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Applied and Environmental Microbiology, June 2008, p. 3336-3341, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02072-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Inactivation of Bacillus anthracis Spores by a Combination of Biocides and Heating under High-Temperature Short-Time Pasteurization Conditions

Sa Xu, Theodore P. Labuza, and Francisco Diez-Gonzalez^*

Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108

Received 11 September 2007/ Accepted 24 March 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85°C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80°C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80°C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80°C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The bioterrorist attack with Bacillus anthracis spores in 2001 in the United States stressed the need for strengthening the biodefense system, especially for the food supply. Bacillus anthracis is one of the potential bioterrorism agents which could be deliberately released into the milk production, processing, and distribution system. This concern has fueled interest in studying new methods for inactivation of B. anthracis spores in milk. Since pasteurization is a standard process in milk plants, it could be adapted to develop a thermal process capable of inactivating B. anthracis spores. Such a process could be easily adopted by high-temperature short-time (HTST) milk pasteurizers in the event of a bioterrorist attack for decontamination and disposal.

A series of thermal inactivation kinetic parameters at 72 to 103°C were determined in our previous study (24). The results of that study confirmed that standard HTST pasteurization processes (e.g., 72°C for 15 s) had little effect on inactivation of B. anthracis spores (11, 12, 24). Six-log reductions in viability were achieved if the spores were heated at 120°C for 16 s. This observation suggests that a thermal process similar to commercial ultra-high-temperature (135 to 140°C for 1 to 2 s) pasteurization could inactivate B. anthracis spores in the event of a deliberate attack by terrorists. However, the limited number of ultra-high-temperature processing plants restricts this application of high-temperature inactivation of spores.

One strategy to inactivate B. anthracis spores at lower temperatures is the use of biocides during thermal treatment. In a related study, the effectiveness of a combination of hydrogen peroxide (HP) and thermal treatment for inactivation of spores of B. anthracis strains was studied using 90 to 95°C combined with HP concentrations ranging from 0.05 to 0.5% (23). The results showed that a combination of heat and HP could be an effective method to inactivate B. anthracis spores in milk. Addition of 0.5% HP to milk containing spores reduced the decimal reduction times (D values) from approximately 10 min to less than 10 s at 90°C. However, lower HP concentrations (0.05 to 0.5%) could not inactivate anthrax spores in a short time (less than 1 min) in the HTST temperature range (72 to 80°C).

The World Health Organization has indicated that sodium hypochlorite (SH), HP, and peroxyacetic acid (PA) can be effective sporicidal agents against Bacillus spores, including B. anthracis spores (http://www.who.int/emc-documents/zoonoses/docs/whoemczdi986_nofigs.html). SH, a chlorine-releasing substance, is a strong oxidizing agent. Its sporicidal efficacy depends on several factors, including the free available chlorine, the temperature, the pH, and the organic matter present. SH was the most effective halogen against Bacillus cereus spores (5).

The most commonly used peroxygens in food production are HP and PA. The sporicidal properties of HP at concentrations ranging from 10 to 41% at 24 to 76°C have been reported previously (18). At 76°C, 26% HP killed 99% of Bacillus subtilis subsp. globigii spores suspended in buffer within 1 min. The temperature and concentration markedly influenced the sporicidal activity, and heating caused HP to produce free radicals (19).

The minimum HP concentration needed to exert sporicidal effects depends on the bacterial strain, pH, and temperature (9).

PA, a stronger oxidizing agent than chlorine compounds, has sporicidal properties, especially under acidic conditions. It decomposes ultimately to HP, acetic acid, and oxygen.

As potential biocides, PA and HP have been used for disinfection of wastewater and indoor surfaces, respectively (15, 20). A combination of PA and SH was reported to be effective for rapidly killing bacterial spores in dairy processing (10). Liquid food contact biocides are considered attractive options because of their low capital costs. Most of the studies that have examined the inactivation of spores have been conducted in a water system and on facility surfaces. However, the presence of organic matter in food may reduce the effectiveness of biocides. Oxidant biocides can react with amino groups of food proteins, which could lead to decreased spore killing. The aim of this study was to evaluate the inactivating effects of biocides and heat in actual food systems.

The objective of this study was to identify combinations and concentrations of SH, HP, and PA that consistently resulted in 6-log reductions in the number of CFU/ml at temperatures ranging from 72 to 85°C. This study identified feasible thermal treatments that could be used in a typical milk plant with a plate and frame heat exchanger system limited to temperatures below 90°C in order to process contaminated milk for disposal.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains.
B. anthracis Sterne strain 7702, avirulent Ames strain ANR-1, and Sterne derivative 9131 were provided by Theresa Kohler, University of Texas-Houston Medical School. Sterne strain 7702 harbors pXO1, which encodes the edema factor toxin, but lacks pXO2 (4, 14, 24). Strain ANR-1 is a pXO2-cured nonencapsulated variant of the Ames strain that harbors pXO1 and pXO2 (21). Plasmidless strain 9131 was obtained in M. Mock's laboratory at the Pasteur Institute by curing strain RP31 from the pXO1 plasmid (7, 13).

Preparation of spore suspensions.
A previously described protocol was used to produce B. anthracis spore suspensions (12, 24). Each spore suspension was stored at 4°C for no more than 2 weeks until it was used. The spore suspensions were observed using a Nikon phase-contrast microscope (Leeds Precision Instruments, Minneapolis, MN) and were verified to contain at least 90% spores. The viability of spores in the suspensions was determined by plating on tryptic soy agar (Acumedia Manufacturers, Inc., Lansing, MI).

Determination of inactivating effects of biocides and heat treatment.
Three different biocides, HP (35%, wt/vol; Sigma-Aldrich Inc., St. Louis, MO), SH (8.4%, wt/vol; Ecolab Inc., St. Paul, MN), and PA (15%, wt/vol; Ecolab Inc., St. Paul, MN), were purchased as stock solutions. The pHs of the HP, SH, and PA stock solutions were 3.5, 12, and 2.3, respectively. Biocide solutions were prepared from the stock solutions on the day of each experiment by dilution of the concentrated materials with sterile distilled water. The different concentrations of the biocides did not cause clotting of milk. The pH of SH was neutralized to 7 in some experiments.

A capillary tube method was used to test spore resistance in an oil bath (24). Biocides were added to milk containing B. anthracis spores and mixed by vortexing (Fisher Inc., New York, NY) to obtain the concentrations studied. Portions (50 µl) of inoculated milk with biocides containing from 10⁹ to 10¹⁰ CFU/ml spores were transferred into capillary tubes (0.8 to 1.1 by 90 mm; catalog no. 34507; Kimble, Vineland, NJ). Sealed capillary tubes were placed in the oil bath set at 72, 80, or 85°C. The tubes were heated within 2 min after addition of biocides. After heating, the residual HP and PA in milk samples was neutralized with 536 U/ml catalase, and each sample was diluted using phosphate-buffered water (Sigma-Aldrich Inc., St. Louis, MO) before the number of viable spores was determined as described previously (24). The enzymatic action of catalase breaks down HP to water and oxygen. The residual SH was neutralized with 10 g/liter sodium thiosulfate (Mallinckrodt Inc., Paris, KY), and then each sample was diluted using phosphate-buffered water.

Statistical analyses.
D values (the times that it took to inactivate 90% of the population) were determined from the linear portions of the survival curves by plotting the log₁₀ survival counts as a function of heating time at each temperature. D values (the times required for a 1-log reduction) were calculated by determining the negative reciprocal of the slopes. We utilized inactivation of 6 log CFU/ml spores as the target reduction. The time necessary to inactivate 6 log CFU/ml B. anthracis spores (TTI6-log) was determined for each treatment consisting of a combination of biocides and heat. For each experiment we used a full factorial design consisting of temperature, strain, and biocide conditions. All the experiments were replicated at least three times with duplicate samples at each time interval for each treatment. Differences in the TTI6-log were analyzed using an analysis of variance procedure and the Duncan multiple range test (version 8.2; SAS, Cary, NC) with a significance level of 0.05.

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The effect of HP on inactivation of B. anthracis spores in milk was determined at 72, 80, and 85°C with HP concentrations ranging from 1,050 to 7,000 ppm (Table 1). At all temperatures, when the spores were heated in milk containing no biocide, the TTI6-log was from 3 to 300 times longer than the TTI6-log at the lowest biocide concentration tested, depending on the strain. At 1,050 ppm HP and 72°C, the TTI6-log for strain 7702 was only 14 min, but it took more than 5 and 18 times longer to observe the same effect on strains ANR-1 and 9131, respectively. Overall, strain 7702 was more sensitive to the combination of heat and HP than strains ANR-1 and 9131. As the concentration of HP increased, the TTI6-log markedly decreased, and at 7,000 ppm HP, an average of 1.0 min was needed to kill spores of strains ANR-1 and 9131. When the temperature was increased to 80°C, the TTI6-log was approximately 2.0 min for these two strains at 2,800 ppm HP. At 85°C, the same concentration produced a similar result in only 1 min.

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TABLE 1. Time needed to inactivate 6 log CFU/ml B. anthracis spores in milk using HP

The inactivating effect of SH on spores of B. anthracis was also determined at 72 and 80°C using concentrations of 840 and 1,260 ppm in milk. With 840 ppm SH at 72°C, the TTI6-log were 12, 6, and 4 min for spores of strains ANR-1, 9131, and 7702, respectively (Table 2). Strain 7702 was again more sensitive to the heat and SH treatment than strains ANR-1 and 9131. Increasing the SH concentration to 1,260 ppm reduced the TTI6-log approximately 10-fold. If the pH of SH was neutralized before the SH was added to milk, the inactivation of spores was enhanced, and the TTI6-log was reduced at least 70% for all spores. When the milk was heated to 80°C, the inactivation of 6 log CFU/ml was at least two times faster than that at 72°C (Table 2). Treatment with neutralized SH also resulted in significantly greater spore inactivation at 80°C than at 72°C.

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TABLE 2. Time needed to inactivate 6 log CFU/ml B. anthracis in milk using SH

Further work was conducted to determine the inactivation of spores by a combination of SH and HP to obtain a 6-log CFU/ml reduction at 72 and 80°C (Table 3). At 72°C, the viability of B. anthracis spores was reduced by 6 log CFU/ml in approximately 50 s with 840 ppm SH and 2,100 ppm HP. The spores of strain ANR-1 were significantly more resistant to the combined treatment than the spores of the other two strains. Similar inactivation effects were observed with 840 ppm SH (pH 7) and 2,100 ppm HP and with 1,260 ppm SH and 1,050 ppm HP. When the temperature was increased to 80°C, the TTI6-log was less than 20 s with 1,260 ppm SH and 1,750 ppm HP. At the same temperature, treatment with 840 ppm SH (pH 7) and 1,750 ppm HP inactivated spores within 30 s.

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TABLE 3. Time needed to inactivate 6 log CFU/ml B. anthracis spores in milk using SH in combination with HP

SH was also tested in combination with PA for inactivation of the spores of B. anthracis strains ANR-1 and 9131 at 72 and 80°C (Table 4). At 72°C, the TTI6-log was approximately 56 s when 840 ppm SH and 75 ppm PA were added to milk. At the same temperature, a mixture of 1,260 ppm SH and 75 ppm PA inactivated 6 log CFU/ml spores in 18 to 24 s. At 80°C, a 6-log CFU/ml reduction in the number of spores was achieved in 30 s by treatment with 840 ppm SH and 75 ppm PA and by treatment with 1,260 ppm SH and 15 ppm PA. Only 15 s was needed to observe the same level of inactivation of both strains with 1,260 ppm SH combined with 75 ppm PA.

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TABLE 4. Time needed to inactivate 6 log CFU/ml B. anthracis spores in milk using SH in combination with PA

The TTI6-log of B. anthracis strain ANR-1 and 9131 spores at 72 and 80°C with mixtures of SH (250 and 420 ppm), HP (350 to 1,100 ppm), and PA (75 and 150 ppm) in milk are shown in Table 5. At 72°C, for all treatments with the three biocides the TTI6-log were less than 2 min, and for two treatments they were less than 50 s. The TTI6-log at 80°C for all six three-biocide treatments was never more than 50 s, and for two combinations the TTI6-log were less than 20 s (250 ppm SH, 700 ppm HP, and 150 ppm PA; 420 ppm SH [pH 7], 1,050 ppm HP, and 75 ppm PA).

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TABLE 5. Time needed to inactivate 6 log CFU/ml B. anthracis viable spores in milk using SH, HP, and PA

The inactivation effects of different biocide combinations on B. anthracis spores was also tested at 22°C to evaluate room temperature effects using strains ANR-1 and 9131 (Table 6). The D values for inactivation by 7,000 ppm HP ranged from 31 to 42 min. The D values were 34 to 60 and 25 to 27 min for 840 ppm SH plus 1,800 ppm HP and for 840 ppm SH plus 75 ppm PA, respectively. These results also suggested that inactivation of 6 log CFU/ml spores could also be achieved in approximately 2 h.

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TABLE 6. Effects of biocides (SH, HP, and PA) on the D values of B. anthracis spores in milk at 22°C

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, strain 7702 was the strain most susceptible to the combination of heat and biocides. This result was in agreement with our previous report which showed that spores of 7702 were more sensitive to treatment with heat (90 to 95°C) and HP than spores of strains ANR-1 and 9131 (23). The spores of these B. anthracis strains had very similar thermal inactivation kinetic parameters (24); however, they had different levels of resistance to combined treatment with heat and biocides. Because strain 7702 was more sensitive, it would not be considered a good indicator strain in thermal/biocide resistance studies; thus, most of our results focused on spores of ANR-1 and 9131.

In this study we observed that neutralization of SH could effectively enhance the inactivation effect of SH/thermal treatment on B. anthracis spores in milk. SH exhibits a dynamic balance in an aqueous system. It partially splits into sodium hydroxide and a hypochlorite anion, while a substantial part is hydrolyzed into hypochlorous acid (pK_a 7.6), which has strong oxidizing power. The hypochlorite ion is a poor disinfectant because of its inability to diffuse through the cell walls of microorganisms (22). Sodium hydroxide would cause the pH of milk to rise. Most of the hypochlorous acid disassociates to form hypochlorite anions at high pH. At a pH above 9 and at 20°C, 96% of the free available chlorine consists of hypochlorite ions, compared to only 8% at pH 6.5 (22). However, 80% of the chlorine is present in the hypochlorous acid form at pH 7.

PA has shown strong inactivation effects on spores (17). It is an equilibrium mixture composed of acetic acid and hydrogen peroxide. Our preliminary results indicated that D values were significantly reduced under HTST conditions after addition of 75 ppm PA compared to thermal treatment alone. However, milk clotting occurred when the concentration of PA was higher than 75 ppm. Coagulation of milk would cause serious problems by clogging the flow in the pasteurizer, thus compromising the effectiveness of thermal/biocide treatment. Although PA was more active at pH 5 than at neutral pH (16), it was not feasible to use PA alone in milk since its low pH would cause the coagulation of milk protein. However, combined treatment with PA and other biocides reduced the TTI6-log to 1 min (Table 7). The activity of PA was reduced only slightly in the presence of organic matter. Thus, PA is a promising agent for inactivating spores in milk systems and could be used in combination with heat and other biocides.

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TABLE 7. Summary of the abilities of HP, SH, and/or PA along with different temperatures to achieve 6-log reductions in the number of CFU/ml of B. anthracis viable spores in milk in a short time

This study evaluated the effects of biocide agents under thermal processing conditions. The TTI6-log were similar for some treatments. Treatment with a combination of 840 ppm SH and 2,100 ppm HP had an inactivation effect on B. anthracis spores at 72°C similar to that of 1,260 ppm SH at pH 7 or a mixture of 840 ppm SH and 75 ppm PA. Mixtures of SH and HP were much more sporicidal than the individual compounds used alone. The ratio of biocides also appears to influence the inactivation activity. In our study, mixtures of two biocides (SH and HP) at ratios of 1:1, 2:1, 3:1 and 5:1 gradually reduced the time needed to inactivate B. anthracis spores. This result was in agreement with a study of B. subtilis spores in distilled water (6). A ratio of SH to HP of 10:1 resulted in more remarkable inactivation of B. subtilis spores than a ratio of 1:1 or 5:1.

The combination of HP with PA has also been found to be synergistic against Bacillus spores (1). The optimum ratios of biocide components were important to the sporicidal effect. In order to reduce the concentration of biocides which need to be used during thermal inactivation of spores, mixtures of several biocides that inactivate B. anthracis spores in a short time were identified. In these treatments with mixtures, lower concentrations of individual biocides resulted in effective inactivation (Table 5).

The inactivating effect of biocides on spores was affected by strain, temperature, concentration, exposure time, medium, and pH (17). Such variations complicate comparisons of the results of different studies. Based upon the inactivating effects of biocide concentrations that we tested in milk with B. anthracis spores (Table 7), the order was as follows: PA > SH > HP. PA has also been reported to be more potent than HP (2, 3). In this work, temperature played an important role by increasing the inactivation activity of biocides. When the heating temperature was increased, the inactivating effect of biocides was also enhanced at the same concentration or pH. If the temperature was lower, higher concentrations were needed to achieve the same inactivation effect. At 840 ppm SH (pH 11), the inactivation time for B. anthracis spores was reduced about threefold when the temperature was increased from 72 to 80°C. Similar results indicated that SH at pH 11 was significantly more effective against spores at 40°C than against spores at 8 or 20°C (17). The spore permeability could be increased by hypochlorite and heat, which led to leakage of dipicolinic acid (8).

A previous study showed that two biocides (0.05% SH and 0.03% PA) inactivated more than 99.9% of a B. subtilis spore suspension after a 30-min exposure at 20°C, but 10% HP was ineffective with spores under similar conditions (17). Compared to our study, significant inactivation occurred at 72°C with 7,000 ppm HP or 1,260 ppm SH. This shows that the effect of SH was reduced in a milk system containing complex organic matter which could react with hypochlorous acid. Thus, the concentration of SH needed to be greater than the concentration in an effective water solution. Our study included multifactorial experiments to determine the effectiveness of inactivation conditions for spores.

In summary, a series of biocide/thermal processing recommendations were identified to achieve a 6-log CFU/ml reduction in viability of B. anthracis spores in milk when they were heated within 1 min at typical pasteurization temperatures (Table 7). The TTI6-log was less than 1 min at 72°C with at least three combinations of biocides. Mixing 75 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80°C, respectively. TTI6-log of less than 20 s were also achieved at 80°C by using at least two mixtures of SH, HP, and PA. This study strongly supports the idea that typical HTST pasteurization facilities could be capable of decontaminating milk containing B. anthracis spores in a short time in combination with low concentrations of biocides for the purpose of disposal. The results did not suggest that the milk could be appropriate for human consumption.

 
This research was supported by the U.S. Department of Homeland Security through grant N-00014-04-1-0659 awarded to the National Center of Food Protection and Defense at the University of Minnesota.

We thank Theresa Koehler (University of Texas—Houston Medical School), who provided B. anthracis strains.

The opinions, findings, conclusions, and recommendations in this publication are those of the authors and do not represent the policy or position of the U.S. Department of Homeland Security.

 
* Corresponding author. Mailing address: Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Ave., St. Paul, MN 55108. Phone: (612) 624-9756. Fax: (612) 625-5272. E-mail: fdiez{at}umn.edu

Published ahead of print on 4 April 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Applied and Environmental Microbiology, June 2008, p. 3336-3341, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.02072-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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