The isolation and characterization of a novel, rapidly growing, scotochromogenic mycobacterial species is reported. Eight independent strains were isolated from clinical specimens from six different countries of the world, two in Iran, two in Italy and one in each of following countries: Greece, the Netherlands, Sweden and the USA. Interestingly, two of the strains were isolated from cerebrospinal fluid. The strains were characterized by rapid growth and presented orange-pigmented scotochromogenic colonies. DNA-based analysis revealed unique sequences in the four regions investigated: the 16S rRNA gene, the rRNA gene internal transcribed spacer 1 and the genes encoding the 65 kDa heat-shock protein and the beta-subunit of RNA polymerase. The phylogenetic analysis placed the strains among the rapidly growing mycobacteria, being most closely related to
Mycobacterium gilvum
. The genotypic and phenotypic data both strongly supported the inclusion of the strains investigated here as members of a novel species within the genus Mycobacterium; the name Mycobacterium iranicum sp. nov. is proposed to indicate the isolation in Iran of the first recognized strains. The type strain is M05T ( = DSM 45541T = CCUG 62053T = JCM 17461T).
A thorough phenotypic and genotypic analysis of 150 strains belonging to the
Mycobacterium terrae
complex resulted in the identification of a number of previously unreported sequevars (sqvs) within the species known to belong to the complex. For the species
Mycobacterium arupense
, three sqvs were detected in the 16S rRNA gene, six sqvs in the hsp65 gene and 15 sqvs in the rpoB gene; in
Mycobacterium senuense
two sqvs were present in each of the three genetic regions; in
Mycobacterium kumamotonense
four, two and nine sqvs were found, respectively, and in
M. terrae
three, four and six sqvs were found, respectively. The inappropriate inclusion of
Mycobacterium triviale
within the
M. terrae
complex was confirmed. The limited utility of biochemical tests and of mycolic acid analyses for the differentiation of the members of
M. terrae
complex was also confirmed. The survey allowed the recognition of three previously undescribed species that were characterized by unique sequences in the 16S rRNA, hsp65 and rpoB genes. Mycobacterium engbaekii sp. nov. (proposed previously 40 years ago but never validly published) was characterized by pink photochromogenic pigmentation and rapid growth; phylogenetically it was related to
Mycobacterium hiberniae
. The type strain of this species, of which eight strains were investigated, is ATCC 27353T ( = DSM 45694T). A cluster of 24 strains was the basis for the description of Mycobacterium heraklionense sp. nov., which has an intermediate growth rate and is unpigmented; nitrate reductase activity is typically strong. Closely related to
M. arupense
with respect to the 16S rRNA gene, M. heraklionense sp. nov. could be clearly differentiated from the latter species in the other genetic regions investigated. The type strain is NCTC 13432T ( = LMG 24735T = CECT 7509T). Mycobacterium longobardum sp. nov., represented in the study by seven strains, was characterized by a unique phylogenetic location within the
M. terrae
complex, clearly divergent from any other species. The type strain is DSM 45394T ( = CCUG 58460T).
Seventy-six nontuberculous mycobacterial isolates obtained from patients living in Greece were analyzed with the GenoType Mycobacterium CM (for common mycobacteria) and AS (for additional species) assays. GenoType correctly identified all but one of the mycobacterial species. For this species, additional probes should be designed and added to the strip.
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