Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR® Green I in a real-time multiplex PCR

2006 ◽  
Vol 20 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Ken J. Yoshitomi ◽  
Karen C. Jinneman ◽  
Stephen D. Weagant
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Shiga Toxin ◽  
E Coli ◽  
Toxin Genes ◽  
Shiga Toxin Genes

Presence and Antimicrobial Resistance of Escherichia coli in Ready-to-Eat Foods in Shaanxi, China

2017 ◽  
Vol 80 (3) ◽  
pp. 420-424 ◽  
Author(s):  
Allah Bux Baloch ◽  
Hua Yang ◽  
Yuqing Feng ◽  
Meili Xi ◽  
Qian Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Shaanxi Province ◽  
Future Research ◽  
E Coli ◽  
Class 1 Integrons ◽  
Toxin Genes ◽  
Class 1 ◽  
Shiga Toxin Genes ◽  
Cooked Meat

ABSTRACT The aim of this study was to determine the presence and characteristics of Escherichia coli in ready-to-eat (RTE) foods. A total of 300 RTE foods samples were collected in Shaanxi Province, People's Republic of China: 50 samples of cooked meat, 165 samples of vegetable salad, 50 samples of cold noodles, and 35 samples of salted boiled peanuts. All samples were collected during summer (in July to October) 2011 and 2012 and surveyed for the presence of E. coli. E. coli isolates recovered were classified by phylogenetic typing using a PCR assay. The presence of Shiga toxin genes 1 (stx1) and 2 (stx2) was determined for these E. coli isolates by PCR, and all isolates were analyzed for antimicrobial susceptibility and the presence of class 1 integrons. Overall, 267 (89.0%) RTE food samples were positive for E. coli: 49 cold noodle, 46 cooked meat, 150 salad vegetable, and 22 salted boiled peanut samples. Of the 267 E. coli isolates, 73.0% belong to phylogenetic group A, 12.4% to group B1, 6.4% to group B2, and 8.2% to group D. All isolates were negative for both Shiga toxin genes. Among the isolates, 74.2% were resistant to at least one antimicrobial agent, and 17.6% were resistant to three or more antimicrobial agents. Resistance to ampicillin (75.6% of isolates) and tetracycline (73.1% of isolates) was most frequently detected; 26.2% of E. coli isolates and 68.8% of multidrug-resistant E. coli isolates were positive for class 1 integrons. All isolates were sensitive to amikacin. Our findings indicate that RTE foods in Shaanxi were commonly contaminated with antibiotic-resistant E. coli, which may pose a risk for consumer health and for transmission of antibiotic resistance. Future research is warranted to track the contamination sources and develop appropriate steps that should be taken by government, industry, and retailers to reduce microbial contamination in RTE foods.

scholarly journals High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes

LWT ◽  
2020 ◽  
Vol 131 ◽  
pp. 109785
Author(s):  
Prashant Singh ◽  
Gabriel Cubillos ◽  
Gabrielle Kirshteyn ◽  
Joseph M. Bosilevac
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Toxin Genes ◽  
Pcr Assays ◽  
Shiga Toxin Genes

Escherichia coli O157:H7 in Retail Ground Beef in Seattle: Results of a One-Year Prospective Study

1999 ◽  
Vol 62 (2) ◽  
pp. 133-139 ◽  
Author(s):  
PHILLIP I. TARR ◽  
NHIEM THANH TRAN ◽  
RICHARD A. WILSON
Keyword(s):  
Escherichia Coli ◽  
Prospective Study ◽  
Shiga Toxin ◽  
Enrichment Culture ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Toxin Genes ◽  
One Year ◽  
Shiga Toxin Genes

Escherichia coli O157:H7 was sought systematically in 1,400 samples of retail ground beef in Seattle in a 1-year prospective study. Sorbitol-nonfermenting, lactose-fermenting, indole-positive colonies isolated after enrichment culture were probed for the presence of Shiga toxin genes. Totals of 67,040 sorbitol-nonfermenting and 66,705 sorbitol-fermenting colonies were characterized, but E. coli O157:H7 was not identified. The sensitivity of this technique was usually sufficient to detect E. coli O157:H7 at a concentration below 1 CFU/g of meat. These data demonstrate that retail ground beef in Seattle is neither frequently nor heavily contaminated with E. coli O157:H7.

Characteristics of Shiga Toxin–Producing Escherichia coli O157 in Slaughtered Reindeer from Northern Finland

2017 ◽  
Vol 80 (3) ◽  
pp. 454-458 ◽  
Author(s):  
Claudio Zweifel ◽  
Lisa Fierz ◽  
Nicole Cernela ◽  
Sauli Laaksonen ◽  
Maria Fredriksson-Ahomaa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Geographic Area ◽  
Sequence Type ◽  
E Coli ◽  
Indirect Transmission ◽  
Toxin Genes ◽  
Domestic Livestock ◽  
Shiga Toxin Genes ◽  
Subtilase Cytotoxin

ABSTRACT Fecal samples collected from 470 slaughtered reindeer 6 to 7 months of age were screened by real-time PCR (after enrichment) for Shiga toxin genes (stx) and then for Escherichia coli serogroup O157. Shiga toxin genes were found frequently (>30% of samples), and serogroup O157 was detected in 20% of the stx-positive samples. From these samples, a total of 25 E. coli O157:H− isolates (nonmotile but PCR positive for fliCH7) were obtained. Twenty-four of these E. coli O157:H− isolates did not ferment sorbitol and originated from one geographic area. These 24 isolates belonged to the multilocus sequence type 11, typical for Shiga toxin–producing E. coli (STEC) O157:H7 and O157:H−, and harbored genes stx1a, stx2c, eae, and hlyA; the stx2c subtype has been associated with high virulence. In contrast, one E. coli O157:H− isolate (multilocus sequence type 11) did ferment sorbitol, lacked Shiga toxin genes, but was positive for eae, hlyA, and sfpA. This isolate closely resembled an STEC that has lost its Shiga toxin genes. Additional examination revealed that reindeer can be colonized by various other STEC isolates; 21 non-O157 STEC isolates belonged to four multilocus sequence types, harbored stx1a (8 isolates) or stx2b (13 isolates), and in the stx2b-positive isolates the recently described new allelic variants (subAB2-2 and subAB2-3) for subtilase cytotoxin were identified. Hence, slaughtered semidomesticated Finnish reindeer might constitute a little known reservoir for STEC O157:H7/H− and other serogroups, and the risk of direct or indirect transmission of these pathogens from reindeer to humans and domestic livestock must not be overlooked.

Rapid and Reliable Detection of Shiga Toxin–Producing Escherichia coli by Real-Time Multiplex PCR

2012 ◽  
Vol 75 (4) ◽  
pp. 643-650 ◽  
Author(s):  
KELLY S. ANKLAM ◽  
KAUSHI S. T. KANANKEGE ◽  
TINA K. GONZALES ◽  
CHARLES W. KASPAR ◽  
DÖRTE DÖPFER
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Real Time ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Health Concern ◽  
E Coli ◽  
Reliable Detection ◽  
Pcr Assays

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin–producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx1 and stx2), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non–E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100% specificity. The detection limits of the assays were 103 or 104 CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 100 CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.

scholarly journals Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea

2008 ◽  
Vol 38 (6) ◽  
pp. 1682-1686 ◽  
Author(s):  
Cleber Jacob Silva de Paula ◽  
José Moacir Marin
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Antimicrobial Agents ◽  
E Coli ◽  
Toxin Genes ◽  
Shiga Toxin Genes

Shiga toxigenic Escherichia coli (STEC) and Attaching and effacing E. coli (AEEC) have been associated with diarrhea illness in dogs. From January to December 2006, 92 E. coli isolates from 25 diarrheic dogs were analyzed, by screening for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twelve isolates were detected by PCR to harbor the Shiga toxin genes (7 the stx 1 (7.6%); 5 the stx 2 (5.4%); and none both of them). Nine (9.8%) of the E. coli isolates studied were eae positive non Shiga toxin-producing. Thirteen (62.0%) isolates, carrying stx or eae gene, also showed a hemolysin production. The strains with virulence genes were also examined for resistance to 12 antimicrobial agents. Resistances to cephalothin (85.7%), streptomycin (81.0%), amoxicillin (71.4%) and gentamicin (71.4%) were predominantly observed.

scholarly journals Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H− Serotype

2003 ◽  
Vol 69 (10) ◽  
pp. 6327-6333 ◽  
Author(s):  
Karen C. Jinneman ◽  
Ken J. Yoshitomi ◽  
Stephen D. Weagant
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Escherichia Coli O157 ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
E Coli ◽  
Pcr Method ◽  
Shiga Toxin Genes

ABSTRACT A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H− uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were ≥1.89, and as few as 6 CFU/reaction could be detected.

Sign in / Sign up

Export Citation Format

Share Document