Postprocessing In Vitro Digestion Challenge To Evaluate Survival of Escherichia coli O157:H7 in Fermented Dry Sausages

Fermented dry sausages, inoculated with Escherichia coli O157:H7 during batter preparation, were submitted to an in vitro digestion challenge to evaluate the extent to which passage through the human gastrointestinal tract could inactivate the pathogenic cells, previously stressed by the manufacturing process. The numbers of surviving E. coli O157:H7 cells remained constant after a 1-min exposure of the finely chopped sausage to synthetic saliva or during the following 120-min exposure to synthetic gastric juice at an initial pH of 2.0. However, significant (P ≤ 0.05) growth of the pathogen (1.03 to 2.16 log₁₀ CFU/g) was observed in a subsequent 250-min exposure to a synthetic pancreatic juice at pH 8.0. In a different set of experiments, fractions from the gastric suspension were transferred into the synthetic pancreatic juice at 30-min intervals to mimic the dynamics of gastric emptying. Concurrently, the pH of the remaining gastric fluid was reduced to 3.0, 2.5, and 2.0 to simulate the gradual reacidification of the stomach contents after the initial buffering effect resulting from meal ingestion. Under these new conditions, pathogen growth during pancreatic challenge was observed for the first few fractions released from the stomach (90 min of exposure [pH 2.5]), but growth was no longer possible in the fractions submitted to the most severe gastric challenge (120 min of exposure [pH < 2.2]).     

Acid tolerance of Escherichia coli O157:H7 and its survival in apple juice

Outbreaks of diarrhoea and haemolytic uraemic syndrome have been associated with the consumption of apple cider and apple juice. The organism implicated in these outbreaks has been Escherichia coli O157:H7, indicating the resistance of the serotype to acidic pH. On comparing the growth of this serotype with a control strain of E. coli, it was found that strain O157:H7 grew well in trypticase soy broth at pH levels ranging from 2.0 to 9.0, while control strains failed to grow at pH levels below 4.0 and above 9.0. The growth of both strains were inhibited by adding 0.05% of either benzoic acid or sorbic acid. Similarly, O157:H7 grew well in both natural (unpasteurized) as well as in pasteurized apple juice and the growth was inhibited by adding 0.1% of either benzoic acid or sorbic acid. Control strains of E. coli failed to grow in either types of apple juice. The possible sources of contamination of natural apple juice with O157:H7 serotype are discussed.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Antibacterial activity of fresh and processed red muscadine juice and the role of their polar compounds on Escherichia coli O157:H7

Aims:  The objectives of this research were to show the anti- Escherichia coli O157:H7 effect of fresh (FRMJ) and processed red muscadine (V itis rotundifolia ) juice (PRMJ) and to discern the active compounds responsible for anti- E . coli O157:H7.
Methods and Results:  Polar and phenolic compounds of FRMJ and PRMJ were analysed by high-performance liquid chromatography. Antibacterial activity of FRMJ, PRMJ, their polar and polyphenol fractions, individual synthetic acids and their mixture with or without sugars were investigated on E . coli O157:H7. FRMJ and PRMJ inactivated ( P  ≤ 0·05) 5-log cocktail cells of E. coli O157:H7 within 4 h at 37°C. Polar fractions that contained malic, tartaric and tannic acids showed strong antimicrobial activity ( P  ≤ 0·05) against E . coli O157:H7. Tannic acid among the synthetic acids showed the highest antimicrobial activity against E. coli O157:H7.
Conclusions:  FRMJ, PRMJ and their polar compounds showed strong anti- E . coli O157:H7 activity.
Significance and Impact of the Study:  Earlier findings have failed to show any anti -E . coli O157:H7 effect of grape juice without adding preservatives. Our findings show that red muscadine juice has natural antibacterial substances and suggest that these can be used as active antimicrobial ingredients against E . coli O157:H7 in nonalcoholic beverages.     

Survival of Escherichia coli O157:H7 in the rhizosphere of maize grown in waste-amended soil

AIMS: To assess whether the persistence of Escherichia coli O157:H7 in soil amended with cattle slurry and ovine stomach content waste is affected by the presence of a maize rhizosphere. METHODS AND RESULTS: Cattle slurry and ovine stomach content waste were inoculated with E. coli O157:H7. Wastes were then applied to soil cores with and without established maize plants. The pathogen survived in soil for over 5 weeks, although at significantly greater numbers in soil receiving stomach content waste in comparison to cattle slurry. Persistence of the pathogen in soil was unaffected by the presence of a rhizosphere. CONCLUSIONS: Other factors may be more influential in regulating E. coli O157:H7 persistence in waste-amended soil than the presence or absence of a rhizosphere; however, waste type did have significant affect on the survival of E. coli O157:H7 in such soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli O157:H7 can be present within animal-derived organic wastes that are routinely spread on land. Introduced measures with regards to such waste disposal may decrease exposure to the organism; however, the persistence of E. coli O157:H7 for considerable periods in waste-amended soil may still pose some risk for both human and animal infection. This study has shown that whilst survival of E. coli O157:H7 in waste-amended soil is not significantly affected by the presence or absence of a maize rhizosphere; it may vary significantly with waste type. This may have implications for land and waste management.     

Escherichia coli acid resistance: tales of an amateur acidophile

Gastrointestinal pathogens are faced with an extremely acidic environment. Within moments, a pathogen such as Escherichia coli O157:H7 can move from the nurturing pH 7 environment of a hamburger to the harsh pH 2 milieu of the stomach. Surprisingly, certain microorganisms that grow at neutral pH have elegantly regulated systems that enable survival during excursions into acidic environments. The best-characterized acid-resistance system is found in E. coli.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

Modeling of pathogen survival during simulated gastric digestion

The objective of the present study was to develop a mathematical model of pathogenic bacterial inactivation kinetics in a gastric environment in order to further understand a part of the infectious dose-response mechanism. The major bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. were examined by using simulated gastric fluid adjusted to various pH values. To correspond to the various pHs in a stomach during digestion, a modified logistic differential equation model and the Weibull differential equation model were examined. The specific inactivation rate for each pathogen was successfully described by a square-root model as a function of pH. The square-root models were combined with the modified logistic differential equation to obtain a complete inactivation curve. Both the modified logistic and Weibull models provided a highly accurate fitting of the static pH conditions for every pathogen. However, while the residuals plots of the modified logistic model indicated no systematic bias and/or regional prediction problems, the residuals plots of the Weibull model showed a systematic bias. The modified logistic model appropriately predicted the pathogen behavior in the simulated gastric digestion process with actual food, including cut lettuce, minced tuna, hamburger, and scrambled egg. Although the developed model enabled us to predict pathogen inactivation during gastric digestion, its results also suggested that the ingested bacteria in the stomach would barely be inactivated in the real digestion process. The results of this study will provide important information on a part of the dose-response mechanism of bacterial pathogens.     

Escherichia coli O157:H7 in free-ranging deer in Nebraska.

In order to determine the prevalence and distribution of the human pathogen, Escherichia coli O157:H7, in free-ranging deer, hunters were asked to collect and submit fecal samples from deer harvested during a regular firearm season (14-22 November 1998). Prior to the season, 47% of the hunters with permits in the southeastern Nebraska (USA) study area indicated a willingness to participate in the study. Approximately 25% of successful hunters in the area submitted deer fecal samples. Escherichia coli O157:H7 was cultured from four (0.25%) of 1,608 total samples submitted. All of the fecal samples that were properly identified (1,426) and all that were positive for E. coli O157:H7 were from white-tailed deer (Odocoileus virginianus). We were unable to detect a statistically significant geographic distribution pattern of E. coli O157:H7. The presence of E. coli O157:H7 in the feces of free-ranging deer has implications not only for hunters, consumers of venison, and others in contact with deer or deer feces, but also for the development of strategies aimed at reducing and/or controlling this pathogen in water sources and domestic livestock.     

Characterization of an Escherichia coli O157:H7 plasmid O157 deletion mutant and its survival and persistence in cattle

Escherichia coli O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans, and its major reservoir is healthy cattle. An F-like 92-kb plasmid, pO157, is found in most E. coli O157:H7 clinical isolates, and pO157 shares sequence similarities with plasmids present in other enterohemorrhagic E. coli serotypes. We compared wild-type (WT) E. coli O157:H7 and an isogenic DeltapO157 mutant for (i) growth rates and antibiotic susceptibilities, (ii) survival in environments with various acidity, salt, or heat conditions, (iii) protein expression, and (iv) survival and persistence in cattle following oral challenge. Growth, metabolic reactions, and antibiotic resistance of the DeltapO157 mutant were indistinguishable from those of its complement and the WT. However, in cell competition assays, the WT was more abundant than the DeltapO157 mutant. The DeltapO157 mutant was more resistant to acidic synthetic bovine gastric fluid and bile than the WT. In vivo, the DeltapO157 mutant survived passage through the bovine gastrointestinal tract better than the WT but, interestingly, did not colonize the bovine rectoanal junction mucosa as well as the WT. Many proteins were differentially expressed between the DeltapO157 mutant and the WT. Proteins from whole-cell lysates and membrane fractions of cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Ten differentially expressed approximately 50-kDa proteins were identified by quadrupole-time of flight mass spectrometry and sequence matching with the peptide fragment database. Most of these proteins, including tryptophanase and glutamate decarboxylase isozymes, were related to survival under salvage conditions, and expression was increased by the deletion of pO157. This suggested that the genes on pO157 regulate some chromosomal genes.     

Role of colanic acid exopolysaccharide in the survival of enterohaemorrhagic Escherichia coli O157:H7 in simulated gastrointestinal fluids

AIM: This study evaluated the production of colanic acid (CA) exopolysaccharide (EPS) by Escherichia coli O157:H7 in relation to the pathogen's ability to survive under acidic conditions simulating the environment in the human gastrointestinal tract. METHODS AND RESULTS: Escherichia coli O157:H7 W6-13 and its CA-deficient mutant M4020 were examined for their resistance to bile salts, and their ability to survive in simulated gastric fluid containing pepsin (pH 2.0) and simulated intestinal fluid containing pancreatin (pH 8.0). The effect of acid adaptation at pH 5.5 on the survival of E. coli O157:H7 in simulated gastric fluid was also determined. The results indicated that the survivability of M4020, under conditions simulating the environment in the human gastrointestinal tract, reduced more drastically than the viability of W6-13. The presence of bile salts had a slight effect on both types of E. coli O157:H7 cells. The loss of CA did not change the ability of M4020 to respond to acid adaptation. CONCLUSION: The EPS CA may serve as a protective barrier to E. coli O157:H7 for its survival in the human gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: The study contributes to a better understanding of the EPS affecting the ability of E. coli O157:H7 to combat acid stress.     

Development and evaluation of a model predicting the survival of Escherichia coli O157:H7 NCTC 12900 in homemade eggplant salad at various temperatures, pHs, and oregano essential oil concentrations

Homemade eggplant salad, a traditional Greek appetizer, was inoculated with Escherichia coli O157:H7 NCTC 12900 supplemented with different concentrations of oregano essential oil (0.0, 0.7, 1. 4, and 2.1% [vol/wt]) and stored at different temperatures (0, 5, 10, and 15 degrees C). The product's pH was adjusted to 4.0, 4.5, or 5. 0 with lemon juice. For each combination of the environmental factors, the bacterial counts were modeled, using the Baranyi model, as a function of time to estimate the kinetic parameters of the pathogen. A reduction of more than 1 log unit in E. coli O157:H7 counts was observed in all cases, and the death rate depended on the pH, the storage temperature, and the essential oil concentration. Separate quadratic models were developed with natural logarithms of the shoulder period and death rate as estimated by the growth model, as a function of temperature, pH, and oregano essential oil concentrations. These were further used to predict the population of E. coli O157:H7 NCTC 12900 from other inoculated eggplant salads at random conditions of temperature, pH, and oregano oil concentration. The predicted values were compared with viable-count measurements for validation.     

The adaptive response of Escherichia coli O157 in an environment with changing pH

AIMS: To predict and validate survival of non-acid adapted Escherichia coli O157 in an environment mimicking the human stomach. METHODS AND RESULTS: Survival was predicted mathematically from inactivation rates at various, but constant pH values. Predictions were subsequently validated experimentally in a pH-controlled fermentor. Contrary to prediction, acid-sensitive cultures of E. coli O157 survived for a long period of time and died as rapidly as acid-resistant cultures. Experimental results showed that in an environment with changing pH, acid-sensitive cultures became acid-resistant within 17 min. Cyclo fatty acids was reported to be a factor in acid resistance. As synthesis of cyclo fatty acids does not require de novo enzyme synthesis and thus requires little time to develop, we analysed the membrane fatty acid composition of E. coli O157 during adaptation. No changes in membrane fatty acid composition were observed. CONCLUSIONS: Acid adaptation of E. coli O157 can occur during passage of the human gastric acid barrier, which can take up to 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of acid-adapted bacteria to survive the human stomach is an important virulence factor. The ability of non-acid adapted E. coli O157 to adapt within a very short period of time under extreme conditions further contributes to the virulence of E. coli O157.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7

AIMS: To determine the effects of outer membrane (OM) permeabilizing agents on the antimicrobial activity of enterocin AS-48 against Escherichia coli O157:H7 CECT 4783 strain in buffer and apple juice. METHODS AND RESULTS: We determined the influence of pH, EDTA, sodium tripolyphosphate (STPP) and heat on E. coli O157:H7 CECT 4783 sensitivity to enterocin AS-48 in buffer and in apple juice. Enterocin AS-48 was not active against intact cells of E. coli O157:H7 CECT 4783 at neutral pH. However, cells sublethally injured by OM permeabilizing agents (EDTA, STPP, pH 5, pH 8.6 and heat) became sensitive to AS-48, decreasing the amount of bacteriocin required for inhibition of E. coli O157:H7 CECT 4783. CONCLUSIONS: The results presented indicate that enterocin AS-48 could potentially be applied with a considerably wider range of protective agents, such as OM permeabilizing agents, with increased efficacy in inhibiting E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control E. coli O157:H7 in combination with other hurdles.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

Inactivation of Escherichia coli O157:H7 in simulated human gastric fluid

Human disease caused by Escherichia coli O157:H7 is a function of the number of cells that are present at potential sites of infection and host susceptibility. Such infectious doses are a result, in part, of the quantity of cells that are ingested and that survive human host defenses, such as the low-pH environment of the stomach. To more fully understand the kinetics of E. coli O157:H7 survival in gastric fluid, individual E. coli O157:H7 strains were suspended in various media (i.e., saline, cooked ground beef [CGB], and CGB containing a commercial antacid product [CGB+A]), mixed at various proportions with simulated human gastric fluid (SGF), and then incubated at 37 degrees C for up to 4 h. The highest inactivation rate among nine E. coli O157:H7 strains was observed in saline. Specifically, the average survival rates in 100:1 and 10:1 proportions of SGF-saline were -1.344 +/- 0.564 and -0.997 +/- 0.388 log(10) CFU/h, respectively. In contrast, the average inactivation rate for 10 E. coli O157:H7 strains suspended in 10:1 SGF-CGB was -0.081 +/- 0.068, a rate that was 12-fold lower than that observed for SGF-saline. In comparison, the average inactivation rate for Shigella flexneri strain 5348 in 100:1 and 10:1 SGF-saline was -8.784 and -17.310, respectively. These latter inactivation rates were 7- to 17-fold higher than those for E. coli O157:H7 strains in SGF-saline and were 4-fold higher than those for E. coli O157:H7 strains in SGF-CGB. The survival rate of E. coli O157:H7 strain GFP80EC increased as the dose of antacid increased from one-half to twice the prescribed dose. A similar trend was observed for the matrix pH over the range of pH 1.6 to 5.7, indicating that pH is a primary factor affecting E. coli O157:H7 survival in SGF-CGB+A. These results can be used in risk assessment to define dose-response relationships for E. coli O157:H7 and to evaluate potential surrogate organisms.     

Evolutionary silence of the acid chaperone protein HdeB in enterohemorrhagic Escherichia coli O157:H7

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli.     

Impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 populations to undercooking and simulated exposure to gastric fluid

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75 degrees C) water followed by lactic acid (2%, 55 degrees C), vacuum packaged, stored at 4 (28 days) or 12 degrees C (16 days), and periodically transferred to aerobic storage (7 degrees C for 5 days). During storage, samples were exposed to sequential heat (55 degrees C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4 degrees C was lower than that of cells on nondecontaminated meat stored at 12 degrees C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12 degrees C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4 degrees C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12 degrees C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.