Whole-genome sequencing as a tool for studying the microevolution of drug-resistant serial Mycobacterium tuberculosis isolates.

Treatment of drug-resistant tuberculosis requires extended use of more toxic and less effective drugs and may result in retreatment cases due to failure, abandonment or disease recurrence. It is therefore important to understand the evolutionary process of drug resistance in Mycobacterium tuberculosis. We here in describe the microevolution of drug resistance in serial isolates from six previously treated patients. Drug resistance was initially investigated through phenotypic methods, followed by genotypic approaches. The use of whole-genome sequencing allowed the identification of mutations in the katG, rpsL and rpoB genes associated with drug resistance, including the detection of rare mutations in katG and mixed populations of strains. Molecular docking simulation studies of the impact of observed mutations on isoniazid binding were also performed. Whole-genome sequencing detected 266 single nucleotide polymorphisms between two isolates obtained from one patient, suggesting a case of exogenous reinfection. In conclusion, sequencing technologies can detect rare mutations related to drug resistance, identify subpopulations of resistant strains, and identify diverse populations of strains due to exogenous reinfection, thus improving tuberculosis control by guiding early implementation of appropriate clinical and therapeutic interventions.

Genetic Diversity and Molecular Epidemiology of Mycobacterium tuberculosis in Roraima State, Brazil.

National border areas are special places for the spread of Mycobacterium tuberculosis (MTB). These regions concentrate vulnerable populations and constant population movements. Understanding the dynamics of the transmission of MTB is fundamental to propose control measures and to monitor drug resistance. We conducted a population-based prospective study of tuberculosis (TB) to evaluate molecular characteristics of MTB isolates circulating in Roraima, a state on the border of Venezuela and Guyana. Eighty isolates were genotyped by IS6110-RFLP (restriction fragment length polymorphism), spoligotyping, and 24-locus mycobacterial interspersed repetitive unit-variable number of repeats tandem (MIRU-VNTR). Drug susceptibility tests were performed by using the proportion method and GeneXpert® MTB/RIF (Cepheid, Sunnyvale, CA). Isolates showing a phenotypic resistance profile were submitted to polymerase chain reaction (PCR) and sequencing. Spoligotyping showed 40 distinct patterns with a high prevalence of Latin-American and Mediterranean (LAM), Haarlem (H), and the "ill-defined" T clades. Mycobacterial interspersed repetitive unit -VNTR and IS6110-RFLP showed clustering rates of 21.3% and 30%, respectively. Drug resistance was detected in 11 (15.1%) isolates, and all were found to have primary resistance; among these, six (8.2%) isolates were streptomycin mono-resistant, four (5.4%) isoniazid mono-resistant, and one (1.3%) multidrug resistant. This is the first study on the molecular epidemiology and drug resistance profile of MTB from Roraima. Herein, we describe high diversity of genetic profiles circulating in this region that may be driven by the introduction of new strain types because of large population flow in this region. In summary, our results showed that analyses of these circulating strains can contribute to a better understanding of TB epidemiology in the northern Brazilian border and be useful to establish public health policies on TB prevention.