Presence of Region of Difference 1 among Clinical Isolates of Mycobacterium tuberculosis from India

Abstract

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

Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosisfrom Tamil Nadu and Pondicherry – south eastern coastal states of India

“…In fact, few isolates we obtained from Hyderabad were found positive for RD1 region in all the isolates (data not shown). This is in concurrence with other reports [ 11 , 4 ].…”

Comparative in-silico proteomic analysis discerns potential granuloma proteins of Yersinia pseudotuberculosis

“…The best known example of a well-studied pathogen and granuloma is Mtb. Experimental studies in Mtb suggested that RD1 locus proteins 28 , ESAT-6 secretion system proteins 29 and proteins of intra-macrophage secretome 30 were mainly involved in Mtb granuloma. Several studies indicated the importance of RD1 locus and ESAT-6 secretion system in Mtb granuloma [71][72][73] .…”

“…A composite interaction network of RD1 locus proteins 28 , ESAT-6 secretion system proteins 29 and intra-macrophagic secretome of Mtb 30 was created using STRING database, at confidence score 0.150. The Mtb orthologs of the proposed seven Yps granuloma proteins were mapped on this interaction network.…”

“…and Yersinia spp respectively.Validation of the role of identified Yps granuloma proteins using experimentally identified Mtb granuloma proteins. Earlier studies have proved that Mtb RD1 locus proteins28 , ESAT-6 secretion system proteins29 and intra-macrophage secreted proteins30 play an important role in the formation and regulation of granuloma. To ascertain the probable involvement of the seven proposed Yps proteins in Yps granuloma…”

Supraclavicular BCG Lymphadenitis Noted at 21 Months after BCG Vaccination Confirmed by a Molecular Method

“…In addition, considering that suspected tuberculous lymphadenitis patients without an identifiable tuberculous contact were subject to further investigations for differential diagnosis of M. bovis BCG, we performed the tar- tuberculosis complex members, enabling us to differentiate different strains belonging to the M. tuberculosis complex and making surveillance of speciesspecific disease suitable 13,14) . This diagnostic method could help to confirm BCG lymphadenitis.…”