Diversity of glpK Gene and Its Effect on Drug Sensitivity in Mycobacterium bovis

Abstract

Background: Glycerol kinase (glpK) is essential for the first step of glycerol catabolism in Mycobacterium tuberculosis. However, Mycobacterium bovis has been known to grow poorly in glycerol media because of a base insertion in the glpK gene.

Methods: We analyzed the glpK gene sequences of 60 clinical M. bovis isolates, and determined the minimum inhibitory concentration of 14 drugs by microdilution method to evaluate the effect of frameshift mutations on drug sensitivity. The effect of M. bovis growth rate on its drug sensitivity was investigated using bacteria grown on glycerol or pyruvate.

Results: A total of 44 (73.33%) clinical M. bovis isolates have frameshift mutations in a homopolymeric tract of 7 cytosines in the glpK gene. 15.00% M. bovis isolates showed phenotypic drug resistance. Glycerol metabolism-deficient M. bovis showed reduced susceptibility to 9 out of 14 tested drugs. Mutations in the glpK gene can lead to impaired growth in glycerol-based media, while the minimal inhibitory concentration values of slow-growing M. bovis were higher.

Conclusion: Mutations in the glpK gene can lead to slowed growth and reduced susceptibility to drugs in M. bovis, which may contribute to the emergence of drug-resistant M. bovis and pose a threat to human health owing to the zoonotic capacity of M. bovis.

Keywords: Mycobacterium bovis; drug resistance; glpK; tuberculosis.

Figure 1

Glycerol promotes the growth of M. bovis glpK 7C HT strain and M. bovis glpK 10C HT strain in vitro. (A–C) Growth curves of M. bovis glpK 7C HT strain, glpK 8C HT strain and glpK 10C HT strain in glycerol or pyruvate medium. (D) Colony formation of the M. bovis glpK 10C HT strain on day 12 of growth in glycerol solid medium. (E) Colony formation of the M. bovis glpK 10C HT strain on day 14 of growth in pyruvate solid medium. (F) The relative expression of mRNA in glycerol and pyruvate medium for the M. bovis glpK 10C HT strain was compared. **Significant at ρ < 0.01; ***Significant at ρ < 0.001; ****Significant at ρ < 0.0001 were determined by unpaired t-test (2 tailed).

Figure 2

The glpK mutation contributes to the anti-tuberculosis drug tolerance capability of M. bovis isolates. (A–F) MIC assay was conducted to investigate the drug sensitivity of different mutants of glpK of M. bovis. The sensitivity of (A) Amikacin, (B) Moxifloxacin, (C) Para-aminosalicylic acid, (D) Rifampin, (E) Ethambutol, (F) Rifabutin, (G) Levofloxacin, (H) Clofazimine, and (I) Linezolid was determined by MIC assay. *Significant at ρ < 0.05; **Significant at ρ < 0.01; ***Significant at ρ < 0.001 were determined by unpaired t-test (2 tailed). Due to the low number of glpK 10C HT and glpK 11C HT strains, they were not analyzed.