Genetic Diversity of Mycobacterium tuberculosis Population in Bulgaria
Résumé
Tuberculosis remains an important public health issue for Bulgaria, a Balkan country located in the world region with contrasting epidemiological situation for tuberculosis. Here, we present results of recent studies on the genetic diversity of Mycobacterium tuberculosis population in Bulgaria, evaluated with various DNA fingerprinting methods (spoligotyping, 24-MIRU-VNTR and IS6110-RFLP typing). The spoligotype-based population structure of M. tuberculosis in Bulgaria was shown to be sufficiently heterogeneous. It is dominated by several worldwide distributed spoligotypes ST53 and ST47 and Balkan-specific spoligotypes ST125 and ST41. The Beijing genotype strains were not found in Bulgaria in spite of close links with Russia in the recent and historical past. Comparison with international database SITVIT2 (Pasteur Institute of Guadeloupe) showed that spoligotype ST53 is found in similar and rather high proportion in the neighboring Greece and Turkey and almost equally distributed across different regions of Bulgaria. Contrarily, ST125 is not found elsewhere and is specific for Bulgaria; furthermore it appears to be mainly confined to the southern part of the country. Novel 15/24-loci format of MIRU-VNTR typing was found to be the most discriminatory tool compared to spoligotyping and IS6110-RFLP typing of M. tuberculosis strains in Bulgaria. Furthermore, VNTR typing was shown useful for resolving ambiguous phylogeny of some spoligotypes, in particular, those classified as LAM/S by bioinformatics approach. In practical terms, a reduced "Bulgaria-specific" 5-locus set (MIRU40, Mtub04, Mtub21, QUB-11b, QUB-26) provided a sufficiently high differentiation and may be preliminarily recommended for a first-line typing of M. tuberculosis isolates in Bulgaria although further studies are needed to validate this scheme. At the same time, a comprehensive secondary subtyping of the clustered isolates should target all 15 discriminatory loci. We additionally investigated molecular basis of drug resistance of the studied strains. Three types of the rpoB mutations were found in 20 of 27 RIF-resistant isolates; rpoB S531L was the most frequent. Eleven (48%) of 23 INH-resistant isolates had katG S315T mutation. inhA -15C>T mutation was detected in one INH-resistant isolate (that also had katG315 mutation) and three INH-susceptible isolates. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. Consequently, rpoB and embB306 mutations may serve for rapid genotypic detection of the majority of the RIF and EMB-resistant strains in Bulgaria; the results on INH resistance are complex and further investigation of more genes is needed. Comparison with spoligotyping and 24-VNTR locus typing data suggested that emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any specific spoligotype or MIRU-VNTR genotype. A local circulation of the particular clones appears to be an important factor to take into consideration in the molecular epidemiological studies of tuberculosis in Bulgaria.