates in some of the beef products. To address this issue, four serogroups were experimentally inoculated together at three different estimated levels (10, 102, and 103 CFU/ml) in all three beef products. Results yielded no significant difference in detecting STEC at the three inoculation levels (10, 102, and 103 CFU/ml) in trim and carcass swabs, but there was a significant difference in detecting STEC at the lowest levels (10 and 102 CFU/ml) in the 80:20 nonirradiated ground beef, and in the detection of STECin irradiated ground beef. The findings from this study could provide industry and government agencies with a tool to evaluate the prevalence and incidence of STEC in beef products and their processing environments.

A gold nanoparticles-assisted multiplex PCR assay for simultaneous detection of Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7

2020 ◽  
Vol 12 (2) ◽  
pp. 212-217 ◽  
Author(s):  
Juan Du ◽  
Shujing Wu ◽  
Liyuan Niu ◽  
Junguang Li ◽  
Dianbo Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gold Nanoparticles ◽  
Listeria Monocytogenes ◽  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Product

Unfunctionalized flower-shaped AuNPs is used as colorimetric sensor for PCR product detection by naked eyes.

scholarly journals Identification, Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaylen M. Capps ◽  
Justin B. Ludwig ◽  
Pragathi B. Shridhar ◽  
Xiaorong Shi ◽  
Elisabeth Roberts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Feedlot Cattle ◽  
Pcr Assay ◽  
Cattle Feces ◽  
Virulence Potential ◽  
Multiplex Pcr Assay ◽  
Human Infections

AbstractShiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

Prevalence of Shiga Toxin–Producing Escherichia coli in Ground Beef and Cattle Feces from King County, Washington

2002 ◽  
Vol 65 (8) ◽  
pp. 1322-1325 ◽  
Author(s):  
MANSOUR SAMADPOUR ◽  
M. KUBLER ◽  
F. C. BUCK ◽  
G. A. DEPAVIA ◽  
E. MAZENGIA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Ground Beef ◽  
Grocery Store ◽  
Grocery Stores ◽  
Fecal Samples ◽  
Slaughter House ◽  
Significant Difference ◽  
Life Threatening ◽  
Retail Grocery

Shiga toxin–producing Escherichia coli (STEC) is increasingly recognized as a common cause of diarrhea. STEC infection is a major public health threat because of its ability to cause serious and potentially life-threatening illnesses. The main reservoirs of STEC are believed to be the intestinal tracts of animals. Several studies have investigated the prevalence of STEC in various food items. The objective of this study was to determine the prevalence of STEC in the Seattle ground beef supply. In addition, the relative amount of STEC contamination between stores was compared, and possible differences between types of ground beef based on fat content (9, 16, and 23%) were investigated. A survey of Stx-I and/or Stx-II genes in fecal samples from cattle at a local slaughter house was also conducted. Of 296 ground beef samples tested from area retail grocery stores, 16.8% were positive for the presence of the toxin genes. Our data showed that there was no statistically significant difference (P > 0.05) in the prevalence of STEC between the ground beef samples of different fat contents and between grocery store chains. Of the 103 cattle fecal samples tested, 19 (18.4%) were found positive for the presence of Stx-I and/or Stx-II genes. The presence of a rather high percentage of STEC in the food supply in the absence of large number of cases suggests that not all STEC lineages are pathogenic for humans.

Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants

2013 ◽  
Vol 62 (12) ◽  
pp. 1823-1827 ◽  
Author(s):  
H. Ciesielczuk ◽  
M. Hornsey ◽  
V. Choi ◽  
N. Woodford ◽  
D. W. Wareham
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Quinolone Resistance ◽  
Pcr Assay ◽  
Resistance Determinants ◽  
Multiplex Pcr Assay ◽  
Susceptible Isolate ◽  
The Uk ◽  
Template Dna

The objective of this study was to develop and validate an expanded multiplex PCR assay for the simultaneous detection of eight plasmid-mediated quinolone-resistance determinants in Enterobacteriaceae. Primers were designed to amplify conserved fragments of qnrABCDS, qepA, oqxAB and aac(6′)-Ib-cr genes and were optimized in uniplex and multiplex PCR assays with control template DNA. The assay was used to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in 174 ciprofloxacin-resistant and 43 ciprofloxacin-susceptible extraintestinal pathogenic Escherichia coli isolates. Each resistance gene could be detected alone and in combination. PMQR determinants were detected in 65 ciprofloxacin-resistant isolates (37 %) and one ciprofloxacin-susceptible isolate (2 %). Prevalences of the identified determinants were: aac(6′)-Ib-cr, 34.5 %; qnrS, 1.1 %; qepA, 1.1 %; and oqxAB, 0.6 %. In conclusion, we developed an eight-target multiplex PCR for the accurate detection of PMQR genes and confirmed that PMQR prevalence remains low among human Escherichia coli clinical isolates in the UK.

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.

Shiga Toxin–Producing Escherichia coli Contamination of Raw Beef and Beef-Based Ready-to-Eat Products at Retail Outlets in Pretoria, South Africa

2020 ◽  
Vol 83 (3) ◽  
pp. 476-484 ◽  
Author(s):  
LIBBY O. ONYEKA ◽  
ABIODUN A. ADESIYUN ◽  
KAREN H. KEDDY ◽  
EVELYN MADOROBA ◽  
AYANDA MANQELE ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
South Africa ◽  
Shiga Toxin ◽  
Plate Count ◽  
Pcr Assay ◽  
Cross Sectional ◽  
Potential Health ◽  
Multiplex Pcr Assay ◽  
Retail Outlets ◽  
Beef Products

ABSTRACT A cross-sectional study was conducted to determine the prevalence of and factors associated with Shiga toxin–producing Escherichia coli (STEC) in raw beef and ready-to-eat (RTE) beef products sold in 31 retail outlets in Pretoria, South Africa, and nearby areas. A total of 463 beef and RTE samples were screened for four STEC virulence genes (stx1, stx2, eaeA, and hlyA) and seven O-serogroups (O113, O157, O26, O91, O145, O111, and O103) with a multiplex PCR assay. The total aerobic plate count (TAPC) per gram was also determined. A total of 38 STEC isolates were recovered and characterized by conventional PCR assay and serotyping. The overall prevalence of STEC in the beef and RTE samples tested was 16.4% (76 of 463 samples; 95% confidence interval, 13 to 20%). The prevalence of STEC differed significantly by product type (P < 0.0001), with the highest prevalence (35%) detected in boerewors (spicy sausage). The STEC prevalences in minced beef, brisket, RTE cold beef, and biltong were 18, 13, 9, and 5%, respectively. The most frequently detected stx gene was stx2 (13%), and STEC serogroups from recovered isolates were detected at the following prevalences: O2, 15%; O8, 12%; O13, 15%; O20, 8%; O24, 3%; O39, 3%; O41, 8%; O71, 3%; O76, 3%; O150, 12%; and O174, 3%. A high proportion (77%) of the samples had TAPCs that exceeded the South African microbiological standards for meat export (5.0 log CFU/g). The prevalence of O157 STEC (16%) and the diversity of non-O157 STEC serogroups found in five common beef-based products from retail outlets in South Africa suggest exposure of raw beef and beef products to multiple contamination sources during carcass processing and/or cutting and handling at retail outlets. These data provide direct estimates of the potential health risk to consumers from undercooked contaminated products and indicate the need to improve sanitary practices during slaughter and processing of beef and beef-based RTE products. A risk-based surveillance system for STEC may be needed. HIGHLIGHTS

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