Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in North India

MATERIALS AND METHODS

RESULTS

Mutations in the hot-spot regions of various loci were characterized. The results are summarized in Table 2. On the basis of the drug susceptibility profile for an isolate, the corresponding loci (representing the drug target gene) were amplified and sequenced. The largest number of samples was obtained from New Delhi, followed by Chandigarh, Ahmedabad, Agra, Bangalore, and Shimla, with a few samples coming from Jaipur and Chennai. Except for Chennai and Bangalore, all the cities are located in North India. We could establish a previous treatment history for patients from whom 94 of the 126 isolates were recovered. These isolates probably represent those with acquired resistance, as the patients had at some time point been given antitubercular drug therapy.

A stretch of 30 amino acids at the center of the amplicon for the rpoB locus was studied. Amino acids 432 to 458 comprised the hot-spot region for mutations. For the sake of comparison, we used the corresponding Escherichia coli numbering, which is amino acids 507 to 533. We identified previously reported mutations as well as certain novel mutations. Codon 531 seemed to be the most vulnerable to mutations, as most rifampin-resistant isolates had this mutation (Fig. 1). Of the 93 rifampin-resistant strains in our study, 28 had the missense mutation Ser531Leu and 8 had the substitution Ser531Trp. The next most common mutations were the amino acid substitutions Asp516Val or Asp516Gly (20 isolates) and His526Tyr, His526Leu, or His526Arg (19 isolates). We found two isolates with Gln510His changes. While all these mutations have been reported earlier, we also found mutations that have not been reported previously. These included Ser509Arg (isolate ICC33), Leu511Val (isolate ICC242), Asn518Thr (isolate ICC107), Ser522Gln (isolate ICC172), Lys527Asn (isolate ICC105), Arg528Pro (isolate ICC129), and Arg528His (isolate ICC213). Most of these mutations occurred less frequently, comprising about 24% of the total mutations in the 94 isolates studied. Other mutations identified in our study were silent mutations at amino acids Leu511 and Leu521. Interestingly, the mutation at position 511 never occurred alone and was present only in isolates with more than one mutation at the rpoB locus.

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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.

An important outcome of these studies is the direct correlation of certain mutations to high MICs. Table 3 lists the isolates, their mutations, and the corresponding MICs at which they remained resistant. Mutations in codons 516 and 521 conferred low-level resistance (MIC, <40 μg/ml) to rifampin, whereas mutations in codons 510, 526, 527, 528, and 531 were seen to confer high levels of resistance (MICs, ≥64 μg/ml). Amino acids 526 to 531 appear to be very important in drug target interactions, and mutations in them result in MICs in the range of 64 μg/ml and above. In a few cases (e.g., for isolates ICC204, ICC257, and ICC128), double mutations were found to have an additive effect on the degree of resistance.

Insertion, deletion, and substitution mutations were mapped in the katG locus in 24 isoniazid-resistant isolates. In the present study we looked for mutations in the 5′ region (nucleotides [nt] 3 to 239) and the midregion (nt 1187 to 1600) of the katG gene, corresponding to amino acid positions 2 to 77 and 395 to 533, respectively. The results are summarized in Fig. 2. A C nucleotide at position 30 was deleted in six of the isolates. This deletion results in chain termination, thereby generating only a short polypeptide of 26 amino acids. Another deletion of a single nucleotide, a G residue at position 109, was observed in two isolates; this deletion would result in the production of a 45-amino-acid truncated polypeptide. Insertions were also observed at nt positions 98 (an A nucleotide) and 185 (a C nucleotide) in four and three isolates, respectively. Both of these insertions cause aberrant chain termination. Ala61Thr, Thr12Pro, Thr11Ala, Asp73Asn, and Asn35Asp missense mutations were observed in this locus in a few of the isolates. These are novel observations, as there are no reports of such mutations occurring in isoniazid-resistant strains from other parts of the world. We were unable to amplify this locus in six of the isolates (isolates ICC14, ICC23, ICC32, ICC85, ICC123, and ICC205), indicating a partial deletion of the gene. A common mutation in all these isolates was Arg463Leu. However, this mutation has been shown to have no direct consequence for drug resistance. To confirm this we sequenced this locus for all 126 isolates included in the study. It was found that the majority of the isolates carried this change. It has been argued previously that this polymorphism in the katG locus might be more important as a marker of evolution than as a marker of resistance (22). Three isoniazid-resistant isolates carried mutations in the ribosomal binding site upstream of the inhA gene. While two isolates showed a C-to-T transition, one had a T-to-A transversion. These mutations have previously been reported by other groups. The present understanding of these mutations is that they probably confer resistance by a drug titration effect.

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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.

Sixty-eight ofloxacin-resistant isolates were analyzed. The hot-spot region of the gyrA gene spanning codons 89 to 95 was sequenced to identify mutations. Most of the isolates showed a single mutation corresponding to the amino acid change Ser95Thr (Fig. 3). The second most common mutation, observed in four isolates, was Asp94Gly or Asp94Ala. Two isolates had an Ala90Val substitution, while one had a silent mutation at this codon. Seven isolates had double mutations, with the S95T change being common to all seven. These mutations were present in MDR isolates for which the MICs of the drugs were high, including the frontline drugs used in antituberculosis therapy. All strains were also checked for mutations in the gyrB locus, which is associated with low levels of resistance. However, we found no mutations in the gyrB loci of these isolates. It has been argued that the S95T mutation does not correlate with drug resistance (22). It therefore appears that the isolates have acquired resistance to ofloxacin via other mechanisms.

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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.

We tested 14 isolates resistant to streptomycin for mutations in the rpsL and rrs loci. In eight strains we found a novel silent mutation at amino acid position 121 in the rpsL locus, where the codon AAA (Lys) was changed to AAG (Lys), but we found no mutations in the rrs genes. To our knowledge, there are no reports of this mutation. The reported mutations at the rpsL locus are generally Leu43Arg, Leu43Thr, or Lys88Arg. We are still not clear about how a mutation at this locus leads to the development of streptomycin resistance. The remaining isolates probably acquired resistance by other means, such as by the development of a permeability barrier or by the production of drug-altering enzymes.

A point to be kept in mind is that the majority of isolates included in the present study were from North India. Our data are therefore inherently biased toward drug-resistant strains from this region and should not be seen as representative for isolates from the whole of India.

DISCUSSION

The mycobacterium uses various mechanisms to evade killing by drugs, including mutations in genes that code for drug target proteins (20), a complex cell wall which blocks drug entry, and membrane proteins that act as drug efflux pumps (6, 14). The objective of the present study was to identify mutations in drug target loci in Indian strains of M. tuberculosis and to identify the different drug resistance genotypes. As in all such studies, the aim was to generate information about the markers associated with drug resistance, polymorphisms in the drug target genes, the association of the level of resistance with particular mutations, etc. Our findings of mutations in the rpoB, katG, and rpsL loci are similar to those reported from other parts of the world, especially the common mutations, which reflect a global pattern (20). Rifampin resistance is often regarded as an excellent surrogate marker for MDR tuberculosis (4, 10), and our study corroborates this hypothesis. The mutation frequency of codon 531 (rpoB) was similar to that reported earlier (13, 17, 19, 20, 21, 25). Significantly, the frequency of mutations (relative to those of other mutations) was higher at codon 516 and lower at codon 526 in Indian isolates compared to those reported elsewhere. We found novel mutations that broaden the range of known mutations at this locus. When taken together, these mutations were detected in a significant number of drug-resistant isolates, a fact that needs to be considered when designing tools for the detection of MDR M. tuberculosis. We found a definite correlation between MICs and the type of mutation in many isolates. As reported by previous investigators (24), mutations at positions 528 and 531 are important in the development of high MICs. Our findings further strengthen the belief that the degree of resistance to rifampin exhibited by an isolate is related to the type of mutation in the rpoB locus.

In isoniazid-resistant isolates, significantly more deletion and insertion mutations than substitution mutations were found, of which a few have been reported previously (11, 19). We observed that almost all isolates studied carried the Arg463Leu substitution, which is also present in isolates that were sensitive to isoniazid. This is in concordance with a report from Sreevatsan et al. (22), who argue that polymorphism at this residue does not contribute to resistance per se but is an important marker for evolutionary genetics. The insertions and deletions in the katG locus invariably resulted in chain truncation and termination, leading to the generation of dysfunctional polypeptides. We found changes in the putative ribosomal binding site of the inhA gene in three isolates. While the exact mechanism of how these mutations confer resistance to isoniazid is not clear, reports (1, 18, 20) indicate that they probably increase the levels of enoyl-acyl carrier protein reductase which in turn leads to resistance via a drug titration mechanism. In isolates with no mutations in the hot-spot region of the gene, the complete sequencing of the gene is being done. However, resistance to isoniazid can also be due to mutations in the ahpC-oxyR and kasA gene loci (7, 16).

Fluoroquinolones comprise the secondary drug regimen in the treatment of tuberculosis. A large number of isolates were resistant to ofloxacin, which could be due in part to the inaccurate diagnosis of tuberculosis as a bacterial infection and fluoroquinolone overuse in the population. Codons 89, 90, 91, 94, and 95 in the gyrA gene have been shown to be polymorphic (20, 23, 26). The most common mutation in ofloxacin-resistant isolates in the present study was Ser95Thr, which reportedly has no direct role in the development of drug resistance, as it also occurs in drug-sensitive strains (22). It seems likely that ofloxacin resistance possibly results due to mutations elsewhere in the gene or the presence of drug efflux pumps.

Mutations in codons 43 and 88 of the rpsL gene generally result in high levels of resistance to streptomycin, while mutations in the loop at codon 530 or the region at codon 915 of the rrs locus are associated with low levels of resistance (3). We did not find any of these mutations in the 14 streptomycin-resistant isolates included in our study. However, we did observe a silent mutation at codon 121 that has not been reported by any other group.

Our study provides valuable data on the different kinds of mutations occurring at various target loci in Indian clinical isolates of M. tuberculosis that enhance our understanding of the molecular mechanisms of drug resistance. The diversity of the polymorphisms exhibited at these loci by the drug-resistant strains indicates the prevalence of a large numbers of drug-resistant strains in this region. Additionally, our data will also assist in the process of designing new molecular biology-based techniques for the diagnosis of MDR tuberculosis. Such methods promise faster detection rates compared to those achieved by methods based solely on culture of the isolates.