Fate of Escherichia coli O157:H7 During Production of Snack Sticks Made from Beef or a Venison/Beef Fat Blend and Directly Acidified with Citric or Lactic Acid

ABSTRACT:  The effectiveness of a typical production process for eliminating Escherichia coli O157:H7 in directly acidified snack sticks made with beef or a venison/beef fat blend was evaluated for formulations of different fat content (10% and 25%) and type of direct acidulant (encapsulated citric or lactic acid). Raw batter inoculated with E. coli O157:H7 to an initial level of approximately 7.1 log CFU/g was stuffed into 21-mm casings and processed according to a thermal-processing schedule typical of those used commercially for directly acidified sausage products (maximum internal product temperature of 68.3 °C, followed by drying and cooling). For both beef and venison/beef fat blend snack sticks, log reductions ranged from 6.2 to > 6.6 CFU/g at the end of processing. Although moderate reductions (1.5 to 2.0 log CFU/g) were achieved as a result of processing to internal product temperatures of 68.3 °C, latter stages of the process (during which products were dried and cooled) factored prominently in the overall lethality of the process, and were essential in achieving the 5-log reduction required by the U.S. Dept. of Agriculture, Food Safety and Inspection Service. The efficacy of the process was not affected ( P ≤ 0.05) by fat content (10% or 25%) or by the type of direct acidulant used (encapsulated citric or lactic acid). Phenol red sorbitol agar was more effective for recovering heat- and acid-injured E. coli O157 than MacConkey sorbitol agar was and, therefore, provided a more conservative estimate of process lethality.     

Thermal inactivation of Escherichia coli O157:H7 in beef treated with marination and tenderization ingredients

Internalization of Escherichia coli O157:H7 in nonintact beef products during mechanical tenderization or during injection of marination and tenderization ingredients is of concern if such products are undercooked. This study tested organic acids (0.2% citric acid and 0.3% acetic acid), potassium and calcium salts (1.8% potassium lactate, 0.63% calcium lactate, 0.86% calcium ascorbate, and 0.23% calcium chloride), and sodium chloride (2.5%) for their influence on thermal destruction of E. coli O157:H7 in ground beef serving as a model system. Ground beef batches (700 g; 5% fat) were mixed with equal volumes (22 ml) of each treatment solution or distilled water and portions (30 g) of treated ground beef were extruded in test tubes (2.5 by 10 cm). A five-strain mixture of E. coli O157:H7 (0.3 ml; 7 log CFU/g) was introduced at the center of the sample with a pipette. After overnight storage (4 degrees C), simulating product marination, samples were heated to 60 or 65 degrees C internal temperature, simulating rare and medium rare doneness of beef, in a circulating water bath. At 65 degrees C, treatments with citric and acetic acid showed greater (P < 0.05) reduction (4 to 5 log CFU/g) of E. coli O157:H7 than all the other ingredients and the control (3 to 4 log CFU/g). Sodium chloride reduced weight losses (16 to 18% compared with 20 to 27% by citric or acetic acid) and resulted in a 4-log reduction in counts during cooking to 65 degrees C. Ingredients such as citric or acetic acid may improve thermal inactivation of E. coli O157:H7 internalized in nonintact beef products, while sodium chloride may reduce cooking losses in such products.     

Bactericidal effects of Lactobacillus reuteri and allyl isothiocyanate on Escherichia coli O157:H7 in refrigerated ground beef

Two naturally occurring antimicrobial agents were tested in packages of refrigerated ground beef for their ability to reduce the viability of Escherichia coli O157:H7 during storage. Allyl isothiocyanate (AITC) and Lactobacillus reuteri were tested separately and together for their action against a cocktail of five strains of E. coli O157:H7 in ground beef held at 4 degrees C for 25 days. Ground beef prepared from whole, raw inside round beef roasts was inoculated with low (3 log CFU/g) or high (6 log CFU/g) levels of the E. coli O157:H7 mixture. The beef was treated with AITC (about 1,300 ppm), L. reuteri, or both, along with 250 mM of glycerol per kg of meat at two levels (3 and 6 log CFU/g) and according to a design that yielded 8 controls plus 10 different treatments. Samples were analyzed for E. coli O157:H7 survivors, numbers of total bacteria, and lactic acid bacteria on days 0 to 25 at 5-day intervals. L. reuteri at both input levels with glycerol killed E. coli O157:H7 at both inoculated levels before day 20. AITC completely eliminated E. coli O157:H7 at the low-inoculum level (3 log CFU/g) and reduced viability >4.5 log CFU/g at the high-inoculum level (6 log CFU/g) by the end of the storage period. The combination of L. reuteri and AITC did not yield an additive effect against E. coli O157:H7 viability. L. reuteri in the presence of glycerol was highly effective against E. coli O157:H7 in ground beef during refrigerated storage (4 degrees C) in modified atmosphere packages. Sensory testing is planned to evaluate effects of treatments.     

Thermal inactivation of acid, cold, heat, starvation, and desiccation stress-adapted Escherichia coli O157:H7 in moisture-enhanced nonintact beef

This study was conducted to compare thermal inactivation of stress-adapted and nonadapted Escherichia coli O157:H7 in nonintact beef moisture enhanced with different brine formulations and cooked to 65°C. Coarsely ground beef was mixed with acid, cold, heat, starvation, or desiccation stress-adapted or nonadapted rifampin-resistant E. coli O157:H7 (eight-strain mixture, 5 to 6 log CFU/g) and a brine solution for a total moisture enhancement level of 10%. The brine treatments included distilled water (control), sodium chloride (0.5% NaCl) plus sodium tripolyphosphate (0.25% STP), or NaCl + STP combined with cetylpyridinium chloride (0.2% CPC), lactic acid (0.3% LA), or sodium metasilicate (0.2% SM). The treated meat was extruded into bags (15 cm diameter), semifrozen (-20°C for 4.5 h), and cut into 2.54-cm (1-in.)-thick portions. Samples were individually vacuum packaged, frozen (-20°C for 42 h), and tempered at 4°C for 2.5 h before cooking. Partially thawed (-1.8 ± 0.4°C) samples were pan broiled to an internal temperature of 65°C. Pathogen counts of partially thawed (before cooking) samples moisture enhanced with brines containing CPC, LA, or SM were 0.7 to 1.1, 0.0 to 0.4, and 0.2 to 0.4 log CFU/g, respectively, lower than those of the control. Compared with microbial count reductions obtained after pan broiling of beef inoculated with nonadapted E. coli O157:H7 cells, count reductions during cooking of meat inoculated with cold and desiccation stress-adapted, acid stress-adapted, and heat and starvation stress-adapted cells indicated sensitization, cross protection, and no effect, respectively, of these stresses on the pathogen during subsequent exposure to heat. Among all stressed cultures, CPC-treated samples (0.8 to 3.6 log CFU/g) and LA-treated samples (0.8 to 3.5 log CFU/g) had the lowest numbers of E. coli O157:H7 survivors after cooking.     

Antibacterial effect of lactoferricin B on Escherichia coli O157:H7 in ground beef.

The antibacterial activity of lactoferricin B on enterohemorrhagic Escherichia coli O157:H7 in 1% peptone medium and ground beef was studied at 4 and 10 degrees C. In 1% peptone medium, 50 and 100 microg of lactoferricin B per ml reduced E. coli O157:H7 populations by approximately 0.7 and 2.0 log CFU/ml, respectively. Studies comparing the antibacterial effect of lactoferricin B on E. coli O157:H7 in 1% peptone at pH 5.5 and 7.2 did not reveal any significant difference (P > 0.5) at the two pH values. Lactoferricin B (100 microg/g) reduced E. coli O157:H7 population in ground beef by about 0.8 log CFU/g (P < 0.05). No significant difference (P > 0.5) was observed in the total plate count between treatment and control ground beef samples stored at 4 and 10 degrees C. The antibacterial effect of lactoferricin B on E. coli O157:H7 observed in this study is not of sufficient magnitude to merit its use in ground beef for controlling the pathogen.     

Inactivation of Escherichia coli O157:H7 in moisture-enhanced nonintact beef by pan-broiling or roasting with various cooking appliances set at different temperatures

This study evaluated inactivation of Escherichia coli O157:H7 in moisture-enhanced restructured nonintact beef cooked to 65 °C using different cooking appliances set at different temperatures. Batches (2 kg) of coarse-ground beef (approximately 5% fat) were mixed with an 8-strain composite (100 mL) of rifampicin-resistant E. coli O157:H7 (6.4 ± 0.1 log CFU/g) and a solution (100 mL) of sodium chloride plus sodium tripolyphosphate to yield concentrations (wt/wt) of 0.5% and 0.25%, respectively, in the final product. Beef portions of 2.54 cm thickness (15 cm dia) were prepared and were vacuum-packaged and frozen (-20 °C, 42 h). Partially thawed (-2.5 ± 1.0 °C) portions were pan-broiled (Presto electric skillet and Sanyo grill) or roasted (Oster toaster oven and Magic Chef kitchen oven) to 65 °C. The appliances were set at, and preheated before cooking to 149 or 204 °C (electric skillet), 149 or 218 °C (grill), 149 or 232 °C (toaster oven), and 149, 204, or 260 °C (kitchen oven). Temperatures of appliances and beef samples were monitored with thermocouples, and meat samples were analyzed for surviving microbial populations. In general, the higher the appliance temperature setting, the shorter the time needed to reach 65 °C, and the higher the edge and surface temperatures of the meat samples. Temperatures of 204 to 260 °C, regardless of appliance, resulted in greater (P < 0.05) pathogen reductions (3.3 to 5.5 log CFU/g) than those obtained at 149 °C (1.5 to 2.4 log CFU/g). The highest (P < 0.05) reduction (5.5 log CFU/g) was obtained in samples cooked in the kitchen oven set at 260 °C. The results should be useful to the food service industry for selection of effective nonintact beef cooking protocols, and for use in risk assessments for nonintact meat products. Practical Application: Results of this study should be useful for developing cooking recommendations to enhance the safety of nonintact beef products, and for use in risk assessments of such products.     

Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation

Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process. A batch culture fermentation was used to produce a maximum biomass of L. reuteri. Then cells were harvested, resuspended in a glycerol-water solution, and anaerobically incubated to produce reuterin. The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials. The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively. In meat decontamination experiments, the surface of cooked pork was inoculated with either L. monocytogenes or E. coli O157:H7 at a level of approximately log10 5 CFU/cm2, incubated for 30 min at 7 degrees C, and decontaminated by exposure to reuterin (500 AU/ml). The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 7 degrees C. After 15 s of exposure to reuterin, viable numbers decreased by 0.45 and 0.3 log10 CFU/cm2 for E. coli O157:H7 and L. monocytogenes, respectively. After 24 h the numbers decreased by 2.7 log10 CFU/cm2 for E. coli O157:H7 and by 0.63 log10 CFU/cm2 for L. monocytogenes. In the same experiment, the combined effect of reuterin and lactic acid was also investigated. Adding lactic acid (5%, vol/vol) to reuterin significantly enhanced (P < or = 0.05) the efficacy of reuterin. No additional effect (P < or = 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid. To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E. coli O157:H7 or L. monocytogenes. Reuterin at a concentration of 100 AU/g resulted in a 5.0-log10 reduction of the viability of E. coli O157:H7 after 1 day of storage at 7 degrees C. Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L. monocytogenes by log10 3.0 cycles after 1 week of storage at 7 degrees C.     

Identification of Escherichia coli O157:H7 meat processing indicators for fresh meat through comparison of the effects of selected antimicrobial interventions

Fresh meat products can become contaminated with the pathogen Escherichia coli O157:H7 during the slaughter process; therefore, an E. coli O157:H7 indicator to verify the effectiveness of process controls in slaughter establishments would be extremely useful. The hides of 20 beef cattle were sampled, and 113 bacterial isolates were obtained. Thirteen of these isolates representing four genera, Escherichia, Enterobacter, Providencia, and Serratia, were selected based on growth and biochemical characteristics similar to those of five clinical strains of E. coli O157:H7. The temperature sensitivity was determined for the individual isolates and the five E. coli O157:H7 strains at 55 and 65 degrees C. D65-values for all 13 isolates were not significantly different from D65-values of the E. coli O157:H7 strains. E. coli isolates were the only isolates whose D55-values were not significantly different from those of the E. coli O157:H7 strains. E. coli isolates P3 and P68 were more resistant to the effects of 55 degrees C than were the other E. coli isolates but were not significantly different from E. coli O157:H7 WS 3331 (P > 0.05). The remaining E. coli isolates (P1, P8, and P14) were not significantly different from E. coli O157:H7 strains ATCC 35150, ATCC 43894, ATCC 43895, and WS 3062 (P > 0.05). Prerigor lean and adipose beef carcass tissue was artificially contaminated with stationary-phase cultures of the five E. coli beef cattle isolates or a cocktail of five E. coli O157:H7 strains in a fecal inoculum. Each tissue sample was processed with the following microbial interventions: 90 degrees C water; 90 degrees C water followed by 55 degrees C 2% lactic acid; 90 degrees C water followed by 20 degrees C 2% lactic acid; 20 degrees C water followed by 20 degrees C 2% lactic acid; 20 degrees C water followed by 20 degrees C 20 ppm chlorine; and 20 degrees C water followed by 20 degrees C 10% trisodium phosphate. The appropriateness of the E. coli isolates as potential E. coli O157:H7 indicators was dependent upon the microbial intervention utilized. For all microbial intervention methods applied irrespective of tissue type, the mean log reductions of at least two E. coli isolates were not significantly different from the mean log reduction of the E. coli O157:H7 cocktail (P > 0.05). Because of the frequent employment of multiple microbial interventions in the cattle industry, no single isolate can realistically represent the effectiveness of all microbial interventions for reduction of E. coil O157:H7. Thus, the use of a combination of E. coli isolates may be required to accurately predict the effectiveness of microbial intervention methods on the reduction of E. coli O157:H7 in beef carcass tissue.     

Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials

Brine solution injection of beef contaminated with Escherichia coli O157:H7 on its surface may lead to internalization of pathogen cells and/or cross-contamination of the brine, which when recirculated, may serve as a source of new product contamination. This study evaluated survival of E. coli O157:H7 in brines formulated without or with antimicrobials. The brines were formulated in sterile distilled water (simulating the composition of freshly prepared brines) or in a nonsterile 3% meat homogenate (simulating the composition of recirculating brines) at concentrations used to moisture-enhance meat to 110% of initial weight, as follows: sodium chloride (NaCl, 5.5%) + sodium tripolyphosphate (STP, 2.75%), NaCl + sodium pyrophosphate (2.75%), or NaCl + STP combined with potassium lactate (PL, 22%), sodium diacetate (SD, 1.65%), PL + SD, lactic acid (3.3%), acetic acid (3.3%), citric acid (3.3%), nisin (0.0165%) + ethylenediamine tetraacetic acid (EDTA, 200 mM), pediocin (11000 AU/mL) + EDTA, sodium metasilicate (2.2%), cetylpyridinium chloride (CPC, 5.5%), or hops beta acids (0.0055%). The brines were inoculated (3 to 4 log CFU/mL) with rifampicin-resistant E. coli O157:H7 (8-strain composite) and stored at 4 or 15 °C (24 to 48 h). Immediate (0 h) pathogen reductions (P < 0.05) of 1.8 to ≥ 2.4 log CFU/mL were observed in brines containing CPC or sodium metasilicate. Furthermore, brines formulated with lactic acid, acetic acid, citric acid, nisin + EDTA, pediocin + EDTA, CPC, sodium metasilicate, or hops beta acids had reductions (P < 0.05) in pathogen levels during storage; however, the extent of pathogen reduction (0.4 to > 2.4 log CFU/mL) depended on the antimicrobial, brine type, and storage temperature and time. These data should be useful in development or improvement of brine formulations for control of E. coli O157:H7 in moisture-enhanced meat products. PRACTICAL APPLICATION: Results of this study should be useful to the meat industry for developing or modifying brine formulations to reduce the risk of E. coli O157:H7 in moisture-enhanced meat products.     

Antimicrobial Activity and Synergistic Effect of Cinnamon with Sodium Benzoate or Potassium Sorbate in Controlling Escherichia coli O157:H7 in Apple Juice

ABSTRACT: Antimicrobial effects of cinnamon, sodium benzoate, potassium sorbate, and combinations were examined against Escherichia coli O157:H7 in apple juice at 8°C and 25°C. E. coli O157:H7 was reduced by 1.6 log colony-forming units (CFU)/mL at 8°C and 2.0 log CFU/mL at 25°C by 0.3% cinnamon. At 8°C, 5.2 log CFU/mL of E. coli O157:H7 was eliminated in 11 d by 0.3% cinnamon with 0.1% sodium benzoate, and in 14 d by 0.3% cinnamon with 0.1% potassium sorbate. At 25°C, 5.3 log CFU/mL E. coli O157:H7 was eliminated in 3 d by the same combinations. A synergistic effect was observed between cinnamon and preservatives against E. coli O157:H7 at 8°C and 25°C.     

Evaluation of Lactic Acid as an Initial and Secondary Subprimal Intervention for Escherichia coli O157:H7, Non-O157 Shiga Toxin-Producing E. coli, and a Nonpathogenic E. coli Surrogate for E. coli O157:H7

Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.     

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.     

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.     

A model study of Escherichia coli O157:H7 survival in fermented dry sausages--influence of inoculum preparation, inoculation procedure, and selected process parameters

The influence of inoculum preparation, inoculation level, and inoculation procedure on Escherichia coli O157:H7 inactivation during the manufacture of fermented sausage was evaluated in a model study. Prior growth in glucose-enriched tryptone soya broth, which provided exposure to mildly acidic conditions (pH 4.8), had no effect on the later survival of E. coli O157: H7 strains 5-1 and ATCC 43894 under extremely acidic conditions (pH 2), but the same strains became sensitive to acidity after 7 days of incubation on the surface of refrigerated beef (as per the normal contamination route from slaughter to further processing). In subsequent sausage production trials, the extent of destruction observed for E. coli O157:H7 strains F-90, 5-1, and ATCC 43894 inoculated directly into the meat batter was unchanged when the inoculation level was decreased from 7.3 to 4.7 log CFU/g, but the level of inactivation was ca. 1 log higher when the surfaces of beef cuts, rather than the batter, were inoculated 7 days prior to processing. Regardless of processing conditions (fermentation to a pH of < or = 5.0 at 24 or 37 degrees C, drying at 14 degrees C to a water activity [a(w)] value of 0.91 or 0.79), strains F-90, 5-1, and ATCC 43894 showed similar survival capacities during the manufacture of sausage. A approximately 2-log reduction in pathogen numbers was generally obtained after samples were dried to an a(w) of 0.91, irrespective of fermentation temperature. The addition of a 5-day predrying holding stage at the fermentation temperature significantly (P < 0.05) increased pathogen inactivation when fermentation was carried out at 37 degrees C (but not when it was carried out at 24 degrees C). However, significant pathogen reductions (4 to 5 log CFU/g) were achieved only for extensively dried products (a(w) = 0.79).     

Inoculation of beef with low concentrations of Escherichia coli O157:H7 and examination of factors that interfere with its detection by culture isolation and rapid methods

Currently used industry testing programs require the ability to detect Escherichia coli O157:H7 in samples of beef trim or ground beef at levels as low as 1 CFU/375 g. We present a reliable protocol for generating a control inoculum for verification testing at this low concentration and evaluate its use. Results show that half of all samples received no cells when 1 CFU was the target concentration and that targets greater than 3 CFU were much more reliable. Detection by culture isolation and two commercial assays, Qualicon BAX-MP and BioControl GDS, detected 94% ± 11%, 92% ± 10%, and 92% ± 7% of samples inoculated with 5.4 CFU (range 1 to 9 CFU), respectively. We also examined the effect of background aerobic plate count (APC) bacteria and fat content effects on the detection of E. coli O157:H7. At APC concentrations below 6 log CFU/g, the rapid methods detected all beef trim samples inoculated with 26 CFU of E. coli O157:H7 per 65 g. At an APC of 6.7 log CFU/g, culture, BAX-MP, and GDS detected 100, 75, and 13%, respectively, of inoculated samples. Neither commercial method detected E. coli O157:H7 in the samples when APC was 7.7 log CFU/g, whereas culture was able to detect 63% of E. coli O157:H7 in the samples when APC was at this concentration. Increased fat content correlated with decreasing recovery of immunomagnetic separation beads, but this was not observed to interfere with detection of E. coli O157:H7.     

Thermal process validation for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ground turkey and beef products

At 55 to 70 degrees C, thermal inactivation D-values for Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes were 19.05 to 0.038, 43.10 to 0.096, and 33.11 to 0.12 min, respectively, in ground turkey and 21.55 to 0.055, 37.04 to 0.066, and 36.90 to 0.063 min, respectively, in ground beef. The z-values were 5.73, 5.54, and 6.13 degrees C, respectively, in ground turkey and 5.43, 5.74, and 6.01 degrees C, respectively, in ground beef. In both ground turkey and beef, significant (P < 0.05) differences were found in the D-values between E. coli O157:H7 and Salmonella or between E. coli O157:H7 and L. monocytogenes. At 65 to 70 degrees C, D-values for E. coli O157:H7, Salmonella, and L. monocytogenes were also significantly (P < 0.05) different between turkey and beef. The obtained D- and z-values were used in predicting process lethality of the pathogens in ground turkey and beef patties cooked in an air impingement oven and confirmed by inoculation studies for a 7-log (CFU/g) reduction of E. coli O157:H7, Salmonella, and L. monocytogenes.     

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.     

Treatments using hot water instead of lactic acid reduce levels of aerobic bacteria and Enterobacteriaceae and reduce the prevalence of Escherichia coil O157:H7 on preevisceration beef carcasses

Lactic acid has become the most commonly used organic acid for treatment of postevisceration beef carcasses. Many processors have also implemented 2% lactic acid washes on preevisceration carcasses. We previously demonstrated that hot water washing and steam vacuuming are effective carcass interventions. Because of the effectiveness of hot water, we compared its use with that of lactic acid as a preevisceration wash in a commercial setting. A commercial hot water carcass wash cabinet applying 74 degrees C (165 degrees F) water for 5.5 s reduced both aerobic plate counts and Enterobacteriaceae counts by 2.7 log CFU/100 cm2 on preevisceration carcasses. A commercial lactic acid spray cabinet that applied 2% L-lactic acid at approximately 42 degrees C (105 to 110 degrees F) to preevisceration carcasses reduced aerobic plate counts by 1.6 log CFU/100 cm2 and Enterobacteriaceae counts by 1.0 log CFU/100 cm2. When the two cabinets were in use sequentially, i.e., hot water followed by lactic acid, aerobic plate counts were reduced by 2.2 log CFU/100 cm2 and Enterobacteriaceae counts were reduced by 2.5 log CFU/100 cm2. Hot water treatments reduced Escherichia coli O157:H7 prevalence by 81%, and lactic acid treatments reduced E. coli O157:H7 prevalence by 35%, but the two treatments in combination produced a 79% reduction in E. coli O157:H7, a result that was no better than that achieved with hot water alone. These results suggest that hot water would be more beneficial than lactic acid for decontamination of preevisceration beef carcasses.     

Effects of using reduced volumes of nonselective enrichment medium in methods for the detection of Escherichia coli O157:H7 from raw beef

Recent work from our laboratory revealed that tryptic soy broth (TSB) was a superior enrichment medium for use in test-and-hold Escherichia coli O157:H7 methods at levels down to a ratio of three volumes of medium to one volume of sample. Lower ratios were examined for their effect on results obtained from culture isolation, the BAX E. coli O157:H7 MP assay, and the Assurance GDS E. coli O157:H7 assay. Ground beef and boneless beef trim were inoculated with a high level (170 CFU/65 g of ground beef and 43 CFU/65 g of trim) and a low level (17 CFU/65 g of ground beef and 4 CFU/65 g of trim) of E. coli O157:H7 and enriched in 3, 1, 0.5, and 0 volumes of TSB. The volume of TSB used did not affect E. coli O157:H7 detection by culture isolation, Assurance GDS detection in ground beef or trim, or the BAX MP assay detection in ground beef. However, BAX MP assay detection of E. coli O157:H7 in beef trim was 50, 42, and 33% positive when enrichment volumes of 0.5x, 1x, and 3x, respectively, were used. Optimum results with all methods were obtained using 1 volume of TSB. We concluded that detection test results can be considered valid as long as enrichment medium is used, even when it is less than the specified 3 or 10 volumes.     

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2 degrees C, and -20 degrees C

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 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) 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 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees 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 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-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.