“Pseudo-Beijing”: Evidence for Convergent Evolution in the Direct Repeat Region of Mycobacterium tuberculosis

Abstract

Background Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern.Methods and FindingsMTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC.ConclusionsWe provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

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

“…Convergent evolution has been described as relatively common when using the direct repeat (DR) region as a genetic marker, and incorrect classification of lineages by spoligotyping was recently evidenced in Brazil (26) and more frequently when the deletion of large blocks of spacers occurs, such as causing misclassification of M. tuberculosis of the Beijing type (46). Therefore, our observation of human isolates with the SIT863 spoligotype characteristic of PINI2, together with the lack of notification of M. pinnipedii as a cause of human TB, lead us to believe that we might be dealing with cases of misclassification.…”

Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

“…However, spoligotyping is well known to show gene conversions, and strains having no genetic relationship sometimes show the same spoligotype (3, 26). Fenner et al (35) reported pseudo-Beijing strains that had a typical Beijing spoligotype even though they actually belonged to the CAS family. This type of confusion seems to occur especially in areas that have a higher prevalence of principal genetic group 1 (PGG1) lineages, including the EAI, CAS, and east Asian lineages, since PGG1 strains usually possess spacers 35 and 36, which are lacking in PGG2 and PGG3 strains (3,36).…”

Mycobacterium tuberculosis Transmission in a Country with Low Tuberculosis Incidence: Role of Immigration and HIV Infection

“…Molecular clusters were defined as a group of completely identical isolates in the spoligotyping and MIRU-VNTR pattern. We determined the main M. tuberculosis lineages by single nucleotide polymorphisms (SNPs) using multiplex real-time PCR with fluorescence-labeled probes (TaqMan; Applied Biosystems) adopted from previous studies (7,13,14,18). Region of difference (RD) deletion PCRs were performed for RD702 and RD711, which define the West African lineages (14).…”