Abstract

Burkholderia pseudomallei causes melioidosis, a potentially fatal disease whose clinical outcomes include rapid-onset septicemia and relapsing and delayed-onset infections. Like other facultative intracellular bacterial pathogens, B. pseudomallei is capable of survival in human phagocytic cells, but unlike mycobacteria, Listeria monocytogenes, and Salmonella serovar Typhimurium, the species has not been reported to survive as an endosymbiont in free-living amebae. We investigated the consequences of exposing Acanthamoeba astronyxis, A. castellani, and A. polyphaga to B. pseudomallei NCTC 10276 in a series of coculture experiments. Bacterial endocytosis was observed in all three Acanthamoeba species. A more extensive range of cellular interactions including bacterial adhesion, incorporation into amebic vacuoles, and separation was observed with A. astronyxis in timed coculture experiments. Amebic trophozoites containing motile intravacuolar bacilli were found throughout 72 h of coculture. Confocal microscopy was used to confirm the intracellular location of endamebic B. pseudomallei cells. Transmission electron microscopy of coculture preparations revealed clusters of intact bacilli in membrane-lined vesicles inside the trophozoite cytoplasm; 5 x 10(2) CFU of bacteria per ml were recovered from lysed amebic trophozoites after 60 min of coculture. Demonstration of an interaction between B. pseudomallei and free-living acanthamebae in vitro raises the possibility that a similar interaction in vivo might affect environmental survival of B. pseudomallei and subsequent human exposure. Endamebic passage of B. pseudomallei warrants further investigation as a potential in vitro model of intracellular B. pseudomallei infection.