Elsevier

Tuberculosis

Volume 96, January 2016, Pages 96-101
Tuberculosis

Drug discovery and resistance
Novel mutations conferring resistance to kanamycin in Mycobacterium tuberculosis clinical isolates from Northern India

https://doi.org/10.1016/j.tube.2015.10.012Get rights and content

Summary

Twenty-nine Kanamycin resistant clinical isolates of Mycobacterium tuberculosis from Northern India were screened to evaluate genetic mutations in rrs gene, eis gene with its promoter, and whiB7 gene along with its 5′UTR. 14 strains (∼48.0%) collectively exhibited mutations in rrs, eis or whiB7 target regions. While the highest frequency of mutations was found in rrs gene, eis and whiB7 loci displayed novel mutations. The novel mutations displayed by eis and whiB7 loci were found to be associated specifically with the Kanamycin resistance as none of the twenty nine Kanamycin sensitive strains harbor them. The inclusion of novel mutations of eis and whiB7 loci will be useful in improving the specificity of future diagnostics.

Introduction

The major obstacle to the effective control of TB is the increasing threat of resistance to the drugs used for its treatment. The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has raised an alarm for optimizing the existing strategies for rapid diagnosis, resistance profile determination and treatment of TB. MDR-TB displays resistance to Isoniazid and Rifampicin, with or without resistance to other first-line drugs. XDR-TB has resistance at least to Isoniazid and Rifampicin, any fluoroquinolone, and any one of the three second-line injectables i.e. Kanamycin (KAN), Amikacin (AMK) and Capreomycin (CAP). Another pattern of resistance, Pre-XDR TB, defined as resistance to Isoniazid, Rifampicin and either a second-line injectable drug or a fluoroquinolone, but not both, needs to be ruled out to forestall the progression of XDR-TB [1].

The association of mutations in genes katG and inhA with isoniazid resistance; rpoB with rifampin; rpsL and rrs (nucleotides 388 to 1084) with streptomycin; embB with ethambutol; pncA with pyrazinamide; rrs (nucleotides 1158 to 1674) and gidB for KAN, AMK and CAP; eis promoter with KAN/AMK; tlyA for CAP and gyrAB for ofloxacin have been well documented in global scenario [2], [3], [4], [5], [6], [7]. In addition, WhiB7 has recently been identified as a transcriptional regulator which regulates the expression of Eis protein of Mtuberculosis and is reported to contribute to low-level KAN resistance indirectly [8], [9].

Although some efforts have been made to study the genetics and single nucleotide polymorphisms (SNPs) related to rrs gene and promoter of eis in M. tuberculosis clinical isolates from Hinduja Hospital, India [2], [10], studies on the second-line drug resistant clinical isolates from Uttar Pradesh, India have not been reported. Therefore, the present study was undertaken with an aim to find the overlap between resistance pattern and genetic mutations in rrs (Rvnr01), eis (Rv2416c) with its promoter, and whiB7 (Rv3197A) along with its 5′UTR in the clinical isolates of Uttar Pradesh, India.

Section snippets

Clinical isolates of M. tuberculosis

Over a period of February 2013 to December 2014, a total of 300 laboratory confirmed M. tuberculosis MDR strains isolated serially were obtained from Tertiary Care Centre Tuberculosis Laboratory, Department of Microbiology, King George Medical University (KGMU), Lucknow, Uttar Pradesh, India and tested for Kanamycin resistance. We obtained 29 KAN resistant isolates from this repository consecutively. The cases from which these isolates were obtained were routinely referred MDR suspects from

Results

In the present study, 29 KAN resistant strains were investigated to evaluate the genetic changes that are accountable for KAN resistance in M. tuberculosis. Twenty-nine KAN sensitive strains were also included for comparison. For all the isolates, MICs were determined. Among KAN resistant isolates, 100% concordance for KAN (ranging from 5 μg/ml to > 20 μg/ml), 93.1% for AMK (ranging from 1 to > 4 μg/ml), and 96.6% for CAP (ranging from 5 to > 20 μg/ml) between MIC and phenotypic drug

Discussion

Misuse of TB drugs in terms of noncompliance, incomplete dose, and misdiagnosis has resulted in escalating the cases of MDR, XDR and TDR tuberculosis. Besides, M. tuberculosis also exerts all its efforts to evade killing by the anti-TB drugs. One of the commonest mechanisms used by M. tuberculosis is mutating its genes to alter their function in order to avoid being targeted by standard first-line or second-line drugs. Attempts to identify such mutations and exploit them as TB diagnostic tools

Acknowledgment

We acknowledge Mr. Shekhar Kumar for his scientific input and critically reading the manuscript.

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