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Abstract
ABSTRACT
The prevalence of nontuberculous mycobacteria (NTM) pulmonary diseases has been increasing
worldwide. NTM consist of approximately 200 species and distinguishing between them
at the subspecies level is critical to treatment. In this study, we sequenced 63 NTM
genomes, 27 of which were newly determined, by hybrid assembly using sequencers from
Illumina and Oxford Nanopore Technologies (ONT). This analysis expanded the available
genomic data to 175 NTM species and redefined their subgenus classification. We also
developed a novel multi-locus sequence typing (MLST) database based on 184 genes from
7547 assemblies and an identification software, mlstverse, which can also be used
for detecting other bacteria given a suitable MLST database. This method showed the
highest sensitivity and specificity amongst conventional methods and demonstrated
the capacity for rapid detection of NTM, 10 min of sequencing of the ONT MinION being
sufficient. Application of this methodology could improve disease epidemiology and
increase the cure rates of NTM diseases.
Multilocus sequence typing (MLST) was proposed in 1998 as a portable sequence-based method for identifying clonal relationships among bacteria. Today, in the whole-genome era of microbiology, the need for systematic, standardized descriptions of bacterial genotypic variation remains a priority. Here, to meet this need, we draw on the successes of MLST and 16S rRNA gene sequencing to propose a hierarchical gene-by-gene approach that reflects functional and evolutionary relationships and catalogues bacteria 'from domain to strain'. Our gene-based typing approach using online platforms such as the Bacterial Isolate Genome Sequence Database (BIGSdb) allows the scalable organization and analysis of whole-genome sequence data.
The genus Mycobacterium contains 188 species including several major human pathogens as well as numerous other environmental species. We report here comprehensive phylogenomics and comparative genomic analyses on 150 genomes of Mycobacterium species to understand their interrelationships. Phylogenetic trees were constructed for the 150 species based on 1941 core proteins for the genus Mycobacterium, 136 core proteins for the phylum Actinobacteria and 8 other conserved proteins. Additionally, the overall genome similarity amongst the Mycobacterium species was determined based on average amino acid identity of the conserved protein families. The results from these analyses consistently support the existence of five distinct monophyletic groups within the genus Mycobacterium at the highest level, which are designated as the “Tuberculosis-Simiae,” “Terrae,” “Triviale,” “Fortuitum-Vaccae,” and “Abscessus-Chelonae” clades. Some of these clades have also been observed in earlier phylogenetic studies. Of these clades, the “Abscessus-Chelonae” clade forms the deepest branching lineage and does not form a monophyletic grouping with the “Fortuitum-Vaccae” clade of fast-growing species. In parallel, our comparative analyses of proteins from mycobacterial genomes have identified 172 molecular signatures in the form of conserved signature indels and conserved signature proteins, which are uniquely shared by either all Mycobacterium species or by members of the five identified clades. The identified molecular signatures (or synapomorphies) provide strong independent evidence for the monophyly of the genus Mycobacterium and the five described clades and they provide reliable means for the demarcation of these clades and for their diagnostics. Based on the results of our comprehensive phylogenomic analyses and numerous identified molecular signatures, which consistently and strongly support the division of known mycobacterial species into the five described clades, we propose here division of the genus Mycobacterium into an emended genus Mycobacterium encompassing the “Tuberculosis-Simiae” clade, which includes all of the major human pathogens, and four novel genera viz. Mycolicibacterium gen. nov., Mycolicibacter gen. nov., Mycolicibacillus gen. nov. and Mycobacteroides gen. nov. corresponding to the “Fortuitum-Vaccae,” “Terrae,” “Triviale,” and “Abscessus-Chelonae” clades, respectively. With the division of mycobacterial species into these five distinct groups, attention can now be focused on unique genetic and molecular characteristics that differentiate members of these groups.
[1
] Department of Infection Metagenomics, Genome Information Research Center, Research
Institute for Microbial Diseases, Osaka University, Suita, Japan
[2
] Department of Infectious, Respiratory, and Digestive Medicine, Graduate School of
Medicine, University of the Ryukyus, Nakagami-gun, Japan
[3
] Laboratory of Pathogen Detection and Identification, International Research Center
for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University,
Suita, Japan
[4
] Division of Clinical Laboratory and Blood Transfusion, University of the Ryukyus
Hospital, Nakagami-gun, Japan
[5
] Integrated Frontier Research for Medical Science Division, Institute for Open and
Transdisciplinary Research Initiatives, Osaka University, Suita, Japan
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