Mycobacterium tuberculosis Complex Mycobacteria as Amoeba-Resistant Organisms

Background: Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii. Methodology/Principal Findings: Using optic and electron microscopy and co-culture methods, we observed that 89 +/- 0.6% of M. canettii, 12.4 +/- 0.3% of M. tuberculosis, 11.7 +/- 2% of M. bovis and 11.2 +/- 0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32 +/- 2% for M. canettii, 26 +/- 1% for M. tuberculosis, 28 +/- 2% for M. bovis and 36 +/- 2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10(-6)) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34x10(4) CFU/mL) than M. tuberculosis (42x10(1) CFU/mL) and M. bovis (35x10(1) CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde. Conclusions/Significance: These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.