Mutations in the rpoB Gene of Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from India

Abstract

Mutations in the 81-bp rifampin resistance-determining region (RRDR) of the rpoB gene were analyzed by DNA sequencing of 50 Mycobacterium tuberculosis clinical isolates (44 resistant and 6 sensitive) from various parts of India. Fifty-three mutations of 18 different kinds, 17 point mutations and one deletion, were observed in 43 of 44 resistant isolates. Three novel mutations and three new alleles within the RRDR, along with two novel mutations outside the RRDR, are reported in this study.Tuberculosis (TB), though curable, still remains a major killer disease worldwide. The magnitude of the problem is reflected in estimates of new cases, which are predicted to number around 10 million in the year 2000 and 12 million by 2005 (7). Global prevalence of infection due to Mycobacterium tuberculosis is 32%. Eighty percent of all new TB cases are found in around 22 countries, with more than half the cases occurring in 5 Southeast Asian countries (6). Thirty percent of the world's TB-infected population is in India. The enormity of the problem has increased with the emergence of multidrugresistant (MDR) strains of M. tuberculosis. Dual infection with human immunodeficiency virus and MDR TB is a virtual death sentence in this era.Rifampin (RIF) resistance serves as a surrogate marker for the detection of MDR TB, as 90% of Rif r isolates are also isoniazid resistant (5). RIF interferes with transcription and elongation of RNA by binding to the DNA-dependent RNA polymerase. It was observed that resistance to RIF follows a single-step, high-level resistance pattern in which mutations occur spontaneously at a frequency of 10 Ϫ9 . The genetic basis for RIF resistance in approximately 95% of the cases is due to mutations in an 81-bp RIF resistance-determining region (RRDR) of the rpoB gene, corresponding to codons 507 to 533 (Escherichia coli numbering system), which codes for the beta subunit of the RNA polymerase of M. tuberculosis.Different groups of workers from diverse regions of the world (4, 8-12, 14, 16, 19, 20, 21, 23, 24) have thus far reported around 65 substitutions, 12 deletions, and 4 insertions in the RRDR of the rpoB gene. Only one of these earlier reports is concerned with Indian isolates, and in that study rpoB mutations were observed in three Rif r isolates (8). Determination of the mutation patterns among large numbers of isolates from different parts of India is essential, since this would help not only in the design of a suitable diagnostic method for rapid detection of MDR TB but also in the identification of any hot-spot regions in the country for proper implementation of TB control programs. It would also help in understanding whether mutated alleles arise independently or due to the spread of a particular genotype. Moreover, it is well known that clinical isolates from southern India are very different from isolates from other parts of the world. The former have lower virulence in guinea pigs (2), higher susceptibility to hydrogen peroxide (13, 18) and thiophene-2-carboxylic acid hydrazide (TCH), lowe...

Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR

“…The mechanism of resistance to RIF involves missense mutations, small deletions or insertions in the rpoB gene encoding the b-subunit of RNA polymerase. Studies from diverse countries have shown that 95-96 % of all RIFresistant isolates have mutations within an 81 bp 'core region' of rpoB (Bártfai et al, 2001;Cavusoglu et al, 2002;Herrera et al, 2003;Mani et al, 2001;Telenti et al, 1993;Yue et al, 2003). In contrast, but perhaps not unexpectedly given its highly complex mechanism of action, the mutations causing INH resistance are located in more than three genes and regions (Barry et al, 1998;Slayden & Barry, 2000).…”

Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

“…Rifampicin resistance is of particular epidemiological importance, since it represents a valuable surrogate marker for multidrug resistant (MDR) tuberculosis strains as more than 90% of rifampicin resistant isolates are also isoniazid resistant [4,5]. Rifampicin interferes with transcription by binding to the beta subunit of DNA-dependent RNA polymerase.…”

CBNAAT: A Novel Tool for Rapid Detection of MTB and Rifampicin Resistance

“…(Mboowa et al, 2014). Mani et al, reported that the codons most commonly involved in these mutations were 531 (53%) and 526 (19%) in a study from South India (Mani et al, 2001). The resistant mutants isolated more frequently in clinical practice have higher mean relative fitness and their prevalence depend on their ability to survive (Billington et al, 1999).…”

“…While, Singhal et al, found 6 strains (6/366) with more than one mutation (Singhal et al, 2015). Probably, mutations continue to arise due to the ability of MTB to adapt to drug exposure (Mani et al, 2001). The results were statically analyzed (p<0.0005).…”