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

The survival of virulent and avirulent Listeria species internalized in cells of a murine macrophage-like cell line, RAW264.7, was monitored. Mouse macrophage cells (ca. 5 X 10(5)/ml) suspended in fresh RPMI medium 1640 containing fetal bovine serum were mixed with 5 X 10(7) to 5 X 10(8) Listeria cells per ml and incubated 1 h at 37 degrees C with CO2-enriched air. Gentamicin (10 mu g/ml) was added to kill bacteria not internalized by the cells. At 2, 4, and 6 h postinfection, 10-mu l amounts of the suspensions were lysed in microtiter plate wells during serial decimal dilution in water. Triplicate dilutions (10 mu l each) were plated on trypticase soy agar, and colonies were counted after 48 h incubation at 35 degrees 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 hemolytic 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.