Discovery of a novel Mycobacterium tuberculosis lineage that is a major cause of tuberculosis in Rio de Janeiro, Brazil

Abstract

The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in approximately 30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RD(Rio). Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RD(Rio) locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RD(Rio) strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RD(Rio) lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RD(Rio) strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RD(Rio) strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RD(Rio) M. tuberculosis.

FIG. 1.

PCR for RD loci in MTC strains from Rio de Janeiro. (A) MTC PCR typing panel result for a typical WT M. tuberculosis strain, showing an uninterrupted arcing pattern. (B) MTC PCR typing panel result for 30% of M. tuberculosis strains in the sample set, showing a failure to amplify from the IS1561′ locus. Lanes: 1, 16S rRNA; 2, cfp32 (Rv0577); 3, IS1561′; 4, Rv1510 (RD4); 5, Rv1970 (RD7); 6, Rv3877 to Rv3878 (RD1); 7, Rv2073c (RD9); 8, Rv3120 (RD12). Unlabeled lanes contain the 100-bp ladder. (C) Composite panel illustrating the representative results of PCR amplification to span the IS1561′-deleted (RD^Rio) locus (panels i and ii) (bridge PCR 1 and bridge PCR 2, respectively), to target the intact locus (IS1561′) (panel iii), and to provide a control amplification (cfp32) (panel iv) (30, 31). The first lane contains a marker (100-bp ladder). H37Rv, M. tuberculosis strain H37Rv; WT strains 1 and 2, two representative M. tuberculosis WT strains; RD^Rio strains 1 and 2, two representative M. tuberculosis RD^Rio strains. Sizes of PCR products (in base pairs) are given on the right.

FIG. 2.

Diagram showing the genes deleted or altered in the derivation of the RD^Rio LSP. The recombination between the homologous pair Rv3346c and Rv3355c formed a new in-frame fusion gene (Rv3346c/55c), and all elements between these two genes were excised, resulting in a deletion of 26.314 kb of contiguous DNA sequence.

FIG. 3.

An MST of spoligotype data from Rio de Janeiro M. tuberculosis LAM strains is consistent with the parsimonious phylogeny of WT and RD^Rio genotype strains. Colors indicate RD^Rio (red) or WT (green) strains. Each circle represents a particular spoligotype, and the size of a circle is relative to the number of strains with that spoligotype. Thicker lines linking circles indicate a 1-spacer difference, and thinner lines indicate a 2-spacer difference, between the spoligotype patterns.

FIG. 4.

An MST of MIRU-VNTR data from Rio de Janeiro M. tuberculosis LAM strains illustrates the genotypic segregation of WT and RD^Rio genotype strains. Colors indicate RD^Rio (red) or WT (green) strains. The size of each circle is relative to the number of strains with a particular MIRU-VNTR type. Each circle represents a single MIRU-VNTR profile. Thicker lines linking circles indicate a single-locus difference, and thinner lines indicate a 2-locus difference, between the profiles. The central RD^Rio node represents strains with the hypothetical founding RD^Rio MIRU-VNTR allele (224226153321) (see Table S7 in the supplemental material).

FIG. 5.

M. tuberculosis RD^Rio strains group together in phylogenetic analysis of IS6110-RFLP fingerprint data. IS6110 Southern blot hybridization and dendrogram construction were performed as described in Materials and Methods using a set of available Rio de Janeiro M. tuberculosis DNA samples. Patient strain numbers are given on the right. RD^Rio patient strain numbers are lightly shaded and boxed; WT LAM patient strain numbers have dark shading; and WT non-LAM patient strain numbers are neither shaded nor boxed. Note that the clonal strains in each separate pair from patient 315 are identical to each other, as are the two strains from patient 38. Interestingly, the IS6110-RFLP types from patient strains 64 and 86 were indistinguishable, and their MIRU-VNTR types were identical, but their spoligotypes differed by 1 spacer (see Table S4 in the supplemental material), suggesting that either they may not in actuality form a directly linked cluster or they may represent an example of strains evolving during transmission. The phylogenetic tree was constructed using Bionumerics software, and the similarity of hybridization patterns was calculated by the Dice coefficient; the Pearson correlation Opt value was 4.00% (range, 0.0% to 100%).