Analysis of mycobacterium species for the presence of a macrolide toxin, mycolactone

Mycobacterium ulcerans is an environmental organism which is responsible for the disease Bunili ulcer, a resistant, severe, necrotizing skin disease. Bunlli ulcer occurs in West and Central Africa, Australia, and Asia, but it appears to be increasing significantly in West Africa. M ulcerans produces a polyketide-derived macrolide, mycolactone, which is required for virulence. Mycobacterium is a large genera with diverse species. Most of these species are non-pathogens present in soil and water. Although mycolactone production has been demonstrated in M ulcerans from many countries, a search for mycolactone in other Mycobacterium species has not been undertaken. We have extracted polyketides from 46 Mycobacterium isolates, analyzed them with thin-layer chromatography (TLC), and tested them in a murine fibroblast cell line (L929) to investigate whether mycolactone or a similar lipid are produced by Mycobacterium species other than M ulcerans. In these studies acetone soluble lipids (ASL) were prepared from a chloroform-methanol extract obtained from 4 fast growing species and 25 slow growing species of Mycobacterium. Among the slow growing species were 16 uncharacterized, species of Mycobacterium isolated from aquatic sources in areas endemic to M ulcerans infection. Biochemical and cell culture assays of these lipids showed that whereas several strains of mycobacteria studied produced toxic lipids when tested in cell culture, none made mycolactone. Whereas mycolactone is toxic at picogram amounts, none of the lipids tested from other Mycobacterium species were active at less than 10 μg amounts, and none produced· a cellular phenotype consistent with mycolactone activity. Two species of Mycobacterium, M scrofulaceum and M kansasii, contained lipids cytopathic to L929 fibroblasts at a concentration of less than 20 μg/μl. In addition, eight of the environmental isolates contained cytopathic lipids. Analysis of the 16S rRNA sequence of these 8 environmental isolates ofmycobacteria with cytopathic ASLs suggest that these 8 environmental isolates are novel species of Mycobacterium. Results from these studies show that although production of cytopathic lipids is relatively common among Mycobacterium species, the production of mycolactone appears so far to be restricted to M ulcerans.