RESUMEN
The cytotoxic effect of culture filtrates from healthy, heat-stressed, and repaired Listeria monocytogenes and L. innocua on the bovine mammary epithelial cell line MAC-T was examined. Culture filtrates were collected from Listeria spp. following treatments which included: (i) 18 h of growth of Listeria at 37°C; (ii) sublethal heat treatment at 56°C for 50 minutes; (iii) repair of the injured cells at 37°C for 7 h; (iv) growth of repaired bacterial cells at 37°C for 36 h; and (v) heat injury at 56°C for 50 min of the cell population obtained after the initial repair and growth. Strains chosen for study included two genetic mutants of L. monocytogenes : a hemolysin-negative mutant, CNL 85/162 (Hly-) and a hemolysin-positive revertant, CNL 85/163 (Hly+). Culture filtrates obtained from Hly- bacteria did not prevent adhesion of the mammary epithelial cells and slightly stimulated their growth. In contrast, culture filtrates from Hly+ bacteria grown for 18 h significantly reduced the ability of MAC-T cells to adhere to the cell culture dishes, prevented the growth of those cells that were attached to the dishes, and caused cell death. Supernatants from Hly+ and Hly- following injury and during repair had no lethal effect on MAC-T cells. The effects of culture medium obtained after growth of the repaired Listeria cells on MAC-T cells were similar to those recorded for medium from the first 18 h growth for both strains, indicating that cells regain virulence potential once they have repaired and reinitiated growth. Culture filtrates obtained from L. monocytogenes Scott A showed results similar to those of Hly+, decreasing adherence and growth of MAC-T cells, while L. innocua culture filtrate had no adverse effect. The results of these experiments suggest that when injured, L. monocytogenes does not demonstrate adverse effects towards MAC-T cells. Once repair is completed and the listeria are growing, activity towards MAC-T cells is restored.
RESUMEN
Two food isolates of Listeria monocytogenes (strains ATCC 51414 and F5027) were sublethally injured by exposure to heat (56°C for 20 min) or to a chlorine sanitizer (Antibac, 100 ppm for 2 min). Percent injury following treatment ranged from 84% to 99%. Injured Listeria were repaired in Listeria repair broth (LRB) at 37°C. Comparison of the repair curves generated by each method indicated that the time for repair was greater for sanitizer-injured cells (14 h) than for heat-injured cells (5 h). Sites of injury were determined by repairing heat- and sanitizer-treated Listeria in LRB supplemented with one of the following inhibitors: rifampicin (10 and 20 µg/ml), chloramphenicol (5 µg/ml), cycloserine D (10 and 20 µg/ml), and carbonyl cyanide m-chlorophenyl-hydrazone(CCCP) (2.5 µg/ml). In both heat- and sanitizer-injured populations, a total inhibition of repair was seen following incubation with rifampicin, chloramphenicol and CCCP. These results clearly indicate a requirement for mRNA, protein synthesis, and oxidative phosphorylation for repair to occur. The cell wall is not a site of damage since cycloserine D had no effect on repair of heat- or sanitizer-injured Listeria . Investigation of damage to the cell membrane showed that stress caused by sublethal heat or sanitizer did not allow proteins or nucleotides to leak into the medium. The recognition of injury and repair in Listeria will lead to improved methods of detection and ultimately to control strategies which prevent outgrowth of this organism in foods.