Simultaneous Immunomagnetic Separation Method for the Detection of Escherichia coli O26, O111, and O157 from Food Samples

2014 ◽  
Vol 77 (1) ◽  
pp. 15-22 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Food Culture ◽  
Cell Detection ◽  
Labor Costs ◽  
Single Strain ◽  
E Coli ◽  
Detection Medium

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef, and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain.

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.

Survival of Escherichia coli O157:H7 in Ground-Beef Patties during Storage at 2, −2, 15 and then −2°C, and −20°C†

1999 ◽  
Vol 62 (11) ◽  
pp. 1243-1247 ◽  
Author(s):  
SUSAN E. ANSAY ◽  
KIM A. DARLING ◽  
CHARLES W. KASPAR
Keyword(s):  
Escherichia Coli ◽  
Frozen Storage ◽  
Ground Beef ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
Control Strain ◽  
Single Strain ◽  
E Coli ◽  
Beef Patties ◽  
Significant Difference

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2°C for 4 weeks, −2°C for 4 weeks, 15°C for 4 h and then −2°C for 4 weeks, and −20°C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 105 CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log10 CFU/g, and pathogen numbers declined 1.9 log10 CFU/g when patties were stored for 4 weeks at 2°C. When patties were stored at −2°C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log10 CFU/g, respectively. Patties stored at 15°C for 4 h prior to storage at −2°C for 4 weeks resulted in 1.6 and 2.7 log10–CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15°C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (−20°C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log10–CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.

scholarly journals Bacteriophages Reduce Experimental Contamination of Hard Surfaces, Tomato, Spinach, Broccoli, and Ground Beef by Escherichia coli O157:H7

2008 ◽  
Vol 74 (20) ◽  
pp. 6230-6238 ◽  
Author(s):  
Tamar Abuladze ◽  
Manrong Li ◽  
Marc Y. Menetrez ◽  
Timothy Dean ◽  
Andre Senecal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Food Samples ◽  
Significant Treatment ◽  
E Coli ◽  
Virulent Strains ◽  
Buffer Control ◽  
And Control ◽  
Phosphate Buffered Saline ◽  
Control Samples

ABSTRACT A bacteriophage cocktail (designated ECP-100) containing three Myoviridae phages lytic for Escherichia coli O157:H7 was examined for its ability to reduce experimental contamination of hard surfaces (glass coverslips and gypsum boards), tomato, spinach, broccoli, and ground beef by three virulent strains of the bacterium. The hard surfaces and foods contaminated by a mixture of three E. coli O157:H7 strains were treated with ECP-100 (test samples) or sterile phosphate-buffered saline buffer (control samples), and the efficacy of phage treatment was evaluated by comparing the number of viable E. coli organisms recovered from the test and control samples. Treatments (5 min) with the ECP-100 preparation containing three different concentrations of phages (1010, 109, and 108 PFU/ml) resulted in statistically significant reductions (P = <0.05) of 99.99%, 98%, and 94%, respectively, in the number of E. coli O157:H7 organisms recovered from the glass coverslips. Similar treatments resulted in reductions of 100%, 95%, and 85%, respectively, in the number of E. coli O157:H7 organisms recovered from the gypsum board surfaces; the reductions caused by the two most concentrated phage preparations were statistically significant. Treatment with the least concentrated preparation that elicited significantly less contamination of the hard surfaces (i.e., 109 PFU/ml) also significantly reduced the number of viable E. coli O157:H7 organisms on the four food samples. The observed reductions ranged from 94% (at 120 ± 4 h posttreatment of tomato samples) to 100% (at 24 ± 4 h posttreatment of spinach samples). The data suggest that naturally occurring bacteriophages may be useful for reducing contamination of various hard surfaces, fruits, vegetables, and ground beef by E. coli O157:H7.

Thermal Inactivation of a Single Strain Each of Serotype O26:H11, O45:H2, O103:H2, O104:H4, O111:H−, O121:H19, O145:NM, and O157:H7 Cells of Shiga Toxin–Producing Escherichia coli in Wafers of Ground Beef†

2013 ◽  
Vol 76 (8) ◽  
pp. 1434-1437 ◽  
Author(s):  
J. B. LUCHANSKY ◽  
A. C. S. PORTO-FETT ◽  
B. A. SHOYER ◽  
J. PHILLIPS ◽  
D. EBLEN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Thermal Inactivation ◽  
Ground Beef ◽  
Sampling Time ◽  
Single Strain ◽  
E Coli ◽  
D Values ◽  
Cooking Times ◽  
Sterile Filter

For each of two trials, freshly ground beef of variable fat content (higher: 70:30 %lean:%fat; lower: 93:7 %lean:%fat) was separately inoculated with ca. 7.0 log CFU/g of a single strain of Escherichia coli serotypes O26:H11, O45:H2, O103:H2, O104:H4, O111:H−, O121:H19, O145:NM, and O157:H7. Next, ca. 3-g samples of inoculated beef were transferred into sterile filter bags and then flattened (ca. 1.0 mm thick) and vacuum sealed. For each temperature and sampling time, three bags of the inoculated wafers of beef were submerged in a thermostatically controlled water bath and heated to an internal temperature of 54.4°C (130°F) for up to 90 min, to 60°C (140°F) for up to 4 min, or to 65.6°C (150°F) for up to 0.26 min. In lower fat wafers, D-values ranged from 13.5 to 23.6 min, 0.6 to 1.2 min, and 0.05 to 0.08 min at 54.4, 60.0, and 65.6°C, respectively. Heating higher fat wafers to 54.4, 60.0, and 65.6°C generated D-values of 18.7 to 32.6, 0.7 to 1.1, and 0.05 to 0.2 min, respectively. In addition, we observed reductions of ca. 0.7 to 6.7 log CFU/g at 54.4°C after 90 min, ca. 1.1 to 6.1 log CFU/g at 60.0°C after 4 min, and 0.8 to 5.8 log CFU/g at 65.6°C after 0.26 min. Thus, cooking times and temperatures effective for inactivating a serotype O157:H7 strain of E. coli in ground beef were equally effective against the seven non-O157:H7 Shiga toxin–producing strains investigated herein.

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

Magnetic Nanoparticle-Antibody Conjugates for the Separation of Escherichia coli O157:H7 in Ground Beef

2005 ◽  
Vol 68 (9) ◽  
pp. 1804-1811 ◽  
Author(s):  
MADHUKAR VARSHNEY ◽  
LIJU YANG ◽  
XIAO-LI SU ◽  
YANBIN LI
Keyword(s):  
Escherichia Coli ◽  
Magnetic Nanoparticles ◽  
Magnetic Nanoparticle ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Capture Efficiency ◽  
Escherichia Coli O157 ◽  
Mechanical Mixing ◽  
E Coli ◽  
Antibody Conjugates

The immunomagnetic separation with magnetic nanoparticle-antibody conjugates (MNCs) was investigated and evaluated for the detection of Escherichia coli O157:H7 in ground beef samples. MNCs were prepared by immobilizing biotin-labeled polyclonal goat anti–E. coli antibodies onto streptavidin-coated magnetic nanoparticles. For bacterial separation, MNCs were mixed with inoculated ground beef samples, then nanoparticle-antibody–E. coli O157:H7 complexes were separated from food matrix with a magnet, washed, and surface plated for microbial enumeration. The capture efficiency was determined by plating cells bound to nanoparticles and unbound cells in the supernatant onto sorbitol MacConkey agar. Key parameters, including the amount of nanoparticles and immunoreaction time, were optimized with different concentrations of E. coli O157:H7 in phosphate-buffered saline. MNCs presented a minimum capture efficiency of 94% for E. coli O157:H7 ranging from 1.6 × 101 to 7.2 × 107 CFU/ml with an immunoreaction time of 15 min without any enrichment. Capture of E. coli O157:H7 by MNCs did not interfere with other bacteria, including Salmonella enteritidis, Citrobacter freundii, and Listeria monocytogenes. The capture efficiency values of MNCs increased from 69 to 94.5% as E. coli O157:H7 decreased from 3.4 × 107 to 8.0 × 100 CFU/ml in the ground beef samples prepared with minimal steps (without filtration and centrifugation). An enrichment of 6 h was done for 8.0 × 100 and 8.0 × 101 CFU/ml of E. coli O157:H7 in ground beef to increase the number of cells in the sample to a detectable level. The results also indicated that capture efficiencies of MNCs for E. coli O157:H7 with and without mechanical mixing during immunoreaction were not significantly different (P &gt; 0.05). Compared with microbeads based immunomagnetic separation, the magnetic nanoparticles showed their advantages in terms of higher capture efficiency, no need for mechanical mixing, and minimal sample preparation.

Incidence of Enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in Retail Fresh Ground Beef, Sprouts, and Mushrooms

2006 ◽  
Vol 69 (2) ◽  
pp. 441-443 ◽  
Author(s):  
M. SAMADPOUR ◽  
M. W. BARBOUR ◽  
T. NGUYEN ◽  
T.-M. CAO ◽  
F. BUCK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Ground Beef ◽  
Food Samples ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Pcr Assays ◽  
Retail Food

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

scholarly journals Improvement of Immunomagnetic Separation for Escherichia coli O157:H7 Detection by the PickPen Magnetic Particle Separation Device†

2006 ◽  
Vol 69 (12) ◽  
pp. 2870-2874 ◽  
Author(s):  
XIANGWU NOU ◽  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
DAYNA M. BRICHTA-HARHAY ◽  
MICHAEL N. GUERINI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogen Detection ◽  
Magnetic Particles ◽  
Magnetic Particle ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
High Background ◽  
E Coli ◽  
Significant Difference

Conventional immunomagnetic separation (IMS) procedures, which use an external magnetic source to capture magnetic particles against the side of a test tube, are labor-intensive and can have poor sensitivity for the target organism because of high background microflora that is not effectively washed away during the IMS process. This report compares the conventional IMS procedure to a new IMS procedure with an intrasolution magnetic particle transfer device, the PickPen. The IMS target for the majority of these studies is Escherichia coli O157:H7 in various types of samples, including cattle feces, hides, carcasses, and ground beef. Comparison of the two IMS methods showed a significant difference (P &lt; 0.05) in the efficiency of detecting E. coli O157:H7 from cattle carcass surface, cattle hide, and cattle fecal samples. No significant improvement (P &gt; 0.05) in E. coli O157:H7 detection was observed when the PickPen IMS procedure was used to isolate this pathogen from ground beef samples. Use of the PickPen IMS greatly increases the throughput of the IMS procedure and may be more compatible with various emerging technologies for pathogen detection. In addition, the efficacy of sequential IMS for multiple pathogens is reported herein.

Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

2005 ◽  
Vol 68 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Perishable Product ◽  
Specificity And Sensitivity

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P &gt; 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

Enumeration of Sublethally Injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli Strain B-41560 Using Selective Agar Overlays versus Commercial Methods

2013 ◽  
Vol 76 (4) ◽  
pp. 674-679 ◽  
Author(s):  
AMANDA R. SMITH ◽  
ALYSHA L. ELLISON ◽  
AMANDA L. ROBINSON ◽  
MARYANNE DRAKE ◽  
SUSAN A. McDOWELL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Food Samples ◽  
Coli Strain ◽  
Milk Formula ◽  
Selective Media ◽  
E Coli ◽  
Infant Milk Formula ◽  
Intervention Measures ◽  
Direct Inoculation

Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 106 to 108 CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid-and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

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