Virulence of Listeria monocytogenes, Listeria seeligeri, and Listeria innocua assayed with in vitro murine macrophagocytosis

Abstract

The survival of virulent and avirulent Listeria species internalized in cells of a murine macrophage-like cell line, RAW264.7, was monitored Mouse macrophage cell (ca. 5 × 105/ml) suspended in fresh RPMI medium 1640 containing fetal bovine serum were mixed with 5 × 107 to 5 x 108 Listeria cells per ml and incubated I h at 37°C with CO2-enriched air. Gentamicin (10 μg/ml) was added to kill bacteria not internalized by the cell. At 2, 4, and 6 h postinfection. 10-μl amounts of the suspensions were lysed in microtiter plate wells during serial decimal dilution in water. Triplicate dilutions (10 μl each) were plated on trypticase soy agar, and colonies were counted after 48 h incubation at 35°C. About 0.1 to 1% of the added hemolytic pathogen L. monocytogenes Scott A and the avirulent nonhemolytic L. innocua were internalized at 2 h. The number of internal L. monocytogenes cells increased significantly by 6 h, but L. innocua cells showed no significant change. A strain of the hemotytic species L. seeligeri behaved like the nonhemolytic L. innocua. This distinction between the intracellular behavior of pathogenic and nonpathogenic species, if a general phenomenon, may be useful as an in vitro virulence assessment parameter.