Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies

Abstract

Because genetically monomorphic bacterial pathogens harbour little DNA sequence diversity, most current genotyping techniques used to study the epidemiology of these organisms are based on mobile or repetitive genetic elements. Molecular markers commonly used in these bacteria include Clustered Regulatory Short Palindromic Repeats (CRISPR) and Variable Number Tandem Repeats (VNTR). These methods are also increasingly being applied to phylogenetic and population genetic studies. Using the Mycobacterium tuberculosis complex (MTBC) as a model, we evaluated the phylogenetic accuracy of CRISPR- and VNTR-based genotyping, which in MTBC are known as spoligotyping and Mycobacterial Interspersed Repetitive Units (MIRU)-VNTR-typing, respectively. We used as a gold standard the complete DNA sequences of 89 coding genes from a global strain collection. Our results showed that phylogenetic trees derived from these multilocus sequence data were highly congruent and statistically robust, irrespective of the phylogenetic methods used. By contrast, corresponding phylogenies inferred from spoligotyping or 15-loci-MIRU-VNTR were incongruent with respect to the sequence-based trees. Although 24-loci-MIRU-VNTR performed better, it was still unable to detect all strain lineages. The DNA sequence data showed virtually no homoplasy, but the opposite was true for spoligotyping and MIRU-VNTR, which was consistent with high rates of convergent evolution and the low statistical support obtained for phylogenetic groupings defined by these markers. Our results also revealed that the discriminatory power of the standard 24 MIRU-VNTR loci varied by strain lineage. Taken together, our findings suggest strain lineages in MTBC should be defined based on phylogenetically robust markers such as single nucleotide polymorphisms or large sequence polymorphisms, and that for epidemiological purposes, MIRU-VNTR loci should be used in a lineage-dependent manner. Our findings have implications for strain typing in other genetically monomorphic bacteria.

Polymorphism in the RD1 locus and its effect on downstream genes among South Indian clinical isolates of Mycobacterium tuberculosis

“…Thus, MIRU 39 should be included in the MIRU-VNTR typing protocol for the south Indian strains where EAI isolates correspond to 84 % of the total circulating strains (Shanmugam et al, 2011). Our findings also suggest that the MIRU polymorphism may vary among geographically diverse clinical isolates, so regionspecific MIRU locus standardization will be necessary to achieve a high discriminatory index as reported previously (Comas et al, 2009). Here, we demonstrated that MIRU 39 occupying the intergenic region between Rv3870 and Rv3871 is present in variable copy numbers ranging from one to six.…”

SNP typing reveals similarity in Mycobacterium tuberculosis genetic diversity between Portugal and Northeast Brazil

“…Contrary to other common markers, such as Spoligotypes, VNTRs or RFLPs, SNPs are found in many sites across the MTBC genomes (in the order of tens of thousands) and can easily preserve traces of the evolutionary background of a sample (Comas et al, 2009). Furthermore, the evolutionary dynamics of MTBC species are particularly benficial for SNP-based phylogenetic analyses: on one hand, the substitution rate of MTBC is considerably low compared to other bacteria, which reduces the chance of convergent evolution in the absence of selection (Achtman, 2008); on the other hand, horizontal gene transfer has very limited expression in MTBC, reducing further the chance of phylogenetic inconguences.…”

“…Spoligotyping has been shown not to be reliable for phylogenetic analysis, because its patterns may not reflect the evolution history of a strain and the "signature" pattern of the strain groups can be ambiguous or uninformative (Comas et al, 2009). To circumvent these problems we used a novel software that relies on an extensive Spolygotype database to assign new Spoligotype patterns to strain groups (SPOTCLUST, Vitol et al, 2006).…”

“…These systems have limited use for phylogenetics and strain identification because, due to targeting polymorphisms with fast substitution rate and using limited number of markers, they may present events of convergent evolution, or homoplasy, (Comas et al, 2009). Furthermore, the use of Spoligotyping to identify genotype profiles absent from the reference database is debatable (but see Vitol et al, 2006).…”

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

“…This reinforces the observation that first-line typing schemes for different countries may require the use of different loci because the population structures of circulating M. tuberculosis strains vary across different parts of the world . Moreover, the relative discriminatory powers of particular VNTR loci vary depending on the strain in question (Murase et al 2008, Comas et al 2009). …”