Drug Susceptibility of Mycobacterium tuberculosis Beijing Genotype and Association with MDR TB

To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype’s association with multidrug-resistant tuberculosis.

VN+) was previously subjected to genome sequencing described by Schürch et al. (Infection, Genetics and Evolution 2011;11:587-597). The M. tuberculosis strains in this study were selected on basis of their diverse genotyping results, as determined by using spoligotyping and IS6110 restriction fragment length polymorphism (RFLP) typing according to the internationally standardized methods (Kamerbeek et al. J Clin Microbiol. 1997;35(4):907-14 and van Embden et al. J Clin Microbiol. 1993;31(2):406-9). The genotypes of the strains were defined as either Beijing or EAI according to their characteristic spoligotypes, following the well-accepted genotype definitions described by Kremer et al. (J. Clin Microbiol. 2004;42(9):4040-9) and Brudey et al. (BMC Microbiol. 2006;6:6-23 VN 2002-1607, VN 1998-2115, and VN 1998-2145 intermediate Beijing SNP type. The EAI strains showed RFLP patterns with either two (VN 2002(VN -1596 or one copy of IS6110. However, the four 1-copy strains could be discriminated on basis of the molecular weight of the IS6110 containing RFLP fragment and/or their spoligo pattern. supplemented with 10% OADC and 0.5% glycerol for 28-35 days at 37°C with 5% CO 2 . The exact incubation time was dependent on the growth rate of the M. tuberculosis genotype strain investigated.

Anti-TB Drugs
The anti-TB drugs assayed were isoniazid (

Minimal Inhibitory Concentration (MIC)
To determine the MIC of the M. tuberculosis genotype strains we used the agar tuberculosis suspension was plated onto solid media containing serial, twofold dilution concentrations of anti-TB drug. After incubation the degree of growth was assessed. The MIC was defined as the lowest concentration of anti-TB drug that resulted in >99% growth inhibition.
MIC determinations were performed in duplicate.

Mutation Frequency (MF)
Determination of the MF of the M. tuberculosis genotype strains was performed using the "critical concentrations" of the anti-TB drug, being 1 mg/L for isoniazid, 1 mg/L for rifampin, 5 mg/L for amikacin and 1 mg/L for moxifloxacin, as defined by the CLSI (Woods suspension were plated onto solid media without drugs and onto solid media containing the "critical concentration" of the respective anti-TB drugs. After incubation, the total numbers of colony forming units (cfu) and numbers of resistant mycobacteria were counted and the MF was calculated. MFs were determined in duplicate. In order to assess the stability of the resistant mutants isolated from the anti-TB drug-containing solid media, 10 colonies were randomly picked and plated onto solid media without anti-TB drug. After incubation, these colonies were plated to check for re-growth on solid media containing the "critical concentration" of the same anti-TB drug.

Time-kill Kinetics
For Beijing-1585 and EAI-1627 a time-kill kinetic assay for rifampin was performed.
In short, M. tuberculosis cultures at low density or at high density were exposed to rifampin at 2fold increasing concentrations, ranging from 0,5 µg/L to 256 mg/L for six days at 37°C. On days 1, 3 and 6, samples were taken for cfu counting provided that the M. tuberculosis suspension did not show visible aggregation.
Page 4 of 4

Selection of Drug-Resistant M. tuberculosis
During the time-kill kinetic assay for Beijing-1585 and EAI-1627 the selection of resistant mutants was performed. In order to detect drug-resistant M. tuberculosis in the low density or the high density cultures, the samples taken after six days of exposure to rifampin were cultured on rifampin-containing solid media. The concentration of rifampin in the solid media was 4-fold the "critical concentration" (4 mg/L rifampin). Resistant M. tuberculosis colonies, able to grow on this medium, were characterized using the GenoType® MTBDRplus assay (Hain Lifescience GmbH, Nehren, Germany), to detect the most common mutations in rpoB gene conferring rifampin-resistance (J Clin Microbiol. 2007;45(8):2635-40).

Mutant Prevention Concentration (MPC)
The MPC of the M. tuberculosis genotype strains was determined using the protocol as tuberculosis culture approximately 10 10 cfu was plated onto solid medium containing 2-fold increasing concentrations of anti-TB drug, ranging from 64 mg/L to 1024 mg/L for isoniazid, rifampin or amikacin and from 1 mg/L to 32 mg/L for moxifloxacin. The MPC was defined as the lowest concentration of anti-TB drug in the solid medium, which prevented growth of M. tuberculosis.