Abstract:
Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of
a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to
have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this
assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by
spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered
from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the
other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two
M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several
isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven
isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the
identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7.
Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position
indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short
timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very
ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the
modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA
screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.
