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

Jurriaan E.M. de Steenwinkel

, Marian T. ten Kate, Gerjo J. de Knegt, Kristin Kremer, Rob E. Aarnoutse, Martin J. Boeree, Henri A. Verbrugh, Dick van Soolingen, and Irma A.J.M. Bakker-Woudenberg

Author affiliations: Erasmus University Medical Center, Rotterdam, the Netherlands (J.E.M. de Steenwinkel, M.T. ten Kate, G.J. de Knegt, H.A. Verbrugh, I.A.J.M. Bakker-Woudenberg); National Institute of Public Health and the Environment Center for Infectious Disease Control (RIVM),; Bilthoven, the Netherlands (K. Kremer, D. van Soolingen); World Health Organization Regional Office for Europe, Copenhagen, Denmark (K. Kremer); Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands (R.E. Aarnoutse, M.J. Boeree, D. van Soolingen)

Abstract

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.

The emergence of Mycobacterium tuberculosis resistance to antituberculosis (anti-TB) drugs is a major public health challenge that is threatening World Health Organization targets set for the elimination of TB (1). Approximately 500,000 cases of multidrug-resistant TB (MDR TB) are diagnosed annually, but the true magnitude of the MDR TB problem is not known because adequate laboratory tools are lacking. Multiple factors contribute to low cure rates, treatment failures, and relapses: poor-quality guidance regarding treatment, HIV co-infection, transmission of resistant forms of TB, underdeveloped laboratory services, and unavailability of alternative drug treatments. However, the evolution of M. tuberculosis is an additional factor that presumably fuels the worldwide problem of emerging resistance. The Beijing genotype is significantly associated with drug resistance (2,3), especially in geographic areas where prevalence of resistance to anti-TB drugs is high, and it is associated with recent TB transmission (2–6). There are also indications that the population structure of M. tuberculosis in areas with a high prevalence of anti-TB drug resistance is changing rapidly toward an increase in Beijing genotype strains (2,6–8).
The World Health Organization target rates for detecting and curing TB in Vietnam have been met; however, the rate of TB infection is not decreasing as expected (4,5). Earlier in this country, the Beijing genotype was strongly correlated with MDR TB and treatment failures (9). Extensive molecular epidemiologic studies showed that the Beijing and East-African/Indian (EAI) genotypes are predominating in Vietnam; each lineage causes ≈40% of the TB cases. According to the single-nucleotide polymorphism typing described by Hershberg et al. (10), the Beijing genotype is a representative of the modern lineage, and the EAI genotype is believed to represent an evolutionary lineage more closely related to the common ancestor of the M. tuberculosis complex.
We compared the in vitro activity of anti-TB drugs against susceptible Beijing and EAI M. tuberculosis isolates from Vietnam and determined the in vitro mutation frequency of these strains during drug exposure. We also determined time-kill kinetics of anti-TB drugs and assessed the emergence of resistant mutants and the concentration range within which resistant mutants and no susceptible mycobacteria were selected. The concentration at which resistant mutants did not emerge (the mutant prevention concentration) was also ascertained. By using this approach, we established an in vitro model for determining differences in the ability of M. tuberculosis strains to resist anti-TB drug treatment.

The Study

Results of a liquid culturing system (BD BACTEC MGIT 960 System; BD Diagnostics, Sparks, MD, US) (for details, see the Technical Appendix

[PDF - 72 KB - 4 pages]) showed that all 5 Beijing and 5 EAI genotype strains were susceptible to isoniazid (INH), rifampin (RIF), moxifloxacin (MXF), and amikacin (AMK). MICs were determined by using the agar proportion method (11), which showed that ranges were small for the Beijing and EAI genotype strains: INH, 0.062–0.125 mg/L; RIF, 0.125–1 mg/L; MXF, 0.125–0.5 mg/L; and AMK, 0.5–2 mg/L. Duplicate values showed only minor differences.