An Improved Rapid Technique for Isolation of Escherichia coli O157:H7 from Foods

1995 ◽  
Vol 58 (1) ◽  
pp. 7-12 ◽  
Author(s):  
STEPHEN D. WEAGANT ◽  
JAMES L. BRYANT ◽  
KAREN G. JINNEMAN
Keyword(s):  
Escherichia Coli ◽  
Comparative Method ◽  
Immunomagnetic Separation ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
Biochemical Tests ◽  
Isolation Technique ◽  
Selective Enrichment ◽  
E Coli ◽  
Polymerase Chain

A newly revised enrichment and agar-plating system was tested for selectivity and sensitivity in recovery of unstressed and cold-stressed Escherichia coli O157:H7 from foods. Various foods inoculated with known levels of enterohemorrhagic E. coli O157:H7 (EHEC) were tested by enrichment for 6 h at 37°C in modified tryptic soy broth (mTSB) base supplemented with vancomycin, cefsulodin and cefixime, referred to as EHEC enrichment broth (EEB). Subsequently, portions were spread-plated on sorbitol–MacConkey agar supplemented with tellurite and cefixime (TCSMAC). Further selective enrichment was also examined using immunomagnetic separation (IMS) from the EEB prior to spread-plating on TCSMAC agar. These methods were compared to a procedure of enrichment in mTSB (supplemented with novobiocin) at 37°C for 24 h followed by spread-plating of decimal dilutions on hemorrhagic colitis 4–methylumbelliferyl–B–D–glucuronide (HC–MUG) agar. The new enrichment isolation technique was found to be sensitive at a level of one EHEC organism per 10 g of food in four food types. This represents an approximate l00-fold to 1,000-fold enhancement in sensitivity over the comparative method for foods with high levels of competitive microflora. These enrichment-isolation protocols also were compared in analysis of naturally contaminated raw or undercooked ground beef samples implicated in foodborne illness. EEB-TCSMAC with and without IMS were combined with rapid biochemical tests, and with O157 latex agglutination and confirmation of toxin genes by polymerase chain reaction (PCR) to provide a completed test within 30 h of initiating testing. The new system was successful in 15 of 17 samples, where only 6 of 17 were found positive by the comparative technique.

Screening Bovine Carcass Sponge Samples for Escherichia coli O157 Using a Short Enrichment Coupled with Immunomagnetic Separation and a Polymerase Chain Reaction–Based (BAX) Detection Step†

2001 ◽  
Vol 64 (10) ◽  
pp. 1610-1612 ◽  
Author(s):  
DONG-HYUN KANG ◽  
GENEVIEVE A. BARKOCY-GALLAGHER ◽  
MOHAMMAD KOOHMARAIE ◽  
GREGORY R. SIRAGUSA
Keyword(s):  
Escherichia Coli ◽  
Culture Method ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Selective Enrichment ◽  
E Coli ◽  
Screening Protocol ◽  
Polymerase Chain ◽  
Culture Positive

A bovine carcass sponge sample screening protocol for detecting Escherichia coli O157:H7 was composed of a short selective enrichment followed by an immunomagnetic separation (IMS) and target detection using the BAX E. coli O157 polymerase chain reaction assay. This screening protocol was compared to a culture-based method for detection of the organism in carcass sponge samples. Enriched samples were subjected to IMS; the bead suspension was divided and plated on selected media or stored at −20°C, then subjected to BAX analysis. The results showed a high degree of agreement between the plating method and the BAX system. Fifty-two of the 59 culture-positive samples were also positive using the BAX system (88.1% sensitivity). Of the 76 samples that appeared negative for the presence of E. coli O157:H7 by the culture method, 66 were determined as negative using the BAX system (86.8% specificity). Four of the 10 samples found negative by the initial culture method and positive by the BAX method were subsequently found to be culture positive upon reanalysis. Based on these data, the BAX system combined with a short, selective enrichment and IMS may be a rapid, reliable, and simple method to screen for E. coli O157:H7 in carcass sponge samples. Our data indicate that optimization and subsequent testing of this protocol for use as a carcass screening tool are warranted.

Virulence Characterization and Molecular Subtyping of Typical and Atypical Escherichia coli O157:H7 and O157:H(−) Isolated from Fecal Samples and Beef Carcasses in Mexico

2015 ◽  
Vol 78 (2) ◽  
pp. 264-272 ◽  
Author(s):  
CLAUDIA NARVÁEZ-BRAVO ◽  
ALEJANDRO ECHEVERRY ◽  
MARKUS F. MILLER ◽  
ARGENIS RODAS-GONZÁLEZ ◽  
M. TODD BRASHEARS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Immunomagnetic Separation ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
High Genetic Diversity ◽  
E Coli ◽  
System A ◽  
Metabolic Genes ◽  
Beef Carcasses

The objective of the study was to characterize virulence genes and subtype Escherichia coli O157:H7 and O157:H(−) isolates obtained from a vertically integrated feedlot slaughter plant in Mexico. A total of 1,695 samples were collected from feedlots, holding pens, colon contents, hides, and carcasses. E. coli O157:H7 detection and confirmation was carried out using conventional microbiology techniques, immunomagnetic separation, latex agglutination, and the BAX system. A total of 97 E. coli O157 strains were recovered and screened for key virulence and metabolic genes using multiplex and conventional PCR. Eighty-eight (91.72%) of the strains carried stx2, eae, and ehxA genes. Ten isolates (8.25%) were atypical sorbitol-fermenting strains, and nine were negative for the flicH7 gene and lacked eae, stx1, stx2, and ehxA. One sorbitol-positive strain carried stx2, eae, tir, toxB, and iha genes but was negative for stx1 and ehxA. Pulsed-field gel electrophoresis (PFGE) analysis yielded 49 different PFGE subtypes, showing a high genetic diversity; however, the majority of the typical isolates were closely related (80 to 90% cutoff). Atypical O157 isolates were not closely related within them or to typical E. coli O157:H7 isolates. Identical PFGE subtypes were found in samples obtained from colon contents, feedlots, holding pens, and carcasses. Isolation of a sorbitol-fermenting E. coli O157 positive for a number of virulence genes is a novel finding in Mexico. These data showed that genetically similar strains of E. coli O157:H7 can be found at various stages of beef production and highlights the importance of preventing cross-contamination at the pre- and postharvest stages of processing.

Occurrence of Shiga Toxin–Producing Escherichia coli O157 in Selected Dairy and Meat Products Marketed in the City of Rabat, Morocco

2004 ◽  
Vol 67 (6) ◽  
pp. 1234-1237 ◽  
Author(s):  
N. BENKERROUM ◽  
Y. BOUHLAL ◽  
A. EL ATTAR ◽  
A. MARHABEN
Keyword(s):  
Escherichia Coli ◽  
Meat Products ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Escherichia Coli O157 ◽  
Selective Enrichment ◽  
E Coli ◽  
Meat Samples ◽  
Antigen Antibody ◽  
The City

Samples of meat and dairy products taken from the city of Rabat, Morocco, were examined for the presence of Escherichia coli O157 by the selective enrichment procedure followed by plating on cefixime–tellurite–sorbitol MacConkey agar and a latex agglutination test. The ability of isolates to produce Shiga toxins (ST1 or ST2) was also tested by an agglutination test using sensitized latex. Dairy samples (n = 44) included different products commonly consumed in the country. Meat samples (n = 36) were taken from traditional butchers because these products are generally marketed in this way. Random samples were taken from each product during the period of January through May. Of the 80 samples tested, 8 (10%) harbored E. coli O157. Four dairy and four meat samples were contaminated (9.1 and 11.1%, respectively). Of 10 E. coli O157 isolates from contaminated samples demonstrating true antigen-antibody agglutination, 5 (50%) produced either ST2 alone or ST2 plus ST1. Four of the five strains (80%) were meat isolates and produced ST2 with or without ST1, and the fifth was a dairy isolate producing ST2.

Effect of Lactobacillus acidophilus Strain NP51 on Escherichia coli O157:H7 Fecal Shedding and Finishing Performance in Beef Feedlot Cattle†

2007 ◽  
Vol 70 (2) ◽  
pp. 287-291 ◽  
Author(s):  
R. E. PETERSON ◽  
T. J. KLOPFENSTEIN ◽  
G. E. ERICKSON ◽  
J. FOLMER ◽  
S. HINKLEY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Acidophilus ◽  
Repeated Measures ◽  
Immunomagnetic Separation ◽  
Feedlot Cattle ◽  
Outcome Variable ◽  
Escherichia Coli O157 ◽  
Fecal Shedding ◽  
Selective Enrichment ◽  
E Coli

A 2-year study was conducted during the summer months (May to September) to test the effectiveness of feeding Lactobacillus acidophilus strain NP51 on the proportion of cattle shedding Escherichia coli O157:H7 in the feces and evaluate the effect of the treatment on finishing performance. Steers (n = 448) were assigned randomly to pens, and pens of cattle were assigned randomly to NP51 supplementation or no supplementation (control). NP51 products were mixed with water and applied as the feed was mixed daily in treatment-designated trucks at the rate of 109 CFU per steer. Fecal samples were collected (n = 3,360) from the rectum from each animal every 3 weeks, and E. coli O157:H7 was isolated by standard procedures, using selective enrichment, immunomagnetic separation, and PCR confirmation. The outcome variable was the recovery of E. coli O157:H7 from feces, and was modeled using logistic regression accounting for year, repeated measures of pens of cattle, and block. No significant differences were detected for gain, intakes, or feed efficiency of control or NP51-fed steers. The probability for cattle to shed E. coli O157:H7 varied significantly between 2002 and 2003 (P = 0.004). In 2002 and 2003, the probability for NP51-treated steers to shed E. coli O157:H7 over the test periods was 13 and 21%, respectively, compared with 21 and 28% among controls. Over the 2 years, NP51-treated steers were 35% less likely to shed E. coli O157: H7 than were steers in untreated pens (odds ratio = 0.58, P = 0.008). This study is consistent with previous reports that feeding NP51 is effective in reducing E. coli O157:H7 fecal shedding in feedlot cattle.

scholarly journals Isolation, Molecular Detection and Antimicrobial Susceptibility Profile of ‎Escherichia coli O157:H7 in Household - reared Small Ruminants in Zaria ‎Metropolis, Kaduna State, Nigeria

2020 ◽  
Vol 17 (4) ◽  
pp. 16-23
Author(s):  
R. O. Yakubu ◽  
M. K. Lawan ◽  
J. K. P. Kwaga ◽  
J. Kabir
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Small Ruminants ◽  
Resistance Index ◽  
Latex Agglutination ◽  
Escherichia Coli O157 ◽  
Biochemical Tests ◽  
Cross Sectional ◽  
E Coli ◽  
Test Kit

Escherichia coli O157:H7 is a zoonotic enteric pathogen of public health significance worldwide. A cross-sectional study was carried out during which 384 faecal samples of household-reared small ruminants and water used in the various houses where the animals are reared were collected. The samples were enriched on tryptone soya broth and cultured on EMB and CT-SMAC to isolate E. coli and E. coli O157:H7 respectively; subjected to conventional biochemical tests and E. coliO157:H7 was confirmed using Wellcolex latex agglutination test kit. E. coli O157:H7 isolates were subjected to antimicrobial susceptibility test and multiplex PCR was carried out to detect the presence of virulence genes stx1, stx2, eaeA and hlyA. The results of the isolation showed isolation rate of E. coli O157:H7 of 4.69% (9/192), 0.52% (1/192) which were obtained from faeces and water samples respectively. The results of the characterisation showed that one of the E. coli O157:H7 isolated harboured the eaeA and hlyA genes but was negative for stx1 and stx2 genes. The highest number of isolates showed resistance to erythromycin (90.9%) while the least was to gentamicin (6.3%). About 97.7% (43/44) of the isolates had multiple antibiotic resistance index greater than 0.2. In conclusion, household-reared small ruminants in the study area were found to be reservoirs of E. coli O157:H7 and humans living within these households are at risk of infection. The multiple antibioticresistance recorded in this study suggests widespread use of antimicrobial drugs in the study area.

Prevalence and Enumeration of Escherichia coli O157 in Steers Receiving Various Strains of Lactobacillus-Based Direct-Fed Microbials

2007 ◽  
Vol 70 (5) ◽  
pp. 1252-1255 ◽  
Author(s):  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
L. M. CHICHESTER ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Acidophilus ◽  
Immunomagnetic Separation ◽  
Feedlot Cattle ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Feeding Period ◽  
Probable Number ◽  
Selective Enrichment ◽  
E Coli

The objective of this research was to evaluate the effect of daily dietary inclusion of specific strains of Lactobacillus acidophilus on prevalence and concentration of Escherichia coli O157 in harvest-ready feedlot cattle. Five hundred yearling steers were housed in pens of 10 animals each. At arrival, steers were randomly allocated to one of five cohorts. Four of the cohorts were fed various strains and dosages of Lactobacillus-based direct-fed microbials throughout the feeding period. Fecal samples were collected from the rectum of each animal immediately prior to shipment to the abattoir. E. coli O157 was detected using selective enrichment and immunomagnetic separation techniques. For positive samples, E. coli O157 concentration was estimated using a most-probable-number (MPN) technique that included immunomagnetic separation. Prevalence varied among the cohorts (P < 0.01). The prevalence in the controls (26.3%) was greater (P < 0.05) than that in cattle supplemented with L. acidophilus strains NP51, NP28, or NP51-NP35 (13.0, 11.0, and 11.0%, respectively). The greatest E. coli O157 concentration was also observed in the controls (3.2 log MPN/g of feces); this concentration was greater (P < 0.05) than that observed in positive animals receiving NP51, NP28, or NP51-NP35 (0.9, 1.1, 1.7 log MPN/g of feces, respectively). Specific strains of Lactobacillus-based direct-fed microbials effectively reduced the prevalence and concentration of E. coli O157 in harvest-ready cattle, whereas others did not. When using direct-fed microbials to reduce carriage of E. coli O157 in cattle, it is important to select appropriately validated products.

scholarly journals Isolation and characterization E. coli O157:H7 from meat &cheese and detection of Stx1, Stx2, hlyA, eaeA by using multiplex PCR

2011 ◽  
Vol 5 (2) ◽  
pp. 15-24
Author(s):  
Shatha T. Ahmed ◽  
Amina N. Jasim ◽  
Ayad M. Ali
Keyword(s):  
Escherichia Coli ◽  
Diagnostic System ◽  
Latex Agglutination ◽  
Diagnostic Methods ◽  
Bacterial Isolates ◽  
Biochemical Tests ◽  
E Coli ◽  
Isolation And Characterization ◽  
Minced Beef ◽  
Polymerase Chain

total of 189 samples were collected from 74 raw uncooked minced beef meat, 115 local white cheeses from 3 different areas in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identified by using morphological diagnostic methods; Samples were cultured on liquid enrichment medium, incubated at 41.5Cº for 6 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 66 non-sorbitol fermenting bacterial isolates were obtained of which 13 were identified as Escherichia coli from (6 meat and 7 cheese samples). By using traditional biochemical tests and Api20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates. Four specific biochemical tests (Cellobiose fermentation, β-Glocuronidase production, KCN and Enterohemolysin production) were done to differentiate serotype O157 differentiation from other NSF bacteria. Only 2 isolates belonging to the serotype O157 were obtained of which one isolate from meat and other isolate from cheese. Latex agglutination test for O157 and H7 showed that the 5 isolates gave positive results with both kits. The Bacterial isolates were identified by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated from various sources by using specific primers in mPCR. The result showed that gene content variety in two E. coli O157:H7 isolates, 1 from meat contain all 4 genes and other isolate from cheese contains 2 genes: Stx1 and hlyA .

scholarly journals Molecular diagnosis of E.coliO157:H7 Which Isolated from Children with Diarrhea by using Multiplex PCR

2010 ◽  
Vol 7 (1) ◽  
pp. 207-215
Author(s):  
Baghdad Science Journal
Keyword(s):  
Escherichia Coli ◽  
Diagnostic System ◽  
Latex Agglutination ◽  
Diagnostic Methods ◽  
Bacterial Isolates ◽  
Biochemical Tests ◽  
E Coli ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Positive Results

A total of 96 stool samples were collected from children with bloody diarrhea from two hospitals in Baghdad. All samples were surveyed and examined for the presence of the Escherichia coli O157:H7 and differentiate it from other Non -Sorbitol Fermenting Escherichia coli (NSF E. coli). The Bacterial isolates were identifed by using morphological diagnostic methods, Samples were cultured on liquid enrichment medium, incubated at 37C? for 24 hrs, and then cultured on Cefixime Tellurite -Sorbitol MacConkey Agar (CT- SMAC). 32 non-sorbitol fermenting bacterial isolates were obtained of which 11 were identified as Escherichia coli by using traditional biochemical tests and API20E diagnostic system without differentiation between serotype O157:H7 and other NSF E. coli isolates . Four special biochemical tests were done for serotype O157:H7 differentiation from other NSF bacteria. Only 3 isolates belonging to the serotype O157:H7 were obtained . Latex agglutination test for O157 and H7 showed that the 3 isolates gave positive results with both tests. The Bacterial isolates were identifed by using Multiplex Polymerase Chain Reaction (MPCR) technology for the presence or absence of 4 genes (Stx1, Stx2, hlyA and eaeA) that encode for main virulence factors to diagnose E. coli O157:H7 isolated By using specific primers in MPCR . The result showed that one E. coli O157:H7 isolates contain all 4 genes , other isolates contain 3 genes: Stx2, hlyA & eaeA.

Effect of a Vaccine Product Containing Type III Secreted Proteins on the Probability of Escherichia coli O157:H7 Fecal Shedding and Mucosal Colonization in Feedlot Cattle†

2007 ◽  
Vol 70 (11) ◽  
pp. 2568-2577 ◽  
Author(s):  
R. E. PETERSON ◽  
T. J. KLOPFENSTEIN ◽  
R. A. MOXLEY ◽  
G. E. ERICKSON ◽  
S. HINKLEY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Humoral Response ◽  
Immunomagnetic Separation ◽  
Environmental Safety ◽  
Feedlot Cattle ◽  
Escherichia Coli O157 ◽  
Selective Enrichment ◽  
E Coli ◽  
Natural Exposure ◽  
Vaccine Product

Preharvest intervention strategies to reduce Escherichia coli O157:H7 in cattle have been sought as a means to reduce human foodborne illness. A blinded clinical trial was conducted to test the effect of a vaccine product on the probability that feedlot steers, under conditions of natural exposure, shed E. coli O157:H7 in feces, are colonized by this organism in the terminal rectum, or develop a humoral response to the respective antigens. Steers (n = 288) were assigned randomly to 36 pens (eight head per pen), and pens were randomized to vaccination treatment in a balanced fashion within six dietary treatments of an unrelated nutrition study. Treatments included vaccination or placebo (three doses at 3-week intervals). Fecal samples for culture (n = 1,410) were collected from the rectum of each steer on pretreatment day 0 and posttreatment days 14, 28, 42, and 56. Terminal rectum mucosal (TRM) cells were aseptically collected for culture at harvest (day 57 posttreatment) by scraping the mucosa 3.0 to 5.5 cm proximal to the rectoanal junction. E. coli O157:H7 was isolated and identified with selective enrichment, immunomagnetic separation, and PCR confirmation. Vaccinated cattle were 98.3% less likely to be colonized by E. coli O157:H7 in TRM cells (odds ratio = 0.014, P < 0.0001). Diet was also associated with the probability of cattle being colonized (P = 0.04). Vaccinated cattle demonstrated significant humoral responses to Tir and O157 lipopolysaccharide. These results provide evidence that this vaccine product reduces E. coli O157:H7 colonization of the terminal rectum of feedlot beef cattle under conditions of natural exposure, a first step in its evaluation as an effective intervention for food and environmental safety.

Comparison of Rapid Enzyme-Linked Immunosorbent Assay and Immunomagnetic Separation Methods for Detection of Escherichia coli O157 in Fecal, Hide, Carcass, and Ground Beef Samples

2007 ◽  
Vol 70 (10) ◽  
pp. 2230-2234 ◽  
Author(s):  
T. W. THOMPSON ◽  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
M. F. MILLER ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Separation Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Perfect Agreement ◽  
Fecal Samples ◽  
E Coli ◽  
Beef Products

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coli O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

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