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BackgroundTuberculosis is endemic to developing countries like India. Though the whole genome sequences of the type strain M. tuberculosis H37Rv and the clinical strain M. tuberculosis CDC1551 are available, the clinical isolates from India have not been studied extensively at the genome level. This study was carried out in order to have a better understanding of isolates from Kerala, a state in southern India.ResultsA PCR based strategy was followed making use of the deletion region primers to understand the genome level differences between the type strain H37Rv and the clinical isolates of M. tuberculosis from Kerala. PCR analysis of patient isolates using RD1 region primers revealed the amplification of a 386 bp region, in addition to the expected 652 bp amplicon. Southern hybridization of genomic DNA with the 386 bp amplicon confirmed the presence of this new region in a majority of the patient isolates from Kerala. Sequence comparison of this amplicon showed close homology with the moaA3 gene of M. bovis. In M. bovis this gene is present in the RvD5 region, an IS6110 mediated deletion that is absent in M. tuberculosis H37Rv.ConclusionThis study demonstrates the presence of moaA3 gene, that is absent in M. tuberculosis H37Rv and H37Ra, in a large number of local isolates. Whether the moaA3 gene provides any specific advantage to the field isolates of the pathogen is unclear. Field strains from Kerala have fewer IS6110 sequences and therefore are likely to have fewer IS6110 dependent rearrangements. But as deletions and insertions account for much of the genomic diversity of M. tuberculosis, the mechanisms of formation of sequence polymorphisms in the local isolates should be further examined. These results suggest that studies should focus on strains from endemic areas to understand the complexities of this pathogen.

ᰔRegion of difference 1 (RD1) was first described by Mahairas et al. (4) as a region that is present in all virulent laboratory and clinical strains of Mycobacterium bovis and Mycobacterium tuberculosis. This region comprises nine genes (Rv3871 to Rv3879c) and spans a 9.5-kb region. In M. bovis BCG, RD1 deletion completely removes seven genes (Rv3872 to Rv3878) and truncates two others (Rv3871 and Rv3879c) (3). Recently Rao et al. (5) reported the total absence of RD1 in clinical isolates from India. Since this region has genes that are important in immunogenicity (ESAT6 and CFP10 genes) and RD1 deletion mutants of M. tuberculosis have been found to be less virulent (3), it was important to study this region in other Indian isolates. In this study, we analyzed 120 M. tuberculosis isolates from different parts of Kerala and 23 other isolates, 14 from west India and 9 from north India (mainly from Mumbai and Agra). All these isolates were characterized by biochemical analysis and IS6110 restriction fragment length polymorphism typing. The IS6110 copy number ranged from 0 to 15, with the majority (70%) representing the "low-copy-number" group. The presence of the RD1 region was checked with three sets of primers. The locations of the primers on the H37Rv genome and the sizes of the expected amplicon and the reference are indicated in Fig. 1.Among the isolates from Kerala, esxB and esxA were conserved in all the isolates (120/120) and Rv3871 and Rv3872 were present in all except 2 (118/120). Rv3878, being present in only 107 of the 120 isolates, was the least conserved among the three regions. In the other 23 isolates, we found that esxA and esxB were present in all but 1 and that the Rv3878 region was present in 20 isolates. But the Rv3871 and Rv3872 genes were present only in seven isolates.Thus, PCR analysis revealed that the entire RD1 region is not absent in field strains from India. Our earlier study on the identification of the moaA3 gene had shown that the RD1 region is present in isolates from Kerala (6). In the other report, Rao et al. (5) had used two sets of primers, one spanning the entire RD1 (9.8-kb) region and the other for amplification of Rv3878. The amplification of the 9.5-kb RD1 region by routine PCR is technically demanding, and truncation of the genes or any mutation at the primer binding site would eliminate the PCR product. But Rv3878 should have been amplified, as our study shows that this gene is reasonably well represented in the isolates from different parts of the country. As all the 30 strains tested by Rao et al. came from a hospital in

In Mycobacterium tuberculosis, genomic variation is generated mainly by insertions and deletions rather than by point mutations.RvD5 is one such deletion in M. tuberculosis H37Rv. Previous studies from our laboratory have shown the presence of moaA3 gene in the RvD5 region in a large number of clinical isolates, that is absent in M. tuberculosis H37Rv and H37Ra. The present study was aimed at investigating the RvD5 locus of the clinical isolates by a detailed PCR analysis. Here we report a new point of insertion of the mobile genetic element, IS6110 in the genome of one clinical isolate of M. tuberculosis. The insertion has disrupted the moaB3 gene, one of the ORFs in the RvD5 region, which is involved in the molybdopterin biosynthetic pathway. This insertion of IS6110 in the moaB3 of the clinical isolate is different when compared to the insertion in the moaB3 gene of M. tuberculosis H37Rv where 4kb RvD5 region has been lost by homologous recombination and only a truncated form of the gene is present. This finding is of relevance since IS6110 is a major element determining the genome plasticity of M. tuberculosis and its numerical and positional polymorphism has always been of special interest.

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