Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates from Ontario, Canada

OBJECTIVES: Ontario bears the greatest burden of tuberculosis in Canada, with 40% of all cases and 60% of multidrug-resistant cases. The purpose of this study was to genotypically characterize isoniazid- and rifampicin-resistant isolates and compare these results with phenotypic drug susceptibility testing data. This is the first Canadian study to examine gene mutations that contribute to multidrug-resistant tuberculosis. METHODS: A total of 751 tuberculosis isolates were tested for drug resistance using phenotypic antimicrobial susceptibility testing methods. Isolates were then characterized using molecular methods. Following DNA extraction, PCR amplification and sequence analysis were performed on the rifampicin resistance region of rpoB, as well as the region surrounding katG315 and the inhA promoter region associated with isoniazid resistance. RESULTS: Eighteen different mutation types were found in the rpoB region of rifampicin-resistant isolates. Isolates with mutations at residues rpoB531 (64.1%), rpoB526 (15.2%) and rpoB516 (8.7%) were the most common. In addition, an insertion was found at residue 514. Three phenotypically rifampicin-resistant isolates (3.3%) were genotypically wild-type. In isoniazid-resistant strains, mutations were found most commonly at katG315 (45.4%) as well as at the inhA promoter region (28.6%). Thirty-nine isolates (25.3%) were phenotypically isoniazid-resistant but genotypically wild-type. The katG315 mutation was statistically associated with multidrug-resistant isolates. CONCLUSIONS: This study expands the knowledge of mutations that potentially contribute to drug resistance in tuberculosis and lays the foundation for developing molecular-based tests to determine drug resistance in clinical tuberculosis isolates.