Connection between two historical tuberculosis outbreak sites in Japan, Honshu, by a new ancestral Mycobacterium tuberculosis L2 sublineage

By gathering 680 publicly available Sequence Read Archives from isolates of Mycobacterium tuberculosis complex (MTBC) including 190 belonging to the lineage 2 Beijing, and using an in-house bioinformatical pipeline, the TB-Annotator, that analyses more than 50 000 characters, we describe herein a new L2 sublineage from 20 isolates found in the Tochigi province, (Japan), that we designate as asia ancestral 5 (AAnc5). These isolates harbour a number of specific criteria (42 SNPs) and their intra-cluster pairwise distance suggests historical and not epidemiological transmission. These isolates harbour a mutation in rpoC, and do not fulfil, any of the modern Beijing lineage criteria, nor any of the other ancestral Beijing lineages described so far. Asia ancestral 5 isolates do not possess mutT2 58 and ogt 12 characteristics of modern Beijing, but possess ancestral Beijing SNPs characteristics. By looking into the literature, we found a reference isolate ID381, described in Kobe and Osaka belonging to the ‘G3’ group, sharing 36 out of the 42 specific SNPs found in AAnc5. We also assessed the intermediate position of the asia ancestral 4 (AAnc4) sublineage recently described in Thailand and propose an improved classification of the L2 that now includes AAnc4 and AAnc5. By increasing the recruitment into TB-Annotator to around 3000 genomes (including 642 belonging to L2), we confirmed our results and discovered additional historical ancestral L2 branches that remain to be investigated in more detail. We also present, in addition, some anthropological and historical data from Chinese and Japan history of tuberculosis, as well as from Korea, that could support our results on L2 evolution. This study shows that the reconstruction of the early history of tuberculosis in Asia is likely to reveal complex patterns since its emergence.