Evaluation of consumer-style cooking methods for reduction of Escherichia coli O157:H7 in ground beef

The objective of this study was to evaluate the thermal inactivation of Escherichia coli O157:H7 in ground beef cooked to an internal temperature of 71.1 degrees C (160 degrees F) under conditions simulating consumer-style cooking methods. To compare a double-sided grill (DSG) with a single-sided grill (SSG), two different cooking methods were used for the SSG: for the one-turnover (OT-SSG) method, a patty was turned once when the internal temperature reached 40 degrees C, and for the multiturnover (MT-SSG) method, a patty was turned every 30 s. Patties (100 g, n = 9) inoculated with a five-strain mixture of E. coli O157: H7 at a concentration of 10(7) CFU/g were cooked until all three temperature readings (for two sides and the center) for a patty were 71.1 degrees C. The surviving E. coli O157:H7 cells were enumerated on sorbitol MacConkey (SMAC) agar and on phenol red agar base with 1% sorbitol (SPRAB). The order of the cooking methods with regard to the cooking time required for the patty to reach 71.1 degrees C was as follows: DSG (2.7 min) < MT-SSG (6.6 min) < OT-SSG (10.9 min). The more rapid, higher-temperature cooking method was more effective (P < 0.01) in destroying E. coli O157:H7 in ground beef. E. coli O157:H7 reduction levels were clearly differentiated among treatments as follows: OT-SSG (4.7 log10 CFU/g) < MT-SSG (5.6 log10 CFU/g) < DSG (6.9 log10 CFU/g). Significantly larger numbers of E. coil O157:H7 were observed on SPRAB than on SMAC agar. To confirm the safety of ground beef cooked to 71.1 degrees C, additional patties (100 g, n = 9) inoculated with lower concentrations of E. coli O157:H7 (10(3) to 10(4) CFU/g) were tested. The ground beef cooked by the OT-SSG method resulted in two (22%) of nine samples testing positive after enrichment, whereas no E. coli O157:H7 was found for samples cooked by the MT-SSG and DSG methods. Our findings suggest that consumers should be advised to either cook ground beef patties in a grill that cooks the top and the bottom of the patty at the same time or turn patties frequently (every 30 s) when cooking on a grill that cooks on only one side.     

Effect of food matrix and cell growth on PCR-based detection of Escherichia coli O157:H7 in ground beef

The purpose of this work was (i) to investigate the feasibility of a previously reported upstream processing method for PCR template preparation to facilitate the detection of Escherichia coli O157:H7 from ground beef and (ii) to assess the impact of cell growth (no growth in the matrix versus growth in the matrix) on molecular detection limits. Two food matrices (autoclaved and raw ground beef) were evaluated in all studies. For no-growth experiments, 10-g meat samples were inoculated with 10(2) to 10(7) CFU/g E. coli O157:H7 and then homogenized. The homogenates were processed to remove large particulates and inhibitors using a two-phase upstream processing method consisting of two sequential centrifugation steps, the second of which used titanous hydroxide to facilitate bacterial immobilization. After upstream processing, sample concentrates were extracted for DNA isolation and amplified by PCR. For growth experiments, 10-g meat samples were inoculated at 1 CFU of E. coli O157:H7 per gram, allowed to grow to 10(2) to 10(7) CFU/g, and then processed for PCR assay. Cell recoveries after upstream processing ranged from 15.9 to 77.6% and were not facilitated by the use of titanous hydroxide, as compared with a saline control (P > 0.05). Bacterial cell recovery and PCR detection limits were similar when comparing autoclaved ground beef and raw ground beef, but cell recoveries were highly variable for raw ground beef samples in which E. coli O157:H7 cells were allowed to grow before processing for detection. Overall, PCR detection limits approximated 10(3) CFU/g of ground beef for all treatments. These results indicate that use of model food systems may not always provide an accurate replication of real-world conditions when evaluating PCR detection limits.     

Collaborative evaluation of detection methods for Escherichia coli O157:H7 from radish sprouts and ground beef

For the evaluation of plating and immunological methods applicable to the detection of Escherichia coli O157:H7 from ground beef and radish sprouts, a collaborative study was conducted. It focused on a comparison of the efficiency of the plating and immunological methods using various plating agars and immuno-kits in combination with enrichment in modified E. coli broth supplemented with novobiocin (mEC + n), and using immunomagnetic separation. The plating media tested were sorbitol MacConkey agar (SMAC), SMAC supplemented with cefixime (0.05 mg/l) and potassium tellurite (2.5 mg/l) (CT-SMAC), and agars containing beta-glucuronidase substrates such as BCM O157 and CHROMagar O157. The immuno-kits used were Now E. coli, Path-Stick O157, VIP, EHEC-Tek ELISA System and Rapiblot E. coli O157. The 20 participating laboratories attempted to detect E. coli O157:H7 in 25 g chilled and frozen samples of ground beef uninoculated and inoculated with E. coli O157:H7 at levels of 138.9 and 23.9 cfu/25 g, and in 25 g chilled and frozen samples of radish sprouts uninoculated and inoculated at levels of 20.4 and 1.7 cfu/25 g. E. coli O157:H7 was recovered well from ground beef by all of the methods except direct plating with SMAC. For radish sprouts, the IMS-plating methods with CT-SMAC, BCM O157 and CHROMagar O157 were most efficient at detecting E. coli O157:H7 in more than 90% of the chilled samples inoculated at the level of 20.4 cfu/25 g. All the methods were less sensitive when applied to similar levels of E. coli O157:H7 in radish sprouts (20.4 cfu/25 g) compared with ground beef (23.9 cfu/25 g) especially if the sprouts were frozen. The sensitivity of the immuno-kits appeared to be similar to the IMS-plating methods, but the specificity was lower. Based on the results, we recommend the IMS-plating method using CT-SMAC and agars containing beta-glucuronidase substrate in combination with static enrichment incubation in mEC + n at 42 degrees C.     

Modification of methods for detection of Escherichia coli O157:H7 on produce

To establish the best detection method for Escherichia coli O157:H7 on produce, the sensitivity, specificity, and recovery values of 3 selective media [sorbitol MacConkey agar containing cefixime and tellurite (CTSMAC), sorbitol MacConkey agar containing cefixime, tellurite, and 5-bromo-4-chloro-3-indoxyl-β-glucuronide (CTB-SMAC), and CHROMagar O157 were compared. Sample preparation methods (pummeling, sonication, and hand-shaking) were compared to determine the optimum procedure for qualitative analysis from cherry tomato. The selectivity and recovery rates of stressed cell of the 3 tested media were in the order of CHROMagar O157>CTBSMAC>CT-SMAC. The recovery rates of hand-shaken tomatoes (10/10) were higher than rates of sonicated (8/10) and pummeled tomatoes (6/10) for inoculated cherry tomatoes in modified E. coli (mEC) broth enriched with novobicin. CHROMagar O157 was the optimum agar medium for detection of E. coli O157:H7. Cherry tomatoes should be treated with hand-shaking to enhance recovery for qualitative analysis.     

Comparison of methods for detection and isolation of cold- and freeze-stressed Escherichia coli O157:H7 in raw ground beef

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at < or =0.5 and < or =2 CFU/g, and samples were then enriched immediately or were stored at 4 degrees C for 72 h or at -20 degrees C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42 degrees C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35 degrees C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42 degrees C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35 degrees C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+n at 35 degrees C, 3.7 times more likely with an initial inoculum of < or = 2.0 CFU/g than with < or = 0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

The use of a fluorescent bacteriophage assay for detection of Escherichia coli O157:H7 in inoculated ground beef and raw milk.

The objective of this study was to develop a fluorescent bacteriophage assay (FBA) for the detection of E. coli O157:H7 in ground beef and raw milk. The FBA is a two step assay that combines immunomagnetic separation, to separate the target organism from mixed culture, with a highly specific fluorescently stained bacteriophage to label the E. coli O157:H7 cells. When used in conjunction with flow cytometry, the FBA was able to detect 2.2 CFU/g of artificially contaminated ground beef following a 6 h enrichment. Between 10(1) and 10(2) CFU/ml of artificially contaminated raw milk were detectable after a 10 h enrichment step. The results show that the FBA is potentially useful as a rapid technique for the preliminary detection of E. coli O157:H7 in food.     

Comparison of two sampling methods for Escherichia coli O157:H7 detection in feedlot cattle

Two sampling methods (rectoanal swabs and rectal fecal grabs) were compared for their recovery of Escherichia coli O157:H7 from feedlot cattle. Samples were collected from 144 steers four times during the finishing period by swabbing the rectoanal mucosa with cotton swabs and immediately obtaining feces from the rectum of each individual steer. The number of steers with detectable E. coli O157:H7 increased from 2 of 144 (1.4%) cattle on arrival at the feedlot to 10 of 144 (6.9%) after 1 month, 76 of 143 (52.8%) after 7 months, and 30 of 143 (20.8%) at the last sampling time before slaughter. Wilcoxon signed-rank tests indicated that the two sampling methods gave different results for sampling times 3 and 4 (P < 0.05) but not for sampling time 2 (P = 0.16). Agreement between the two sampling methods was poor (kappa < 0.2) for three of the four sampling times and moderate (kappa = 0.6) for one sampling time, an indication that in this study rectoanal swabs usually were less sensitive than rectal fecal grabs for detection of E. coli O157:H7 in cattle. Overall, the herd of origin was not significantly associated with E. coli O157:H7 results, but the weight of the steers was. Further investigation is needed to determine the effects of potential confounding factors (e.g., size and type of swab, consistency of feces, site sampled, and swabbing technique) that might influence the sensitivity of swabs in recovering E. coli O157:H7 from the rectoanal mucosa of cattle.     

食品中大肠杆菌O157:H7的几种检测方法的比较

目的评估平板分离培养法、免疫磁珠分离(IMS)法、VIDAS全自动酶标免疫测试系统、BAX全自动病原菌检测系统及环介导等温扩增(LAMP)技术在食品中检验肠出血型大肠埃希菌O157∶H7的特异性、敏感性。方法使用平板分离培养法、免疫磁珠分离法、VIDAS法、BAX法及LAMP法对人工制备的染菌猪肉样本进行检测,并对这几种方法进行比较。结果 BAX法和LAMP法的检出率最高,分别是89.1%和85.9%,免疫磁珠法和VIDAS法检出率次之,分别是75.0%和78.1%,传统分离培养法为43.8%。结论 BAX法和LAMP法具有快速、高效、特异性好、敏感性高的特点,可快速筛选食品中可能存在的肠出血型大肠埃希菌O157∶H7。     

原料乳中大肠杆菌O157:H7的快速检测

建立了一种快速检测原料乳中大肠杆菌O157:H7的PCR技术.该方法利用过滤富集菌体后的PCR技术来检测原料乳中大肠杆菌O157:H7,先对人工污染大肠杆菌O157:H7的原料乳进行离心脱脂,然后添加EDTA-2Na获得澄清乳液,最后通过0.45 μm微膜过滤收集菌体,整个过程只需6 h左右即可完成.检测灵敏度高达10-mL-1.这种方法在传统检测方法的基础上做了有效改进,使得原料乳中的大肠杆菌O157:H7的检测能够快速、准确、灵敏的进行.     

Presence of Escherichia coli O157:H7 in ground beef and ground baby beef meat

A total of 114 beef and baby beef samples were examined. The samples included ground baby beef, mixed ground baby beef and pork, and chopped and shaped meat. The samples were analyzed from 30 different grocery stores in Zagreb, Croatia. The object of this study was to evaluate the prevalence of Escherichia coli O157:H7 in the samples that can enhance the potential risk of outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. The results in all tested samples of E. coli O157:H7 were negative. A single sample was positive in a latex agglutination test using antiserum to O157:H7. It was identified as Proteus vulgaris at the Pasteur Institute, Paris, France. This result correlates positively with cross-contamination with Yersinia enterocolitica 09, Brucella abortus, Salmonella type N, and Pseudomonas maltophila.     

Rapid detection of Escherichia coli O157:H7 in ground beef using a fiber-optic biosensor.

A portable fiber-optic biosensor was used to detect Escherichia coli O157:H7 in seeded ground beef samples. The principle of the system is a sandwich immunoassay using cyanine 5 dye-labeled polyclonal anti-E. coli O157:H7 antibodies for generation of a specific fluorescent signal. Signal acquisition is effected by launching light from a 635-nm diode laser into a dual tapered 600-microm silica fiber. Fluorescent molecules within approximately 100 nm of the fiber surface are excited by the evanescent field, and a portion of the emission recouples into the fiber. A photodiode allows for quantitation of the collected emission light at wavelengths of 670 to 710 nm. Biotin-avidin interactions are used to attach polyclonal antibodies specific for E. coli O157:H7 to the final 7.5 cm of the fiber probe. The biosensor was able to detect E. coli O157:H7 to 3 to 30 CFU/ml in seeded ground beef samples. The reaction was highly specific. Signals with Listeria monocytogenes, Salmonella Typhimurium, or E. coli nonO157:H7 were 2 to 3% of those observed with a similar concentration of E. coli O157:H7. Assays were conducted at or near real-time with results obtained within 20 min of sampling.     

Modifications to methods for the enumeration and detection of injured Escherichia coli O157:H7 in foods

Reliable methods are required for the detection and enumeration of potentially injured E. coli O157 in food in the presence of outnumbering competing bacteria. Selective agents can prevent or inhibit the recovery and subsequent multiplication of injured cells and direct inoculation, either into selective enrichment broths or onto selective agar plates is still used in many methods for E. coli O157 detection and enumeration. When compared with tryptone soya agar (TSA), sorbitol MacConkey agar (SMAC) was shown to underestimate the concentration of viable E. coli O157:H7 subjected to low pH and high NaCl concentration. Using a resuscitation stage on TSA followed by membrane transfer to SMAC improved recovery to levels obtained on TSA. The membrane method was used to monitor the numbers of artificially contaminated E. coli O157:H7 during the fermentation of a meat product and demonstrated better survival when compared to counts on SMAC. Six rapid methods for the detection of E. coli O157 in food (BAX E. coli O157, Reveal 8 E. coli O157-H7 screening test, VIP EHEC, VIDAS E. coli O157 (ECO), EHEC-Tek and Tecra E. coli O157 visual immunoassay), were evaluated using beetburgers, parsley and fermented meat artificially contaminated with injured cells. Methods using direct selective enrichment, with or without an elevated incubation temperature gave false-negative results. The incorporation of a non-selective pre-enrichment medium improved the detection rates of these assays by up to ten fold.     

Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction

A method for quantitative detection of Escherichia coli O157:H7 based on the polymerase chain reaction (PCR) was developed. The method used the NIH Image 1·61 software program to quantitatively analyse the intensity of the fluorescent image of the amplified PCR product. Based on the PCR with SLT1 and SLT2 primers used separately, a log-linear relationship between the numbers of cfu of E. coli O157:H7 inoculated into ground beef and the intensity of the PCR products was achieved with and without enrichment. Without enrichment, 150 cfu of E. coli O157:H7 per gram of ground beef were detected. In contrast, the detection limit decreased to 1·2 cfu g⁻¹ of ground beef using SLT1 and SLT2 primers after 4·5 h of enrichment using modified EC broth with 20 μg ml⁻¹ of novobiocin.     

Evaluation of Escherichia coli O157:H7 growth media for use in test-and-hold procedures for ground beef processing

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 degrees 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.     

Efficacy of enrichment broths in the recovery of freeze-injured Escherichia coli O157:H7 in inoculated ground beef by PCR

Escherichia coli O157:H7 strains ATCC 35150 and ATCC 43894 and five pooled isolates from beef and pork freeze injured at -25 degrees 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 degrees 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 degrees 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 degrees 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.     

QCM immunosensor with nanoparticle amplification for detection of Escherichia coli O157:H7

A quartz crystal microbalance (QCM) immunosensor was developed and evaluated for detection of Escherichia coli O157:H7. The immunosensor was fabricated by self-assembling of protein A and affinity-purified anti-E. coli O157:H7 antibodies on the gold electrode of an AT-cut piezoelectric quartz crystal. To enhance the sensitivity of the QCM immunosensor, nanoparticle-antibody conjugates, which were prepared using streptavidin-conjugated nanoparticles (145 nm diameter) and biotinylated anti-E. coli antibodies, were used for signal amplification. After the binding of E. coli O157:H7 cells with the antibodies immobilized on the electrode, nanoparticle-antibody conjugates were introduced as mass amplifiers. Compared to the direct detection of E. coli O157:H7, the binding of the nanoparticle conjugates further resulted in a decrease in resonant frequency and an increase in resonant resistance, and the detection sensitivity was improved by five orders of magnitude by lowering the detection limit from 10⁷ to 10² CFU/mL. The sensor specificity and nonspecific adsorption of nanoparticle-antibody conjugates were also investigated.     

Magnetic nanoparticle-antibody conjugates for the separation of Escherichia coli O157:H7 in ground beef

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 x 10(1) to 7.2 x 10(7) 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 x 10(7) to 8.0 x 10(0) 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 x 10(0) and 8.0 x 10(1) 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 > 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.     

Monitoring Escherichia coli O157:H7 in inoculated and naturally colonized feedlot cattle and their environment

On-farm methods of monitoring Escherichia coli O157:H7 were assessed in 30 experimentally inoculated steers housed in four pens over a 12-week period and in 202,878 naturally colonized feedlot cattle housed in 1,160 pens on four commercial Alberta feedlots over a 1-year period. In the challenge study, yearling steers were experimentally inoculated with 10(10) CFU of a four-strain mixture of nalidixic acid-resistant E. coli O157:H7. After inoculation, shedding of E. coli O157:H7 was monitored weekly by collecting rectal fecal samples (FEC), oral swabs (ORL), pooled fecal pats (PAT), manila ropes (ROP) orally accessed for 4 h, feed samples, water, and water bowl interface. Collection of FEC from all animals per pen provided superior isolation (P < 0.01) of E. coli O157:H7 compared with other methods, although labor and animal restraint requirements for fecal sample collection were high. When one sample was collected per pen of animals, E. coli O157:H7 was more likely to be detected from the ROP than from the FEC, PAT, or ORL (P < 0.001). In the commercial feedlot study, samples were limited to ROP and PAT, and E. coli O157:H7 was isolated in 18.8% of PAT and 6.8% of ROP samples. However, for animals that had been resident in the feedlot pen for at least 1 month, isolation of E. coli O157:H7 from ROP was not different from that from PAT (P = 0.35). Pens of animals on feed for <30 days were six times more likely to shed E. coli O157:H7 than were animals on feed for >30 days. However, change in diet did not affect shedding of the organism (P > 0.23) provided that animals had acclimated to the feedlot for 1 month or longer. Findings from this study indicate the importance of introduction of mitigation strategies early in the feeding period to reduce transference and the degree to which E. coli O157:H7 is shed into the environment.