ABSTRACT
A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and -8 and -15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997-2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.
Subject(s)
Databases, Nucleic Acid , Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bacterial Proteins/genetics , Catalase/genetics , DNA Probes/genetics , DNA-Directed RNA Polymerases , Genetic Markers/genetics , Humans , Isoniazid/pharmacology , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/drug effects , Point Mutation , Polymerase Chain Reaction/methods , Promoter Regions, Genetic , Rifampin/pharmacology , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
OBJECTIVES: Three Mycobacterium tuberculosis genetic loci--rpoB and katG genes and the fabG1(mabA)-inhA operon promoter region--were studied to reveal the mutations associated with rifampicin and isoniazid resistance. METHODS: Four hundred and twelve isolates of M. tuberculosis from different regions of the Russian Federation were collected during 1997-2005. A matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based minisequencing method was used for the detection of mutations. RESULTS: Thirteen different variants of single mutations in codons 533, 531, 526, 516, 513 and 511 of the rifampicin resistance-determining region of the rpoB gene as well as the TTG insertion in the 514a position were found among the rifampicin-resistant isolates. Single nucleotide substitutions in codons 531, 526 and 516 (64.8%, 10.3% and 7.7%, respectively) were the most prevalent mutations. Codon 526 was shown to be the most variable of all. No mutations were detected in rpoB genes for 29 (10.7%) of the rifampicin-resistant isolates. 76.9% of the isoniazid-resistant isolates carried single mutations in codon 315 of the katG gene. For another 12.9% of them, double mutations in the katG gene and the fabG1(mabA)-inhA promoter region were revealed. No mutations were detected in 8.2% of the isoniazid-resistant isolates. CONCLUSIONS: Molecular analysis of the loci of rpoB and katG genes and the inhA promoter region of 412 M. tuberculosis clinical isolates from various parts of the Russian Federation was carried out. The new MALDI-TOF MS-based method may be used for rapid and accurate monitoring of the spread of drug resistance.
