Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India

Abstract

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.

FIG. 1.

Summary of mutations at codons 508 to 532 in the rpoB gene. The wild-type sequence and amino acids are shown in the middle frame. Nucleotide changes are marked with arrows in the top frame, and the corresponding amino acid changes are denoted in the bottom frame. The amino acids are subscripted with numbers that indicate the number of isolates harboring the change. Changes marked with orange lines (dotted arrows) are novel mutations; silent mutations are marked with blue lines (dashed arrows). Codons 531, 526, and 516 exhibit high degrees of polymorphism. Codons 509, 511, 522, 527, and 528 show novel mutations.

FIG. 2.

Summary of mutations in the katG gene. Deletions are indicated by lines with a minus sign, while insertions are depicted by dashed lines with a plus sign. Solid lines show the substitutions. Codon 463 exhibited the highest degree of polymorphism, followed by the deletion at nucleotide 30.

FIG. 3.

Summary of missense mutations in the gyrA locus. Nucleotide changes are indicated on top of the wild-type sequence, and the corresponding amino acid changes are shown at the bottom. The most common mutation in this locus is Ser95Thr.