Method for the Detection of Priority Shiga Toxin–Producing Escherichia coli in Beef Trim

2013 ◽  
Vol 76 (10) ◽  
pp. 1689-1696 ◽  
Author(s):  
GEORGE HUSZCZYNSKI ◽  
MARTINE GAUTHIER ◽  
SAM MOHAJER ◽  
ALEXANDER GILL ◽  
BURTON BLAIS
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
Turnaround Time ◽  
Direct Plating ◽  
E Coli ◽  
Agar Media ◽  
Pcr Techniques ◽  
Positive Results

A method has been developed for the detection in beef trim of priority Shiga toxin–producing E. coli (STEC) strains, defined as E. coli possessing the virulence factors stx1 and/or stx2 and intimin (eae), with O serogroups O26, O45, O103, O111, O121, O145, or O157. The method is based on recovery of the target bacteria by overnight enrichment in a broth optimized for recovery of O157 and non-O157 STEC, followed by screening using multiplex PCR techniques targeting (i) stx1, stx2, and eae (STE PCR) and (ii) gene sequences associated with the seven priority O serogroups (Poly O PCR), and then direct plating of broth samples positive in both STE and Poly O PCR onto Rainbow agar. Colonies on agar media were screened batchwise for STEC by the STE PCR, and presumptive isolates were characterized using a multiplex PCR and cloth-based hybridization array system targeting key virulence and O serogroup-specific markers. Using one representative strain of each priority O serogroup individually inoculated in beef trim samples, the method exhibited a limit of detection approaching 1 to 2 viable STEC cells per 65 g. None of the uninoculated trim samples produced positive results with either of the screening PCR procedures or on analysis of colonies recovered on plating media. STEC-negative samples were readily identified by screening PCR within 24 h, with a turnaround time of fewer than 4 days for confirmation of positives. The inclusivity and exclusivity characteristics of the screening PCR techniques were verified using a total of 65 different priority STEC strains: 24 nonpriority STEC, 15 non-STEC bacteria, and only those strains bearing the targeted characteristics produced screening PCR-positive results.

Latex Agglutination Assays for Detection of Non-O157 Shiga Toxin–Producing Escherichia coli Serogroups O26, O45, O103, O111, O121, and O145†

2012 ◽  
Vol 75 (5) ◽  
pp. 819-826 ◽  
Author(s):  
MARJORIE B. MEDINA ◽  
WEILIN L. SHELVER ◽  
PINA M. FRATAMICO ◽  
LAURIE FORTIS ◽  
GLENN TILLMAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Latex Agglutination ◽  
Polystyrene Latex ◽  
Optimum Conditions ◽  
E Coli ◽  
Agar Media ◽  
Positive Results

Latex agglutination assays utilizing polyclonal antibodies were developed for the top six non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups. Rabbit antisera were affinity purified through protein A/G columns, and the isolated immunoglobulins (IgGs) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 0.20 to 0.28 mg/ml in a 1% latex suspension. Optimum conditions for the agglutination assay consisted of utilizing 20 μl of latex-IgG reagent containing 2.0 to 2.8 μg IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly after mixing the colonies with the latex-IgG, indicating STEC strains. More than 100 target and nontarget strains were tested in more than 3,000 test replicates. All target organisms produced positive results, but three antisera (anti-O26, anti-O103, and anti-O145) cross-reacted with some other STECs. The anti-O103 and anti-O145 latex reagents cross-reacted with O26 strains, and the anti-O26 cross-reacted with O103 strains. The latex-IgG reagents are stable for at least 1 year and are easy to prepare. These agglutination assays can be used for identification of presumptive non-O157 STEC colonies from agar media. The techniques used to prepare the latex reagents also can be utilized for testing other STEC serogroups, other E. coli serotypes, or other pathogens to ensure safe foods to consumers.

Validation of Pepperoni Process for Control of Shiga Toxin–Producing Escherichia coli

2012 ◽  
Vol 75 (5) ◽  
pp. 838-846 ◽  
Author(s):  
KATHLEEN A. GLASS ◽  
CHARLES W. KASPAR ◽  
JEFFREY J. SINDELAR ◽  
ANDREW L. MILKOWSKI ◽  
BRIAN M. LOTZ ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
The Other ◽  
Direct Plating ◽  
E Coli ◽  
Log Reduction ◽  
Three Samples ◽  
Processing Steps

The objective of this study was to compare the survival of non-O157 Shiga toxin–producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC strains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.

Low Prevalence of Salmonella and Shiga Toxin–Producing Escherichia coli in Lymph Nodes of Australian Beef Cattle

2017 ◽  
Vol 80 (12) ◽  
pp. 2105-2111 ◽  
Author(s):  
Gavin Bailey ◽  
Long Huynh ◽  
Lachlan Govenlock ◽  
David Jordan ◽  
Ian Jenson
Keyword(s):  
Escherichia Coli ◽  
Lymph Nodes ◽  
Shiga Toxin ◽  
Limit Of Detection ◽  
Ground Beef ◽  
Plate Count ◽  
Salmonella Spp ◽  
Live Animal ◽  
E Coli ◽  
Low Prevalence

ABSTRACT Salmonella contamination of ground beef has been viewed as originating from the surface of carcasses. Recent studies have identified lymph nodes as a potential source of Salmonella contamination because these tissues play an active role in containment of pathogens in the live animal and because some lymph nodes are unavoidably present in manufacturing beef trimmings or primal cuts that may be incorporated into ground beef. A survey was conducted of the microbiological status of lymph nodes from Australian cattle at the time of slaughter to determine the prevalence of microbiological contamination. Sets of lymph nodes (n = 197), consisting of the superficial cervical (prescapular), prepectoral, axillary, presternal, popliteal, ischiatic, subiliac (precrural), coxalis, and iliofemoralis (deep inguinal), were collected from five geographically separated Australian abattoirs over a period of 14 months. Samples were tested for the presence of Salmonella spp. and Shiga toxin–producing Escherichia coli by BAX PCR assay. Aerobic plate count, E. coli, and coliforms were enumerated with a lower limit of detection of 80 CFU per node. The observed prevalence of Salmonella within peripheral lymph nodes was 0.48% (7 of 1,464). Two of the seven lymph nodes in which Salmonella organisms were detected came from the same animal. Grass-fed, grain-fed, and cull dairy cattle were all found to have detectable Salmonella in lymph nodes. All Salmonella detections occurred during cooler months of the year. No Shiga toxin–producing E. coli were detected. Aerobic microorganisms were detected above the limit of quantification in 3.2% of nodes (median count 2.24 log per node), and E. coli was detected in 0.8% of nodes (median count 3.05 log per node). The low prevalence of Salmonella and low concentration of aerobic microorganisms in Salmonella-positive lymph nodes of Australian cattle at the time of slaughter suggest that the likelihood of lymph nodes contributing significantly to the presence of Salmonella in ground beef is low.

Molecular Detection of wzx1 and wzy Genes in Multi Drugs Resistance E. coli Isolates

2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Mona Al-Terehi ◽  
Saba Saadoon Khazaal ◽  
Haidar J Muhammed ◽  
Russul Hikmat Behjet
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Gene Cluster ◽  
Molecular Detection ◽  
E Coli ◽  
Pcr Products ◽  
New Finding ◽  
O Antigens ◽  
Positive Results

Escherichia coli have been genetically changes in Iraqi environments, the present study was carried out to detection E coli isolates sero-group using o-antigens gene cluster, tow genes were amplified used multiplex PCR, the results of present study show that present isolates have more than one plasmid in different size (500-700 bp). PCR results show about 100% of isolates were had a positive results of PCR products of O45 wzx1 gene (451 bp) while 25% of isolates were a positive PCR products of O45 wzy1 gene (255 bp) and about 25% of isolates have tow genes. A new finding in present study that 50% of isolates have other copy of O45 wzx1 gene, these results concluded that it may be important in Iraqi isolates identification and classification however we need more information about genes sequencing.

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 Presence and characteristics of sorbitol-negative Escherichia coli O157 in healthy sheep faeces

2008 ◽  
Vol 52 (No. 7) ◽  
pp. 301-307 ◽  
Author(s):  
H. Turutoglu ◽  
D. Ozturk ◽  
L. Guler ◽  
F. Pehlivanoglu
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shiga Toxin ◽  
Human Pathogens ◽  
Isolation Rate ◽  
E Coli ◽  
Awassi Sheep ◽  
Healthy Sheep

The presence of sorbitol-negative <i>Escherichia coli</i> O157 was investigated in healthy Awassi sheep faeces from 175 randomly selected animals in Burdur province of Turkey. Out of 175 animals, 16 (9.1%) were faecal shedding of sorbitol-negative <i>E. coli</i> O157. Out of the 15 flocks included in the study, 7 (47%) had at least one sheep positive for sorbitol-negative <i>E. coli</i> O157. The isolation rate of sorbitol-negative <i>E. coli</i> O157 ranged from 8.3 to 60% among the animals tested in the flocks. A total of 16 ovine sorbitol-negative <i>E. coli</i> O157 strains were characterized by a multiplex PCR. Results showed that 6 (37.7%) strains carried <i>stx1</i> gene, 3 (18.8%) <i>stx2</i> gene and 1 (6.3%) both <i>stx1</i> and <i>stx2</i> genes. Intimin (<i>eaeA</i>) gene was detected in 4 (25%) of the strains. None of the strains encoding for <i>stx</i> genes was positive for <i>eaeA</i> gene. The results demonstrate that the majority of sorbitol-negative <i>E. coli</i> O157 strains (62.5%) isolated from Awassi sheep in Burdur province of Turkey are Shiga toxin-producing <i>E. coli</i> that have a potential as human pathogens.

scholarly journals Multiplex PCR for Enterotoxigenic, Attaching and Effacing, and Shiga Toxin-Producing Escherichia coli Strains from Calves

1998 ◽  
Vol 36 (6) ◽  
pp. 1795-1797 ◽  
Author(s):  
Sophia M. Franck ◽  
Brad T. Bosworth ◽  
Harley W. Moon
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shiga Toxin ◽  
Shiga Toxins ◽  
E Coli ◽  
Heat Stable ◽  
Genes Encoding

A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2. This multiplex PCR was specific and sensitive. It will be useful for identification of E. coli strains which cause diarrhea in calves.

Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Proteinase K ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

Antagonistic activity of Bacillus subtilis strains isolated from various sources

2018 ◽  
Vol 8 (2) ◽  
pp. 354-364
Author(s):  
A. N. Irkitova ◽  
A. V. Grebenshchikova ◽  
A. V. Matsyura
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Wild Strain ◽  
Fish Farming ◽  
Antagonistic Activity ◽  
Antagonistic Effect ◽  
E Coli ◽  
Long Period ◽  
Test Culture ◽  
Agar Media

<p>An important link in solving the problem of healthy food is the intensification of the livestock, poultry and fish farming, which is possible only in the adoption and rigorous implementation of the concept of rational feeding of animals. In the implementation of this concept required is the application of probiotic preparations. Currently, there is an increased interest in spore probiotics. In many ways, this can be explained by the fact that they use no vegetative forms of the bacilli and their spores. This property provides spore probiotics a number of advantages: they are not whimsical, easily could be selected, cultivated, and dried. Moreover, they are resistant to various factors and could remain viable during a long period. One of the most famous spore microorganisms, which are widely used in agriculture, is <em>Bacillus subtilis</em>. Among the requirements imposed to probiotic microorganisms is mandatory – antagonistic activity to pathogenic and conditional-pathogenic microflora. The article presents the results of the analysis of antagonistic activity of collection strains of <em>B. subtilis</em>, and strains isolated from commercial preparations. We studied the antagonistic activity on agar and liquid nutrient medias to trigger different antagonism mechanisms of <em>B. subtilis</em>. On agar media, we applied three diffusion methods: perpendicular bands, agar blocks, agar wells. We also applied the method of co-incubating the test culture (<em>Escherichia coli</em>) and the antagonist (or its supernatant) in the nutrient broth. Our results demonstrated that all our explored strains of <em>B. subtilis</em> have antimicrobial activity against a wild strain of <em>E. coli</em>, but to varying degrees. We identified strains of <em>B. subtilis</em> with the highest antagonistic effect that can be recommended for inclusion in microbial preparations for agriculture.</p><p><em><br /></em><em></em></p>

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