Abstract:

Objective: To measure the susceptibility of clinical isolates of Mycobacterium kansasii (M.kansasii) to isoniazid (INH), and investigate the mechanism that causes the differences. Methods: The standard strain of Mycobacterium tuberculosis H37Rv (MTB, ATCC27294) and M.kansasii (ATCC12478), as well as 35 clinical strains of M.kansasii isolated from patients infected with nontuberculous mycobacteria, all of which come from Beijing Chest Hospital affiliated to Capital Medical University. The changes of minimum inhibitory concentration (MIC) of INH in vitro before and after the addition of effluvium pump inhibitors, including carbonyl cyanide 3-chlorophenzone, N, N'-dicyclohexylcarbodiimide, verapamil and reserpine, were measured by microplate method and their correlation was evaluated. Sequences of known genes associated with INH resistance were identified to understand the correlation between gene sequence polymorphisms and MIC values in clinical isolates. Catalase-peroxidase (KatG) of MTB and M.kansasii were overexpressed, the affinity of KatG proteins to INH was tested with the Biolayer Interferometry (BLI) technique, and the peroxide-peroxidase activity of KatG proteins was tested with enzyme-linked immunoassay. Results: 5.7% (2/35), 71.4% (25/35), 2.9% (1/35) and 20.0% (7/35) of the 35 clinical strains of M.kansasii had MIC to INH at 0.5 μg/ml, 1 μg/ml, 4 μg/ml and >32 μg/ml, respectively. After adding appropriate amounts of carbonyl cyanide 3-chlorophenzone, N, N'-dicyclohexylcarbodiimide, verapamil and reserpine, 5 (14.3%), 2 (5.7%), 9 (25.7%) and 0 strains showed more than 50% reduction in MIC values. The katG, inhA, kasA, ahpC and oxyR gene sequences of all clinical isolates were consistent with reference strain of M.kansasii. The dissociation constant between INH and KatG proteins derived from M.kansasii (K_D=1.45×10^-4 mol/L) was slightly lower than that from MTB (K_D=7.44×10^-4 mol/L), but the specific peroxidase activity of KatG proteins from MTB (318.69 U/mg) was higher than that from M.kansasii (87.62 U/mg). Conclusion: Clinical isolates M.kansasii showed variable INH susceptibility in vitro, but neither the known INH resistance-related gene polymorphisms nor the efflux pump was the main mechanism of this intra-specious difference in INH susceptibility. The higher susceptibility of MTB to INH than that of M.kansasii may be related to the stronger peroxidase activity of its KatG proteins.

Key words: Mycobacterium kansasii, Mycobacterium tuberculosis, Isoniazid, IDrug resistance, microbial