MycobacteriologyCharacterization of mutations in multi- and extensive drug resistance among strains of Mycobacterium tuberculosis clinical isolates in Republic of Korea
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.
References (39)
- et al.
Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait
Tuberculosis
(2007) - et al.
Extensively drug-resistant tuberculosis
Lancet Infect Dis
(2009) The action of antituberculosis drugs in short-course chemotherapy
Tubercle
(1985)- et al.
Mutation detection and accurate diagnosis of extensively drug-resistant tuberculosis: report from a tertiary care center in India
J Clin Microbiol
(2011) - et al.
The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol
Proc Natl Acad Sci USA
(1996) - et al.
Molecular detection of mutations associated with first and second-line drug resistance compared with conventional drug susceptibility testing in Mycobacterium tuberculosis
Antimicrob Agents Chemother
(2011) - et al.
Global trends in resistance to antituberculosis drugs
N Engl J Med
(2001) - et al.
Risk factors for multidrug resistant tuberculosis in Europe: a systematic review
Thorax
(2006) - et al.
Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot
Mol Microbiol
(1993) - et al.
embB gene mutations associated with ethambutol resistance in Indian strains of Mycobacterium tuberculosis
Curr Sci
(2006)
High level of cross-resistance between kanamycin, amikacin, and capreomycin among Mycobacterium tuberculosis isolates from Georgia and a close relation with mutations in the rrs gene
Antimicrob Agents Chemother
Drug-resistant tuberculosis in Korea, 1994
Int J Tuberc Lung Dis
Novel mutations within the embB gene in ethambutol-susceptible clinical isolates of Mycobacterium tuberculosis
Antimicrob Agents Chemother
Extensively drug resistant tuberculosis: 2 years of surveillance in Iran
Clin Infect Dis
Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis
Antimicrob Agents Chemother
Molecular analysis of cross-resistance to capreomycin, kanamycin, amikacin, and viomycin in Mycobacterium tuberculosis
Antimicrob Agents Chemother
Genetic alterations in streptomycin-resistant Mycobacterium tuberculosis: mapping of mutations conferring resistance
Antimicrob Agents Chemother
Interaction of antibiotics with functional sites in 16S ribosomal RNA
Nature
Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis
Antimicrob Agents Chemother
Cited by (0)
- 1
These two authors are equally contributed.