Perfil genético por sequenciamento e genotipagem de cepas multirresistentes do <i>Mycobacterium tuberculosis</i> provenientes de diversos estados brasileiros

Abstract

Multidrug resistant (MR) tuberculosis is a major threat to public health due to the greater complexity, cost and side effects of treatment. Few studies have described the molecular epidemiology of MR <i>Mycobacterium tuberculosis</i> isolates in Brazil. In this study, we investigated the genetic diversity and mutations associated with drug resistance of 99 MR and 7 non-MR isolates collected in a 8-year period from 12 Brazilian states. This investigation was performed by restriction fragment length polymorphism analysis of the IS6110 insertion element (IS6110-RFLP), spoligotyping and sequencing of the rpoB and katG genes that, when mutated, confer resistance to isoniazid and rifampin, respectively. Mutations in rpoB and katG were found in 90.9% and 93% of the MR isolates analyzed, respectively. For the rpoB gene, 91.9% of the mutations were found in the 81-bp RRDR region Fifty-one (51.5%), 23 (23.3%) and 11 (11.1%) MR isolates had mutations in codons 531, 526 and 516, respectively. In the katG gene, mutations were found in 93% of the MR isolates analyzed. Seven isolates had mutations only in the first region analyzed (katG1). Codon 315 of the second katG gene region analyzed (katG2) had mutations in 82.8% of the MR isolates, mostly being a Ser315Thr substitution. Mutations were found in 30.3% of the MR isolates in the katG1 region and the majority was a codon 4 deletion. Spoligotyping detected 5 different M. tuberculosis families (with their subfamilies) in the MR isolates in this study. The most common were: LAM (46%), T (17%) and H (12%). We observed that one of the families, the EAI5, carries mutations in codon 463 of the katG gene, which does not occur for the other isolates. In addition, among our isolates we identified an isolate belonging to the Beijing family (highly virulent), but this fact is not alarming since this was only one case. We described new alleles for the rpoB and katG genes. With the exception of the X2 family, we identified an initial region of the katG gene with high frequency of mutations in the MR isolates. IS6110-RFLP analysis demonstrated that 25 isolates formed 11 genotypic groups while 74 showed a unique pattern of bands. This high rate of polymorphism indicates independent acquisition of resistance among our isolates. Concerning the H family, an inversion in the frequency of mutations occurred in the rpoB gene with codon 516 being the most mutated one followed by codons 526 and 531. The results of this study provide useful data for better understanding the spectrum of mutations of the MR isolates from patients in Brazil. Our results are also useful for the development of diagnostic tests for MR tuberculosis and to aid in tracking global transmission of this disease.