Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls

Abstract

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-micron-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.

FIG. 1

Growth of the M. avium serotype 4 strain, L. pneumophila JR32, and E. coli HB101 in cocultures with A. polyphaga. Each coculture experiment was performed three times in Acanthamoeba buffer at 33°C. The values shown are the mean numbers of CFU (± standard deviations) determined at zero time and at 1, 7, and 14 days after coincubation was started.

FIG. 2

Growth of the M. avium serotype 4 strain, L. pneumophila JR32, and E. coli HB101 when they were separated from A. polyphaga by a 0.1-μm-pore-size polycarbonate membrane (parachamber). Each parachamber experiment was performed three times in Acanthamoeba buffer at 33°C. The values shown are the mean numbers of CFU (± standard deviations) determined at zero time and at 1, 7, and 14 days after inoculation.

FIG. 3

Transmission electron micrograph of M. avium serotype 4 bacilli within cytoplasmic vesicles of an A. polyphaga trophozoite after 2 days of coincubation. Bar = 1 μm.

FIG. 4

Differential live-dead fluorescence staining of M. avium serotype 4 bacilli within vesicles of A. polyphaga trophozoites after 2 days of coincubation. Green fluorescence indicates live bacterial cells that have an intact membrane.

FIG. 5

Transmission electron micrograph of a mature A. polyphaga cyst containing M. avium serotype 4 bacilli (arrow) within the double cell wall (note that the outer cell wall is divided and surrounds the bacterial cells). Bar = 1 μm.