rpoB gene sequencing and spoligotyping of multidrug-resistant Mycobacterium tuberculosis isolates from India

Multi drug-resistant Mycobacterium tuberculosis (MDR TB) has been well studied in outbreaks in settings of low endemicity in developed countries. However, the characteristics of MDR TB in the community with high endemicity such as India have not been well investigated. Mutations in the 81-bp rifampicin resistance-determining region of the rpoB gene were analyzed by DNA sequencing of 187 M. tuberculosis clinical isolates (149 resistant and 38 sensitive) from different parts of India. 146-Point mutations and two insertions were found in 146 of 149 resistant isolates in seven codons. The most common mutations were in codons 531 (59%), 526 (22%), and 516 (11.5%). Mutations were not found in three (2%) of the resistant isolates. N-terminal sequencing in these isolates showed no mutation at codon V176. None of the drug-susceptible isolates showed any mutation in the 437-bp rpoB gene segment sequenced. Genotypic analysis revealed a total of 80 different spoligotypes. A unique pattern was found in 65 (43.6%) isolates, whereas 84 (56.4%) were in 15 clusters. Comparison with an international spoligotype database showed ST26, Delhi type (18.1%), ST1, Beijing type (9.4%), and ST11 (5.4%), as the most common. The majority of isolates in the Beijing genotype (13/14) were associated with mutation 531TTG and similar drug-resistance patterns while other major clusters showed that the nature and frequency of occurrence of mutations in the rpoB gene were independent of spoligopatterns.