GENTAMICIN KILLS INTRACELLULAR LISTERIA-MONOCYTOGENES

The purpose of the experiments described here was to test whether memb rane-impermeant antibiotics present in the extracellular milieu could kill bacteria within macrophages. For this, mouse macrophage hybrids a nd elicited mouse peritoneal macrophages first were allowed to phagocy tose the facultative intracellular bacterium Listeria monocytogenes. T he cells were incubated with or without gentamicin, and their bacteric idal activity was measured. The results show that gentamicin caused no rmally nonbactericidal macrophages to kill L. monocytogenes. In additi on, gentamicin caused listericidal cells to kill significantly more ba cteria. To determine whether gentamicin accumulated within macrophages during culture, we tested whether lysates of macrophage hybrids cultu red for 72 h in gentamicin-containing medium and then washed could kil l Listeria cells. When cultured,vith 50 to 100 mu g of gentamicin per ml, but not when cultured with 0 to 5 mu g of gentamicin per ml, cell lysates were extremely listericidal, demonstrating the presence of int racellular gentamicin. Because gentamicin does not penetrate cell memb ranes, we hypothesized that it can be internalized by the cell through pinocytosis and can enter the same intracellular compartment as does phagocytosed L. monocytogenes. To test this, macrophages which had pha gocytosed L. monocytogenes mere incubated with the fluorochrome lucife r yellow to trace pinocytosed medium. About half of the Listeria cells within the macrophages were surrounded by lucifer yellow, indicating delivery of pinocytosed fluid, which could contain antibiotics, to pha gosomes containing bacteria. The experiments described here indicate t hat membrane-impermeant antibiotics can enter macrophages and kill int racellular bacteria. Thus, the use of gentamicin in macrophage bacteri cidal assays can interfere with the results and interpretation of expe riments designed to study macrophage bactericidal activity.