CORE Metadata, citation and similar papers at core.ac.uk
Provided by Elsevier - Publisher Connector
ORIGINAL ARTICLE 10.1111/j.1469-0691.2004.01034.x
Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum C. Arnold1, L. Westland1,2, G. Mowat3, A. Underwood1, J. Magee3 and S. Gharbia1
1Genomics, Proteomics and Bioinformatics Unit, Centre for Infections, Health Protection Agency, London, UK, 2Hogeschool van Utrecht, Faculteit Natuur & Techniek, Institute of Life Sciences and Chemistry, Utrecht, The Netherlands and 3Regional Centre for Mycobacteriology, Health Protection Agency Newcastle Laboratory, Newcastle, UK
ABSTRACT The rapid technique of pyrosequencing was used to examine 123 samples (in the form of DNA extracts and inactivated sputum) of Mycobacterium spp. Of 99 Mycobacterium tuberculosis samples investigated for single-nucleotide polymorphisms (SNPs), 68% of isoniazid-resistant isolates analysed had an AGC fi ACC mutation in katG at codon 315, resulting in the Ser fi Thr substitution associated previously with isoniazid resistance. Of the rifampicin-resistant isolates, 92% showed SNPs in rpoB at codons 516, 531 or 526. Inactivated sputum samples and DNA extracts could both be analysed by pyrosequencing, and the method was able to differentiate rapidly between the closely related species of the M. tuberculosis complex (M. tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti and Mycobacterium microti), except between M. tuberculosis, M. canetti and one of two M. africanum strains. This low-cost, high-throughput technique could be used as a rapid screen for drug resistance and as a replacement for some of the time-consuming tests used currently for species identification. Keywords Antibiotic susceptibility testing, identification, Mycobacterium tuberculosis complex, pyrosequencing, resistance Original Submission: 5 April 2004; Revised Submission: 2 September 2004; Accepted: 11 September 2004 Clin Microbiol Infect 2005; 11: 122–130
amikacin, and fluoroquinolones such as ofloxacin INTRODUCTION and ciprofloxacin, are used as second-line agents. It has been estimated that one-third of the global Drug resistance in M. tuberculosis occurs fol- population is infected with Mycobacterium tuber- lowing spontaneous mutation in target genes culosis, with c. 9 million cases of active tubercu- such as katG, rpoB and inhA [3]. The mechanisms losis (TB) and > 2 million deaths occurring each and the dominant mutations resulting in the drug year [1,2]. These numbers are increasing, as is the resistance profiles have been well-documented number of individuals infected with strains resist- [3–5]. Isoniazid resistance is often associated with ant to first-line anti-TB drugs. In the UK, the mutation in a gene encoding catalase-peroxidase current first-line treatment is a 2-month course of (katG), most frequently a Ser315Thr mutation [6]. isoniazid, rifampicin and pyrazinamide, often Rifampicin inhibits transcription by binding to with the addition of ethambutol, followed by the RNA polymerase b-subunit, encoded by the isoniazid and rifampicin for a further 4–6 months. rpoB gene [7], with resistance resulting from Aminoglycosides such as streptomycin and mutations in the rifampicin resistance-determin- ing region of rpoB, an 81-bp region encoding amino-acids 507–533. The most common (70%) Corresponding author and reprint requests: C. Arnold, mutations are at codons 526 and 531, and Genomics, Proteomics and Bioinformatics Unit, Health Pro- tection Agency, 61 Colindale Avenue, London NW9 5HT, UK are associated with high levels of resist- E-mail: [email protected] ance (> 100 mg ⁄ L). Less frequent mutations,