Background Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.Methodology/Principal FindingsOne culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5′ end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3′ end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.ConclusionStrain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactoneinduced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging, devastating, difficult-to-treat skin disease reported in many countries, mostly in tropical areas (16), and BU has become the third most common mycobacterial infection in humans after tuberculosis and leprosy. BU assumes various nonulcerative clinical forms that can progress to ulcers.M. ulcerans (26, 41, 47, 59), M. tuberculosis (24, 38, 42), M. marinum (25,57), and M. haemophilum (23) are known to have cytotoxic activity, M. ulcerans being the most cytotoxic of all known mycobacteria. The M. ulcerans toxin is a mycolactone, a unique polyketide lipid exotoxin that has a potent destructive activity for cells that provokes the extensive necrotic lesions characteristic of BU (26). Mycolactone is considered to be the major virulence factor of this pathogen (1, 26, 41).Intracellular parasites are defined on the basis of their lifestyles in the infected hosts and of the types of immune responses elicited (7, 37): they live and multiply predominantly within host cells, typically macrophages, and therefore are able to survive and grow within cultured macrophages. The host immune response against intramacrophage parasites involves mechanisms of cell-mediated immunity (CMI) accompanied by delayed-type hypersensitivity (DTH).Genetic analysis places M. ulcerans very close to M. marinum and M. tuberculosis (71), two species of mycobacteria that are typical intracellular p...

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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 ...

Mycobacterium ulcerans causes Buruli ulcer, an ulcerative skin disease in tropical and subtropical areas. Despite restricted genetic diversity, mycobacterial interspersed repetitive unit–variable-number tandem repeat analysis on M. ulcerans revealed 3 genotypes from different African countries. It is the first time this typing method succeeded directly on patient samples.