avium) 2.6

± 2.2 vacuoles Exocyst M. chimaera 3.6 ± 2.6 vacuoles Exocyst, cytoplasm M. intracellulare 4.6 ± 4.8 vacuoles Exocyst, Endocyst M. colombiense 5.7 ± 6.2 vacuoles Exocyst, cytoplasm M. arosiense 9.4 ± 15.2 vacuoles Exocyst Moreover, we observed that all MAC species can survive within such A. polyphaga cyst. This occurrence did not merely result from the potential contamination of the amoeba by extra-amoebal mycobacteria, since we destroyed any MAC organism left on the surface of cysts by incubating the cysts in HCl, a method previously demonstrated to kill remaining trophozoites, immature cysts and extra-amoebal M. avium [21]. We checked the efficacy of this process by incubating the rinsing buffer on Middlebrook and found no growth of mycobacteria, which indicated Erismodegib price that the HCl had indeed destroyed any extracystic MAC organisms. The fact that all of the MAC species survived in the exocyst may be relevant to the persistence of these organisms

in the environment despite adverse conditions. Non-tuberculous mycobacteria, including M. avium, have been shown to persist up to 26 months in drinking water systems despite filtration and ozonation [45]. Also, M. intracellulare and other non-tuberculous mycobacteria have been shown to be protected against 15 mg/liter of free-chlorine for 24 hours by entrapment within A. polyphaga cysts [3]. Therefore, free-living amoeba cysts may be a “”Trojan horse”" for MAC organisms and check details protect them from adverse environmental conditions, including high concentrations of chlorine, as previously reported for other environmental mycobacteria. Conclusion The Tangeritin data presented herein on MAC species illustrate that survival within the amoebal exocyst is a significant feature of environmental mycobacteria. This particular location, preserving mycobacteria from adverse environment, nevertheless allow them to rapidly escape from the amoebal cyst. The mechanisms for such unique location remain to be established in environmental mycobacteria. Methods Mycobacterium strains M. avium subsp. avium ATCC 25291T, M. chimaera DSM 446232T,

M. colombiense CIP 108962T, M. arosiense DSM45069T [33], M. marseillense CSURP30T, M. timonense CSURP32T and M. bouchedurhonense CSURP34T [35] reference strains that were previously identified by 16S rRNA and rpoB gene sequencing [34] were subcultured on Middlebrook 7H10 agar (Becton Dickinson, Le Pont de Claix, France) for 7 days at 30°C under a 5% CO2 atmosphere. Cells were washed in 1.5 ml phosphate buffered saline (PBS), pH 7.3, by centrifugation at 8,600 g, and the inoculum was adjusted to 106 bacteria/ml in PBS. Infection of amoeba The A. polyphaga strain Linc-AP1 was obtained from T. J. Rowbotham, Public Health Laboratory, Leeds, United Kingdom and cultured at 28°C for 3 days in 150 cm3 culture flasks (Corning, New York USA) that contained 30 ml PYG broth [46]. Amoebal cells were harvested by centrifugation at 500 g for 10 min.