Mycobacterium insubricum sp. nov

1 Mycobacteriology Reference Center, Microbiology Laboratory, Careggi Hospital, Florence, Italy 2 Department of Pulmonary Diseases, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands 3 DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 4 Cytogenetics and Genetics Unit, Careggi Hospital, Florence, Italy 5 National Mycobacteria Reference Laboratory, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Correspondence Enrico Tortoli e.tortoli{at}libero.it Five independent strains, isolated from clinical samples but probably not responsible for disease, revealed phenotypic and genotypic features that appeared to exclude their belonging to any of the recognized Mycobacterium species. The strains, which are non-pigmented rapid growers, presented a cell-wall lipid pattern resembling those of Mycobacterium brumae and Mycobacterium fallax . Sequencing of the 16S rRNA, hsp65 and rpoB genes and the 16S–23S rRNA gene internal transcribed spacer (ITS) revealed that the strains are clearly distinct from every other Mycobacterium species. While the 16S rRNA and rpoB genes were characterized by a single sequevar, two sequevars were detected in hsp65 and three in the ITS. The divergence shown in the latter region was striking, in which only two short regions (less than 150 bp in all) were comparable with other mycobacteria, apart from Mycobacterium monacense and Mycobacterium gilvum . The PCR restriction analysis pattern of the novel strains also differs from any reported to date. The name Mycobacterium insubricum sp. nov. is proposed for the novel species; the type strain is FI-06250 T (=DSM 45132 T =CIP 109609 T ). Abbreviations: ITS, internal transcribed spacer; MIC, minimal inhibitory concentration; PRA, PCR restriction analysis The GenBank/EMBL/DDBJ accession numbers for the sequences of the 16S rRNA gene and ITS of strains FI-06250 T and FI-06215 are EU605695 and EU605694 ; that for the 16S rRNA gene sequence of FI-05244 is DQ142672 ; those for the hsp65 gene sequences of strains FI-06250 T , FI-06215 and FI-05244 are EF584487 –EF584489; those for the ITS sequences of FI-05244 and NLA000701900 are EF584486 and EU624330 ; and those for rpoB gene sequences of FI-06250 T , M. fallax DSM 44179 T , M. brumae CIP 103465 T and M. triviale IP140330 01 are EU022519 and EU999023 –EU999025. Phylogenetic trees based on hsp65 and rpoB gene sequences of selected mycobacterial species and the fatty ac