Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan

Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606. IS2404 was identified by complete sequencing of a previously described repetitive DNA segment fromM. ulcerans. This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida(AsIs1), Escherichia coli (H repeat element), Vibrio cholerae (VcIS1), and Porphyromonas gingivalis(PGIS2). The second IS, IS2606, was discovered by sequence analysis of a HaeIII fragment of M. ulcerans genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS1554from M. tuberculosis. Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti(ISRm3), Burkholderia cepacia(IS1356), Corynebacterium diphtheriae, andYersinia pestis. PCR screening of DNA from 45 other species of mycobacteria with primers for IS2404 confirm that this element is found only in M. ulcerans. However, by PCR, IS2606 was also found in Mycobacterium lentiflavum, another slow-growing member of the genusMycobacterium that is apparently genetically distinct fromM. ulcerans. Testing the sensitivity of PCR based on IS2404 and IS2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations ofM. ulcerans among large numbers of other environmental mycobacteria.

Mycobacterium bovis BCG is one of the most commonly administered vaccines. Complications, including disseminated BCG disease, are rare but increasingly reported in immunodeficient children. There is growing recognition of the importance of differences between BCG vaccine strains. We determined the susceptibilities of five genetically distinct BCG vaccine strains to 12 antituberculous drugs.Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine is one of the most commonly administered vaccines worldwide. It is a live attenuated vaccine derived from M. bovis, and for historical reasons a number of genetically different BCG vaccine strains are in use today (3,17). In immunocompetent individuals local adverse reactions such as injection site abscess and lymphadenitis occur in less than 1% of vaccinees (21) and disseminated BCG disease is extremely rare (12). However, in individuals with congenital or acquired immunodeficiency the risk of local complications and disseminated BCG disease after immunization is considerably greater (7). Recent reports from regions with a high prevalence of human immunodeficiency virus suggest that BCG-related complications are becoming increasingly common (7). The appropriate treatment of local adverse reactions and disseminated BCG disease remains controversial, and the influence of the particular BCG vaccine strain or its susceptibility pattern is not considered (6,8,9). There are only limited data on the susceptibility of BCG to different antituberculous drugs and even less on the influence of genetic differences between BCG vaccine strains on susceptibility.BCG vaccine strains were kindly provided by their manufacturers as follows: BCG-Bulgaria (SL 222 Sofia), BB-NCIPD Ltd., Sofia, Bulgaria; BCG-Denmark (SSI 1331), Statens Serum Institute, Copenhagen, Denmark; and BCG-Japan (Tokyo 172) Japan BCG Laboratory, Tokyo, Japan. BCG-Connaught (Sanofi Pasteur, Toronto, Canada) and BCG-Medac (RIVM from 1173-P2; Medac, Hamburg, Germany) were purchased. Freeze-dried BCG vaccine strains were dissolved in 0.5 ml of 0.9% saline (10 ml for BCG-Medac) and cultured using the MB/BacT system (Biomérieux, Durham, NC) according to manufacturer's instructions and subsequently subcultured on solid media (Lowenstein-Jensen and Brown & Buckle agar) for antimicrobial susceptibility testing. Susceptibility testing was done using the Bactec MGIT (mycobacterial growth indicator tube) 960 system (Becton Dickinson, MD) according to the manufacturer's instructions. Lyophilized streptomycin, isoniazid, rifampin (rifampicin), and ethambutol (Bactec MGIT SIRE kit; Becton Dickinson, MD) were each reconstituted and used according to the manufacturer's instructions. Amikacin, capreomycin, ethionamide, kanamycin, and ofloxacin (all Sigma), ciprofloxacin (Bayer), and rifabutin (Farm Italia, Carlo Erba, Research Laboratories) were dissolved and used at the recommended concentrations (Table 1) (11). For susceptibility testing of all drugs except pyrazinamide, the drugcontaining tubes were inoculated with a suspension pre...

Infection with M genavense may be responsible for more than 10% of disseminated nontuberculous mycobacterial infections in patients with human immunodeficiency virus infection. Its clinical presentation and response to treatment are similar to those of infection with M avium-intracellulare complex.

Mycobacterium ulcerans is an environmental bacterium which causes chronic skin ulcers. Despite significant epidemiological evidence to suggest that water is the source of infection, the organism has never been identified in the environment. Environmental water samples were collected from a small town in which an outbreak of 29 cases had occurred in a 3-year period. These were examined by mycobacterial culture and PCR amplification. Similar to previous studies, M. ulcerans was not cultured from the water samples. However, five samples were positive for M. ulcerans by PCR. These samples were collected from a swamp and a golf course irrigation system within the outbreak area. This is the first time that M. ulcerans has been demonstrated to be present in the environment and supports the postulated epidemiology of disease due to this organism.