A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n ؍ 39), M. marinum (n ؍ 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.Mycobacterium ulcerans causes Buruli ulcer (BU), a disease that represents, after tuberculosis and leprosy, the third most common mycobacterial disease in humans (23,27). BU has been observed in many tropical areas, but most patients have come from Central and West Africa and Australia (5,11,27). Epidemiologically, the disease is associated with riverine and swampy terrains (27). BU is a devastating disease characterized by necrotizing, ulcerative lesions of subcutaneous tissues and the overlying skin. The main specific genomic characteristics of M. ulcerans are the IS2404 element (34) and a 174-kb plasmid(s) that houses the genes for mycolactone, a polyketide toxin (39). M. marinum causes infections in humans and in fish (7,31). Large outbreaks of infection due to M. marinum have been described in association with swimming pools (swimming pool granuloma) (42) and fish tanks (fish tank granuloma) (13,14,17,19). M. ulcerans and M. marinum, once cultured, are readily identified by conventional mycobacterial characterization methods (46). Various DNA-based techniques have been used to type mycobacteria (6,33,35). Such studies have demonstrated a close taxonomic relationship between M. ulcerans and M. marinum, although M. ulcerans harbors IS2404 and M. marinum does not (34). Recently, however, IS2404 was found in an unusual mycobacterial isolate with phenotypic properties closely related to those of M. marinum (4).Previous studies showed limited genotypic diversity in M. ulcerans, especially among isolates from a given geographic region. PCR-restriction profile analyses of 16S rRNAs showed four different genotypic profiles of M. ulcerans, i.e., the ...