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.     

Evaluation of various antimicrobial interventions for the reduction of Escherichia coli O157:H7 on bovine heads during processing

The effectiveness of electrolyzed oxidizing water, FreshFx, hot water, DL-lactic acid, and ozonated water was determined using a model carcass spray-washing cabinet. A total of 140 beef heads obtained from a commercial processing line were inoculated with Escherichia coli O157:H7 on the cheek areas. Each head was exposed to a simulated preevisceration wash and then had antimicrobial wash treatments. Hot water, lactic acid, and FreshFx treatments reduced E. coli O157:H7 on inoculated beef heads by 1.72, 1.52, and 1.06 log CFU/cm2, respectively, relative to the simulated preevisceration wash. Electrolyzed oxidizing water and ozonated water reduced E. coli O157:H7 less than 0.50 log CFU/cm2. Hot water, lactic acid, and FreshFx could be used as decontamination washes for the reduction of E. coli O157:H7 on bovine head and cheek meat.     

Acid tolerance of acid-adapted and nonacid-adapted Escherichia coli O157:H7 strains in beef decontamination runoff fluids or on beef tissue

This study assessed the acid tolerance response (ATR) of stationary phase, acid-adapted (tryptic soy broth [TSB]+1% glucose) or nonacid-adapted (glucose-free TSB) Escherichia coli O157:H7 strains (ATCC43889, ATCC43895, ATCC51658 and EO139), grown individually or in a mixed culture, prior to inoculation of beef or meat decontamination runoff (washings) fluids (acidic [pH 4.95] or nonacidic [pH 7.01]). The inoculated beef was left untreated or treated by dipping for 30s in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C). Inoculated beef samples and washings were stored aerobically at 4 or 15 degrees C for 6d, and at set intervals (0, 2, and 6d) were exposed (for 0, 60, 120, and 180min) to pH 3.5 (adjusted with lactic acid) TSB plus 0.6% yeast extract. Overall, there were no significant (P0.05) differences in responses of cultures prepared as individual or mixed strains. Decontamination of meat did not affect the subsequent ATR of E. coli O157:H7 other than resulting in lower initial pathogen levels exposed to acidic conditions. In this study, E. coli O157:H7 appeared to become more tolerant to acid following incubation in acidic washings of sublethal pH (4.89-5.22) compared to nonacidic washings (pH 6.97-7.41) at 4 degrees C or in both types of washings incubated at 15 degrees C. The ATR of the pathogen inoculated into washings was enhanced when cells were previously acid-adapted and incubated at 4 degrees C. Similarly, the ATR on meat was increased by previous acid-adaptation of the inoculum in broth and enhanced by storage at 4 degrees C. Populations on treated meat were consistently lower than those on untreated meat during storage and following exposure to acid. Although on day-0 there were no significant (P0.05) differences in ATR between acid-adapted and nonacid-adapted populations on meat, acid-adapted cells displayed consistently higher resistance through day-6. This suggests that acid-adapted E. coli O157:H7 introduced on meat may become resistant to subsequent lactic acid exposure following storage at 4 degrees C.     

Effects of vacuum or modified atmosphere packaging in combination with irradiation for control of Escherichia coli O157:H7 in ground beef patties

The efficacy of controlling Escherichia coli O157:H7 in ground beef patties by combining irradiation with vacuum packaging or modified atmosphere packaging (MAP) was investigated. Fresh ground beef patties were inoculated with a five-strain cocktail of E. coli O157:H7 at 5 log CFU/g. Single patties, packaged with vacuum or high-CO(2) MAP (99.6% CO(2) plus 0.4% CO), were irradiated at 0 (control), 0.5, 1.0, or 1.5 kGy. The D(10)-value for this pathogen was 0.47 ± 0.02 kGy in vacuum and 0.50 ± 0.02 kGy in MAP packaging. Irradiation with 1.5 kGy reduced E. coli O157:H7 by 3.0 to 3.3 log, while 0.5 and 1.0 kGy achieved reductions of 0.7 to 1.0, and 2.0 to 2.2 log, respectively. After irradiation, the numbers of survivors of this pathogen on beef patties in refrigerated storage (4°C) did not change significantly for 6 weeks. Temperature abuse (at 25°C) resulted in growth in vacuum-packaged patties treated with 0.5 and 1.5 kGy, but no growth in MAP packages. This study demonstrated that combining irradiation with MAP was similar in effectiveness to irradiation with vacuum packaging for control of E. coli O157:H7 in ground beef patties during refrigerated storage. However, high-CO(2) MAP appeared to be more effective after temperature abuse.     

Effect of acid adaptation on survival of Escherichia coli O157:H7 in meat decontamination washing fluids and potential effects of organic acid interventions on the microbial ecology of the meat plant environment.

The acid tolerance of Escherichia coli O157:H7 may be pH inducible. Correspondingly, organic acid meat decontamination washing fluids may enhance the establishment of acid-adapted E. coli O157:H7 strains in packing plants, especially in mixtures with water washings from meat that may be of sublethal pH. Acid-adapted and nonadapted cultures of a rifampin-resistant derivative of the acid-resistant E. coli O157:H7 strain ATCC 43895 were tested to evaluate their survival in meat-washing fluids over a wide pH range. The cultures were exposed (10(5) CFU/ml) to acidic (2% lactic acid. 2% acetic acid, or a mixture of the two with water washings at ratios of 1/1, 1/9, or 1/99 [vol/vol]) or nonacid (water) meat washings for up to 14 days at 4 or 10 degrees C storage. E. coli O157:H7 survived in water washings, but the low storage temperatures and predominant natural microbiota synergistically inhibited its growth. Compared with acid-adapted populations, nonadapted populations displayed greater potential for survival and a tendency to initiate growth in water meat washings at 10 degrees C. The pathogen survived in most of the acid washings throughout storage (14 days), sometimes with minimal population reductions. Overall. nonadapted populations declined faster than acid-adapted populations, while the declines increased as the acid concentration and temperature of storage increased and were more dramatic in lactate, compared to acetate, washings. Acid-containing washings were selective for growth of lactic acid bacteria and yeasts. indicating that organic acid treatments may alter the microbial ecology of meat plant environments and potentially that of the meat. These results should be considered when selecting decontamination technologies for meat.     

High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef

The influence of natural background flora under aerobic and anaerobic incubation on the growth of Escherichia coli O157:H7 in ground beef was investigated. The background flora from eight different commercial ground beef were added to ground beef spiked with E. coli O157:H7 and stored either aerobically or anaerobically at 12 degrees C. The results showed that the presence of a large number of background bacteria in the ground meat inhibited the growth of E. coli O157:H7 both aerobically and anaerobically. Inhibition was more pronounced under anaerobic conditions. The background floras consisted mainly of lactic acid bacteria of which approximately 80% were Lactobacillus sakei. These results show the importance of the natural background flora in meat for inhibition of growth of E. coli O157:H7.     

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.     

Survival of Escherichia coli O157:H7 and Salmonella on chill-stored vacuum or carbon dioxide packaged primal beef cuts

The ability of two Escherichia coli O157:H7 strains (E27, a cattle isolate, and B6-914 gfp-91, a fluorescent marker strain) and two Salmonella serotypes (S. typhimurium and S. brandenberg) to survive on chilled preservatively packaged primal beef cuts was examined. Each of the strains was inoculated separately at two dilution levels (10(3) and 10(5) cfu g(-1)) onto 500 g beef steaks, packaged under vacuum or 100% carbon dioxide, and stored, with uninoculated controls, for 6 weeks at - 1.5 degrees C, then for 2 weeks at 4 degrees C. Bacterial numbers were determined by dilution and incubation at 37 degrees C for 24 h on either Sorbitol McConkey Agar or Xylose Lysine Desoxycholate Agar for E. coli O157:H7 and Salmonella samples, respectively. Counts were corrected for background growth and their accuracy checked using immunological tests. Fluorescent E. coli O157:H7 B6-914 gfp-91 was also counted under ultra-violet light. No significant changes in numbers of the E. coli O157:H7 or Salmonella strains occurred during storage at either - 1.5 or 4 degrees C packaged under either vacuum or carbon dioxide. The ability of these pathogens to survive standard preservative packaging conditions is different from that reported from their generic counterparts and therefore a cause for public health concern.     

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.     

Control of Escherichia coli O157:H7 with sodium metasilicate

Three intervention strategies-trisodium phosphate, lactic acid, and sodium metasilicate--were examined for their in vitro antimicrobial activities in water at room temperature against a three-strain cocktail of Escherichia coli O157:H7 and a three-strain cocktail of "generic" E. coli. Both initial inhibition and recovery of injured cells were monitored. When 3.0% (wt/wt) lactic acid, pH 2.4, was inoculated with E. coli O157:H7 (approximately 6 log CFU/ml), viable microorganisms were recovered after a 20-min exposure to the acid. After 20 min in 1.0% (wt/wt) trisodium phosphate, pH 12.0, no viable E. coli O157:H7 microorganisms were detected. Exposure of E. coli O157:H7 to sodium metasilicate (5 to 10 s) at concentrations as low as 0.6%, pH 12.1, resulted in 100% inhibition with no recoverable E. coli O157:H7. No difference in inhibition profiles was detected between the E. coli O157:H7 and generic strains, suggesting that nonpathogenic strains may be used for in-plant sodium metasilicate studies.     

乳酸菌对肠出血性大肠杆菌O157:H7抑制作用的研究

为探讨乳酸菌对肠出血性大肠杆菌O157：H7 ATCC43895(E．Coli O157：H7)的抑制作用，在培养基上进行了研究。将E．Coli O157：H7与干酪乳杆菌干酪亚种、植物乳杆菌、发酵乳杆菌、乳酸乳球菌和瑞士乳杆菌同时接种在培养基中，E．Coli O157：H7的活性不受影响；将E．Coli O157：H7接种到培养了24h的乳酸茵培养液中，E．Coli O157：H7活性显下降。以乳酸调整的低pH值对E．Coli O157：H7有一定的杀灭作用。本研究表明：乳酸菌的代谢产物乳酸对E．Coli O157：H7有杀灭作用。     

Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach

Escherichia coli O157:H7 contaminated spinach has recently caused several outbreaks of human illness in the USA and Canada. However, to date, there has been no study demonstrating an effective way to eliminate E. coli O157:H7 in spinach. Therefore, this study was conducted to investigate the effect of chemical sanitizers alone or in combination with packaging methods such as vacuum and modified atmosphere packaging (MAP) on inactivating E. coli O157:H7 in spinach during storage time. Spinach inoculated with E. coli O157:H7 was packaged in four different methods (air, vacuum, N(2) gas, and CO(2) gas packaging) following treatment with water, 100 ppm chlorine dioxide, or 100 ppm sodium hypochlorite for 5 min at room temperature and stored at 7+/-2 degrees C. Treatment with water did not significantly reduce levels of E. coli O157:H7 in spinach. However, treatment with chlorine dioxide and sodium hypochlorite significantly decreased levels of E. coli O157:H7 by 2.6 and 1.1 log(10)CFU/g, respectively. Levels of E. coli O157:H7 in samples packaged in air following treatments grew during storage time, whereas levels were maintained in samples packaged in other packaging methods (vacuum, N(2) gas, and CO(2) gas packaging). Therefore there were significant differences (about 3-4 log) of E. coli O157:H7 populations between samples packed in air and other packaging methods following treatment with chemical sanitizers after 7 days storage. These results suggest that the combination of treatment with chlorine dioxide and packaging methods such as vacuum and MAP may be useful for improving the microbial safety of spinach against E. coli O157:H7 during storage.     

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.     

Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms

This study evaluated resistance to sanitizing solutions of Escherichia coli O157:H7 cells forming biofilms on stainless steel coupons exposed to inoculated meat decontamination runoff fluids (washings). A previously acid-adapted culture of a rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated in unsterilized or sterilized combined hot-water (85 degrees C) and cold-water (10 degrees C) (50/50 [vol/vol]) composite water (W) washings (pH 6.29 to 6.47) and in W washings mixed with 2% acetic acid (pH 4.60 to 4.71) or in 2% lactic acid W washings (pH 4.33 to 4.48) at a ratio of 1/99 (vol/vol). Stainless steel coupons (2 by 5 by 0.08 cm) were submerged in the inoculated washings and stored for up to 14 days at 15 degrees C. Survival of E. coli O157:H7 was determined after exposure (0 to 60 s for cells in suspension and 0 to 300 s for attached cells) to two commercial sanitizers (150 ppm peroxyacetic acid and 200 ppm quaternary ammonium compound) at 2, 7, and 14 days. E. coli O157:H7 attached more rapidly to coupons submerged in washings containing the natural flora than to those without. The attached cells were more resistant to the effects of the sanitizers than were the cells in suspension, and survival was highest in the presence of the natural flora. Attached cells in the presence of dilute acid washings were more sensitive to subsequent sanitizer treatments than were cells generated in the presence of W washings. Under the conditions of this study, cells of E. coli O157:H7 in W washings were more sensitive to acidic (peroxyacetic acid) than to alkaline (quaternary ammonium) sanitizers during storage. These results suggest that meat processing plants that apply no decontamination or that use only water washings of meat should consider using acidic sanitizers to enhance biofilm removal. Plants that apply both water and acidic washings may create a sublethal acid-stressing environment in the runoff fluids, sensitizing biofilm cells to subsequent sanitizing treatments.     

Survival and growth of Escherichia coli O157:H7, Yersinia enterocolitica and Salmonella enteritidis on decontaminated and untreated meat

Decontamination of meat or carcasses may have an effect in reducing the number of pathogens. Recontamination with other pathogens during cutting or packaging may, however, result in higher growth on decontaminated than on untreated meat due to the lack of competing non-pathogenic microorganisms. In this study we compared the growth of pathogens during storage at 10°C (worst case condition) on untreated meat and meat that had been decontaminated by steam vacuuming combined with spraying with 0.2 M lactic acid. Salmonella enteritidis inoculated on chicken multiplied quickly and reached log 7 cfu per cm(2) after 4 days of aerobic storage at 10°C, but growth was not significantly higher on decontaminated than on untreated chicken. The number of Yersinia enterocolitica inoculated on decontaminated pork skin reached log 9 cfu per cm(2) after 5 days of aerobic storage at 10°C. Overall, growth on vacuum-packed decontaminated and untreated pork under the same conditions was not significantly different, although there tended to be less growth on the untreated samples. The number of Escherichia coli O157:H7 on decontaminated beef increased by nearly 3 log cycles after 5 days of aerobic storage at 10°C compared to only a 1 log cycle increase on untreated beef. For the vacuum-packed beef, growth of E. coli O157:H7 on the fresh meat was very slow, while there was about a 3 log increase on the decontaminated beef. A higher average growth on the decontaminated beef was also found in an experiment with a very low inoculum (27 cfu per cm(2)). During storage of vacuum-packed samples there was multiplication of E. coli O157:H7 on the decontaminated beef, but virtually none on the untreated beef. This study shows that multiplication of S. enteritidis on chicken and Y. enterocolitica on pork skin was not significantly higher on decontaminated compared to untreated meat. The increased multiplication of E. coli O157:H7 on decontaminated beef, especially when vacuum-packed, gives cause for concern. Preventive measures might be a strict HACCP approach to the handling of the decontaminated meat before packaging or use of a protective culture of lactic acid bacteria.     

Acid tolerance of acid-adapted and nonadapted Escherichia coli O157:H7 following habituation (10 degrees C) in fresh beef decontamination runoff fluids of different pH values

This study evaluated survival of Escherichia coli O157:H7 strain ATCC 43895 during exposure to pH 3.5 following its habituation for 2 or 7 days at 10 degrees in fresh beef decontamination waste runoff fluid mixtures (washings) containing 0, 0.02, or 0.2% of lactic or acetic acids. Meat washings and sterile water (control) were initially inoculated with approximately 5 log CFU/ml of acid- and nonadapted E. coli O157:H7 cells cultured (30 degrees C, 24 h) in broth with and without 1% glucose, respectively. After 2 days, E. coli O157:H7 survivors from acetate washings (pH 3.7 to 4.7) survived at pH 3.5 better than E. coli O157:H7 survivors from lactate washings (pH 3.1 to 4.6), especially when the original inoculum was acid adapted. Also, although E. coli O157:H7 habituated in sterile water for 2 days survived well at pH 3.5, the corresponding survivors from nonacid water meat washings (pH 6.8) were rapidly killed at pH 3.5, irrespective of acid adaptation. After 7 days, E. coli O157:H7 survivors from acetate washings (pH 3.6 to 4.7) continued to resist pH 3.5, whereas those from lactate washings died off. This loss of acid tolerance by E. coli O157:H7 was due to either its low survival in 0.2% lactate washings (pH 3.1) or its acid sensitization in 0.02% lactate washings, in which a Pseudomonas-like natural flora showed extensive growth (> 8 log CFU/ml) and the pH increased to 6.5 to 6.6. Acid-adapted E. coli O157:H7 populations habituated in water washings (pH 7.1 to 7.3) for 7 days continued to be acid sensitive, whereas nonadapted populations increased their acid tolerance, a response merely correlated with their slight (< 1 log) growth at 10 degrees C. These results indicate that the expression of high acid tolerance by acid-adapted E. coli O157:H7 can be maintained or enhanced in acid-diluted meat decontamination waste runoff fluids of pH levels that could permit long-term survival at 10 degrees C. Previous acid adaptation, however, could reduce the growth potential of E. coli O157:H7 at 10 degrees C in nonacid waste fluids of high pH and enriched in natural flora. These conditions might further induce an acid sensitization to stationary E. coli O157:H7 cells.     

Effect of different storage conditions on E. coli O157:H7 and the indigenous bacterial microflora on lamb meat

Lamb chops inoculated with 2.23-2.83 log cfu/g of E. coli O157:H7 strain NCTC 12900 were packed in air (AP), vacuum (VP), and two modified atmospheres (MAP) consisting of 100% CO2 and a commercial mixture of 35% CO2/35% O2/30% N2. All samples (initial total counts <3.5 log cfu/g) were stored in a commercial cold storage facility set at 4 degrees C and one AP trial also at 12+/-1 degrees C in a temperature controlled incubator. Pathogen and indigenous flora evolution, physicochemical and sensory changes, surface packages temperature and MAP gas composition were monitored throughout the lamb meat shelf life. Temperature monitoring revealed that during chilled storage packed chops exceeded 7 degrees C about 3% of the time for periods of 10-20 min at 6 h intervals corresponding to defrosting cycles. In AP samples under these conditions, the E. coli O157:H7 strain had an overall increase of 0.48 log cfu/g by day 12. This increase, which may be regarded as an artefact of the sampling procedure, might also be a response to fluctuating temperatures. Regardless of rapid proliferation of the background microflora on AP lamb meat kept at 12+/-1 degrees C, the pathogen significantly increased by 2.35 log cfu/g after nine days. There was a slight decrease (0.20 log cfu/g) of the pathogen numbers after four weeks cold storage in VP despite a significant increase in lactic acid bacteria (LAB). With a relatively small outgrowth of LAB, chilled storage in 100% and 35% CO2 resulted in significant differences compared to similar conditions in air (decrease from initial numbers of 0.80 and 0.45 log cfu/g, respectively). Our data confirm the importance of effective temperature control to prevent pathogen growth on raw meat and also that contaminated meat remains hazardous regardless of refrigeration and protective packaging. Further studies are needed to determine the behaviour of E. coli O157:H7 at temperatures that fluctuate around the minimum for growth.     

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.     

Fate of inoculated Escherichia coli O157:H7, cultured under different conditions, on fresh and decontaminated beef transitioned from vacuum to aerobic packaging

This study evaluated the behavior of Escherichia coli O157:H7 during aerobic storage, after storage in vacuum packages, on beef inoculated with cultures prepared (35 degrees C, 24 h) in tryptic soy broth without dextrose (TSB), nonacid hot water carcass decontamination runoff fluids (washings; pH 6.0; WASH), cells from biofilms formed on stainless steel coupons in WASH (WETB), or WETB dried (25 degrees C, 12 h) before harvesting of cells (DRYB). These inocula were applied to fresh beef pieces (40 cm2), which were then left untreated or treated by immersion in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C; hot water/lactic acid [HW/LA]), for 30 s each. Inoculated samples were vacuum packaged and stored at 0 (30, 60, or 90 days), 4 (7 or 14 days), or 12 degrees C (4 or 8 days) and subsequently transferred to retail packages for aerobic storage at 7 degrees C for 5 days. Populations of E. coli O157:H117, regardless of inoculum type, remained generally unchanged (P > 0.05) after aerobic storage (7 degrees C, 5 days) of untreated or HW/LA-treated beef samples previously stored in vacuum packages at 0 or 4 degrees C. However, reductions in E. coli O157:H7 levels were generally obtained when vacuum packaged, untreated beef samples previously stored at 12 degrees C were transitioned to aerobic conditions. Additionally, despite similar (P > 0.05) levels of E. coli O157:H7 cells of TSB, WASH, WETB, and DRYB origin on vacuum-packaged, untreated samples after 8 days of storage at 12 degrees C, subsequent aerobic storage resulted in larger (P < 0.05) reductions of cells of WETB and DRYB origin than for cells of TSB and WASH origin. For HW/LA-treated beef previously stored at 12 degrees C in vacuum packages, populations of E. coli O157:H7 remained largely unchanged after aerobic storage in retail packages. Results thus indicated that aerobic storage of beef (7 degees C, 5 days) previously stored in vacuum packages at 0 or 4 degrees C did not lead to E. coli O157:H7 population changes, whereas transition from vacuum packages stored under mildly abusive temperature (12 degrees C) to aerobic storage may have caused injury and death to the pathogen.