Survival and Heat Resistance of Salmonella enterica and Escherichia coli O157:H7 in Peanut Butter

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2011, p. 8434 8438 Vol. 77, No. 23 0099-2240/11/$12.00 doi:10.1128/aem.06270-11 Copyright 2011, American Society for Microbiology. All Rights Reserved. Survival and Heat Resistance of Salmonella enterica and Escherichia coli O157:H7 in Peanut Butter Yingshu He, 1 Dongjing Guo, 1 Jingyun Yang, 2 Mary Lou Tortorello, 3 and Wei Zhang 1 * Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, Illinois 60501 1 ; University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 2 ; and U.S. Food and Drug Administration, Bedford Park, Illinois 60501 3 Received 22 July 2011/Accepted 24 September 2011 Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment. Salmonella enterica is the leading cause of hospitalizations (28%) and deaths (35%) attributed to known bacterial pathogens transmitted by foods in the United States (15). This pathogen is capable of surviving extended starvation and desiccation stresses and has caused major disease outbreaks associated with foods of low water activity (a w ), such as peanut butter (a w, 0.3 to 0.5) (13). Several recent outbreaks of salmonellosis, including two linked to peanut butter in 2007 and 2008, have brought attention to the persistence of Salmonella in low-a w foods (2 4). Peanut butter is produced from roasted peanuts with moisture content of 1% (a w, 0.3); the extremely low water activity in peanut butter precludes active growth of spoilage and pathogenic microorganisms (10). The stresses of starvation and desiccation encountered by S. enterica throughout peanut harvest and peanut butter production may induce stress response mechanisms for cross-protection against subsequent heat stress (13, 14). Several studies of S. enterica survival and heat resistance in peanut butter have been published (1, 10, 12, 16). Burnett et al. (1) reported the population of a five-serotype S. enterica cocktail decreased by 4.14 to 4.5 log and 2.86 to 4.28 log in different peanut butter and peanut butter spreads over a 24-week incubation period when incubated at 5 C and 21 C, respectively. Park et al. (12) did a similar study using a 3-strain cocktail of S. enterica serovar Tennessee in five commercial brands of peanut butter. About 0.15- to 0.65-log and 0.34- to 1.29-log reductions were observed over a 14-day incubation period at 4 C and 22 C, respectively. Shachar and Yaron (16) compared the relative levels of heat resistance of three S. enterica serotypes (i.e., Agona, Enteritidis, and Typhimurium) in artificially inoculated peanut butter and reported a ca. 3.2-log reduction at 90 C. Ma et al. (10) compared the thermal inactivation rates of S. Tennessee, Enteritidis, Typhimurium, and Heidelberg in * Corresponding author. Mailing addresses: Institute for Food Safety and Health, 6502 South Archer Road, Bedford Park, IL 60501. Phone: (708) 563-2980. Fax: (708) 563-1873. E-mail: zhangw@iit.edu. Present address: University of Georgia, Athens, GA 30602. Supplemental material for this article may be found at http://aem.asm.org/. Published ahead of print on 30 September 2011. commercial peanut butter and found 120 min was required to achieve a 7-log reduction of S. Tennessee at 90 C. The main objective of this study was to compare the survival and heat resistance rates of S. enterica and its close relative, Escherichia coli O157:H7, in different peanut butter products with various compositions of fat, carbohydrate, protein, and water activity. Comparison between S. enterica and E. coli O157:H7 in peanut butter has rarely been reported in the literature (8, 18) possibly because E. coli O157:H7 has not been frequently linked to food-borne illness outbreaks associated with low-a w food. Instead of using a fresh bacterial culture for evaluating heat resistance, as done in most published studies, we focused on the heat resistance of stressed bacterial cells after a 30-day incubation in peanut butter. The extended exposure to the low-a w environment prior to heat treatment simulated the desiccation and starvation stresses that S. enterica typically encounters during peanut harvesting and processing (13); therefore, our challenge studies based on stressed bacterial cells may provide a more realistic evaluation of the heat resistance of the bacteria. S. enterica strains representing five different serotypes were selected to make a five-strain cocktail for evaluation. These strains included S. Tennessee strain K4643 (a human isolate from the 2006 peanut butter outbreak in the United States) (3), S. Enteritidis strain BSS-1045 (an isolate from contaminated raw almonds) (6, 9), S. Typhimurium strain LT2, S. Anatum strain 6802 (an isolate from contaminated raw peanuts), and S. Oranienburg strain 1839 (an isolate from contaminated raw pecans). For comparison, five E. coli O157:H7 strains representing five different clades were selected to make an E. coli cocktail. These strains included strain Sakai (clade 1), strain 93-111 (clade 2), strain EDL-933 (clade 3), strain TW07941 (clade 6), and strain TW14359 (clade 8) (11). Individual cultures of bacterial strains were prepared by transfer of single colonies into brain heart infusion (BHI) broth at 37 C for 16 to 18 h. Cell pellets were collected by centrifugation and resuspended in 5 ml phosphate-buffered saline(pbs) buffer (ph 6.8) to an adjusted optical density (OD) of 0.5. Equal volumes of each bacterial culture were mixed to prepare the 5-strain cocktails. The cocktails were washed twice in PBS, and after centrifugation at 4,000 g for 20 min at 4 C, cell pellets 8434

VOL. 77, 2011 S. ENTERICA AND E. COLI O157 SURVIVAL IN PEANUT BUTTER 8435 TABLE 1. Formulations of four different peanut butters used in this study Formulation (peanut butter) a w Ingredient content (wt %) Fat Sodium Carbohydrate Protein Other Regular (A) 0.4 33.33 0.58 41.67 22.22 2.20 Organic (B) 0.4 50.00 0.25 21.88 25.00 2.87 Reduced fat (C) 0.7 6.25 0.59 40.63 12.50 40.03 Organic, no salt (D) 0.4 50.00 0.00 21.88 25.00 3.12 were air dried at room temperature for 20 min prior to inoculation into peanut butter. Four different commercial peanut butter products from a single manufacturer were used for this study (Table 1). These peanut butter products varied in formulations of fat, sodium, carbohydrate, protein, and water activity. Bacterial pellets were resuspended in 7.5 ml peanut oil (a w, 0.3), and the suspension was transferred to 100 g peanut butter in a sterile 250-ml glass beaker. The contents were vigorously stirred using a sampler spatula for 15 min. The water activity of the peanut butters was determined using an Aqualab Rapid Read a w meter (Pullman, WA) prior to and after the inoculation. An obvious advantage of using peanut oil as a resuspension intermediate, as opposed to an aqueous buffer, was that the a w throughout the inoculation process remained below 0.4. By maintaining the cells in a low-a w environment, any effects on survival and heat resistance could be ascribed to the low-a w stress and not to stresses that would occur in the transfer from an aqueous buffer. The initial inoculation levels were adjusted to approximately 10 8 CFU/g (high level) or 10 4 CFU/g (medium level). Homogenous distribution of the inoculum was shown by the similar bacterial cell counts that were obtained from 1-g samples taken from different locations in the inoculated peanut butter. The inoculated samples were stored at 4 or 25 C for up to 30 days to monitor the population dynamics and survival rates of the bacteria in the different peanut butters. Four grams of the inoculated sample was taken immediately after inoculation (as an untreated control) and then at 1, 3, 6, 9, 14, 20, and 30 days for plate counting on the nonselective brain heart infusion (BHI; Sigma) medium and on the selective media xylose-lysine-deoxycholate (XLD; Sigma) for S. enterica and sorbitol-macconkey (SMAC; Sigma) for E. coli O157:H7. After the 30-day incubation, 4 g of inoculated peanut butter was aseptically transferred into zipper seal sample bags, compressed to a layer measuring approximately 0.2 mm in thickness, and submerged in a water bath for heat treatment at 72 or 90 C. The come-up time to reach the treatment temperature was determined to be approximately 4 s. The heattreated samples were taken at 4 s, 10 min, 20 min, 30 min, and 1 h and immediately cooled on ice until assayed by plate counting. Bacterial survival rates were calculated by enumerating viable cell counts on BHI and selective media. For comparison, we also inoculated the peanut butters with fresh overnight bacterial cultures at two different levels (4 and 8 log CFU/g), followed by immediate heat treatments. Peanut butter inoculated with peanut oil was used as a negative control. Experiments under all treatments and conditions were repeated six times using independent cultures. For statistical analysis, all plate counts were converted to log 10 -CFU/g values for comparison. D values (time to reduce a bacterial population by 90% or 1 log 10 at a given temperature) were calculated by using the Bigelow model (7) based on the equation log [N(t)/N(0)] ( 1/D)t, where N(t) was the viable plate count at time t and N(0) was the initial count before treatment. Each data set was also modeled using the Weibull model (5, 17), S t e t where S(t) N(t)/N(0), is the scale parameter and is the shape parameter. We used the following equation to estimate and, as previously described (10): ln [ ln S(t)] ln t ln. Differences in D value,, or of different bacterial cultures in the same peanut butter were tested using general linear models. Statistical analyses were performed using Microsoft Excel 2007 package (Microsoft Corporation, Redmond, WA), the R statistical package (version 2.12.0), and SAS version 9.2 (SAS Institute, Inc., Cary, NC). As shown in Fig. 1, incubation of S. enterica and E. coli O157:H7 in regular peanut butter A (a w, 0.4) with 33.33% fat and 41.67% carbohydrate resulted in less than a 1-log reduction of the total bacterial population at both 4 and 25 C. In contrast, incubation in organic peanut butters B and D (a w, 0.4), with more fat (50%) but less carbohydrate (21.88%), led to an increased log reduction for both S. enterica and E. coli O157 over the 4-week storage period. E. coli O157:H7 showed greater levels of inactivation than S. enterica in peanut butter. The higher storage temperature (25 C) appeared to have a significant bactericidal effect on both species in organic peanut butters with higher fat but lower carbohydrate contents, consistent with previous studies (1, 12). For instance, the S. enterica population dropped by 2.5 to 3 log and E. coli O157:H7 by 3.2 to 5 log after the 4-week storage. For peanut butter C with reduced fat (6.25%) and higher water activity (a w, 0.7), S. enterica showed a significantly higher log reduction at 25 C than at 4 C (P 0.05). These results collectively suggest that less carbohydrate and higher storage temperature are more effective at reducing bacterial populations in peanut butter with a w of 0.4. After a closer look at the behaviors of S. enterica and E. coli O157:H7 in the four tested peanut butters over the 30-day storage period (Fig. 2), we found that the survival rates of these two pathogen species differed significantly (P 0.05) in peanut butters A, B, and C at both 4 and 25 C, whereas no significant difference was detected in peanut butter D. The presence of 0.25% salt in peanut butter B led to greater than a 5-log reduction of E. coli O157:H7 at 25 C, compared to a 3.2-log reduction in peanut butter D without salt. Interestingly, throughout the 30-day storage, differences in bacterial survival rates became more significant, as indicated by P values from 0.05 to 0.01, between the following sample-treatment pairs: S25A-S4A, S25C-S4C, S25D-S25B, E4D-S4D, and E25D- S25D. The altered statistical significance reflected the influence of various storage temperatures and peanut butter formulations on the survivability of S. enterica and E. coli O157:H7 in these peanut butter products. Following the 30-day storage, the spiked peanut butters were subjected to heat treatment at 72 or 90 C. As shown in Fig. 1,

8436 HE ET AL. APPL. ENVIRON. MICROBIOL. Downloaded from http://aem.asm.org/ FIG. 1. Log 10 reduction of S. enterica and E. coli O157:H7 (compared to an untreated control) in different peanut butters over a 30-day incubation period at 4 C or 25 C (first column) and subsequent thermal treatment at 72 C (second column) or 90 C (third column). S(4), S. enterica incubated at 4 C; S(25), S. enterica incubated at 25 C; E(4), E. coli O157:H7 incubated at 4 C; E(25), E. coli O157:H7 incubated at 25 C. on January 15, 2019 by guest longer heat treatments generally resulted in higher log reductions of pathogens in all peanut butters. At the low a w of 0.4, a 1-h heat treatment at 72 C achieved less than a 2-log reduction of both S. enterica and E. coli O157:H7; whereas at 90 C, 5.5- to 7.1-log reductions were achieved. Interestingly, stressed bacteria previously incubated at 25 C were more heat resistant than those previously incubated at 4 C. This was probably because, even though more bacteria died during the prior incubation at 25 C, the smaller number of survivors became more protected in a certain way and therefore they were more resistant to the subsequent heat stress. For peanut butter C, in particular, S. enterica cells stored for 30 days at 25 C showed a 2.9-log greater reduction and 3.5-log lesser reduction after a 1-h heat treatment at 90 C, compared to cells that had been stored at 4 C. As shown in Table 2, significant differences in D values for inactivating S. enterica were detected among regular, organic, and reduced fat peanut butters. Compared to the heat treatment of peanut butter spiked with fresh culture, inactivation of stressed S. enterica after 30-day storage required significantly

VOL. 77, 2011 S. ENTERICA AND E. COLI O157 SURVIVAL IN PEANUT BUTTER 8437 Downloaded from http://aem.asm.org/ FIG. 2. Heat maps that show pairwise comparisons of statistical differences between the viable cell counts of S. enterica and E. coli O157:H7 (log 10 CFU/g) in different peanut butters at different sampling times over a 30-day incubation period. S, S. enterica;e,e. coli O157:H7; 4, incubated at 4 C; 25, incubated at 25 C; A, B, C, and D, four different peanut butter products. Orange, P 0.05; red, 0.01 P 0.05; dark red, P 0.01. The white background color indicates that no comparison was made or no statistical difference was detected. more time (P 0.05). For instance, it required an average of 5.89 to 8.84 min to kill 90% of stressed S. enterica cells in organic peanut butter D at 90 C, whereas it only took about 2.15 to 2.33 min to achieve the same for the fresh culture. Although much longer times (e.g., 40 to 60 min) and higher temperatures (e.g., 160 to 180 C) are typically used in peanut butter production (i.e., peanut roasting), the large differences in D values between the fresh and stressed bacterial cultures reflected the effects of physiological status on heat sensitivity and highlighted the need for choosing appropriate bacterial culture conditions for validating thermal inactivation parameters. Different inoculation levels of the overnight culture (i.e., at 10 4 or 10 8 CFU/g) did not show a significant impact on the D values in peanut butter in this study. Using the Weibull model, we predicted the minimum time (min) required for achieving 1- to 7-log reductions of S. enterica in different peanut butter formulations based on our experimental data (see Table S1 in the supplemental material). These predictions provided some idea of the relative levels of heat resistance of different bacterial cultures in different peanut butter products. For example, 30 min at 72 C or 10 min at 90 C is predicted to kill 90% of S. enterica in peanut butter; however, more time would be required to achieve 5-log reductions, as reported previously (10). It was also noted that to achieve the same log reduction for stressed S. enterica after a 30-day incubation at 25 C required longer heat treatment than any other conditions. In addition, a higher water activity (a w, 0.7) allowed much faster thermal inactivation of S. enterica in peanut butter. Again, the higher carbohydrate or lower fat content in regular peanut butter appeared to better protect S. enterica from heat stress compared to the organic high-fat, low-carbohydrate peanut butter formulation. In summary, our results showed that (i) peanut butter formulation and storage temperature have a significant impact on the survival and heat resistance of S. enterica and E. coli O157: H7; (ii) a significant difference was found between S. enterica and E. coli O157:H7 in their survivability in peanut butter; and (iii) stressed S. enterica cells were significantly more heat re- on January 15, 2019 by guest

8438 HE ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. D values of S. enterica in different peanut butters calculated based on the first-order kinetics Thermal treatment temp and peanut butter Mean SD D value, min (r 2 ) a 30-day culture Overnight culture 4 C 25 C 10 8 CFU/g 10 4 CFU/g 72 C A 18.38 0.64 (0.98) Aa 16.94 3.05 (0.96) Aa 10.42 1.70 (0.99) Ab 12.00 2.80 (0.96) Ab B 13.83 3.12 (0.98) Ba 15.42 2.19 (0.90) Aa 6.46 0.25 (0.93) Bb 4.64 1.11 (0.97) Bb C 3.91 0.21 (0.91) Ca 8.83 0.43 (0.86) Bb 2.27 0.35 (0.99) Ca 3.62 0.63 (0.97) Ba D 12.66 2.43 (0.97) Ba 22.69 9.61 (0.92) Cb 7.24 0.3 (0.94) ABc 6.98 0.05 (0.98) Bc 90 C A 5.08 1.32 (0.97) Aa 5.40 0.48 (0.98) Aa 3.55 0.41 (0.99) Aa 3.44 0.35 (1.00) Aa B 5.33 1.80 (0.99) Aab 7.26 3.76 (0.98) ABa 2.71 1.17 (0.96) Ab NA C 3.59 0.17 (0.84) Aa 9.91 0.62 (0.89) Bb NA NA D 5.89 1.09 (0.98) Aab 8.84 2.83 (0.96) ABa 2.33 0.70 (0.98) Ab 2.15 0.80 (0.98) Ab a D values were calculated based on 3 independent biological replicates. Coefficients of determination (r 2 ) are shown in parentheses. Different capital letters indicate significant differences (P 0.05) among D values of the same bacterial cultures in different peanut butters (columns); different lowercase letters indicate significant differences (P 0.05) among D values of different bacterial cultures in the same peanut butter (rows). NA, not applicable. sistant than fresh cells. It should be noted that some limitations inherent in the nature of our experimental design did exist. For instance, we used bacterial cocktails instead of individual strains; therefore, we were unable to differentiate the serotypeor strain-specific characteristics in survival and heat resistance. In addition, we used preformulated commercial peanut butter, making it difficult to separate the effects of individual food components such as carbohydrate, fat, and salt on the bacterial behavior in peanut butter. 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