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Applied and Environmental Microbiology, February 2008, p. 1111-1116, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01292-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Behavior of Bacillus anthracis Strains Sterne and Ames K0610 in Sterile Raw Ground Beef{triangledown}

Mark L. Tamplin,1,2* Robert Phillips,3 Tod A. Stewart,1 John B. Luchansky,1 and Lynda C. Kelley4

Microbial Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038,1 Australian Food Safety Centre of Excellence/University of Tasmania, Hobart, Tasmania 7001, Australia,2 Microbiology Division, Russell Research Center, Food Safety Inspection Service, Office of Public Health and Science, United States Department of Agriculture, 950 College Station Road, Athens, Georgia 30605,3 Office of Food Defense and Public Health Response, Richard Russell Research Center, Food Safety Inspection Service, United States Department of Agriculture, 950 College Station Road, Athens, Georgia 306054

Received 11 June 2007/ Accepted 23 November 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The behavior of Bacillus anthracis Sterne spores in sterile raw ground beef was measured at storage temperatures of 2 to 70°C, encompassing both bacterial growth and death. B. anthracis Sterne was weakly inactivated (–0.003 to –0.014 log10 CFU/h) at storage temperatures of 2 to 16°C and at temperatures greater than and equal to 45°C. Growth was observed from 17 to 44°C. At these intermediate temperatures, B. anthracis Sterne displayed growth patterns with lag, growth, and stationary phases. The lag phase duration decreased with increasing temperature and ranged from approximately 3 to 53 h. The growth rate increased with increasing temperature from 0.011 to 0.496 log10 CFU/h. Maximum population densities (MPDs) ranged from 5.9 to 7.9 log10 CFU/g. In addition, the fate of B. anthracis Ames K0610 was measured at 10, 15, 25, 30, 35, 40, and 70°C to compare its behavior with that of Sterne. There were no significant differences between the Ames and Sterne strains for both growth rate and lag time. However, the Ames strain displayed an MPD that was 1.0 to 1.6 times higher than that of the Sterne strain at 30, 35, and 40°C. Ames K0610 spores were rapidly inactivated at temperatures greater than or equal to 45°C. The inability of B. anthracis to grow between 2 and 16°C, a relatively low growth rate, and inactivation at elevated temperatures would likely reduce the risk for recommended ground-beef handling and preparation procedures.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Since the terrorist attacks of 2001, risk managers have recognized that the food system may be vulnerable to attack with biological agents not usually considered hazards to the food supply. This awareness has led to an increased need to understand how atypical food-borne pathogens behave in different food matrices, including foods whose intentional contamination could lead to widespread human exposure. Bacillus anthracis is an atypical food-borne pathogen with a high level of interest as a threat agent. This gram-positive facultative anaerobic spore-forming bacterium has been used in acts of terrorism and is considered to be a potential agent of bioterrorism and biowarfare against civilian populations (5, 16).

Anthrax presents three clinical forms in human infection: inhalation, cutaneous, and gastrointestinal. Contamination of food would likely lead to cutaneous and/or gastrointestinal anthrax. Gastrointestinal anthrax, which is seen clinically as either intestinal or oropharyngeal anthrax, has been described in persons eating undercooked meat (10). The mortality rate for gastrointestinal anthrax can be greater than 50%, a rate that is likely exacerbated by misdiagnoses at early onset of the disease. Most cases have been reported in Africa, central and Southeast Asia, and the Middle East, areas of the world where anthrax is endemic in food animals (1, 7, 15). Cases in the United States are rare due to continuous animal vaccinations, animal inspections, and postslaughter carcass inspections, which enhance the safety of the food supply. These practices would not detect intentional contamination of postslaughter and processed food.

While various studies have determined the viability of B. anthracis spores after exposure to different environmental conditions, such as freezing, heating, drying, and changes in pH, such research has most often been conducted with laboratory media or with buffers and may not accurately represent organism behavior in marketplace foods. Such differences have been illustrated for a variety of pathogens in food. For example, Escherichia coli O157:H7 does not display a lag phase when grown in raw ground beef at 10°C, whereas in microbiological broth, it has a lag phase of greater than 48 h (19).

In the present study, we examined the fate of B. anthracis in raw ground beef, a food that is produced in bulk and consumed by millions of people each day. We hypothesize that B. anthracis behavior differs from that of more common pathogens associated with raw ground beef. The resulting information and models provide risk managers with greater certainty in estimating the impact of B. anthracis on food safety.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacteria.
B. anthracis Sterne (avirulent) and Ames K0610 (virulent) were selected for these studies. The Sterne strain was produced in 1937 and lacks the pXO2 plasmid that encodes the poly-{gamma}-D-glutamic acid capsule (21, 22). The Ames K0610 strain possesses both the pXO1 and pXO2 virulence plasmids. Spores of B. anthracis Sterne and Ames K0610 strains were produced essentially as described by Novak et al. (14) and provided to the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service Microbial Food Defense and Emergency Response Branch, Athens, GA, by the U.S. Army Dugway Proving Grounds, UT. Both stock preparations were suspended in sterile deionized water. B. anthracis Sterne was shipped in a refrigerated container to the Eastern Regional Research Center (ERRC), Agricultural Research Service, USDA, from the Food Safety and Inspection Service-Microbial Outbreaks and Special Projects Laboratory (MOSPL) by overnight courier. After arrival, the preparation was portioned into 10-ml aliquots and stored at 5°C for up to 3 months. The aliquots were tested initially and at approximately weekly intervals to measure the concentrations of spores and vegetative cells. This was done by heating the suspension at 75°C for 20 min and comparing counts on tryptic soy agar (TSA) (Difco Laboratories, Detroit, MI) to those of an unheated spore preparation. The spore preparations were also examined by phase-contrast microscopy to measure the proportion of refractile (spores) and nonrefractile (germinated) cells. Although brain heart infusion (BHI) agar (Difco) is commonly used to enumerate B. anthracis, we cultured the cells on TSA after determining that there were no significant differences between the media. Experiments with B. anthracis Sterne and Ames K0610 were conducted at the ERRC and MOSPL facilities, respectively.

Ground beef.
The ERRC and MOSPL facilities used two different batches of ground beef (batches A and B, respectively) that were purchased from local retail outlets in Pennsylvania and Georgia, respectively. After purchase, the ground-beef samples were separated into 90-g portions, placed in 400-ml-capacity stomacher bags (Koch Industries, Kansas City, MO), vacuum sealed at 105 Pa, frozen, shipped to a commercial irradiation facility, and sterilized with 42 kGy of ionizing irradiation from a Cs137 source. After irradiation, the frozen samples were shipped to the respective laboratories and stored at –20°C for up to 3 months. Proximate composition analyses were conducted with both batches of ground beef by a commercial food-testing laboratory.

Inoculation and incubation of B. anthracis spores in raw ground beef.
Prior to experimentation, bags of sterile ground beef were thawed overnight at 5°C or at room temperature (~25°C) for ~1 to 2 h. After the thawing, the refrigerated spore preparations were diluted in 10-fold serial increments with sterile peptone water (PW) (0.1% proteose peptone [Difco] in deionized water). The dilutions were used to enumerate the spore preparation and to prepare the inoculum. Next, 10 ml of the appropriate spore dilution were added to a stomacher bag containing 90 g of sterile ground beef. The inoculum was mixed into the ground beef by external hand massage and then stomached for 2 min. Next, 3-g portions of the inoculated ground beef were transferred to 100-ml-capacity stomacher bags (Spiral Biotech, Norwood, MA). The stomacher bags were vacuum sealed at 9.50 x 104 Pa (Multivac A300/16 vacuum-packaging unit; Sepp Haggemüller KG, Wolfertschwenden, Germany). For some experiments, the spore inoculum was heated at 75°C for 20 min before the ground-beef samples were inoculated to compare the behavior of heat-shocked and non-heat-shocked spores.

For experiments with the Sterne strain, the inoculated 3-g ground-beef samples were stored at 2, 5, 8, 10, 12, 14, 15, 16, 17, 18, 20, 22, 25, 30, 35, 37, 40, 42, 43, 44, 45, 50, 60, and 70°C and at 10, 15, 25, 30, 35, 40, and 70°C for the Ames strain. In independent experiments, the effect of the difference in fat content between the two batches of ground beef on their growth was measured at 20°C using the Sterne strain. In another independent experiment, growth kinetics were compared in vacuum-packaged and opened (i.e., loosely taped) packages of ground beef at 20 and 37°C.

Each incubator was fitted with a Dickson model FT121 or D100 temperature data recorder (Dickson, Addison, IL) attached to a thermocouple. The temperature recorder was calibrated against a National Institute of Standards and Technology-certified thermometer (ERTCO Precision Ever Ready Thermometer Co., West Patterson, NJ).

Sampling.
At each time interval, two bags containing 3-g samples of inoculated ground beef were removed from the incubator. A minimum of two independent experiments were performed for each test condition. For studies conducted at >45°C, the samples were immediately placed in an ice water slurry to reduce thermal inactivating effects prior to sampling. Twenty-seven milliliters of PW was added to the 3-g ground-beef samples, and the contents were stomached for 1 min. Next, 50 µl of the whole stomached sample and/or 10-fold serial PW dilutions was plated in duplicate on TSA. The TSA plates were incubated overnight at 37°C or, in a few instances, at room temperature (~25°C), and the CFU were measured using an image capture system (Protocol; Synbiosis, Frederick, MD) or by manual counting. Data were transferred to an Excel spreadsheet (Microsoft Corp., Redmond, WA) for computational analyses.

All of the experiments were conducted with sterile raw ground beef, eliminating the inhibitory effects of the native ground-beef microbial flora. In some experiments, variation in colony morphology was noted at lower storage temperatures. In order to confirm the identities of these colonies, representative colonies were tested with an API 50 CHB/E test strip by following the manufacturer's instructions (bioMérieux, Inc., Durham, NC). In brief, colonies were picked with a sterile loop and streaked on TSA to produce a bacterial lawn. The plates were incubated overnight at 37°C, the surface growth was swabbed, and then the cell density was adjusted with 0.85% NaCl to a McFarland 2 turbidity standard. The suspension was added to the API CHB/E cupules, and the strip was incubated at 37°C. The results were recorded at 24 and 48 h. We also observed the morphology of colonies on Polymyxin B-lysozyme-EDTA-Thallous acetate (PLET) agar and TSA supplemented with 5% sheep blood (Gibson Laboratories Inc., Lexington, KY). PLET is a selective medium for B. anthracis, and sheep blood agar is used to measure hemolysis.

Modeling techniques.
DMFit software (courtesy of J. Baranyi, Institute of Food Research, Norwich, United Kingdom) was used to fit the D model (2, 3) to the kinetic data. The D model was used to estimate the lag phase duration (LPD [h]), growth rate (GR [log10 CFU/h]), and maximum population density (MPD [log10 CFU/g]). The model was also used to fit inactivation kinetics, where "shoulders" are referred to as LPD, linear reduction as negative GR, and "tails" as MPD. Secondary models were produced using TableCurve 2D (SPSS Inc., Chicago, IL) containing preset and customized algorithms.

Statistical tests.
Student's t test was used to measure differences among the means at a 95% confidence level. Mean separations were performed using the pairwise least significant difference method.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Proximate analysis of ground-beef samples.
Ground-beef samples were tested for levels of moisture, protein, fat, ash, and carbohydrate. The primary differences between ground beef batches A and B were in moisture content (68.7 versus 57.7%, respectively) and fat (11.1 versus 25.5%, respectively) (Table 1). The average pH of the ground beef was 5.9 (batch A, pH 5.8; batch B, pH 6.0). Previous studies had shown that higher levels of fat in ground beef resulted in a lower MPD and a lower GR for E. coli O157:H7 (19). However, this earlier finding did not extend to B. anthracis, as described below.


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  		TABLE 1. Proximate analysis of ground-beef samples

 
Growth of B. anthracis Sterne and Ames K0610 in sterile raw ground beef.
B. anthracis replicated in sterile raw ground beef at storage temperatures of 17 to 44°C (Table 2). This temperature range is narrower than that reported in a previous study, in which spores germinated at 8 to 45°C over a pH range of 5 to 9 (1). Higher storage temperatures were associated with an increased GR, decreased LPD, and a lower MPD.


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  		TABLE 2. Growth parameter values for B. anthracis Sterne in sterile raw ground beef at storage temperatures of 17 to 44°Ca

 
To confirm that an increase in CFU resulted from vegetative-cell growth and not from enhanced recovery of spores, we heat treated each sample at 75°C for 20 min and then compared the number CFU on TSA to that of a non-heat-treated ground sample. At selected time intervals, heat-shocked ground-beef samples yielded few, if any, CFU on TSA, indicating that the increase in CFU during storage of the ground beef resulted from vegetative-cell growth.

However, this interpretation was challenged when we observed similar results with samples that had been heat shocked immediately after addition of the spore inoculum to the raw ground beef. These findings indicated that the ground-beef matrix exerted an inactivating effect on spores and that this method could not be used to differentiate spores versus vegetative cells. Similar results were observed independently in the two laboratories.

Other studies have shown that heat shocking spores leads to spore germination and to more rapid outgrowth than in non-heat-shocked spores. We did not observe a significant difference in LPD, GR, or MPD for heat-shocked B. anthracis Sterne spores added to raw ground beef and stored at 37°C. Although no differences were observed at 37°C, it is possible that the growth parameters may be altered at other storage temperatures.

GR.
The GR significantly increased from 0.011 log10 CFU/h at 17°C to a maximum rate of 0.496 log10 CFU/h at 37°C, followed by a reduction to 0.045 log10 CFU/h at 44°C (Table 2 and Fig. 1). This GR profile can be modeled with the extended Ratkowsky square root model (equation 1) (18). The GRs for Ames K0610 at 25, 30, 35, and 40°C were within, or close, to the 95% confidence intervals for the Sterne secondary model (Fig. 1).

Formula 1(1)
where k = GR, b = 0.0302, c = 0.7095, Tmin = 14.0483, Tmax = 43.6745, and R2 = 0.97. Tmin and Tmax are the minimum and maximum temperatures, respectively, at which growth is observed.


Figure 1
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  		FIG. 1. GRs of B. anthracis Sterne ({blacklozenge}) and Ames K0610 ({square}) as a function of sterile raw ground beef storage temperature from 17 to 44°C. Secondary-model predictions (solid line) with upper and lower 95% confidence intervals (dashed lines) are shown.

 
LPD.
Researchers have proposed that bacterial LPD is related to the time required for cells to adjust physiologically to a new environment (2, 3). In the case of spore-forming organisms, LPD would also include the time required for spores to germinate and the time required for the resulting vegetative cells to adjust to growth in the food matrix. Baranyi et al. (3) developed a dynamic growth model that includes a mechanistic parameter for LPD, and this model was used to describe LPD. For the majority of bacterial pathogens, the LPD has been shown to decrease with increasing temperature, and a similar trend was observed for B. anthracis Sterne and Ames K0610. The LPD significantly decreased from 53 h at 18°C to 6.8 h at 44°C (Table 2 and Fig. 2). This distribution of LPD can be described with an exponential model (equation 2).

Formula 2(2)
where a = 2.3573, b = 3.47E8, c = 11.0396, and R2 = 0.96.


Figure 2
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  		FIG. 2. LPDs of B. anthracis Sterne ({blacklozenge}) and Ames ({square}) as a function of sterile raw ground beef storage temperature from 17 to 44°C. Secondary-model predictions (solid line) with upper and lower 95% confidence intervals (dashed lines) are shown.

 
The observed LPD for Ames K0610 was within or close to the 95% confidence intervals for the Sterne secondary model (Fig. 2).

MPD.
The MPD represents the highest concentration that a microbial population attains in an environment. The distribution of the B. anthracis Sterne MPD as a function of temperature was described by a cubic model, with the MPD progressively decreasing with higher temperature (equation 3). For example, at 20°C, the MPD was 7.8 log10 CFU/g, whereas at 44°C it was 5.9 log10 CFU/g (Table 2 and Fig. 3). The cubic model has also been used to describe the change in the E. coli O157:H7 MPD as a function of temperature (20).

Formula 3(3)
where a = 7.9314, b = –1.9E–5, and R2 = 0.82.


Figure 3
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  		FIG. 3. MPDs of B. anthracis Sterne ({blacklozenge}) and Ames ({square}) as a function of sterile raw ground beef storage temperature from 17 to 44°C. Secondary-model predictions (solid line) with upper and lower 95% confidence intervals (dashed lines) are shown.

 
Unlike the GR and LPD, the observed MPD for the Ames K0610 strain was not within or close to the 95% confidence intervals of the Sterne model (Fig. 3). Strain Ames K0610 displayed MPDs that were 0.95, 1.35, and 1.65 log10 CFU higher than the MPDs for Sterne at 30, 35, and 40°C, respectively. The lower MPDs for Sterne could have resulted from a limited number of replication cycles and/or a density-dependent phenomenon. To test these hypotheses, we inoculated batch A sterile raw ground beef with 3 or 5 log10 CFU/g of the Sterne strain and then measured the MPD at 25°C. The results showed that at either inoculum level, an MPD of ~7.2 log10 CFU/g was observed (data not shown). Therefore, we suggest that the lower-than-expected MPD in ground beef was due to a density-dependent mechanism, and additional research is warranted to define the cause(s).

In addition to potential strain variation, differences in fat content between the two batches of ground beef could have resulted in the different MPDs and GRs. We found that when the Sterne strain was grown in both batches of ground beef at 20°C, the Sterne MPD was significantly lower (~1 log CFU/g) in batch A. In addition, at 20°C, the GR was lower in batch A (0.04 log CFU/h) than in batch B (0.07 log CFU/h), although not significantly different. Similar studies testing the Ames strain in batches A and B were not performed.

The effects of fat on the MPD and GR were not expected based on previous studies of E. coli O157:H7 growth in sterile raw ground beef. Using three different fat levels (i.e., 5, 12, and 16%), investigators found that the MPD and GR increased with decreasing fat concentration (19). These results indicate that compositional differences in the two batches of ground beef, perhaps other than fat, may affect the B. anthracis GR and MPD. Further studies are warranted to define specific components of ground beef that influence B. anthracis survival.

We also measured the GRs and LPDs for B. anthracis Sterne in vacuum-packaged and opened packages of ground beef at 20 and 37°C and found that the GR was not significantly higher. For example, at 20°C, the GR was 0.089 log CFU/h for opened packages versus 0.068 log CFU/h for vacuum-packaged ground beef. Similarly, there was no significant difference in lag time between the two preparations (37.8 versus 36.7 h).

At storage temperatures of 10 to 17°C, the ground-beef samples yielded a variant colony form that was transparent with a glossy or slighted matted surface. This typically occurred at storage times of >100 h. The variant morphotype retained the same rough edge as the control B. anthracis Sterne strain. With longer incubation time, some ground-beef samples yielded only the transparent variant. Such patterns of change in colony morphology were observed at 10, 14, 15, 16, and 17°C. The stability of the variant form was tested by inoculating BHI broth with a transparent colony isolated from 17°C ground beef stored for 288 h and then incubating the BHI broth at 17°C or 37°C. In general, colonies maintained the translucent morphology through 288 h on TSA and PLET agars. The B. anthracis Sterne control and the variant morphotype did not show hemolysis on 5% sheep blood agar. Biochemical tests using the API system identified the translucent variant only to the level of Bacillus sp., indicating that it varied from the parent strain in some metabolic traits.

Inactivation of B. anthracis Sterne and Ames K0610 in sterile raw ground beef.
At 2 to 16 and ≥45°C, B. anthracis Sterne viability decreased through 195 h of storage (Fig. 4). Sterne spores displayed either an immediate linear reduction or a "shoulder" followed by linear reduction. Table 3 lists the average inactivation rate at each low storage temperature, showing that there was relatively low variation (i.e., –0.0031 to –0.0144; µ = –0.0066; standard deviation = 0.0038) over the range of conditions. The inactivation rate increased as the temperature increased, although this was not significant. A "shoulder" was observed only at temperatures of ≤10°C, with durations ranging from not detected to 59 h.


Figure 4
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  		FIG. 4. Inactivation rates of B. anthracis Sterne ({blacklozenge}) and Ames K0610 ({square}) as a function of sterile raw ground beef storage temperature from 2 to 16°C.

 

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  		TABLE 3. Average survival rates and LPDs for B. anthracis Sterne and Ames K0610 in sterile raw ground beef at storage temperatures of 2 to 16°Ca

 
These results were consistent with previous findings that B. anthracis spore viability decreases in pasteurized milk by approximately 1 log10 CFU at 5 to 9°C over 24 h of storage (4). However, the studies with unpasteurized milk contrast with our findings in that B. anthracis spore viability remained relatively unchanged in unpasteurized milk over 24 h of storage at 5 to 9, 22, or 37°C.

Since there was little change in the viability of the stock B. anthracis spore preparation stored in deionized water at 5°C, the observed inactivation in ground beef at that temperature was unexpected. It could have resulted from factors in ground beef that trigger germination events, leading to vegetative cells that were inactivated at low temperatures. For example, L-alanine has been shown to cause strong and rapid germination of B. anthracis spores (7, 11). However, as discussed above, our ability to differentiate vegetative cells from spores using heat treatment was complicated by the fact that spores were easily heat inactivated.

Various studies have established the thermal resistance of B. anthracis spores; however, there are few reports about spore inactivation kinetics in food matrices. Montville and colleagues (12) reported that the heat inactivation kinetics of B. anthracis were not unusual compared with eight strains of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis but that there was variation in D and z values among the strains when they were suspended in different matrices, including buffer, milk, and orange juice. The D value refers to the decimal reduction time or the time necessary to reduce bacterial counts by 90%; the Z value refers to the temperature change necessary to change the D value by a factor of 10. In saline solution, D values for B. anthracis spores vary from 2.5 to 7.5 min and 1.7 to 4.2 min at 90 and 95°C, respectively (13). Purdue et al. (17) reported that milk pasteurization temperatures (i.e., 63°C for 30 min or 72°C for 15 s) have little effect on B. anthracis Sterne spore viability.

Although the primary objectives of this research did not include measuring high-temperature inactivation kinetics, we did compare spore inactivation in PW and raw ground beef at 45 and 70°C in an attempt to understand why we were not able to selectively inactivate vegetative cells versus spores in ground-beef preparations. Table 4 shows the change in B. anthracis Sterne counts when spores were heated at both temperatures for up to 3 h. Interestingly, the ground-beef matrix caused a greater reduction in spore viability than PW. The results showed that ground beef caused a 2.2- to 5.2-fold-greater change in log10 CFU than PW at 45°C and 4.7- to 6.9-fold greater at 70°C. In separate experiments conducted at 70°C with batch B ground beef, Ames K0610 spore viability was also reduced more in ground beef than in PW (not shown).


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  		TABLE 4. Changes in viable counts for B. anthracis Sterne at temperatures of 45 and 70°C for 1, 2, and 3 h

 
In further experiments to define the inactivating effect of raw ground beef on B. anthracis spores, we added B. anthracis Sterne spores to PW, to raw ground beef, and to an equal mixture of PW and raw ground beef (not shown). The inactivation rate showed a concentration-dependent effect on spore viability, in that the rate for the 50% PW-ground-beef mixture (–0.272 log CFU/h) was approximately half the differences of the rates in 100% PW (–0.005 log CFU/h) and 100% ground beef (–0.573 log CFU/h).

Since B. anthracis counts were measured in the liquid phase of the diluted and stomached ground-beef sample, we hypothesized that large numbers of spores could have bound to clumps of ground beef, thus producing the marked reductions observed at 45 and 70°C. To test this idea, we inoculated a duplicate set of PW and ground-beef samples, heated the preparations at 45 and 70°C, and then sonicated them for 30 s to more thoroughly disperse the spores prior to enumeration. The results showed that there was no difference between the sonicated and nonsonicated samples, supporting the observation that ground beef has an inactivating effect on B. anthracis spore viability.

These experiments demonstrate that B. anthracis is able to grow in sterile raw ground beef over a temperature range of 17 to 44°C and that the GR is moderately (0.021 log CFU/h) higher in ground beef that is exposed to an ambient atmosphere. At temperatures of ≤16°C, viability is weakly reduced at a rate of –0.003 to –0.014 log10 CFU/h. Although the spores are relatively resistant to the short-term effects of high temperature, they are markedly reduced in ground beef at sustained temperatures of ≥45°C.

Based on other published studies, the kinetics of spore growth and inactivation may be affected by strain variation and by the methods used to produce the spore crop. Researchers have shown that spores of B. cereus, a close relative of B. anthracis, are more resistant to heat when the sporulation temperature is increased from 20° to 45°C (9). Other researchers have suggested that prolonged storage of spores leads to a loss of calcium from the spore coat and greater heat sensitivity (6).

The Sterne strain appears to be a reasonable surrogate for the Ames strain in measurements of GR and LPD in raw ground beef, based on observations over a broad range of temperature for two different batches of ground beef by two independent laboratories. Additional research with B. anthracis Sterne may accelerate information about the behavior of virulent B. anthracis strains, particularly considering that there are many fewer laboratories capable of working with highly virulent strains.

In a hypothetical contamination scenario, if beef trims were contaminated with B. anthracis spores, then the grinding process would likely distribute the spores over a large volume of product (8). Considering the typical lower storage temperatures (5 to 10°C) of ground beef in commercial and home settings, B. anthracis spores would not be expected to reproduce but to slowly die off. This weak inactivating effect would likely be amplified by the inhibitory effects of the numerous spoilage organisms typically found in retail ground beef, which were not studied in the present experiments. Subsequent cooking would also cause a sharp reduction in spore and vegetative-cell viability. However, further research is warranted to quantify the inactivation kinetics for retail and consumer cooking methods.


   ACKNOWLEDGMENTS
 
We thank Tanishia Lawson, Ethel Richards, and Jessica Phillips for their valuable contributions to this research and James Lindsay for insightful discussions.

This research was supported, in part, by the National Biodefense Analysis and Countermeasures Center and the USDA Food Safety and Inspection Service.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.


   FOOTNOTES
 
* Corresponding author. Mailing address: Australian Food Safety Centre of Excellence/University of Tasmania, Hobart, TAS 7001, Australia. Phone: (613) 6226 6378. Fax: (613) 6226 7450. E-mail: mark.tamplin{at}utas.edu.au Back

{triangledown} Published ahead of print on 14 December 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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Applied and Environmental Microbiology, February 2008, p. 1111-1116, Vol. 74, No. 4
0099-2240/08/$08.00+0     doi:10.1128/AEM.01292-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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