Viability of Escherichia coli O157:H7 during manufacturing and storage of a fermented,semidry soudjouk-style sausage

Soudjouk-style batter was inoculated with a five-strain mixture of Escherichia coli O157:H7 at about 7.6 log10 CFU/g in each of two trials. The sticks were fermented and dried at 22 degrees C and 50% relative humidity (RH) for 3 days and then at 9 degrees C and 40% RH for 18 h. After being flattened to about 1.25 cm, the sticks were conditioned at 38 degrees C and 70% RH or at 22 degrees C and 50% RH for about 3 days. After the latter conditioning treatment, sticks either were cooked to an internal temperature of 63 degrees C or received no heat treatment. Final mean pH values after conditioning at 22 degrees C and 50% RH for soudjouk manufactured with a starter culture and dextrose (1.0%) and for soudjouk manufactured without a starter culture were about 4.9 and 6.0, respectively. For soudjouk produced with a starter culture, pathogen numbers were reduced by 4.53 and 0.88 log10 CFU/g after conditioning at 38 degrees C and 70% RH and at 22 degrees C and 50% RH, respectively. For soudjouk produced via natural fermentation, pathogen numbers were reduced by 1.39 and 0.09 log10 CFU/g after conditioning at 38 degrees C and 70% RH and at 22 degrees C and 50% RH, respectively. Cooking reduced pathogen numbers to below the levels detectable by direct plating (<1.0 log10 CFU/g) and by enrichment for soudjouk produced with a starter culture and also reduced pathogen numbers by 6.28 log10 CFU/g for soudjouk produced via natural fermentation. However, cooking also resulted in an unacceptable product. In general, the reduction in pathogen numbers achieved by storage at ambient temperature (25 degrees C) was greater than that achieved by storage at cooler temperatures (4 and 15 degrees C), particularly for soudjouk prepared with a starter culture (for which a final pH value of 4.8 and a 6.4-log10 reduction were obtained after 21 days at 25 degrees C) rather than for that prepared without a starter culture (for which a final pH value of 6.1 and a 2.6-log10 reduction were obtained after 21 days at 25 degrees C). These results indicate that naturally fermented old-country-type sausage may allow the survival of E. coli O157:H7 in the absence of controlled fermentation, postfermentation cooking, and/or an ambient-storage processing step.     

Modeling the survival of Escherichia coli O157:H7 in uncooked, semidry, fermented sausage

The data collected from studies to monitor inactivation of Escherichia coli O157:H7 in uncooked fermented salami were used to develop models to describe survival of the organism. Three models were developed that included different variables to best describe E. coli O157:H7 reduction. Model A included the variables water activity (a(w)), pH, time, and quadratic variables pH and time. Model B separated the processing stages into fermentation and drying. The fermentation included the variables pH and temperature x time (ttarea) and interaction between the two variables. The drying stage was modeled using the variables time and a(w) and interaction between the two. Model C looked at variables a(w) and the time at pH 5.3 to achieve a 2-D log reduction of E. coli O157:H7 and the interaction between the variables. The variables selected for inclusion in all the models were significant at the P < 0.0001 level. The predicted values for all models correlated well to the observed values with R2 of 0.888, 0.828, 0.836, and 0.818 for models A. Bferm, Bdrying, and C, respectively. The models were validated using data from a trial not used to develop the model. In general the predicted reduction in E. coli O157:H7 count in uncooked fermented salami was greater than for the observed E. coli O157:H7 log reductions for all models, but the relation between the two was linear. The results demonstrate that modeling can be a useful tool in assessing manufacturing practices for uncooked fermented salami processes.     

Frequency of Escherichia coli O157:H7 in Turkish cattle

In this study, five abattoirs in Istanbul were visited between January 2000 and April 2001. During these visits, 330 cattle were selected by a systematic sampling method. Cattle were examined clinically and breed, age, and sex were recorded. Rectal swabs were taken immediately after slaughter. Immunomagnetic separation was performed, and sorbitol-negative colonies were selected on sorbitol MacConkey agar with cefixime and tellurite (CT-SMAC agar). These colonies were checked for 4-methylenebelliferyl-beta-D-glucuronide, indol, rhamnose, and urease activity and motility. Serotypes of bacteria were determined by using antisera specific for Escherichia coli O157 and H7. All cattle selected were clinically healthy. Of 88 sorbitol-negative colonies selected on CT-SMAC agar, isolates from only 14 (4.2%) cattle reacted with anti-O157, and 13 of these isolates also reacted with anti-H7. E. coli O157:H7 was isolated from all breeds, but the numbers of isolates were largest for Holstein and Swiss Brown cows. E. coli O157:H7 was most frequently isolated from 2-year-old cattle. Similarly, it was most frequently isolated from male cattle. E. coli O157:H7 was isolated from cattle slaughtered in four of the five abattoirs studied.     

A note on Escherichia coli O157:H7 serotype in Turkish meat products

255 minced beef, 101 soudjouk and 50 uncooked hamburger samples were analyzed for the presence of Escherichia coli O157:H7 serotype. m-EC and LST broths were used as selective enrichment media and SMAC agar was used as a selective isolation medium. A total of 3 E. coli O157 were isolated by conventional culture techniques; one from each of minced beef, uncooked hamburger and soudjouk but none were identified as the H7 serotype. For determination of selective media-growing cohabitant bacteria, 2645 isolates were obtained from SMAC agar. Results showed that E. coli type 1, Hafnia alvei and Citrobacter freundii were dominant competitive flora. As selective enrichment broth-growing cohabitant microflora existed at higher levels, it was too difficult to isolate E. coli O157 from these mixed flora. Therefore, conventional methods are not suitable for these types of products, because of isolation difficulties and failure to confirm.     

Development of a dose-response relationship for Escherichia coli O157:H7

E. coli O157:H7 is an emerging food and waterborne pathogen. The development of acceptable guidelines for exposure to this organism based on quantitative microbial risk assessment requires a dose response curve. In this study, a prior animal study was used to develop a dose response relationship. The data was adequately fit by the beta-Poisson dose response relationship. This relationship was validated with reference to two outbreaks of this organism. It was found that the low dose extrapolation of the animal data using the beta-Poisson relationship provided estimates of risk concordant with those noted in the outbreaks. The fitted dose response relationship in conjunction with population estimates of the prevalence of E. coli O157:H7 illness indicates that the overall exposure is quite low in the US.     

Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir

The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P<0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment.     

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.     

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.     

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.     

Experimental use of a gas sensor-based instrument for differentiation of Escherichia coli O157:H7 from non-O157:H7 Escherichia coli field isolates

The rapid and economical detection of human pathogens in animal and food production systems would enhance food safety efforts. An instrument based on gas sensors coupled with an artificial neural network (ANN) was developed for the detection of and differentiation between laboratory isolates of Escherichia coli O157:H7 and non-O157:H7 E. coli. The purpose of this study was to use field isolates of E. coli to further evaluate the sensor system. This gas sensor-based, computer-controlled detection system was used to monitor gas emissions from 12 isolates of E. coli O157:H7 and 8 non-O157:H7 E. coli isolates. A standard concentration of each isolate was grown in 10 ml of nutrient broth at 37 degrees C for 16 h, and gas sampling was carried out every 5 min. Readings were continuously plotted to generate gas signatures. A back-propagation ANN algorithm was used to interpret the gas patterns. By analysis of the response of the ANN, the sensitivity and specificity of the instrument were calculated. Detectable differences between the gas signatures of the E. coli O157:H7 isolates and the non-O157:H7 isolates were observed. The instruments degree of sensitivity was high for E. coli O157:H7 isolates, but a lower degree of accuracy was observed for non-O157:H7 isolates because of increased strain variation. The sensitivity of the detection system was improved by the normalization of the data generated from the gas sensors. Because of its ability to detect differences in gas patterns, this instrument has a broad range of potential food safety applications.     

Development of a medium for differentiation between Escherichia coli and Escherichia coli O157:H7.

A new medium (Escherichia coli O157:H7 medium: EOH) was developed for differentiation between E. coli and E. coli O157:H7. The EOH medium was compared with sorbitol MacConkey agar (SMAC), which is the most popular medium to enumerate E. coli O157:H7. Several combinations of 35 dyes were evaluated to develop the new medium. Indigo carmine (0.03) g/liter) and phenol red (0.036 g/liter) were found as the best combination for differentiation between E. coli O157:H7 and E. coli and added to the basal agar medium (SMAC medium excluding neutral red and crystal violet) for EOH medium. On the dark blue EOH medium, E. coli produced a yellow color with clear zone, whereas E. coli O157:H7 produced a red color without clear zone. For differentiation between E. coli and E. coli O157:H7, EOH has much better potential than SMAC. Furthermore. the red color produced by normal E. coli in SMAC may mask the light gray color produced by E. coli O157: H7, whereas the yellow color with clear zone did not mask the red color without clear zone in the EOH medium. The recovery numbers of E. coli O157:H7 from inoculated ground beef, pork, and turkey were not significantly different between SMAC and EOH media (P > 0.05). The recovery rates of heat- and cold-injured E. coli O157:H7 also were not significantly different (P > 0.05).     

Cross-contamination of lettuce with Escherichia coli O157:H7

Contamination of produce by bacterial pathogens is an increasingly recognized problem. In March 1999, 72 patrons of a Nebraska restaurant were infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7, and shredded iceberg lettuce was implicated as the food source. We simulated the restaurant's lettuce preparation procedure to determine the extent of possible EHEC cross-contamination and growth during handling. EHEC inoculation experiments were conducted to simulate the restaurant's cutting procedure and the subsequent storage of shredded lettuce in water in the refrigerator. All lettuce pieces were contaminated after 24 h of storage in inoculated water (2 x 10(9) CFU of EHEC per 3 liters of water) at room temperature or at 4 degrees C; EHEC levels associated with lettuce increased by > 1.5 logs on the second day of storage at 4 degrees C. All lettuce pieces were contaminated after 24 h of storage in water containing one inoculated lettuce piece (approximately 10(5) CFU of EHEC per lettuce piece) at both temperatures. The mixing of one inoculated dry lettuce piece with a large volume of dry lettuce, followed by storage at 4 degrees C or 25 degrees C for 20 h resulted in 100% contamination of the leaves tested. Microcolonies were observed on lettuce stored at 25 degrees C, while only single cells were seen on leaves stored at 4 degrees C, suggesting that bacterial growth had occurred at room temperature. Three water washes did not significantly decrease the number of contaminated leaves. Washing with 2,000 mg of calcium hypochlorite per liter significantly reduced the number of contaminated pieces but did not eliminate contamination on large numbers of leaves. Temperature abuse during storage at 25 degrees C for 20 h decreased the effectiveness of the calcium hypochlorite treatment, most likely because of bacterial growth during the storage period. These data indicate that storage of cut lettuce in water is not advisable and that strict attention must be paid to temperature control during the storage of cut lettuce.     

Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.     

Inhibition of Escherichia coli O157:H7 in ripening dry fermented sausage by ground yellow mustard

Compounds generated by the enzymatic hydrolysis of glucosinolates naturally present in mustard powder are potently bactericidal against Escherichia coli O157:H7. Because E. coli O157:H7 can survive the dry fermented sausage manufacturing process, 2, 4, and 6% (wt/wt) nondeheated (hot) mustard powder or 6% (wt/wt) deheated (cold) mustard powder were added to dry sausage batter inoculated with E. coli O157:H7 at about 7 log CFU/g to evaluate the antimicrobial effectiveness of the powders. Reductions in E. coli O157:H7 populations, changes in pH and water activity (aw), effects on starter culture (Pediococcus pentosaceus and Staphylococcus carnosus) populations, and effects of mustard powder on sausage texture (shear) were monitored during ripening. Nondeheated mustard powder at 2, 4, and 6% in dry sausage (0.90 aw) resulted in significant reductions in E. coli O157:H7 (P < 0.05) of 3.4, 4.4, and 6.9 log CFU/g, respectively, within 30 days of drying. During fermentation and drying, mustard powder did not affect P. pentosaceus and S. carnosus activity in any of the treatments. Extension of drying to 36 and 48 days reduced E. coli O157:H7 by >5 log CFU/g in the 4 and 2% mustard powder treatments, respectively. The 6% deheated mustard powder treatment provided the most rapid reductions of E. coli O157:H7 (yielding <0.20 log CFU/g after 24 days) by an unknown mechanism and was the least detrimental (P < 0.05) to sausage texture.     

Survival of Escherichia coli O157:H7 in dry sausage fermented by probiotic lactic acid bacteria

Probiotic Lactobacillus rhamnosus GG, L rhamnosus E‐97800, L rhamnosus LC‐705 and commercial Pediococcus pentosaceus were studied for their ability to inhibit the growth of Escherichia coli O157:H7 in dry sausage. The strains were able to produce technologically high‐quality dry sausage. The number of E coli O157:H7 decreased from approximately 5 to approximately 2 log cfu g⁻¹ It was concluded that the above‐mentioned strains and the commercial culture were equally effective in inhibiting E coli O157:H7. © 2000 Society of Chemical Industry     

Predicting survival of Escherichia coli O157:H7 in dry fermented sausage using artificial neural networks

The Canadian Food Inspection Agency required the meat industry to ensure Escherichia coli O157:H7 does not survive (experiences > or = 5 log CFU/g reduction) in dry fermented sausage (salami) during processing after a series of foodborne illness outbreaks resulting from this pathogenic bacterium occurred. The industry is in need of an effective technique like predictive modeling for estimating bacterial viability, because traditional microbiological enumeration is a time-consuming and laborious method. The accuracy and speed of artificial neural networks (ANNs) for this purpose is an attractive alternative (developed from predictive microbiology), especially for on-line processing in industry. Data from a study of interactive effects of different levels of pH, water activity, and the concentrations of allyl isothiocyanate at various times during sausage manufacture in reducing numbers of E. coli O157:H7 were collected. Data were used to develop predictive models using a general regression neural network (GRNN), a form of ANN, and a statistical linear polynomial regression technique. Both models were compared for their predictive error, using various statistical indices. GRNN predictions for training and test data sets had less serious errors when compared with the statistical model predictions. GRNN models were better and slightly better for training and test sets, respectively, than was the statistical model. Also, GRNN accurately predicted the level of allyl isothiocyanate required, ensuring a 5-log reduction, when an appropriate production set was created by interpolation. Because they are simple to generate, fast, and accurate, ANN models may be of value for industrial use in dry fermented sausage manufacture to reduce the hazard associated with E. coli O157:H7 in fresh beef and permit production of consistently safe products from this raw material.     

Behavior of Escherichia coli O157:H7 in leafy vegetables

Leafy vegetables, including lettuce and spinach, have been implicated in several outbreaks of foodborne disease caused by Escherichia coli O157:H7, a pathogen of increasing public health significance because of the severity of the gastrointestinal illness and long-term, chronic sequelae that can result from infection. A definitive association between the consumption of leafy vegetables and human disease provides implicit evidence of transfer from animal sources to field crops and retail commodities, including minimally processed or fresh-cut products. Understanding the behavior of E. coli O157:H7 in leafy vegetables during production, after harvest, in storage, during processing, and in packaged fresh-cut products is essential for the development of effective control measures. To this end, previous research on the fate of the species at each step in the production of market-ready leafy vegetables is reviewed in this study. Several critical gaps in knowledge are identified, notably uncertainty about the location of contaminating cells on or in plant tissues, behavior in packaged products stored at low temperatures, and the influence of environmental stresses on growth and infectivity.     

Fate of Escherichia coli O157:H7 during silage fermentation

The survival characteristics of Escherichia coli O157:H7 in silage derived from contaminated grass were investigated. The survival of other enteric bacteria was also investigated to determine if E. coli O157:H7 demonstrates enhanced acid tolerance in comparison. Samples of chopped grass were treated as follows: (i) no additive (control); (ii) inoculation with E. coli O157:H7 to a final concentration of log10 4.0 CFU g(-1); (iii) addition of an 85% solution of formic acid at 3.0 ml kg(-1) grass; and (iv) addition of both E. coli O157:H7 and formic acid, at the above concentrations. Treated 6-kg grass samples were packed into laboratory silos, sealed, and stored at 15 degrees C for up to 180 days. Individual replicate silos were removed from storage periodically and subjected to microbiological and chemical analyses. Chemical analyses of the silage samples indicated that lactic acid-dominant fermentations, with a rapid drop in pH, occurred. Numbers of enteric bacteria decreased from log10 7.0 to 8.0 CFU g(-1) to undetectable levels within 19 days' storage. E. coli O157:H7 did not survive the silage fermentation process, with numbers declining from approximately log10 4.0 CFU g(-1) to undetectable levels within 19 days of ensiling. The pattern of decline in numbers of E. coli O157:H7 was the same as that for the enteric bacteria, indicating that under the conditions tested, the acid tolerance of E. coli O157:H7 was not significantly different from the acid tolerance of other enteric bacteria. This study found that E. coli O157:H7 did not survive a good silage fermentation process, indicating that properly ensiled grass that is correctly stored is unlikely to be a vector for the transmission of the pathogen among cattle.