Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

Abstract

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in 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-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD Rio , and fbpC, the insertion of IS6110 at a site specific to 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 belong to the LAM genotype. Most of these isolates (n ‫؍‬ 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(؊17)T single-nucleotide polymorphism (SNP) 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 an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers. M. tuberculosis is the predominant cause of human TB worldwide, but M. africanum and M. bovis remain important agents of human disease in certain geographical regions (9). The MTBC species share identical 16S rRNA sequences, and recent studies have improved our knowledge on the genetic diversity, host range, epidemiological aspects, and differences in pathogenicity and virulence among the species of the complex (10, 11). Based on the various genotyping techniques, like spoligotyping (12), restriction fragment length polymorphism (RFLP) (13), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing (14), and whole-genome sequencing (15), M. tuberculosis strains have been subdivided into lineages and families. The most geographically widespread family worldwide (spoligotyping) is the Latin American Mediterranean (LAM) lineage, which is part of the heterogeneous Euro-American lineage, one of seven M. tuberculosis lineages (16). The LAM family strains are widespread across all five continents, with a marked incidence in

Clonal expansion across the seas as seen through CPLP-TB database: A joint effort in cataloguing Mycobacterium tuberculosis genetic diversity in Portuguese-speaking countries

“…Interestingly, the SIT863/PINI2 strains underwent diversification at locus 154 (154_2). The phylogenetic positioning of SIT863/PINI2 in the RD115-sublineage is congruent with previous data on its SNP barcode lineage (4.3.3) as a RD115 sub-lineage branch (Coll et al, 2014; Dalla Costa et al, 2015). Nevertheless, some phylogenetic incongruences were noted as other unrelated strains, e.g., Beijing or T1, were included in this branch, thus exemplifying the problem of MIRU-VNTR convergence.…”

“…Thirty-one isolates were also found to form a monophyletic branch containing the 154_1 allele as a marker for the RD115 sublineage. This branch included, among others, the “ pinnipedii -like” strains SIT863/PINI2 which has been shown by some of us to in fact belong to the LAM lineage (Dalla Costa et al, 2015). Interestingly, the SIT863/PINI2 strains underwent diversification at locus 154 (154_2).…”

Genomic epidemiology of Mycobacterium tuberculosis in Santa Catarina, Southern Brazil

“…LAM5 spoligotype is also the most reported in Rio Grande do Sul, another state in Southern Brazil, however belonging to SIT93 34 . A parallel situation has been reported with SIT863 MDR-TB strains in Rio Grande do Sul, that was initially identified in prison establishments but is now responsible for the majority of MDR-TB cases in that state 37,41 . The presence of isolates from inmate individuals in the TB transmission chains supports the dissemination of strains between the general population and prison establishments and although the directionality is unclear at this point, the latter along with the entire prison system may act as reservoir of specific strains and promote a wider dispersion of specific clones 42 .…”

A highly rifampicin resistant Mycobacterium tuberculosis strain emerging in Southern Brazil

“…The 12nt insertion results in a duplication of four amino acids (QNNP) and was firstly described by Perizzolo et al, (2012)[5]. Following studies analyzing MDR strains from Rio Grande do Sul[6–9] also reported strains carrying on the 12nt insertion, and to our knowledge, this insertion was not described in any other region worldwide.…”