Membrane Damage and Viability Loss of Escherichia coil K - i 2 and Salmonella Enteritidis in Liquid Egg by Thermal Death Time Disk

Bacterial injury, including leakage of intracellular substance and viability loss, of Escherichia cvii K-12 (ATCC 23716) and Salmonella Enteritidis (ATCC 13076) inoculated in liquid egg white and liquid whole egg was determined by thermal death time disk. E. coli K-12 and Salmonella Enteritidis were inoculated in liquid egg white and liquid whole egg to a final count of 7.8 log CFU/mI and were thermally treated with thermal death time disks at room temperature (23°C), 54, 56, 58, and 60°C from 0 to 240 s. Sublethal injury, leakage of intracellular substances, and viability loss of E. co/i K12 and Salmonella Enteritidis was investigated by plating 0.1 ml on selective trypticase soy agar containing 3% NaCI, 5% NaCl, sorbitol MacConky agar, and xylose lysine sodium tetradecylsulfate and nonselective trypticase soy agar. No significant (P > 0.05) differences on percent injury or viability loss for E. co/i K-12 and Salmonella populations were determined in all samples treated at 23°C. Sublethal injury occurred in E. co/i and Salmonella populations at 54°C or above for 120 s. Viability losses for both bacteria averaged 5 log at 54°C or above for 180 s, and the surviving populations were below detection (<10 CFU/ ml). Thermal treatment at 40°C and above led to membrane damage, leakage, and accumulation of intracellular ATP from 2 to 2.5 log fglml and UV-absorbing substances of 0.1 to 0.39 in the treated samples. These results indicate similar thermal injury/damage on both E. co/i and Salmonella membranes as determined by the amount of inactivation, viability loss, and leakage of intracellular substances of bacteria. Microbial contamination of eggs as well as its economic implications to the poultry industry has been reported (6, 38). Salmonella is the most frequently reported cause of foodborne outbreaks of gastroenteritis in the United States (8, 9). Diseases caused by Salmonella (salmonellosis) have been steadily increasing as a public health problem in the United States since reporting began in 1943 (30). In recent years, Salmonella in eggs has emerged as a major concern for consumers and health agencies, and the risk of salmonellosis is greater when the egg is used as an ingredient in a food (32). Liquid eggs, homogenized as whole egg or separated into white and yolk, are used as ingredients in a wide variety of processed products, including bakery and confectionary products, drinks, infant products, dressings, and noodles (15). A 1-year survey of 20 egg-breaking plants in the United States found 6 to 20% of unpasteurized liquid egg samples to contain Salmonella Enteritidis (19). The presence of Salmonella Enteritidis in liquid egg was implicated as the source of salmonellosis infection (5, 32). Increase in the incidence of human food poisoning caused by Salmonella Enteritidis was reported in Europe and the United States (3, 8, 9, 26). However, pasteurized egg products have not been * Author for correspondence. Tel: 215 233-6427; Fax: 215 233 6406; E-mail: [email protected]. t Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. involved in any egg-associated outbreaks of gastroenteritis (8, 9, 26). The reason can he attributed to the U.S. Department of Agriculture (35) requirement that liquid egg be heated at 60°C and held for no less than 3.5 min to achieve a reduction of more than 3.0 log CFU/rnl Salmonella (15). Physical and chemical treatments are used in food processing to eliminate or at least reduce the presence of pathogenic and spoilage microorganisms in foods (1. 4, 13). The use of thermal processing to inactivate foodborne pathogens in food systems has been reported (28, 29, 37). Thermal processing of juice and liquid egg to inactivate foodborne pathogens might impair the characteristic flavor of the juice (1) and lead to coagulation of liquid egg (11, 13, 31). Quality attributes of liquid egg product are heat sensitive, and the time-temperature margins for a thermal pasteurized liquid egg free of bacteria are narrow. Therefore, the use of a minimum heat treatment process that would ensure food safety is needed. In designing such process treatment, care must be taken to factor in even distribution of heat in a short period to minimize bacterial resistance. Thermal resistance of bacteria in food systems that use capillary tubes and thermal death time (TDT) glass tubes have been reported and were found to have limited use for food samples with low viscosity (23. 24, 27). Similarly, temperature could not be monitored accurately in the capillary tubes or the come-up time related to thermal lag in large glass tubes (25). In this study, we monitored inactivation L ° ri lr,i.\H 7i' . W rHI:IN1 \[. IA( I IV\I Io 01 0 \(IIRI,\ IN LIQLIO LG of Escherichia co/i and Salmonella Enteritidis in liquid egg with a new aluminum TDT disk developed at Washington State University (Pullman, Wash.) (16). The TDT disk was attached with a type K thermocouple at the center to monitor treatment temperature within the disk. The volume of the TDT disk chamber is 1.2 ml and was constructed to allow rapid heating of samples in water or oil baths. The close, compact nature of this TDT disk provides near isothermal conditions necessary for the study of thermal death kinetics of microorganisms. In an earlier study from our laboratory, a thermal resistance of E. co/i K-12 to Salmonella Enteritidis in liquid egg with the use of this new TDT disk device was investigated and reported (16). In this study, we concentrated our effort in understanding the thermal effect of the disk treatment on sublethal injury, viability loss, and leakage of intracellular materials from the bacterial cells. Leakage of bacterial intracellular ATP and UVabsorbing substances as a function of membrane damage was also investigated with a bioluminescence ATP assay and spectrophotometer. The bioluminescence ATP assay has been used to measure direct membrane damage of Gandida albicans treated with ketoconazole and tioconazole (2) and Listeria monocytogenes treated with nisin, ampicillin, and streptomycin (34). MATERIALS AND METHODS Test strains and preparation of inocula. E. co/i K12 (ATCC 23716) and Salmonella Enteritidis (ATCC 13076) from the U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center culture collection, were used in this study. Individual cell cultures were maintained on trypticase soy agar (TSA) at 4°C. Before use, the cells were inoculated by loop in tryptic soy broth (TSB; Remel, Inc., Lenexa. Kans.) with incubation at 37°C for 16 to 18 h with shaking. A 0.1-ml cell aliquot was transferred to 20 ml of TSB and incubated at 37°C for 24 h. The overnight cell suspensions were centrifuged at 3,000 X g for 10 min at 5°C. The cell pellets were washed with an equal volume (100 ml) of sterile phosphate-buffered saline (PBS; pH 7.2) solution. Finally, the washed cells were resuspended in PBS and used as the inoculum. The inoculum was maintained at 5°C for less than 30 min until used to inoculate commercial pasteurized and laboratory-prepared liquid egg at 7.0 log CFU/mI as determined by plate count. Preparation of liquid egg samples. Commercial pasteurized liquid egg white (PLEW), without preservatives, and fresh Grade A whole eggs were purchased from a local grocery store. Surfaces of the whole eggs were sanitized with 70% alcohol, rinsed with sterile denionized water, and allowed to dry inside a biosafety cabinet for I Ii. The shells were broken and the contents emptied into a Stomachcr bag, and the contents were pummeled in Stomacher model 400 (Dynatech Laboratories, Alexandria, Va.) at 250 rpm for 30 s. The pH of the PLEW was 8.20, whereas the pH of our laboratory-prepared liquid whole egg (LWE) ranged from 7.50 to 7.65. To determine the initial bacterial load of each type of liquid eggs. 0.1 ml of the samples were plated in duplicate on ISA, and selective agar plates (sorbitol MacConkey agar [SMACI, xylose lysine sodium tetradecylsulfate [XLT4J, and ISA plates containing 3 and 5% NaCl [TSA3 and TSA5]; Difco, Beeton Dickinson, Sparks, Md.) incubated at 37°C for 24 h to determine the number of colony-forming units. Thermal inactivation of bacteria. The inside and outside temperatures of the TDT disks were monitored and recorded with a Fluke 54 II thermometer (Everett. Wash.) for all experiments. Before each use, the TDT disks were dipped in sanitizer (Coverage Plus. E. R. Squibb & Sons, Inc., St. Louis, Mo.) for 2 mm according to the manufacturer's instructions, rinsed five times with sterile water, and then air dried. An aliquot (1 ml) of PLEW and LWE inoculated with E. co/i or Salmonella Enteritidis was added to each TDT disk, The disks were hermetically sealed and then submerged completely in a water bath (Isotemp 110, Fisher Scientific. Pittsburgh, Pa.), with temperatures set at 52, 54, 56, 58, or 60 0.1°C. The samples were pulled from the water bath at 0 to 240 s and were immediately immersed in an ice water bath to cool before opening. Survival, injury, and inactivation of treated E. co/i or Salmonella Enteritidis in liquid egg samples were determined by plating on a range of agar plates mentioned below. Leakage of bacteria, intracellular UV-absorbing materials, and ATP related to membrane damage were investigated. The controls were E. coli and Salmonella Enteritidis populations in liquid egg samples inside the TDT disks and placed in a water bath maintained at room temperature (--23°C) for the same amount of time. Enumeration of viable E. co/i and Salmonella Enteritidis. The surviving population of each bacterium and injury resulting from the TDT disk treatment was investigated with the use of selective plates: SMAC. XLT4, TSA3. and TSA5 (Difco, Becton Dickinson). Sublethal injury resulting from the TDT disk treatments was monitored with the use of differential plating methods on nonselective versus selective agar plates, and the difference in viable populations enumerated was considered injured cells. Percent injury was calculated with the formula (I — colonies on selective agar/counts on nonselective agar) X 100. Aliquots (0.1 ml) of inoculated liquid egg in TDT disks treated at different temperatures for a specific time were plated on a range of agar plates as stated above. Also, the number of colony forming units per milliliter on nonselective and selective agar media was used to calculate the viability loss, which is defined as the difference (log CFU per milliliter) in bacteria between control and heat-treated TDT disk samples (18). Treated and untreated inoculated liquid eggs were stored at 5°C for 24 h and then plated on nonselective and selective afar media to determine the effect of treatment and immediate cold storage on cell viability. Untreated inoculated liquid eggs were used as positive controls for each experiment, and uninoculated TDT heat-treated liquid eggs were used as the negative controls. All agar plates were incubated at 25 and 37°C for 48 h. Leakage of bacterial intracellular UV-absorbing materials. To quantify the intracellular UV materials released from E. co/i K-12 and Salmonella Enteritidis, the extracts from treated and untreated cells containing the UV materials were measured at 260 and 280 nni with a spectrophotometer (DUR 530. Beckman Coolter, Fullerton. Calif.) according to published reports (7. 33. 37). Bioluminescence ATP assay. Extracellular (somatic) ATP content of commercial pasteurized and laboratory-prepared liquid egg before and after inoculation with E. co/i and Salmonella Enteritidis inside TDT disks was determined. Portions (0.1 nil) of PLEW and LWE samples were mixed with 0.1 ml of luciferinluciferase (Sigma. St. Louis. Mo.) and the somatic ATP content of the samples was measured in relative light units (RLIJs) with an ATP bioluminescent assay kit (Turner Design. Sunnyvale. Calif.). The generated light signal (RLU) was measured with a TD20/20 (DL Ready) luminometer (Turner Design) after 3 s of delay time and 14 s of integration time. Assays of standard amounts of