Abstract
Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.
Highlights
Non-tuberculous mycobacteria (NTM) are a ubiquitous group of diverse environmental mycobacteria related to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB)[1,2,3,4,5,6,7]
The nontuberculous mycobacteria (NTM)-NET published a survey of NTM patient isolates12: “A total of 20 182 patients had NTM species cultured from pulmonary samples in these centres in 2008; 91.3% (n=18 418) of the isolates were identified to species/complex level; the remaining 1764 isolates (8.7%) were not identified beyond Mycobacterium species other than M. tuberculosis complex
We propose a distance metric based on the concatenated alignment of the multi-locus sequence typing (MLST) loci and evaluate how this can be used in the absence of average nucleotide identity (ANI) for classification
Summary
Non-tuberculous mycobacteria (NTM) are a ubiquitous group of diverse environmental mycobacteria related to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB)[1,2,3,4,5,6,7]. A significant number of NTMs cause clinical disease. A total of 91 different NTM species were encountered in this survey.” While this was an important survey of NTM geographical distribution the low number of NTM species identified and the inability to classify some samples emphasizes the need for more classification resources. There are a large and growing number of nontuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. The ANI comparisons likewise can assist in the classification of version 2
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