Virulence of Culture Filtrate from Heat-Injured and Repaired Listeria Strains: Assay on Bovine Mammary Epithelial (MAC-T) Cells

Abstract

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.