Outbreak of Escherichia coli O157:H7 infections associated with nonintact blade-tenderized frozen steaks sold by door-to-door vendors

Steaks have not been recognized as an important vehicle of Escherichia coli O157:H7 infection. During 11 to 27 June 2003, the Minnesota Department of Health (MDH) identified four O157 infection cases with the same pulsed-field gel electrophoresis (PFGE) subtype. All four case patients consumed brand A vacuum packed frozen steaks sold by door-to-door vendors. The steaks were blade tenderized and injected with marinade (i.e., nonintact). Information from single case patients in Michigan and Kansas identified through PulseNet confirmed the outbreak. The MDH issued a press release on 27 June to warn consumers, prompting a nationwide recall of 739,000 lb (335,506 kg) of frozen beef products. The outbreak resulted in six culture-confirmed cases (including one with hemolytic uremic syndrome) and two probable cases in Minnesota and single confirmed cases in four other states. The outbreak PFGE subtype of O157 was isolated from unopened brand A bacon-wrapped fillets from five affected Minnesota households. A fillet from one affected household was partially cooked in the laboratory, and the same O157 subtype was isolated from the uncooked interior. The tenderizing and injection processes likely transferred O157 from the surface to the interior of the steaks. These processing methods create new challenges for prevention of O157 infection. Food regulatory officials should reevaluate safety issues presented by nonintact steak products, such as microbiologic hazards of processing methods, possible labeling to distinguish intact from nonintact steaks, and education of the public and commercial food establishments on the increased risk associated with undercooked nonintact steaks. Information on single cases of O157 infection in individual states identified through PulseNet can be critical in solving multistate outbreaks in a timely manner.     

Ozone treatment for reduction of Escherichia coli 0157:H7 and Salmonella serotype typhimurium on beef carcass surfaces

The effectiveness of an aqueous ozone treatment in reducing Escherichia coli O157:H7 and Salmonella serotype Typhimurium on hot carcass surfaces was determined with the use of a model carcass spray cabinet. Carcass surface regions were removed from carcasses and inoculated with feces containing 10(6) to 10(7) CFU each of E. coli O157:H7 and Salmonella Typhimurium per g and were then exposed to a water wash or to a water wash followed by a sanitizing ozone treatment. Water washes were applied at 28 degrees C beginning at a pressure of 10 lb/in2 and gradually increasing to 400 lb/in2. Ozone treatment was carried out by spraying surfaces with an aqueous ozone solution (80 lb/in2 at 28 degrees C) containing 95 mg of ozone per liter. Pathogen reductions achieved with ozone treatment were not significantly different from those achieved with a water wash alone. In addition, ozone treatment did not reduce E. coli O157:H7 or Salmonella Typhimurium contamination that was spread over the carcass surface as a result of the water wash. Under the conditions of this study, the aqueous ozone treatment applied resulted in no significant improvement over a water wash in reducing pathogens on beef carcass surfaces.     

食品中大肠杆菌0157: 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0157: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0157∶H7.     

Bacteriophage-based bioluminescent bioreporter for the detection of Escherichia coli 0157:H7

The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli 0157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli 0157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP0-luxl to detect several strains of E. coli 0157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PPOI-luxl and E. coli 0157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within I h when exposed to 10(4) CFU/ml of E. coli 0157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 10(4) CFU/ml of E. coli 0157:H7 in 2 h after a 6-h preincubation.     

Cryotolerance of Escherichia coli 0157:H7 in Laboratory Media and Food

ABSTRACT: The present study compared the cryotolerance of E. coli 0157:H7 strains with nonpathogenic strains of E. coli. Cold shocked (exposed to 10°C for 6 h) and non-cold shocked (held at 20°C) cultures were frozen at -18°C for up to 240 h in brain heart infusion broth, apple juice, frozen yogurt, and ground beef. The E. coli 0157:H7 strains showed the greatest cold shock effect and cryotolerance. The cold shocked E. coli 0157:H7 strains showed a 25 to 35% increase in their ability to survive frozen storage for 24 h at -18°C compared to non-cold shocked cells. The corresponding value for non-O157 strains was only about 5%. The food matrix changed the cold shock response in all investigated strains. The largest cold shock effect was observed with broth cultures. Cryotolerance of E. coli was not observed in frozen yogurt and ground beef. The effect of prior cold shock was most pronounced in E. coli 0157:H7 strains after 24 h of freezing.     

Enzyme-linked immunomagnetic electrochemical detection of live Escherichia coli 0157:H7 in apple juice

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 x 10(3) cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.     

失活/存活模型在大肠杆菌0157:H7风险分析中的应用

目的 研究出口分割鸡肉中大肠杆菌0157:H7的控制措施.方法 建立了出口分割鸡肉中大肠杆菌0157:H7在速冻过程中的失活模型和在-18℃下的存活模型,通过风险分析提出控制措施.结果 利用所建模型定量描述了出口分割鸡肉从成品到消费过程中大肠杆菌0157:H7带菌量的变化,得出摄入1份鸡肉感染大肠杆菌0157:H7病的风险.结论 本研究提出的控制措施可提高出口分割鸡肉的安全性.     

失活/存活模型在大肠杆菌0157:H7风险分析中的应用

目的 研究出口分割鸡肉中大肠杆菌0157:H7的控制措施.方法 建立了出口分割鸡肉中大肠杆菌0157:H7在速冻过程中的失活模型和在-18℃下的存活模型,通过风险分析提出控制措施.结果 利用所建模型定量描述了出口分割鸡肉从成品到消费过程中大肠杆菌0157:H7带菌量的变化,得出摄入1份鸡肉感染大肠杆菌0157:H7病的风险.结论 本研究提出的控制措施可提高出口分割鸡肉的安全性.     

Escherichia coli O157:H7 shedding in vaccinated beef calves born to cows vaccinated prepartum with Escherichia coli O157:H7 SRP vaccine

Extensive research, intervention equipment, money, and media coverage have been directed at controlling Escherichia coli O157:H7 in beef cattle. However, much of the focus has been on controlling this pathogen postcolonization. This study was conducted to examine the performance, health, and shedding characteristics of beef calves that were vaccinated with an E. coli O157:H7 SRP bacterial extract. These calves had been born to cows vaccinated prepartum with the same vaccine. Cows and calves were assigned randomly to one of four treatments: (i) neither cows nor calves vaccinated with E. coli O157:H7 SRP (CON), (ii) cows vaccinated with E. coli O157:H7 SRP prepartum but calves not vaccinated (COWVAC), (iii) calves vaccinated with E. coli O157:H7 SRP but born to cows not vaccinated (CALFVAC), (iv) cows vaccinated with E. coli O157:H7 SRP prepartum and calves also vaccinated (BOTH). Calves born to vaccinated cows had significantly higher titers of anti-E. coli O157:H7 SRP antibodies (SRPAb) in circulation at branding time (P < 0.001). Upon entry to the feedlot, overall fecal E. coli O157:H7 prevalence was 23 % among calves, with 25 % in the CON treatment group, 19 % in the CALFVAC group, 32 % in the COWVAC group, and 15 % in the BOTH group (P > 0.05). Fecal shedding of E. coli O157 on arrival to the feedlot was not correlated with fecal shedding at slaughter (Spearman's rho = -0.02; P = 0.91). No significant effects of cow or calf E. coli O157:H7 SRP vaccination treatment were found on feedlot calf health or performance (P > 0.05), prevalence of lung lesions or liver abscess (P > 0.05), or morbidity, retreatment, or mortality numbers (P > 0.05). The findings of this study indicate that the timing of vaccination of calves against E. coli O157:H7 may be an important consideration for maximizing the field efficacy of this vaccine.     

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.     

Seasonal prevalence of Escherichia coli O157:H7 in beef cattle feces

Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.     

Rapid and simultaneous quantitation of Escherichia coli 0157:H7, Salmonella, and Shigella in ground beef by multiplex real-time PCR and immunomagnetic separation

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 10(2) to 10(9) CFU/ml for E. coli O157:H7, 10(3) to 10(9) CFU/ml for Salmonella, and 10(1) to 10(8) CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10(5) CFU/g for E. coli O157:H7, 10(3) CFU/g for Salmonella, and 10(4) 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 10(3) 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 0157:H7, Salmonella, and Shigella in food.     

Temperature and NaCl Affect Growth and Survival of Escherichia coli 0157:H7 in Poultry-Based and Laboratory Media

In tryptic soy broth (TSB) and a poultry extract broth (PB) with 0 to 10% (w/v) NaCl incubated at 37°C, growth of E. coli 0157:H7 was inhibited at 28% NaCl whereas at 10°C, growth was inhibited at 24% NaCl in TSB and at 26% NaCl in PB. The bacterium did not grow at 4°C. Increased NaCl-sensitivity observed at 10°C was a bacteriostatic effect that was ineffective with increasing incubation temperature. At 10°C, E. coli 0157:H7 was more salt-tolerant in PB than in TSB, although PB growth rates were lower. Findings suggest that PB may be a more suitable medium for testing E. coli 0157:H7 in poultry products. Cells of E. coli 0157:H7 that were exposed to refrigeration (4°C) and/or NaCl for 24 days did not grow on MacConkey agar with 1% sorbitol.     

Thermal inactivation of Escherichia coli O157:H7 in ground beef supplemented with sodium lactate

A study was conducted to investigate the antimicrobial effect of sodium lactate (NaL) (0, 1.5, 3.0, and 4.5%) on the survival of Escherichia coli O157:H7 in 93% lean ground beef. Samples inoculated with a mixture of four strains of E. coli O157:H7 (10(7) to 10(8) CFU/g) were subjected to immersion heating in a water bath stabilized at 55, 57.5, 60, 62.5, or 65 degrees C. Results of statistical analysis indicated that the heating temperature was the only factor affecting the decimal reduction times (D-values) of E. coli O157:H7 in 93% lean ground beef. The change in temperature required to change the D-value (the z-value) was determined as 7.6 degrees C. The thermal resistance of this organism was neither affected by the addition of NaL nor by the interactions between NaL and temperature. Adding NaL to ground beef to reduce the thermal resistance of E. coli O157:H7 is therefore not recommended.     

Evaluating lethality of beef roast cooking treatments against Escherichia coli O157:H7

Added salt, seasonings, and phosphates, along with slow- and/or low-temperature cooking impart desirable characteristics to whole-muscle beef, but might enhance Escherichia coli O157:H7 survival. We investigated the effects of added salt, seasoning, and phosphates on E. coli O157:H7 thermotolerance in ground beef, compared E. coli O157:H7 thermotolerance in seasoned roasts and ground beef, and evaluated ground beef-derived D- and z-values for predicting destruction of E. coli O157:H7 in whole-muscle beef cooking. Inoculated seasoned and unseasoned ground beef was heated at constant temperatures of 54.4, 60.0, and 65.5°C to determine D- and z-values, and E. coli O157:H7 survival was monitored in seasoned ground beef during simulated slow cooking. Inoculated, seasoned whole-muscle beef roasts were slow cooked in a commercial smokehouse, and experimentally determined lethality was compared with predicted process lethality. Adding 5% seasoning significantly decreased E. coli O157:H7 thermotolerance in ground beef at 54.4°C, but not at 60 or 65.5°C. Under nonisothermal conditions, E. coli O157:H7 thermotolerance was greater in seasoned whole-muscle beef than in seasoned ground beef. Meeting U.S. Government (U.S. Department of Agriculture, Food Safety and Inspection Service, 1999, Appendix A) whole-muscle beef cooking guidance, which targets Salmonella destruction, would not ensure ≥6.5-log CFU/g reduction of E. coli O157:H7 in ground beef systems, but generally ensured $ 6.5-log CFU/g reduction of this pathogen in seasoned whole-muscle beef. Calculations based on D- and z-values obtained from isothermal ground beef studies increasingly overestimated destruction of E. coli O157:H7 in commercially cooked whole-muscle beef as process severity increased, with a regression line equation of observed reduction = 0.299 (predicted reduction) + 1.4373.     

Process control and sampling for Escherichia coli O157:H7 in beef trimmings

Raw beef producers currently face the problem of Escherichia coli O157:H7 surface contamination of beef carcasses that can lead to product adulteration. Although carcass interventions are in place, elimination of E. coli O157:H7 from every potential hiding place on the surfaces of a beef carcass is not technologically feasible. Therefore, E. coli O157:H7 on beef carcasses might further contaminate the surfaces of beef trimmings. With the use of case scenarios from nine commercial processing facilities, we present a process control and statistical sampling approach for monitoring beef trimmings to divert contaminated lots of the trimmings from the raw ground beef supply chain.     

Antimicrobial effect of herb extracts against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella typhimurium associated with beef

The effects of plant extracts against pathogenic bacteria in vitro are well known, yet few studies have addressed the effects of these compounds against pathogens associated with muscle foods. A series of experiments was conducted to determine the effectiveness of a commercially available, generally recognized as safe, herb extract dispersed in sodium citrate (Protecta One) or sodium chloride (Protecta Two) against Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes associated with beef. In the first experiment, E. coli O157:H7, Salmonella typhimurium, and L. monocytogenes inoculated onto beef and subjected to surface spray treatments with 2.5% solutions of Protecta One or Protecta Two were not affected by immediate application (day 0) of the herbal extracts. However, after 7 days of storage at 4 degrees C, E. coli O157:H7 was reduced by >1.3 log10 CFU/cm2 by Protecta Two; L. monocytogenes was reduced by 1.8 and 1.9 log10 CFU/cm2 by Protecta One and Protecta Two, respectively; Salmonella typhimurium was not reduced >0.3 log10 CFU/cm2 by either extract by day 7. In the second experiment, 2.5% Protecta Two (wt/vol or wt/wt) added to inoculated lean and adipose beef trim, processed, and packaged as ground beef chubs (80% lean, 20% adipose), did not reduce pathogen populations >0.5 log10 CFU/g up to 14 days at 4 degrees C. In the third experiment, surface spray treatments of beef with 2.5% lactic acid or 2.5% solutions of Protecta One or Protecta Two, vacuum packaged, and stored up to 35 days at 4 degrees C did reduce E. coli O157:H7, L. monocytogenes, and Salmonella Typhimurium slightly. These studies suggest that the use of herb extracts may afford some reductions of pathogens on beef surfaces; however, the antimicrobial activity may be diminished in ground beef by adipose components.     

Viability of acid-adapted Escherichia coli O157:H7 in ground beef treated with acidic calcium sulfate

The effects of lactic acid, acetic acid, and acidic calcium sulfate (ACS) on viability and subsequent acid tolerance of three strains of Escherichia coli O157:H7 were determined. Differences in tolerance to acidic environments were observed among strains, but the level of tolerance was not affected by the acidulant to which cells had been exposed. Cells of E. coli O157:H7 adapted to grow on tryptic soy agar acidified to pH 4.5 with ACS were compared to cells grown at pH 7.2 in the absence of ACS for their ability to survive after inoculation into ground beef treated with ACS, as well as untreated beef. The number of ACS-adapted cells recovered from ACS-treated beef was significantly (alpha = 0.05) higher than the number of control cells recovered from ACS-treated beef during the first 3 days of a 10-day storage period at 4 degrees C, suggesting that ACS-adapted cells might be initially more tolerant than unadapted cells to reduced pH in ACS-treated beef. Regardless of treatment of ground beef with ACS or adaptation of E. coli O157:H7 to ACS before inoculating ground beef, the pathogen survived in high numbers.