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Applied and Environmental Microbiology, January 2006, p. 672-679, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.672-679.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Impact of Inoculum Preparation and Storage Conditions on the Response of Escherichia coli O157:H7 Populations to Undercooking and Simulated Exposure to Gastric Fluid

Jarret D. Stopforth,1 Panagiotis N. Skandamis,1 Laura V. Ashton,1 Ifigenia Geornaras,1 Patricia A. Kendall,2 Keith E. Belk,1 John A. Scanga,1 Gary C. Smith,1 and John N. Sofos1*

Department of Animal Sciences,1 Department of Food Science and Human Nutrition, Colorado State University, Fort Collins, Colorado 805232

Received 23 June 2005/ Accepted 1 November 2005


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ABSTRACT
 
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°C) water followed by lactic acid (2%, 55°C), vacuum packaged, stored at 4 (28 days) or 12°C (16 days), and periodically transferred to aerobic storage (7°C for 5 days). During storage, samples were exposed to sequential heat (55°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°C was lower than that of cells on nondecontaminated meat stored at 12°C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12°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°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°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.


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INTRODUCTION
 
Although decontamination interventions are used to treat carcass surfaces, limited work has been conducted to evaluate the fate of pathogens surviving in residual decontamination runoff fluids (washings [WASH]) and on subsequently recontaminated carcasses or meat. Research has demonstrated that Escherichia coli O157:H7 can survive for up to 14 days in a mixture (1:1) of lactic acid and water washings (23, 27, 28). Escherichia coli O157:H7 inoculated into lactic acid washings recovered directly from beef rinsing without diluting was reduced by ≥4 logs within 2 days of storage at 4 or 10°C (22, 23). In addition, Stopforth et al. (27, 28) indicated that E. coli O157:H7 populations were able to form biofilms on stainless steel coupons submerged in beef decontamination runoff fluids; these cells were more resistant to sanitizers than those suspended in the washings. The results also demonstrated that acid washings may alter the microbial ecology of meat plant environments (27, 28).

Exposure of cells in biofilms to stressful environments such as adverse temperature and pH, desiccation (low water activity), and sublethal concentrations of sanitizers associated with food processing may result in stress-hardened cells that may be able to survive subsequent antimicrobial treatments or processing stresses (21). Furthermore, detachment of even one cluster of such biofilms may recontaminate previously unadulterated or decontaminated product with sufficient organisms to comprise an infectious dose and as such enter the food supply (25-28). More importantly, previous exposure to adverse environmental and processing conditions may alter pathogen tolerance to subsequent consumer-related stresses including heat from cooking and acid upon consumption (21).

The ability of pathogens to survive in adverse conditions plays a crucial role in food-borne disease, and indeed the emergence of E. coli O157:H7 as a food-borne pathogen may be partly due to its increased acid tolerance (3, 17). A major requirement for food-borne pathogenicity is the ability of cells to survive the acidic gastrointestinal environment (10), and as such it is expected that pathogens with a high acid tolerance may have a lower infective dose and vice versa. An important concern relative to consumers is the potential for incomplete cooking of beef products, especially ground beef, where the pathogen may be introduced below the product surface and, as such, potentially survive inadequate cooking and increase the risk of food-borne illness. An additional concern is that incomplete cooking of contaminated product may result in selection of hardy pathogen cells which may have increased ability to survive in the acidic conditions encountered during consumption and digestion, especially among at-risk populations such as the elderly, the young, and the immunocompromised.

Therefore, the objective of this study was to determine the fate of E. coli O157:H7 cultures originating under different conditions (i.e., tryptic soy broth [TSB], WASH, moist biofilms [WETB], and dried biofilms [DRYB]), inoculated on fresh lean beef, and exposed to sequential hot (75°C) water and lactic acid (2%; 55°C) decontamination or left nondecontaminated, prior to vacuum and vacuum-to-aerobic storage and exposure to sequential heat (55°C) and simulated gastric fluid (pH 1.0, HCl) stresses.


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MATERIALS AND METHODS
 
Bacterial strains.
Four rifampin-resistant (100 µg/ml) derivatives of Escherichia coli O157:H7 (ATCC 43895, ATCC 43889, ATCC 51658, and EO139 [venison jerky isolate provided by M. P. Doyle, University of Georgia, Griffin]), developed in our laboratory according to procedures described by Hardin et al. (14) were used in this study. Stock cultures of each strain were maintained at –70°C in TSB (Difco, Becton Dickinson Co., Sparks, MD) containing 100 µg/ml of rifampin (Sigma Chemical Company, St. Louis, MO) and 20% glycerol (Mallinckrodt Baker, Inc., Paris, KY). Working cultures were stored on tryptic soy agar (TSA; Difco) slants containing 100 µg/ml rifampin (TSA+rif) at 4°C and were activated by transferring a loopful of each individual strain from TSA+rif slants to TSB without dextrose (TSB-G; Difco) containing 100 µg/ml rifampin and incubating at 35°C for 24 h. The activated strains were subcultured once in TSB-G supplemented with 100 µg/ml of rifampin to ensure that cultures retained their antibiotic resistance, as well as streaked on sorbitol MacConkey agar (Difco) supplemented with cefixime-tellurite (Dynal Inc., Lake Success, NY; SMAC-CT) and incubated at 35°C for 24 h. Colonies from the SMAC-CT plates were tested using the RIM E. coli O157:H7 agglutination test (Remel, Lenexa, KS) to confirm the presence of the O157 antigen. The four cultures obtained from subculturing were mixed and centrifuged at 4,629 x g (Eppendorf model 5810 R; Brinkmann Instruments Inc., Westbury, NY) for 15 min at 4°C. The resulting pellets were washed in 10 ml of sterile phosphate-buffered saline (PBS; pH 7.4; 0.2 g KH2PO4, 1.5 g Na2HPO4 · 7H2O, 8.0 g NaCl, and 0.2 g KCl in 1 liter distilled water) and centrifuged for a second time, and the final pellet was resuspended in 10 ml PBS and serially diluted in 0.1% buffered peptone water (BPW; Difco) to yield approximately 6 log CFU/ml. It should be noted that a composite of strains was used with the knowledge that strains may respond differently to stresses, and it is likely that research investigating stress responses of only one strain may not always provide the most accurate evaluation of the worst-case scenario; however, the potential for one or more strains to predominate in any inoculum preparation is possible and should be considered when interpreting results.

Inoculum preparation.
The four-strain composite culture was used to inoculate various substrates in order to prepare the inocula under representative environmental conditions (inoculum origin). The control inoculum (TSB) was obtained by inoculating (5 log CFU/ml) TSB-G with the composite E. coli O157:H7 culture (1 ml), and, after overnight incubation at 35°C, the resulting cell suspension was diluted (1,000-fold) in sterilized maximum-recovery diluent (MRD; 1.0 g peptone [Difco] and 8.5 g sodium chloride [Fisher Scientific, Fair Lawn, NJ] in 1 liter distilled water) (18).

To simulate pathogen populations potentially surviving in meat decontamination runoff fluids, spray-washing runoff fluids (washings) from decontamination of beef carcasses at a local commercial slaughtering plant using water (84°C at spraying) were collected and stored in 10-liter sterile bottles (Nalgene; Nalge Co., Rochester, NY) for ≤30 days at –30°C. The washings were thawed (at 4°C overnight) prior to use and were passed through four layers of cheesecloth three times to remove large beef tissue particles. Following initial filtering, the washings were passed through Fisherbrand filter paper (Qualitative P8; Fisher Scientific, Houston, TX) four times using a Buchner funnel under vacuum to remove remaining small beef particles. The resulting fluid was filter sterilized through 500-ml Stericups (0.22-µm GV Durapore membrane; Millipore Corporation, Bedford, MA) under vacuum. After preparation, the meat washings were plated on TSA to ensure sterility. Suspended populations in WASH were prepared by inoculating the E. coli O157:H7 composite (1 ml; 5 log CFU/ml) in 40 ml of the filtered meat washings and, after incubation at 35°C for 24 h, diluting the resulting cell suspension (1,000-fold) in sterilized MRD. Previous research (28) revealed that the presence of the natural flora in meat washings resulted in enhanced resistance of E. coli O157:H7 to the antimicrobial effects of the substrate; thus, in order to produce a monoculture inoculum, the washings were filtered to remove the natural flora in this study.

Pathogen cells simulating origination from meat decontamination runoff fluids and attached to meat processing equipment surfaces were prepared by submersing stainless steel (type 304, no. 2b finish, 0.08 mm thick) coupons (2 by 5 cm) in inoculated washings. Cleaned stainless steel coupons (23-25) were individually placed in an upright position in sterile centrifugation tubes (85 ml, 28.5-mm outside diameter by 104 mm long; Nalgene) containing 40-ml aliquots of the sterilized washings. The washings were then inoculated (5 log CFU/ml) with the composite E. coli O157:H7 culture (1 ml) and incubated at 35°C for 24 h.

Two types of biofilm cells were prepared based on conditions prior to harvesting of cells; namely, cells from DRYB and WETB. DRYB cells were obtained by subjecting the stainless steel coupons to air drying for 12 h after removal from the overnight incubation in washings and prior to harvesting, while WETB cells were harvested immediately after incubation without a drying step. Cells from WETB and DRYB were harvested from the stainless steel by placing coupons in a centrifuge tube containing 45 ml of sterile MRD and 10 glass beads (4 mm in diameter; Fisher) to aid in removal of attached cells. The tube was then vortexed (3,200 rpm; Vortex-Genie 2; Scientific Industries Inc., Bohemia, NY) for 2 min to remove cells attached to coupons, after which the coupons were rinsed with 1 ml sterile distilled water to collect cells loosely associated with the coupon before the process was repeated with four more coupons in the same tube in an effort to concentrate cells. Counts on TSA+rif after incubation but prior to dilution for inoculation of beef pieces were 8.1, 8.7, 5.3, and 4.5 log CFU/cm2 for TSB, WASH, WETB, and DRYB inocula, respectively. These high cell populations determined on TSA+rif originated from sterile substrates inoculated with rifampin-resistant cultures in the absence of added rifampin. This indicated that the inocula maintained their resistance (5, 16) and could be used to inoculate meat to be stored at 4 and 12°C, even without added rifampin, in order to avoid changes in the substrate.

Inoculation and decontamination of fresh lean beef.
For each of two separate replicates conducted in this study, fresh untreated beef top rounds were obtained from a local commercial slaughtering plant, stored at 4°C, and used within 72 h postmortem. For use in the studies, the beef was cut into 5- by 2.5- by 1-cm (total surface area of 40 cm2) pieces. Separate beef pieces were inoculated with 200 µl of the TSB, WASH, WETB, or DRYB inocula on one side, and cells were allowed to attach for 15 min at 4°C before the same procedure was repeated on the reverse side. For inoculation, the original TSB and WASH cultures were diluted 1,000-fold, while the WETB and DRYB cultures were directly inoculated following concentration of the cultures as described in the section above. Inoculum levels (TSA+rif) achieved on meat were 3.0, 3.9, 2.8, and 1.6 log CFU/cm2 for TSB, WASH, WETB, and DRYB inocula, respectively. The levels of the pathogen from TSB and WETB inocula approached the target (3 log CFU/cm2), whereas WASH resulted in a level approximately 1 log CFU/cm2 higher than the target and the DRYB level was limited due to cell injury caused by the drying process (our unpublished data).

Following inoculation, the meat was either left untreated or exposed to sequential decontamination treatments (decontaminated) comprising hot water (distilled, 75°C) for 30 s followed by 2% lactic acid (55°C, pH 2.14) (Mallinckrodt Baker, Inc.) for 30 s. The treatments were applied by dipping 20 beef pieces in 1 liter of each fluid. After application of the decontamination treatments, individual beef pieces were placed into sterile 24-oz Whirl-Pak filter bags (Nasco, Fort Atkinson, WI), and samples were either analyzed immediately or placed in an additional vacuum bag (15 by 20 cm, 3-mil standard barrier, nylon/polyethylene vacuum pouch; Koch, Kansas City, MO), vacuum sealed (Hollymatic Corp., Countryside, IL), and stored at 4 (for 28 days) or 12°C (for 16 days). Samples from each inoculum (TSB, WASH, WETB, or DRYB) and treatment (untreated or decontaminated) were analyzed on day 0; days 7, 14, and 28 of storage at 4°C; and days 4, 8, and 16 of storage at 12°C. The number of samples analyzed at each time and for each inoculum type were (i) three samples for time-zero analysis, (ii) three samples for 20 min of heat exposure, and (iii) three samples for 20 min of heat exposure and subsequent acid exposure (90 min) (Fig. 1).


Figure 1
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  		FIG. 1. Diagrammatic layout detailing (A) inoculation of untreated meat with a four-strain composite of Escherichia coli O157:H7 prepared under different environmental conditions, TSB, WASH, WETB, or DRYB, left untreated, or decontaminated by sequential dipping (30 s each) in hot water (75°C) and lactic acid (2%, 55°C), followed by vacuum packaging and storage, and (B) exposure to heat (55°C; 20 min) followed by simulated gastric fluid (pH 1.0; 90 min) of samples of the above treatments after vacuum or aerobic storage.

Transfer of vacuum-packaged refrigerated beef to aerobic storage.
Triplicate samples were removed from 4°C vacuum storage at days 7 and 14 and from 12°C vacuum storage at days 4 and 8, placed on retail foam trays (7.5 by 12.5 cm; Pactiv, Lake Forest, IL) covered with air-permeable film (Omnifilm; Pliant Corporation, Uniontown, OH), and stored at 7°C for 5 days. When samples were removed from aerobic storage, they were placed into a sterile 24-oz Whirl-Pak filter bag (Nasco) for microbiological analyses or were sealed in vacuum bags (Koch), as described above, in preparation for further stress exposure.

Exposure of fresh beef to heat.
In order to assess the effect of exposure to sublethal heat or incomplete heating, samples sealed in vacuum bags were submersed for 20 min in a 55°C water bath (Isotemp 228; Fisher). Two sets of samples representing each inoculum type by treatment combination were submersed. One set of samples was submersed for 20 min and analyzed microbiologically immediately after heating to determine the survival of bacterial populations on the beef. The second set of samples was also submersed in the 55°C water bath for 20 min, and then it was exposed to a simulated gastric fluid (Fig. 1). In all cases, bags removed from the water bath were immediately placed on ice to stop the heat treatment.

Exposure of beef to simulated gastric fluid.
To assess the susceptibility of E. coli O157:H7 to inactivation at a low pH, similar to the stomach environment, use was made of a simulated gastric fluid (4, 9). The simulated gastric fluid in this experiment was adjusted to pH 1.0 using HCl (analytical reagent, approximately 37%; Mallinckrodt). To test the acid tolerance of E. coli O157:H7 populations surviving on beef pieces previously submersed in water (55°C; 20 min), 50 ml of simulated gastric fluid (GF; 25°C) was added to the bags containing the samples followed by pummeling, using a Masticator (IUL Instruments, Barcelona, Spain), for 2 min. Although 37°C may have been more representative of body temperature to simulate consumption, sampling during acid exposure was performed at ambient temperature. Samples were exposed to GF for 90 min, and at 30-min intervals aliquots of the samples were removed from the bags for determination of survivors (Fig. 1).

Determination of populations on inoculated samples that were not exposed to heat (55°C) or GF served as controls, or as time zero of exposure, to determine initial populations. Control samples received 40 ml of sterilized MRD, as it allowed for a more sensitive detection level compared to the 50 ml of GF (at pH 1.0), which was selected based on preliminary investigations to achieve a pH simulating the stomach (approximately pH 2) after addition of the GF to beef samples.

Microbiological and physical analyses of samples.
For microbiological analysis of time-zero (initial level) and 20-min (heat-treated) samples, 40 ml of sterilized MRD was added to the Whirl-Pak bag (Nasco) containing the beef pieces and homogenized (Masticator; IUL Instruments) for 2 min. The GF-treated samples were analyzed directly from the bag containing GF. A portion (1 ml) of the homogenized samples was serially diluted in 9 ml 0.1% buffered peptone water, and appropriate dilutions were plated onto TSA for determination of total bacterial counts and onto TSA+rif for the selective enumeration of inoculated rifampin-resistant E. coli O157:H7 populations. Colonies were counted after incubation at 35°C for 48 h. The detection limit was estimated as 0 log CFU/cm2 for time-zero (initial level) and 20-min (heat-treated) samples, based on the assumption that plating 1 ml of the initial sample homogenate could yield at least 1 CFU on the agar plate. The detection limit for the GF-treated samples was estimated as 0.1 log CFU/cm2 (log 1.25 CFU/cm2) based on the same procedure described above, except for the dilution created by homogenizing beef with a surface area of 40 cm2 in 50 ml of GF (log [1 CFU ml–1 x 50 ml/40 cm2]). Considering the count of 25 CFU per plate to be the minimum acceptable, the detection limit would be 1.5 log CFU/cm2; however, enumeration below this level in this study was included to provide an estimation of populations recovered.

The pH of homogenized control samples was measured after each microbiological analysis, while that of samples exposed to GF was randomly measured after the 90-min exposure period due to time constraints. The pH was measured using a digital pH meter (UltraBasic, UB-10; Denver Instrument, Arvada, CO) with a glass pH electrode (Denver Instrument). Partial biochemical characterization of randomly selected colonies on countable agar plates was performed by observing colony morphology and testing for Gram, catalase, and oxidase reactions (28).

Statistical analysis.
Two replicate experiments (different meat and cultures) were conducted with three samples tested per treatment at each sampling time in each replicate. Microbiological data were converted to log CFU/cm2 before being analyzed. Values for the mean log and standard deviation of each set of bacterial counts were calculated on the assumption of a log-normal distribution of microbial counts. Data were separated by storage temperature, as the days of analyses were different based on the expected growth rates at different temperatures, and thus they were not compared statistically. For the 12°C data set, analysis of fixed effects using the GLM procedure of SAS version 8.2 (24) indicated that log CFU/cm2 populations were dependent on type of medium (TSA or TSA+rif) (F statistic = 126.16; P < 0.0001), inoculum type (TSB, WASH, WETB, or DRYB) (F statistic = 49.25; P < 0.0001), treatment (untreated or decontaminated) (F statistic = 3,073.16; P < 0.0001), day of analysis (0, 4, 8, 9, 13, or 16) (F statistic = 301.25; P < 0.001), and time of analysis (time zero and 20 [heat], 30 [GF], 60 [GF], or 90 min [GF]) (F statistic = 684.96; P < 0.0001). For the 4°C data set, analysis of fixed effects using the GLM procedure of SAS indicated that log CFU/cm2 populations were dependent on type of medium (TSA or TSA+rif) (F statistic = 273.61; P < 0.0001), inoculum type (TSB, WASH, WETB, or DRYB) (F statistic = 27.00; P < 0.0001), treatment (untreated or decontaminated) (F statistic = 643.87; P < 0.0001), day of analysis (0, 7, 12, 14, 19, or 28) (F statistic = 9.04; P < 0.001), and time of analysis (time zero and 20 [heat], 30 [GF], 60 [GF], or 90 min [GF]) (F statistic = 815.06; P < 0.0001). Data for each temperature of incubation were thus further separated for each medium by treatment combination, and data regarding viable populations were evaluated using a 4 by 6 by 5 (inoculum type, day of analysis, time of analysis, respectively) factorial design. For each medium-by-temperature combination, individual fixed effects and up-to-three-way interactions were evaluated with analysis of variance using the model y = x1 + x2 + x3 + x1x2 + x1x3 + x2x3 +x1x2x3 in the GLM procedures of SAS, where x1 represents inoculum type, x2 represents day of analysis, and x3 represents time of analysis. Least-square means were separated using a protected pairwise t test of SAS. All differences were reported at a significance level of a P value of 0.05.


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RESULTS AND DISCUSSION
 
In the present study, fresh beef was contaminated with E. coli O157:H7, decontaminated with hot water and lactic acid, stored in vacuum packages at 4 or 12°C before being transferred to aerobic storage, and exposed to heat and subsequently to acid. The application of heat in this study was selected to simulate incomplete cooking (undercooking) of beef that could subsequently be exposed to acidic conditions representing ingestion by an individual. Exposure of food to heat is one of the oldest methods of preservation or preparation for consumption (6). There are several different methods to cook meat, and although the main objective is to achieve a specific internal temperature, the type of product, the rate and type of heating, the type of equipment used, and several other factors may affect the cooking process (1). Furthermore, various factors may influence the heat and/or acid resistance of microorganisms such as their previous environment (i.e., inoculum preparation), metabolic phase, storage temperature, and nutrient availability (20). Most food-borne outbreaks involving infection with E. coli O157:H7 through consumption of hamburger meat are thought to be due to undercooking (7). The potential for E. coli O157:H7 to survive cooking and mount an acid tolerance response under the acidic conditions faced in the human digestive tract is important as it may have severe public health consequences.

The initial pH of the nondecontaminated meat inoculated with E. coli O157:H7 ranged from 4.99 to 5.60 and that of the decontaminated meat from 4.74 to 5.47. Although the pH of the meat samples generally increased during storage, increases did not appear to be substantial (data not shown). The pH of the meat homogenate exposed to simulated gastric fluid for 90 min ranged from 1.76 ± 0.13 to 2.23 ± 0.22 (data not shown).

In general, the recovery of bacterial populations from fresh beef tissue after exposure to stresses, during storage, was up to 2 log CFU/cm2 higher (P < 0.05) in certain cases with general growth media (TSA) (data not shown) than on the selective medium (TSA+rif) (initial levels in Tables 1 to 4). The higher (up to 2 log cycles) levels of total bacterial populations (TSA) during storage, especially on nondecontaminated beef, compared with the pathogen populations (TSA+rif) were most likely due to increases in psychrophilic/psychrotrophic natural flora associated with fresh beef. Due to the predominance of natural flora on TSA plates, data regarding changes in pathogen populations on beef during storage and survival following exposure to heat and acid stress are discussed with reference to TSA+rif counts (Tables 1 to 4). Rifampin-resistant strains were used in this study to specifically distinguish the responses of the inoculated pathogen from that of the natural flora. It should be noted that initial laboratory investigation was conducted to determine if rifampin-resistant strains maintained their resistant properties during prolonged incubation at a temperature (i.e., 15°C) unfavorable for E. coli O157:H7 growth. Results revealed that resistant strains inoculated (at approximately 1.5 log CFU/ml) into TSB without rifampin and incubated at 15°C grew to 2.7, 3.6, and 7.3 log CFU/ml by days 2, 4, and 7, respectively, on TSA and to 2.6, 4.0, and 7.1 log CFU/ml, respectively, on TSA+rif, indicating maintenance of their rifampin-resistant properties under an unfavorable condition.


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  		TABLE 1. E. coli O157:H7 (TSA+rif) populations inoculated onto untreated fresh beef, stored under vacuum at 4°C for 28 days, and periodically transferred to aerobic storage at 7°C for 5 days


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  		TABLE 4. E. coli O157:H7 (TSA+rif) populations inoculated onto fresh beef which was then decontaminated, stored under vacuum at 12°C for 16 days, and periodically transferred to aerobic storage at 7°C for 5 days

Decontamination of fresh beef inoculated with TSB, WASH, WETB, and DRYB cultures resulted in pathogen reductions of 1.2, 2.5, 2.5, and 0.8 log CFU/cm2, respectively, compared to populations on untreated beef (Tables 1 to 4). There was no obvious trend in pathogen reduction by decontamination; however, it appeared that, if the reductions are expressed as percentages compared to initial levels, TSB and DRYB cultures survived better than WASH and WETB cultures. This observation may be explained by the extremes of stress conditions faced by the two populations. Populations prepared in TSB may have faced the least amount of stress compared to populations prepared in nutrient-limited washings, those detached from stainless steel, and those exposed to desiccation. Thus, TSB cultures may have been more tolerant of decontamination than WASH and WETB cultures. On the other hand, DRYB cultures faced stress conditions of nutrient limitation, attachment, desiccation, and subsequent detachment of cells, which may have allowed such cells to resist the stress of decontamination better than WASH or WETB cultures.

In this study, it appeared that pathogen cultures prepared in TSB and in WASH cultures were, in general, more resistant to heat and acid stresses than those grown as biofilms and detached prior to inoculation (Tables 1 to 4). It should be noted that cells from WETB cultures were always more resistant to stresses than those from DRYB cultures, which may have been due to lower initial levels of DRYB populations exposed to stresses and increased sensitivity of DRYB cultures in comparison with all other cultures due to the severe desiccation and starvation stresses faced during preparation of this inoculum (Tables 1 to 4). Although in some cases the lower heat and acid tolerance of cells derived from biofilms may have been due to lower initial counts than those from TSB or WASH inocula, this observation was not consistent for all conditions studied.

Cells from TSB and WASH cultures inoculated on meat samples that were untreated and stored at 4°C were consistently more (P < 0.05) resistant to heat and acid stresses than those originating from WETB and DRYB cultures (Table 1). In this case it appears that higher initial pathogen levels from TSB and WASH inocula were responsible for increased resistance to stresses compared to those derived from a biofilm. Furthermore, it was apparent that inocula originating from TSB were more resistant (P < 0.05) to heat and acid than those from WASH (Table 1). Ultimately, however, storage of beef at 4°C contributed positively to inactivation of cells during heat and acid exposure, and this was probably due to the lack of growth of the pathogen at 4°C (Table 1). When cells originating from decontaminated meat stored at 4°C were exposed to the heat and acid stresses, low levels of survivors of TSB and WASH origin were periodically obtained following the heat treatment (Table 2). These surviving cells, however, were completely inactivated following only 30 min of exposure to GF. Thus, decontamination, which reduced initial levels of the pathogen, and storage of meat at 4°C, which prevented growth of surviving populations, enhanced their inactivation when exposed to the heat and acid stresses.


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  		TABLE 2. E. coli O157:H7 (TSA+rif) populations inoculated onto fresh beef, which was then decontaminated, stored under vacuum at 4°C for 28 days, and periodically transferred to aerobic storage at 7°C for 5 days

In contrast to results obtained for inoculated cell cultures on meat stored at 4°C, growth of the pathogen during storage at 12°C, under vacuum, on nondecontaminated meat was accompanied by increased heat and acid tolerance, pointing to development of a resistance to stresses over time (Tables 1 and 3). In general, heat exposure (55°C; 20 min) of cell cultures on meat stored for 16 days at 12°C, under vacuum, did not (P > 0.05) reduce pathogen levels compared to cells stored on meat for 4 or 8 days (Table 3). Furthermore, pathogen populations on meat stored for 8 and 16 days at 12°C were more resistant to exposure to GF than populations at similar initial pathogen levels on meat stored for only 4 days. This raises potential safety concerns for the risk of E. coli O157:H7 contamination if temperature abuse is not prevented and subsequently consumers undercook the product.


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  		TABLE 3. E. coli O157:H7 (TSA+rif) populations inoculated onto untreated fresh beef, stored under vacuum at 12°C for 16 days, and periodically transferred to aerobic storage at 7°C for 5 days

In contrast to the trend observed with cell cultures on product stored at 4°C, pathogen populations on meat inoculated with WETB cultures and stored at 12°C were more resistant to sequential stresses than, or as resistant as, cultures from TSB and WASH (Tables 1 and 3). This may be due to the fact that WETB cultures were formed under stressing conditions associated with biofilm formation and as such were able to generate populations more resistant to stresses than those from TSB or WASH. This would not have been observed at 4°C, where the pathogen was unable to replicate.

Decontamination not only reduced levels of the cells attached to beef immediately following its application but also inhibited growth at 12°C (Table 4). Moreover, decontamination reduced the heat and acid tolerance of the pathogen, which may have been a result of reduced population levels exposed to the stresses. These findings support the use of decontamination of fresh beef with hot water and lactic acid. These decontamination treatments not only resulted in an immediate decrease in pathogen levels but may have also caused cell injury as indicated by the lack of subsequent growth of surviving populations or increases of only up to 1.5 log CFU/cm2 after 16 days of storage at 12°C and also decreased the pathogen's resistance to heat and acid conditions. In almost all cases, regardless of inoculum history and day of storage, E. coli O157:H7 on decontaminated beef was reduced to <0.1 log CFU/cm2 when exposed to sequential heat (55°C) and acid (pH 1.0) conditions (Table 4).

An important phenomenon identified in this study was the remarkable reduction in pathogen populations on decontaminated, and especially on nondecontaminated, beef upon transfer from anaerobic storage in vacuum packages at 12°C to aerobic storage in retail packages at 7°C for 5 days (Tables 3 and 4). Moreover, the transfer of beef from anaerobic conditions during storage at 12°C to aerobic conditions resulted in a decrease in the pathogen's heat and acid tolerance (Tables 3 and 4). It is hypothesized that the sensitization and reduction of the E. coli O157:H7 cells brought about by the transfer of meat from a low-oxygen environment to an oxygen-rich environment may be due to oxidative damage of cells (2). Considering that there is a reduction of the pathogen in the absence of the adverse heat and acid stresses, sensitization is not dependent on the inimical processes of heat and acid. Pathogen decreases may be explained by self-destruction brought about when the exponentially growing cells (12°C) were transferred from the low-oxygen vacuum packages to the oxygen-rich retail packages (2). This finding was not observed in beef stored at 4°C, indicating that cells not actively growing may be unaffected by the oxidative damage brought about by transfer of meat from vacuum to aerobic storage conditions. Furthermore, investigations using laboratory media (12, 13) have indicated that the heat resistance of E. coli O157:H7 was higher when grown under anaerobic conditions compared to that when grown under aerobic conditions, indicating that low-oxygen environments may protect cells against stress responses. Recent work suggests that expression of the general stress response master regulator (RpoS) is activated under aerobic environments when glucose availability is limited (15, 19). Furthermore, expression of PoxB, an E. coli pyruvate oxidase that catalyzes decarboxylation of pyruvate to acetate and CO2 (8, 11), is known to be dependent on RpoS and plays an important role in the transition between anaerobic and aerobic environments (8). However, since RpoS expression is not favored in anaerobic environments with limited glucose (nutrient) availability, such as meat in this experiment, this would imply that PoxB expression is not favored, and hence the transition between anaerobic and aerobic storage may not have favored the pathogen.

In summary, storage of beef inoculated with E. coli O157:H7 at chill temperatures, such as 4°C, may cause cells to become resistant to potential stresses faced by transfer from vacuum storage at 4°C to aerobic storage at 7°C. Temperature abuse of vacuum-packaged beef, as opposed to vacuum-packaged beef transferred to aerobic storage prior to cooking and consumption, may pose a higher risk of infection to consumers. In this study, growth of E. coli O157:H7 on whole beef muscle stored under temperature abuse conditions, such as 12°C, resulted in an increase in the heat and acid tolerance of the pathogen, making it a potential food safety risk for consumers not properly cooking whole-muscle cuts. Under the conditions of this study, planktonic pathogen cells from incubation in tryptic soy broth and meat decontamination runoff fluids were, in general, more resistant than those grown as biofilms and detached prior to inoculation of fresh beef exposed to heat and acid stresses. Decontamination of fresh beef resulted in cell reductions and injury that inhibited subsequent growth of the surviving populations, even at 12°C, as well as in a decrease in the pathogen's resistance to subsequent heat and acid conditions. Transfer of temperature-abused (12°C) meat contaminated with E. coli O157:H7 from a low-oxygen environment to an oxygen-rich environment resulted in potential oxidative damage that triggered self-destruction of the cells. This observation implies that temperature abuse of vacuum-packaged beef may pose a higher risk to consumers than vacuum-packaged beef shifted to aerobic storage prior to cooking and consumption. Overall, heat and acid tolerance of E. coli O157:H7 may be influenced by inoculum history and to a greater extent by temperature of storage and transfer of beef from anaerobic conditions in vacuum packages to aerobic conditions in retail packages.


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ACKNOWLEDGMENTS
 
This study was supported by the United States Department of Agriculture Cooperative State Research, Education and Extension Service and by the Colorado State University Agricultural Experiment Station.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Animal Sciences, Colorado State University, 1171 Campus Delivery, Fort Collins, CO 80523-1171. Phone: (970) 491-7703. Fax: (970) 491-0278. E-mail: John.Sofos{at}colostate.edu Back


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Applied and Environmental Microbiology, January 2006, p. 672-679, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.672-679.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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