Kathleen T. Rajkowski

Reduction of Salmonella spp. and strains of Escherichia coli O157:H7 by gamma radiation of inoculated sprouts.

There have been several recent outbreaks of salmonellosis and infections with Escherichia coli O157:H7 linked to the consumption of raw sprouts. Use of ionizing radiation was investigated as a means to reduce or to totally inactivate these pathogens, if present, on the sprouts. The radiation D value, which is the amount of irradiation in kilograys for a 1-log reduction in cell numbers, for these pathogens was established using a minimum of five doses at 19 +/- 1 degrees C. Before inoculation, the sprouts were irradiated to 6 kGy to remove the background microflora. The sprouts were inoculated either with Salmonella spp. cocktails made with either meat or vegetable isolates or with E. coli O157:H7 cocktails made with either meat or vegetable isolates. The radiation D values for the Salmonella spp. cocktails on sprouts were 0.54 and 0.46 kGy, respectively, for the meat and vegetable isolates. The radiation D values for the E. coli O157:H7 cocktails on sprouts were 0.34 and 0.30 kGy, respectively, for the meat and vegetable isolates. Salmonella was not detected by enrichment culture on sprouts grown from alfalfa seeds naturally contaminated with Salmonella after the sprouts were irradiated to a dose of 0.5 kGy or greater. Ionizing radiation is a process that can be used to reduce the population of pathogens on sprouts.

Alfalfa seed germination and yield ratio and alfalfa sprout microbial keeping quality following irradiation of seeds and sprouts.

Foods can be treated with gamma radiation, a nonthermal food process, to inactivate foodborne pathogens and fungi, to kill insects on or in fruits and vegetables, and to increase shelf life. Gamma irradiation is especially well suited for these treatments because of its ability to penetrate commercial pallets of foods. Irradiated fruits, vegetables, poultry, and hamburger have been received favorably by the public and are now available in supermarkets. The use of irradiation on fresh alfalfa sprouts was studied to determine its effect on keeping quality as related to aerobic microbial load. After an irradiation dose of 2 kGy, the total aerobic count decreased from 10(5-8) to 10(3-5) CFU/g, and the total coliform counts decreased from 10(5-8) to 10(3-0) CFU/g. The results showed that the sprouts maintained their structure after irradiation, and the keeping quality was extended to 21 days, which is an increase of 10 days from the usual shelf life. The effect of various doses of irradiation on alfalfa seeds as measured by percent germination and yield ratio (wt/wt) of sprouts was determined. There was little effect on the percent germination, but as the dose increased, the yield ratio of alfalfa sprouts decreased. As the length of growing time increased, so did the yield ratio of the lower dose irradiated seeds (1 to 2 kGy). The irradiation process can be used to increase the shelf life of alfalfa sprouts, and irradiating alfalfa seeds at doses up to 2 kGy does not unacceptably decrease the yield ratio for production of alfalfa sprouts.

Irradiation D-Values for Escherichia coli O157:H7 and Salmonella sp. on Inoculated Broccoli Seeds and Effects of Irradiation on Broccoli Sprout Keeping Quality and Seed Viability†

Like alfalfa sprouts, broccoli sprouts can be a vehicle for bacterial pathogens, which can cause illness when they are consumed. The gamma irradiation process was used to reduce numbers of bacterial pathogens on broccoli sprouts and seeds, and the effect of this process on the seeds was studied. The irradiation destruct values for Salmonella sp. and for strains of Escherichia coli O157:H7 inoculated on broccoli seeds were determined. Results obtained in this study indicate that a dose of 2 kGy reduced total background counts for broccoli sprouts from 10(6) to 10(7) CFU/g to 10(4) to 10(5) CFU/g and increased the shelf life of the sprouts by 10 days. Yield ratio (wt/wt), germination percentage, sprout length, and thickness were measured to determine the effects of various irradiation doses on the broccoli seeds. Results show a decreased germination percentage at a dose level of 4 kGy, whereas the yield ratio (wt/wt), sprout length, and thickness decreased at the 2-kGy dose level. The radiation doses required to inactivate Salmonella sp. and strains of E. coli O157:H7 were higher than previously reported values. D-values, dose required for a 1-log reduction, for the nonvegetable and vegetable Salmonella sp. isolates were 0.74 and 1.10 kGy, respectively. The values for the nonvegetable and vegetable isolated strains of Escherichia coli O157:H7 were 1.43 and 1.11 kGy, respectively. With the irradiation process, a dose of up to 2 kGy can extend the shelf life of broccoli sprouts. A dose of > 2 kGy would have an adverse effect on the broccoli seed and decrease the yield of broccoli sprouts.

Inactivation of Escherichia coli O157:H7 and Salmonella by Gamma Irradiation of Alfalfa Seed Intended for Production of Food Sprouts†

Inonizing irradiation was determined to be a suitable method for the inactivation of Salmonella and Escherichia coli O157:H7 on alfalfa seed to be used in the production of food sprouts. The radiation D (dose resulting in a 90% reduction of viable CFU) values for the inactivation of Salmonella and E. coli O157:H7 on alfalfa seeds were higher than the D-values for their inactivation on meat or poultry. The average D-value for the inactivation of Salmonella on alfalfa seeds was 0.97 +/- 0.03 kGy; the D-values for cocktails of meat isolates and for vegetable-associated isolates were not significantly different. The D-values for nonoutbreak and outbreak isolates of E. coli O157:H7 on alfalfa seeds were 0.55 +/- 0.01 and 0.60 +/- 0.01 kGy, respectively. It was determined that the relatively high D-values were not due to the low moisture content or the low water activity of the seed. The D-values for Salmonella on alfalfa seeds from two different sources did not differ significantly, even though there were significant differences in seed size and water activity. The increased moisture content of the seed after artificial inoculation did not significantly alter the D-value for the inactivation of Salmonella. The results of this study demonstrate that 3.3- and 2-log inactivations can be achieved with a 2-kGy dose of ionizing radiation, which will permit satisfactory commercial yields of sprouts from alfalfa seed contaminated with E. coli O157:H7 and Salmonella, respectively.

Efficacy of Washing and Sanitizing Trailers Used for Swine Transport in Reduction of Salmonella and Escherichia coli

Healthy pigs can carry Salmonella in their intestine and may shed this pathogen because of stresses incurred during transportation, contaminating trailer floors and bedding material. If not cleaned and sanitized between trips, trailers and bedding have the potential to infect other farms, the abattoir environment, or other animals with Salmonella. Floors and bedding material from pig trailers were sampled to determine the efficacy of the abattoir-developed washing and sanitizing regime on the level of Salmonella before and after a single haul. Escherichia coli levels were an indicator of high contamination. The study also determined the effect of ambient temperature (during four seasons) and of the distance the pigs traveled in the haulers (> 500 miles or < 500 miles) on bacterial levels. Salmonella was isolated from 80% of the bedding material tested. Of the 188 floor samples taken, 41.5% were positive for Salmonella before washing, and 2.7% were positive after washing and sanitizing. E. coli was isolated from all bedding material and floor samples before washing, but washing and sanitizing significantly decreased levels (P < 0.05) by 2 logs. There was no significant difference (P > 0.05) in the number of Salmonella- or E. coli-positive trailers attributable to distance traveled or season of the year. These results demonstrate that washing and sanitizing the trailers after each load significantly reduced levels of Salmonella and its possible spread by the contaminated trailer and bedding, which ultimately could promote improvement in food safety.

Characteristics of selected strains of Bacillus cereus

Identification techniques for Bacillus cereus are unsettled even though there is an increased awareness of the organisms potential public health implications. Biochemical and morphological characteristics of 17 strains of B. cereus , including 10 isolated from confirmed foodborne outbreaks, were studied by routine methods. Of the characteristics attributed to B. cereus , two strains were negative for the Voges-Proskauer reaction and nitrate reduction; three did not utilize salicin; and five exhibited rhizoidal growth on nutrient agar. Heat resistance of the strains was determined using the serum bottle technique. In demineralized water D- values at 100°C ranged from 0.6 to 27 min, with z-values from 7.4 to 14.5°C. Mean growth constant (k/h) determined turbidimetrically in nutrient broth at 15, 21, 25, 35, 40, 45 and 50°C was 0.15, 0.39, 0.89, 1.54, 1.99, 2.54 and 2.08, respectively. No single feature typified pathogenic strains. At 7 or 11°C, sixteen strains produced hemolysin on blood agar plates, whereas at 45°C, only two strains were hemolytic. Phospholipase activity measured on egg yolk agar plates was evident for three strains at 7°C, for all strains at 35°C, and for only two strains at 45°C.

Simple technique to determine heat resistance of Bacillus stearothermophilus spores in fluid systems.

A simple, rapid, highly reproducible procedure was developed to determine heat resistance of Bacillus stearothermophilus spores in milk and soy protein-based formulas at temperatures > 100 C. Plating efficiencies on different media and heat activation temperatures were also studied. The procedure involved use of a serum bottle to which was added formula. The bottle was closed with a rubber septum and sealed air-tight with a crimped aluminum cap. The formula was agitated during heating in a thermostatically controlled oil bath, using a wrist action shaker. When the formula attained the desired temperature, a spore suspension was injected through the rubber septum, using a high-pressure GLC syringe. At selected time intervals, a portion was withdrawn from the bottle, using a sterile GLC syringe. The number of surviving spores was determined by plating on Trypticase Soy agar, which yielded significantly higher spore recovery count than did Trypticase Soy broth fortified with 1.5% agar with and without starch, or Dextrose Tryptone agar. The serum bottle procedure yielded higher D values than did the capillary tube procedure. The difference was significant where p = 0.05 but not where p = 0.01. With the serum bottle procedure, D values for spores in the milk protein base formula were 18.46, 3.56 and 1.13 min at 115, 121 and 125 C, respectively. In the soy protein base formula, D values were 26.1, 3.64 and 1.26 min, respectively. The z values were 7.7 and 7.6 Centigrade degrees (13.86 and 13.68 Fahrenheit degrees). Maximum heat activation of the spore was at 95 C for 10 min in milk protein base formula and at 100 C for 5 min in the soy product.

Radiation D10-values on thawed and frozen catfish and tilapia for finfish isolates of Listeria monocytogenes.

With the popularity of catfish and tilapia in the healthy diet, the consumption and harvesting of farm-raised finfish have increased. Since 1987 the pathogenic bacterium Listeria monocytogenes has been isolated from seafood, particularly farm-raised catfish in the United States. Seafood isolates of L. monocytogenes are now available. In order to maintain the raw finfish product, nonthermal interventions to remove bacterial pathogens need to be evaluated using these isolates. A nonthermal intervention process, irradiation, was used to determine the destruct values of the L. monocytogenes seafood isolates along with a nonpathogenic Listeria strain and an L. monocytogenes strain previously studied. The irradiation destruct values were obtained for each individual isolate inoculated on raw and frozen catfish or tilapia irradiated at 4 or -10 degrees C. The Dradiation values obtained for L. monocytogenes inoculated on raw or frozen catfish did not differ (P > 0.05) from the values obtained for strains inoculated on the raw or frozen tilapia. The Dradiation-values ranged from 0.48 to 0.85 kGy, with an average of 0.62 +/- 0.09 kGy, which is typical for Listeria. The data obtained have identified a multi-isolate cocktail that can be used for future radiation inactivation studies for L. monocytogenes inoculated on finfish.

Recovery and survival of Escherichia coli O157:H7 in reconditioned pork-processing wastewater.

The pathogen Escherichia coli O157:H7 has been recovered from various water sources and food samples. The growth potential of this bacterium in nutrient-limited, reconditioned wastewater from a pork-processing plant was determined over a temperature range of 4 to 46 degrees C. Even though the biological oxygen demand of the wastewater was <2 mg/liter, results of bioassays for assimilable organic carbon and the coliform growth response of the water suggested that sufficient nutrients were present to support limited bacterial growth. A three-strain mixture of E. coli O157:H7 grew over the temperature range of 10.2 to 29.4 degrees C. Bioassays appear to be a good indicator of the ability of this wastewater to support growth of this pathogen. Statistically higher levels of bacterial growth (P < 0.05) were detected on a nonselective medium (tryptic soy agar) than on a selective medium (sorbitol-MacConkey agar), suggesting that stress or injury of the bacterium occurs when the organism is exposed to the nutrient-limited conditions of the wastewater. These results indicate that E. coli O157:H7 can survive and grow in this particular nutrient-limited wastewater, suggesting a potential hazard if this water becomes contaminated with this pathogen.

Thermal inactivation of Escherichia coli O157:H7 and Salmonella on catfish and tilapia.

Thermal inactivation kinetics of individual cocktails of Escherichia coli O157:H7, or of Salmonella meat isolates or seafood isolates were determined in catfish and tilapia. Determinations were done at 55, 60 and 65 °C using a circulating-water bath and calculated using linear regression analysis. Salmonella seafood and meat isolates D-10 values on the finfish were the same and ranged from 425 to 450, 27.1 to 51.4, 2.04-3.8 s (z = 4.3 °C) at 55, 60 and 65 °C, respectively. The E. coli O157:H7 D-10 values ranged from 422 to 564, 45.2 to 55.5 and 3.3-4.2 s (z = 4.3 °C) at 55, 60 and 65° C, respectively. The only statistical difference (P ≤ 0.05) was found when comparing the D-10 values for E. coli O157:H7 at 55 °C on catfish and tilapia. The other D-10 values for the Salmonella at all temperatures and E. coli O157:H7 at 60 and 65 °C on the catfish or tilapia showed no statistical difference. D-10 values for the catfish and tilapia were significantly lower than the reported values in other food systems, but the z-values were within the literature reported range. These D-10 values can be used to determine cooking parameters of finfish.