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.

Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67–5.47, p<0.001). Stratifying our analysis by the two phylogenetic lineages of M. africanum (i.e. MTBC Lineages 5 and 6) revealed that this association was mainly driven by Lineage 5 (also known as M. africanum West Africa 1). Our findings suggest interactions between the genetic diversity of MTBC and human diversity, and offer a possible explanation for the geographical restriction of M. africanum to parts of West Africa.

BackgroundThere is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007–2014.MethodologyMycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpoß PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively.ResultsAmong 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively.ConclusionThis study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

BackgroundMycobacterium africanum comprises two phylogenetic lineages within the M. tuberculosis complex (MTBC) and is an important cause of human tuberculosis (TB) in West Africa. The reasons for this geographic restriction of M. africanum remain unclear. Here, we performed a prospective study to explore associations between the characteristics of TB patients and the MTBC lineages circulating in Ghana.MethodWe genotyped 1,211 MTBC isolates recovered from pulmonary TB patients recruited between 2012 and 2014 using single nucleotide polymorphism typing and spoligotyping. Associations between patient and pathogen variables were assessed using univariate and multivariate logistic regression.ResultsOf the 1,211 MTBC isolates analysed, 71.9 % (871) belonged to Lineage 4; 12.6 % (152) to Lineage 5 (also known as M. africanum West-Africa 1), 9.2 % (112) to Lineage 6 (also known as M. africanum West-Africa 2) and 0.6 % (7) to Mycobacterium bovis. Univariate analysis revealed that Lineage 6 strains were less likely to be isoniazid resistant compared to other strains (odds ratio = 0.25, 95 % confidence interval (CI): 0.05–0.77, P < 0.01). Multivariate analysis showed that Lineage 5 was significantly more common in patients from the Ewe ethnic group (adjusted odds ratio (adjOR): 2.79; 95 % CI: 1.47–5.29, P < 0.001) and Lineage 6 more likely to be found among HIV-co-infected TB patients (adjOR = 2.2; 95 % confidence interval (CI: 1.32–3.7, P < 0.001).ConclusionOur findings confirm the importance of M. africanum in Ghana and highlight the need to differentiate between Lineage 5 and Lineage 6, as these lineages differ in associated patient variables.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1725-6) contains supplementary material, which is available to authorized users.

Ethnopharmacological relevanceTuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants.Material and methodsThe microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis.ResultsResults of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3 µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8).ConclusionThe overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis.

SummaryWe spoligotyped and screened 1490 clinical Mycobacterium tuberculosis complex strains from Northern and Greater Accra regions of Ghana against INH and RIF using the microplate alamar blue phenotypic assay. Specific drug resistance associated genetic elements of drug resistant strains were analyzed for mutations. A total of 111 (7.5%), 10 (0.7%) and 40 (2.6%) were mono-resistant to INH, RIF, and MDR, respectively. We found the Ghana spoligotype to be associated with drug resistance (INH: 22.1%; p = 0.0000, RIF: 6.2%; p = 0.0103, MDR: 4.6%; p = 0.0240) as compared to the Cameroon spoligotype (INH: 6.7%, RIF: 2.4%, MDR: 1.6%). The propensity for an isolate to harbour katG S315T mutation was higher in M. tuberculosis (75.8%) than Mycobacterium africanum (51.7%) (p = 0.0000) whereas the opposite was true for inhApro mutations; MAF (48.3%) compared to MTBSS (26.7%) (p = 0.0419). We identified possible novel compensatory INH resistance mutations in inhA (G204D) and ahpCpro (-88G/A and -142G/A) and a novel ndh mutation K32R. We detected two possible rpoC mutations (G332R and V483G), which occurred independently with rpoB S450L, respectively. The study provides the first evidence that associate the Ghana spoligotype with DR-TB and calls for further genome analyses for proper classification of this spoligotype and to explore for fitness implications and mechanisms underlying this observation.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches.

This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acidtrimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCEDiseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.