Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.

BackgroundThis study aimed to compare the prevalence of Mycobacterium tuberculosis complex (MTBc) lineages between direct genotyping (on sputum) and indirect genotyping (on culture), to characterize potential culture bias against difficult growers.Methodology/Principal findingsSmear-positive sputa from consecutive new tuberculosis patients diagnosed in Cotonou, (Benin) were included, before patients had started treatment. An aliquot of decontaminated sputum was used for direct spoligotyping, and another aliquot was cultured on Löwenstein Jensen (LJ) medium (90 days), for indirect spoligotyping. After DNA extraction, spoligotyping was done according to the standard method for all specimens, and patterns obtained from sputa were compared versus those from the derived culture isolates. From 199 patient’s sputa, 146 (73.4%) yielded a positive culture. In total, direct spoligotyping yielded a pattern in 98.5% (196/199) of the specimens, versus 73.4% (146/199) for indirect spoligotyping on cultures. There was good agreement between sputum- and isolate derived patterns: 94.4% (135/143) at spoligotype level and 96.5% (138/143) at (sub)lineage level. Two of the 8 pairs with discrepant pattern were suggestive of mixed infection in sputum. Ancestral lineages (Lineage 1, and M. africanum Lineages 5 and 6) were less likely to grow in culture (OR = 0.30, 95%CI (0.14 to 0.64), p = 0.0016); especially Lineage 5 (OR = 0.37 95%CI (0.17 to 0.79), p = 0.010). Among modern lineages, Lineage 4 was over-represented in positive-culture specimens (OR = 3.01, 95%CI (1.4 to 6.51), p = 0.005).Conclusions/ SignificanceAncestral lineages, especially M. africanum West African 1 (Lineage 5), are less likely to grow in culture relative to modern lineages, especially M. tuberculosis Euro-American (Lineage 4). Direct spoligotyping on smear positive sputum is effective and efficient compared to indirect spoligotyping of cultures. It allows for a more accurate unbiased determination of the population structure of the M. tuberculosis complex.Trial registrationClinicalTrials.gov NCT02744469

47Human tuberculosis is caused by members of the Mycobacterium tuberculosis Complex 48 (MTBC). The MTBC comprises several human-adapted lineages known as M. 49 tuberculosis sensu stricto as well as two lineages (L5 and L6) traditionally referred to as 50 M. africanum. Strains of L5 and L6 are largely limited to West Africa for reasons 51 unknown, and little is known on their genomic diversity, phylogeography and evolution. 52 Here, we analyzed the genomes of 365 L5 and 326 L6 strains, plus five related genomes 53 that had not been classified into any of the known MTBC lineages, isolated from patients 54 from 21 African countries. 55 Our population genomic and phylogeographical analyses show that the unclassified 56 genomes belonged to a new group that we propose to name MTBC Lineage 9 (L9). While 57 the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely 58 ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, 59 we found important differences between L5 and L6 strains with respect to their 60 phylogeographical substructure, genetic diversity and association with drug resistance. 61In conclusion, our study sheds new light onto the genomic diversity and evolutionary 62 history of M. africanum, and highlights the need to consider the particularities of each 63 MTBC lineage for understanding the ecology and epidemiology of tuberculosis in Africa 64 and globally. 65 3 MAIN TEXT 66 67 Introduction 68 Tuberculosis (TB) causes more human deaths than any other infectious disease, and it is 69 among the top ten causes of death worldwide (1). Among the 30 high TB burden 70 countries, half are in Sub-Saharan Africa (1). Africa also comprises the highest number 71 of countries with the highest TB mortality (1). TB in humans and animals is caused by 72 the Mycobacterium tuberculosis Complex (MTBC) (2), which includes different lineages, 73 some referred to as Mycobacterium tuberculosis sensu stricto (Lineage 1 to Lineage 4 and 74 Lineage 7) and others as Mycobacterium africanum (Lineage 5 and Lineage 6), a recently 75 discovered Lineage 8 (3), as well as different animal-associated ecotypes such as M. 76 bovis, M. pinnipedii, or M. microti among others (4, 5). Among the human-associated 77 MTBC lineages, some are geographically widespread and others more restricted (6). The 78latter is particularly the case for Lineage (L) 7 that is limited to the Horn of Africa (7, 8), 79 and L5 and L6 that are mainly found in West Africa (9). L5 and L6 differ substantially 80 from the other lineages of the MTBC with respect to metabolism and in vitro growth (10, 81 11). Several mutations in different genes of the electron transport chain and central carbon 82 metabolic pathway can explain metabolic differences between L5 and L6 and the other 83 lineages (12). L5 and L6 are also less virulent than other lineages in animal models, and 84 appear to transmit less efficiently in clinical settings (13, 14). Even though L5 and L6 are 85 mostly restricted to Wes...