Survey of 150 strains belonging to the Mycobacterium terrae complex and description of Mycobacterium engbaekii sp. nov., Mycobacterium heraklionense sp. nov. and Mycobacterium longobardum sp. nov.

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 27353(T) ( = DSM 45694(T)). 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 13432(T) ( = LMG 24735(T) = CECT 7509(T)). 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 45394(T) ( = CCUG 58460(T)).