Pathogenetic mechanisms of the intracellular parasite Mycobacterium ulcerans leading to Buruli ulcer.

The necrotising skin infection Buruli ulcer is at present the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. Buruli ulcer is an emergent disease that is predominantly found in humid tropical regions. There is no vaccine against Buruli ulcer and its treatment is difficult. In addition to the huge social effect, Buruli ulcer is of great scientific interest because of the unique characteristics of its causative organism, Mycobacterium ulcerans. This pathogen is genetically very close to the typical intracellular parasites Mycobacterium marinum and Mycobacterium tuberculosis. We review data supporting the interpretation that M ulcerans has the essential hallmarks of an intracellular parasite, producing infections associated with immunologically relevant inflammatory responses, cell-mediated immunity, and delayed-type hypersensitivity. This interpretation judges that whereas M ulcerans behaves like the other pathogenic mycobacteria, it represents an extreme in the biodiversity of this family of pathogens because of its higher cytotoxicity due to the secretion of the exotoxin mycolactone. The acceptance of the interpretation that Buruli ulcer is caused by an intracellular parasite has relevant prophylactic and therapeutic implications, rather than representing the mere attribution of a label with academic interest, because it prompts the development of vaccines that boost cell-mediated immunity and the use of chemotherapeutic protocols that include intracellularly active antibiotics.

Buruli ulcer.

Buruli ulcer is an indolent necrotizing disease of the skin, subcutaneous tissue, and bone that is caused by Mycobacterium ulcerans. Buruli ulcer is presently the third most common mycobacterial disease of humans, after tuberculosis and leprosy, and the least understood of the three. The disease remained largely ignored by many national public health programs, but more recently, it has been recognized as an emerging health problem, primarily due to its frequent disabling and stigmatizing complications. The contribution discusses various aspects of Buruli ulcer, including its geographic distribution, incidence, and prevalence; mode of transmission, pathogenesis, and immunity; clinical manifestations; laboratory diagnosis; differential clinical diagnosis; and treatment.

First cultivation and characterization of Mycobacterium ulcerans from the environment.

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 FINDINGS: One 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. CONCLUSION: Strain 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.

New data onthe ultrastructure of the membrane of Mycobacterium leprae

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown