The effect of prior heat shock on thermotolerance of Listeria monocytogenes and Salmonella typhimurium in broth culture was determined. Bacteria were grown at the permissive temperature of 35°C, sublethally heated at 35 (control), 42, 48, and 52°C (nonpermissive control) for various times, and inactivated at either 57.8 or 52°C. The induction of increased thermotolerance by heat shock, although consistent within each experiment, was generally not significant for L. monocytogenes; the increase was significant for S. typhimurium. Temperature shift experiments with L. monocytogenes suggested that induced thermotolerance was not long lived unless the shock temperature was maintained. Exposure to temperatures above the range for normal cell growth (the normal of Arrhenius range) (13) leads to progressive loss of bacterial viability. When bacteria are shifted for a short period from lower to higher temperatures within or slightly above their normal growth range, a degree of protection against the lethal effects of a subsequent shift to a higher temperature or an acquired thermotolerance is achieved (17,20,26).Several investigators (20, 29) concluded that acquired thermotolerance develops during the brief incubation period at elevated but nonlethal temperature as a result of heat shock response, i.e., induction of a specific set of proteins known as heat shock proteins that mediate recovery to stress-induced damage (16,20,26). A direct cause-and-effect relationship between heat shock protein synthesis and acquired thermotolerance, however, is controversial (23, 26).The effect of heat shock response on food safety may be important because certain foods are thermally processed to ensure safety (11). Mackey and Derrick (17, 18) pioneered such studies with Salmonella spp. in broth, liquid whole egg, and reconstituted dried milk and concluded that marginal heat treatments may not be adequate in certain instances. Recent foodborne outbreaks of listeriosis were epidemiologically linked to the consumption of dairy products and meats; hence, the thermal processing parameters of these foods are being reexamined (5,8). This study was undertaken to determine the degree of acquired thermotolerance conferred on Listeria monocytogenes and Salmonella typhimurium by heat shock response and the kinetics and duration of its induction.(Results of this study were presented at the 12th Food Microbiology Research Conference, Chicago, Ill., 8 to 12 November 1989.) L. monocytogenes F5069 was obtained from Robert Weaver, Centers for Disease Control, Atlanta, Ga. This strain belongs to serotype 4b and was isolated from raw bovine milk (3, 4). S. typhimurium 42ScBs was obtained from J. G. Bradshaw, Food and Drug Administration, Cincinnati, Ohio. The strain was isolated from 2% lowfat milk, epidemiologically linked to the 1983 Chicago milk outbreak, and carries the outbreak strain plasmid profile (22).

Thermal resistance of intracellular and freely suspended Listeria monocytogenes that was associated with a milkborne outbreak of listeriosis was studied by using the sealed tube and slug flow heat exchanger methods. Test temperatures for the former method were 57.

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (107/ml) were suspended in sterile milk and heated at 71.7°C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 0.5 s; FDA, D = 1.4 + 0.3 s; USDA, D = 0.6 0.2 s; CE, D < 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells: NSB, D = 2.7 ± 0.8 s; FDA, D = 1.3 ± 0.4 s; USDA, D = 0.7 ± 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25°C for 7 days) failed to recover and multiply during experimental CEs (4°C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols. Recovery by the NSB procedure (68 cells per ml) was compromised by background flora. The above data suggest that any cells surviving high-temperature, short-time pasteurization will be injured and unable to multiply either during cold storage of milk or in the FDA or USDA systems. Thus, L. monocytogenes cells recovered in finished pasteurized milk products by these detection methods probably represent uninjured environmental contaminants.

The thermal resistance of Listeria monocytogenes associated with a milk-borne outbreak of listeriosis was determined in parallel experiments by using freely suspended bacteria and bacteria internalized by phagocytes. The latter inoculum was generated by an in vitro phagocytosis reaction with immune-antigen-elicited murine peritoneal phagocytes. The heat suspension medium was raw whole bovine milk. Both suspensions were heated at temperatures ranging from 52.2 to 71.7°C for various periods of time. Mean D values for each temperature and condition of heated suspension revealed no significant differences. The extrapolated D71.7rc (161F) value for bacteria internalized by phagocytes was 1.9 s. Combined tube and slug-flow heat exchanger results yielded an estimated D71.7'c value of 1.6 s for freely suspended bacteria. The intracellular position did not protect L. monocytogenes from thermal inactivation.

Full scare commercial pasteurization equipment operated at 72–73°C with a holding time of 15–16 s was used to determine the ability of commercial thermal processing to inactivate Listeria monocytogenes strain Scott A. Three methods of providing L. monocytogenes concentration in raw milk were employed: freely suspended (extra-cellular), inside bovine phagocytes (in vitro procedure), and inside bovine phagocytes in experimentally infected cows (in vivo). Three enrichment methods were used to assay for L. monocytogenes after pasteurization: cold enrichment (4°C, 28 d), selective enrichment of Lovett et al. (FDA procedure) (17), and the USDA-FSIS two stage enrichment procedure. In addition, a 1-L sample taken just before the vacuum breaker was incubated undiluted in the original sample container (4°C, 4 weeks). None of the four assay methods could detect Listeria in the pasteurized milk.