Whole Genome Sequencing of Mycobacterium tuberculosis clinical isolates recovered in South Brazil with genotypic convergence at the Direct Repeat Locus.. Multidrug resistant Mycobacterium tuberculosis from south Brazil

We recently detected the spoligotype of Mycobacterium pinnipedii, a species of the M. tuberculosis Complex, in the sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24 MIRU-VNTR typing, verification of TbD1, RD9, pks15/1, RDRio, fbpC (AG85), the insertion of IS6110 at a site specific for the M. tuberculosis Latin American Mediterranean (LAM) lineage and whole genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically of the LAM genotype. Most of these isolates (n=8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of rpoA, rpoB, rpoC, katG and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG and one of these (11%) also presented SNP C (-17) T in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of a M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at modest frequency, and highlights that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.