Mycobacteriology
Characterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea

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Abstract

In order to characterize molecular mechanisms of first- and second-line drug resistance in Mycobacterium tuberculosis and to evaluate the use of molecular markers of resistance, we analyzed 62 multidrug-resistant, 100 extensively drug-resistant, and 30 pan-susceptible isolates from Korean tuberculosis patients. Twelve genome regions associated with drug resistance, including katG, ahpC, and inhA promoter for isoniazid (INH); embB for ethambutol (EMB), rpoB for rifampin (RIF), pncA for pyrazinamide (PZA), gyrA for fluoroquinolones; rpsL, gidB, and rrs for streptomycin; rrs and eis for kanamycin (KM); rrs and tylA for capreomycin (CAP); and rrs for amikacin (AMK) were amplified simultaneously by polymerase chain reaction, and the DNA sequences were determined. We found mutations in 140 of 160 INH-resistant isolates (87.5%), 159 of 162 RIF-resistant isolates (98.15%), 127 of 143 EMB-resistant isolates (88.8%), 108 of 123 ofloxacin-resistant isolates (87.8%), and 107 of 122 PZA-resistant isolates (87.7%); 43 of 51 STM-resistant isolates (84.3%), 15 of 17 KM-resistant isolates (88.2%), and 14 of 15 (AMK and CAP)-resistant isolates (93.3%) had mutations related to specific drug resistance. In addition, the sequence analyses of the study revealed many novel mutations involving these loci. This result suggests that mutations in the rpoB531, katGSer315Thr, and C-15T in the inhA promoter region, and gyrA94, embB306, pncA159, rpsL43, and A1401G in the rrs gene could serve as useful markers for rapid detection of resistance profile in the clinical isolates of M. tuberculosis in Korea, with potentials for the new therapeutic benefits in actual clinical practice.

Introduction

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB). It is estimated that about one-third of the world's population is considered to be latently infected with Mycobacterium tuberculosis, and 10% of these persons will develop active disease at some point in their lifetimes. Due to the lack of effective vaccine and the current need for the more potent anti-tuberculosis drugs which can shorten the duration of therapy, it is not clear how the disease can ever be controlled in the countries where it is truly endemic. In recent years, the control of TB has become a global challenge due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). The emergence of multidrug resistant TB (MDR-TB), i.e., M. tuberculosis strains, resistant to at least isoniazid (INH) and rifampicin, is of great concern, because it requires the use of second-line drugs that are difficult to procure, have more side effects and are more expensive than the first-line regimen (Espinal et al., 2001). Hence, the detection and treatment of drug-susceptible or single drug-resistant TB is important in optimizing strategies to prevent the emergence of MDR-TB and its transmission (Masjedi et al., 2006). XDR-TB isolates are resistant to isoniazid and rifampicin with additional bacillary resistance to any fluoroquinolone, and to at least 1 of the 3 injectable second-line drugs (amikacin, kanamycin, and capreomycin) (Jassal and Bishai, 2009).

The global increase in drug resistance, particularly MDR-TB, reflects, at least in part, inappropriate use of anti-TB drugs during the treatment course of TB patients with drug susceptible strains (Espinal et al., 2001). Additional factors such as immigration, sex, age, HIV infection, and socioeconomic factors have been shown to be associated with the increased prevalence of MDR-TB (Faustini et al., 2006). The World Health Organization has documented that MDR-TB is becoming extensively widespread today. Almost half of the global MDR-TB cases reported from heavily populated areas of China and India (WHO, 2010). The rapid determination of the resistance profile of an isolate can facilitate selection of an appropriate drug regimen and preclude development of additional drug resistances. Rapid detection of resistances can be best achieved with molecular diagnostic approaches, particularly, in developing countries where access to culture facilities is limited. Such strategies require a detailed understanding of the molecular basis of drug resistance.

The present study was undertaken to characterize mutations prevalent in clinical isolates from Korea with respect to various drug resistance target loci. We used DNA sequencing to detect resistance to both the first-line and the second-line anti-tuberculosis drugs. These include isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), amikacin (AMK), capreomycin (CAP), kanamycin (KAN), and ofloxacin (OFX). Twelve loci were sequenced: rpoB (for resistance to RIF); katG, ahpC, and inhA (INH); pncA (PZA), embB (EMB), gyrA (OFX), and gidB; and rpsL, rrs, eis, and tlyA (SM, KAN, AMK, and CAP). The loci studied were rpoB (RNA polymerase B subunit), katG (catalase-peroxidase), inhA (mycolic acid biosynthetic pathway enzyme), ahpC (alkyl hydroperoxide reductase), rrs (16S rRNA), rpsL (ribosomal protein S12), eis (aminoglycoside acetyltransferase), gidB (7-methylguanosine methyltransferase), embB (arabinosyltransferase), tylA (a putative rRNA methyltransferase), pncA (pyrazinamidase), and gyrA (DNA gyrase A-subunit).

Section snippets

Mycobacterium tuberculosis clinical strains

M. tuberculosis-resistant strains were selected from sputum cultures of tuberculosis patients registered in public health centers of South Korea from 2009 to 2010. Multiple isolates from the same patient were avoided. A total of 30 pan-susceptible, 100 XDR and 62 MDR strains were used in this study. All strains were obtained from the Korea Mycobacterium Resource Center.

Drug susceptibility test

The drug susceptibility of the M. tuberculosis isolates was determined by the absolute concentration method with

Mutations in rpoB gene for rifampicin resistance

To identify the mutations associated with rifampicin resistance, we examined the 439 bp region of rpoB gene, including the 81-bp, 27-amino acid (codons 507–533) hyper-variable RRDR. The vast majority (159/162; 98.2%) of RFP-resistant isolates harbored at least 1 mutation within the rpoB gene, while 3 of the RFP-resistant isolates lacked such a mutation (Table 2). A total of 23 non-synonymous single nucleotide polymorphisms, frame shift mutations causing one 9-bp deletion, and one 10-bp deletion

Discussion

The ever increasing burden of anti-tuberculosis drug resistance is a serious concern in both developing and developed countries. Mycobacterium tuberculosis continues to evolve and adopts various mechanisms to evade killing in response to the selection pressure exerted by anti-tuberculosis drugs, including mutations in genes that code for the specific drug target proteins (Morris et al., 1995, Ramaswamy et al., 2000). The objective of the present study was to identify mutations in drug target

Acknowledgments

This study is supported by Korean Centers for Disease Control and Prevention (2011-E4400100) and partially funded by Research Grants from DAIICHI-SANKYO Corporation in Japan. Special thanks to Ms. Ji Hye Kim of the Department of Pulmonary and Critical Care Medicine, Ajou University School of Medicine, and Hae Sun Hwang of the Department of Life Science, Ewha Woman’s University, for the preparation of the manuscript.

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