Distinct genotypic profiles of the two major clades of Mycobacterium africanum

Abstract

Background: Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis (TB) and a member of the M. tuberculosis complex (MTC). Additional MTC species that cause TB in humans and other mammals include Mycobacterium africanum and Mycobacterium bovis. One result of studies interrogating recently identified MTC phylogenetic markers has been the recognition of at least two distinct lineages of M. africanum, known as West African-1 and West African-2.

Methods: We screened a blinded non-random set of MTC strains isolated from TB patients in Ghana (n = 47) for known chromosomal region-of-difference (RD) loci and single nucleotide polymorphisms (SNPs). A MTC PCR-typing panel, single-target standard PCR, multi-primer PCR, PCR-restriction fragment analysis, and sequence analysis of amplified products were among the methods utilized for the comparative evaluation of targets and identification systems. The MTC distributions of novel SNPs were characterized in the both the Ghana collection and two other diverse collections of MTC strains (n = 175 in total).

Results: The utility of various polymorphisms as species-, lineage-, and sublineage-defining phylogenetic markers for M. africanum was determined. Novel SNPs were also identified and found to be specific to either M. africanum West African-1 (Rv1332(523); n = 32) or M. africanum West African-2 (nat(751); n = 27). In the final analysis, a strain identification approach that combined multi-primer PCR targeting of the RD loci RD9, RD10, and RD702 was the most simple, straight-forward, and definitive means of distinguishing the two clades of M. africanum from one another and from other MTC species.

Conclusion: With this study, we have organized a series of consistent phylogenetically-relevant markers for each of the distinct MTC lineages that share the M. africanum designation. A differential distribution of each M. africanum clade in Western Africa is described.

Figure 1

The composite MTC PCR typing panel. Illustrated is the MTC PCR typing panel output pattern for A) a typical M. tuberculosis strain, B) a secondary pattern seen in some Cameroon genotype M. tuberculosis strains from Ghana, C) a typical M. africanum West African-1 strain, D) a typical M. africanum West African-2 strain, and E) a typical M. bovis strain [7]. PCR products and the 100-bp ladder (unlabelled lanes) were visualized by agarose gel electrophoresis and ethidium bromide staining. Lanes: 1, 16S rRNA; 2, cfp32 (Rv0577); 3, MiD3 (IS1561'); 4, RD4 (Rv1510); 5, RD7 (Rv1970); 6, RD1 (Rv3877-Rv3878); 7, RD9 (Rv2073c); 8, RD12 (Rv3120). WA - West African.

Figure 2

Summary diagram and phylogenetic postitions of the genomic markers interrogated against the Ghana MTC strain collection. Shown are the various major divisions of the MTC segregated according to the presence or absence of inter-species-, intra-species-, and sublineage-specific polymorphisms. Circles are placed at points in evolutionary history beyond which each strain that was evaluated possessed a consistent set of polymorphisms. The nodes are numbered in the figure as follows to denote: 1. RD12^can, 3' cfp32 deletion; 2. TbD1, narGHJI ^-215; 3. pks15/1 (7-bp deletion), katG⁴⁶³; 4. undefined deletion at the RD^Rio/MiD3 locus; 5. gyrA⁹⁵; 6. RD9, gyrB¹⁴⁵⁰; 7. RD713, TbD1¹⁹⁷, aroA²⁸⁵, Rv1332⁵²³; 8. RD711; 9. RD7, RD8, RD10, pks15/1 (6-bp deletion), katG²⁰³, 3'cfp32³¹¹, RD13¹⁷⁴, PPE55²¹⁴⁸, PPE55²¹⁵⁴; 10. Rv1510¹¹²⁹; 11. RD701, RD702, hsp65⁵⁴⁰, nat⁷⁵¹; 12. rpoB¹¹⁶³; 13. rpoB¹⁰⁴⁹; 14. RD1^das; 15. mmpL6⁵⁵¹; 16. MiD3; 17. RD12, RD13; 18. RD4; 19. RD1^BCG. Lineages that include strains from the Ghana collection are terminated with arrowheads. Note that distances are arbitrary and do not reflect the number of phylogenetically relevant polymorphisms present at each juncture. TbD1-positive M. tuberculosis is also known as "ancient" M. tuberculosis and TbD1-negative M. tuberculosis is also known as "modern" M. tuberculosis [1]. WA - West African.

Figure 3

Map of sub-Saharan West Africa illustrating the differential geographic distribution by country of the M. africanum clades. Current evidence suggests that only M. africanum West African-1 is found in Eastern-West Africa (Cameroon, and Nigeria; black) and M. africanum West African-2 alone is found in Western-West Africa (the Gambia, Guinea-Bissau, and Senegal; speckled), but that the two clades overlap in Central-West Africa (Côte D'Ivoire, Ghana, Niger, and Sierra Leone; grey).