Prevalence and Numbers of Escherichia coli O157 on Bovine Hides at a Beef Slaughter Plant

2005 ◽  
Vol 68 (4) ◽  
pp. 660-665 ◽  
Author(s):  
S. B. O[apos]BRIEN ◽  
G. DUFFY ◽  
E. CARNEY ◽  
J. J. SHERIDAN ◽  
D. A. McDOWELL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Early Stage ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Hemolysin Gene ◽  
Peptone Water ◽  
Encoding Gene ◽  
Genetic Profiles

In this study, we investigated the prevalence and numbers of Escherichia coli O157 on bovine hides. Samples (n = 1,500) were collected over a 17-month period (30 samples per week) by sponge swabbing approximately 122-cm2 areas of the bovine rump of slaughtered cattle at an early stage of carcass processing (first legging). Sponge samples (n = 1,500) were stomached in buffered peptone water supplemented with novobiocin, directly plated on sorbitol MacConkey with Cefixime tellurite (SMAC-CT), enriched for 24 h, extracted by immunomagnetic separation, and plated onto SMAC-CT agar. Presumptive E. coli O157 colonies from SMAC-CT plates were confirmed by PCR for the presence of eaeA, hlyA, fliCh7, vt1, vt2, and portions of the rfb (O-antigen encoding) region of E. coli O157. Overall, E. coli O157 was recovered from 109 samples (7.3%) at concentrations ranging from less than 0.13 to 4.24 log CFU/100 cm2. PCR analysis revealed a wide diversity of genetic profiles among recovered isolates of verocytotoxigenic E. coli. Of the isolates recovered, 99 of 109 contained the attaching and effacing gene (eaeA) and the hemolysin gene (hlyA), and 78 of 109 had the flagellar H7 antigen–encoding gene (fliCh7). Only 6 of 109 isolates contained both verotoxin-producing genes (vt1 and vt2); 91 of 109 contained the vt2 gene only, whereas 1 of 109 contained the vt1 gene only. The remaining 11 of 109 contained neither vt1 nor vt2.

scholarly journals Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

2012 ◽  
Vol 1 (2) ◽  
Author(s):  
Hilda Nyati ◽  
Annet Heuvelink ◽  
Caroliene Van Heerwaarden ◽  
Ans Zwartkruis
Keyword(s):  
Escherichia Coli ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Immunomagnetic Beads ◽  
Tryptone Soya Broth ◽  
Detection Rates ◽  
E Coli ◽  
Peptone Water ◽  
Cattle Faeces

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

Efficacy of Enrichment Broths in the Recovery of Freeze-Injured Escherichia coli O157:H7 in Inoculated Ground Beef by PCR

2003 ◽  
Vol 66 (10) ◽  
pp. 1911-1915 ◽  
Author(s):  
W. C. LIONBERG ◽  
L. RESTAINO ◽  
E. W. FRAMPTON ◽  
W. M. BARBOUR
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Automated System ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
E Coli ◽  
Cultural Isolation ◽  
Peptone Water ◽  
Chromogenic Agar

Escherichia coli O157:H7 strains ATCC 35150 and ATCC 43894 and five pooled isolates from beef and pork freeze injured at −25°C in beef infusion were used to inoculate ground beef. Samples (25 g each) were added to 225 ml of buffered peptone water with vancomycin, cefsulodin, and cefixime (BPW-VCC), 225 ml of modified EC broth plus novobiocin (mEC+n), and 225 ml of R&F enrichment broth (R&F-EB) and aerobically incubated at 41 to 42°C. After 6, 7, 8, and 24 h of incubation, levels of E. coli O157:H7 recovered from each broth by a PCR assay with the BAX automated system as well as by conventional enrichment with the use of nonaerated mEC+n incubated at 35°C for 24 h were compared with levels recovered by cultural isolation with immunomagnetic separation and plating on BCM E. coli O157:H7 chromogenic agar. For ground beef inoculated with a mean of 4.23 ± 1.00 total cells (74% freeze injured) per 25 g, after 6 h the PCR assay identified 72.7, 57.6, and 66% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive, whereas the recovery rates after 7 and 8 h exceeded 90%, with the rate for R&F-EB being 100%. For ground beef inoculated with a mean of 1.50 ± 0.56 total cells (80% freeze injured) per 25 g, after 6 h the PCR assay identified 47.6, 19.1, and 9.5% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive. These values increased to 81.0, 61.9, and 52.4% after 7 h and to 95.2, 61.9, and 71.4% after 8 h. After 24 h, only 55 to 60% of the samples at both inoculum levels tested positive by PCR with conventional enrichment and incubation, whereas >95% of the samples tested positive with R&F-EB aerated at 41 to 42°C. Culture results for R&F-EB and mEC+n after 7 and 8 h of incubation were closely correlated with presumptive positive PCR results.

scholarly journals Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

2012 ◽  
Vol 1 (2) ◽  
Author(s):  
Hilda Nyati ◽  
Annet Heuvelink ◽  
Caroliene Van Heerwaarden ◽  
Ans Zwartkruis
Keyword(s):  
Escherichia Coli ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Immunomagnetic Beads ◽  
Tryptone Soya Broth ◽  
Detection Rates ◽  
E Coli ◽  
Peptone Water ◽  
Cattle Faeces

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

scholarly journals Evaluation of Escherichia coli O157:H7 Growth Media for Use in Test-and-Hold Procedures for Ground Beef Processing†

2006 ◽  
Vol 69 (5) ◽  
pp. 1007-1011 ◽  
Author(s):  
MICHAEL N. GUERINI ◽  
TERRANCE M. ARTHUR ◽  
STEVEN D. SHACKELFORD ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Ground Beef ◽  
Threshold Level ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Peptone Water

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7—Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX), BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42°C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.

Contamination of Intact Apples after Immersion in an Aqueous Environment Containing Escherichia coli O157:H7†

1999 ◽  
Vol 62 (5) ◽  
pp. 444-450 ◽  
Author(s):  
R. L. BUCHANAN ◽  
S. G. EDELSON ◽  
R. L. MILLER ◽  
G. M. SAPERS
Keyword(s):  
Escherichia Coli ◽  
Outer Core ◽  
Core Region ◽  
Effect Of Temperature ◽  
Aqueous Environment ◽  
Escherichia Coli O157 ◽  
Dye Uptake ◽  
E Coli ◽  
Golden Delicious ◽  
Peptone Water

The extent and location of Escherichia coli O157:H7 contamination after intact apples were immersed in cold (2°C) 1% peptone water containing approximately 3 × 107 CFU/ml was assessed using four apple varieties, Golden Delicious, McIntosh, Red Delicious, and Braeburn. Room temperature and refrigerated apples were used to determine the effect of temperature differential on E. coli infiltration. The highest levels of E. coli were associated with the outer core region of the apple, followed by the skin. Apples were subsequently treated by immersing them for 1 min in 2,000 mg/liter sodium hypochlorite, followed by a 1-min tapwater rinse. This treatment reduced pathogen levels by 1- to 3-log cycles but did not eliminate the microorganism, particularly from the outer core region. While E. coli was not detected in the inner core of most apples, warm fruit immersed in cold peptone water occasionally internalized the pathogen. The frequency and extent of internalization of the pathogen was less when cold apples were immersed in cold peptone water. Subsequent dye uptake studies with Golden Delicious apples indicated that approximately 6% of warm apples immersed into a cold dye solution accumulated dye via open channels leading from the blossom end into the core region. However, dye uptake did not occur when the dye solution was warmer than the apple.

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.

Simultaneous Enrichment of Shiga Toxin–Producing Escherichia coli O157 and O26 and Salmonella in Food Samples Using Universal Preenrichment Broth

2009 ◽  
Vol 72 (10) ◽  
pp. 2065-2070 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
JUNKO SAKATA ◽  
TETSUYA HARADA ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Food Samples ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Significant Difference ◽  
Peptone Water ◽  
Radish Sprouts ◽  
Pork Samples ◽  
Better Than

Universal preenrichment broth (UPB) was compared with modified Escherichia coli broth with novobiocin (mEC+n) for enrichment of Shiga toxin–producing E. coli O157 and O26, and with buffered peptone water (BPW) for preenrichment of Salmonella enterica. Ten strains each of the three pathogens were inoculated into beef and radish sprouts following thermal, freezing, or no treatment. With regard to O157 and O26, UPB incubated at 42°C recovered significantly more cells from inoculated beef than UPB at 35°C and from radish sprout samples than UPB at 35°C and mEC+n. With regard to Salmonella, UPB incubated at 42°C was as effective as UPB at 35°C and BPW at recovering cells from beef and radish sprout samples. No significant difference was noted between the effectiveness of UPB at 42°C and UPB at 35°C or BPW in the recovery of Salmonella from 205 naturally contaminated poultry samples. By using UPB at 42°C, one O157:H7 strain was isolated from the mixed offal of 53 beef samples, 6 cattle offal samples, and 50 pork samples all contaminated naturally, with no pathogen inoculation. The present study found that UPB incubated at 42°C was as effective as, or better than, mEC+n for enrichment of O157 and O26 and comparable to BPW for preenrichment of Salmonella. These findings suggest that a great deal of labor, time, samples, and space may be saved if O157, O26, and Salmonella are enriched simultaneously with UPB at 42°C.

scholarly journals Development and Characterization of a Fluorescent-Bacteriophage Assay for Detection of Escherichia coli O157:H7

1999 ◽  
Vol 65 (4) ◽  
pp. 1397-1404 ◽  
Author(s):  
Lawrence Goodridge ◽  
Jinru Chen ◽  
Mansel Griffiths
Keyword(s):  
Escherichia Coli ◽  
Flow Cytometry ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Lower Detection ◽  
Unreliable Method ◽  
Direct Epifluorescent Filter Technique ◽  
Bacterial Concentrations

ABSTRACT In this paper we describe evaluation and characterization of a novel assay that combines immunomagnetic separation and a fluorescently stained bacteriophage for detection of Escherichia coliO157:H7 in broth. When it was combined with flow cytometry, the fluorescent-bacteriophage assay (FBA) was capable of detecting 104 cells/ml. A modified direct epifluorescent-filter technique (DEFT) was employed in an attempt to estimate bacterial concentrations. Using regression analysis, we calculated that the lower detection limit was between 102 and 103cells/ml; however, the modified DEFT was found to be an unreliable method for determining bacterial concentrations. The results of this study show that the FBA, when combined with flow cytometry, is a sensitive technique for presumptive detection of E. coliO157:H7 in broth cultures.

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.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

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