Mycobacterium tuberculosis Beijing Type Mutation Frequency

In Response: We explain the differing frequencies of mutation to rifampin resistance mentioned by Werngren (1). First, the strains of Mycobacterium tuberculosis that we tested differed from those previously tested (2). Second, we used different rifampin concentrations in subculture plates. For Beijing strain 2002–1585, Bergval et al. (3) found a mutation frequency of 4–24 × 10−8 at a subculture concentration of 8 mg/L, whereas we found a mutation frequency of 3–4 × 10−3 at a subculture concentration of 1 mg/L and a lower mutation frequency at 2 mg/L. Thus, the concentration of drugs in subculture plates is crucial to mutation frequency assays. Absent a subculture concentration standard, we applied rifampin at 1 mg/L (4) because bacteria growing at this concentration are considered resistant to rifampin. Our mutation frequency and time-kill kinetics assay results are not contradictory because in the time-kill kinetics assays, the subculture rifampin concentration was 4 mg/L. 
 
We performed no classical fluctuation assays. We compared the Beijing genotype with the East African/Indian genotype to learn how M. tuberculosis strains differed in their capacity to withstand antituberculosis drug treatment. For reference strain H37Rv, mutation frequency was 1.5 × 10−6, higher than that found with higher subculture concentrations. 
 
With regard to the 3 other issues, our drug-susceptibility testing of mutants showed a stable rifampin-resistant phenotype. We agree that these bacteria might represent preexisting mutants selected during drug exposure in a certain drug concentration window. By using different concentrations in subculture plates in our mutation frequency assay, we detected such preexisting mutants. Heteroresistance probably does not explain our observations because in our time-kill kinetics experiments, the whole mycobacterial population decreased over time in a drug concentration-dependent way, and regrowth of a drug-resistant subpopulation was not observed. 
 
By not sticking to the fixed test conditions as used in the classical drug-susceptibility assays, research leads to highly interesting findings. One can conclude that serendipity flourishes with variation.


Mycobacterium tuberculosis Beijing Type Mutation Frequency
To the Editor: A striking finding in the study by de Steenwinkel et al. (1) is the high frequency of mutation to rifampin resistance by 2 Mycobacterium tuberculosis Beijing strains, which might play a role in the association between the Beijing strains and multidrug-resistant tuberculosis. Earlier reported frequency of mutation to rifampin resistance by M. tuberculosis has been 10 −8 CFU (2,3), including the Beijing genotype (3,4). Of note, the Beijing 2002-1585 strain, for which frequency of mutation to rifampin resistance is 10 -3 CFU (1 mutant/1,000 CFU), showed a moderate frequency of 10 −8 CFU in another study (4). We think that a mutation frequency increase of 100,000× is remarkably high. In contrast, rifampinresistant mutants of the Beijing 1585 strain did not emerge in low-density cultures (5 × 10 5 CFU/mL) used for time-kill kinetics experiments, al-though frequency of mutation to rifampin resistance was determined to be 10 -3 CFU.
Mutation frequency is determined by fluctuation assays. To exclude preexisting mutants, which would bias the mutation frequency by so-called jackpots, a series of low-inoculum cultures is typically used (5). However, for unknown reasons, de Steenwinkel et al. used only 1 highdensity culture of 10 10 CFU of each strain to determine mutation frequency. This strategy is not recommended because mutations can occur early or late, resulting in substantial mutation frequency fluctuation between test episodes. A strain with known mutation rates should preferably be included to rule out possible technical errors.
We propose the following explanations for the remarkable results: 1) the rifampin concentration for selecting mutants might have been too low, enabling growth of some colonies of drug-susceptible bacteria; 2) rifampin mutants arose early or preexisted in the cultivation of Beijing strains 1585 and 1607, producing jackpots; or 3) the 2 Beijing isolates might contain rifampin-resistant subpopulations (heteroresistance). The capacity of the Beijing strain to develop and, especially, transmit multidrug-resistant tuberculosis remains to be further analyzed.  ) found a mutation frequency of 4-24 × 10 −8 at a subculture concentration of 8 mg/L, whereas we found a mutation frequency of 3-4 × 10 −3 at a subculture concentration of 1 mg/L and a lower mutation frequency at 2 mg/L. Thus, the concentration of drugs in subculture plates is crucial to mutation frequency assays. Absent a subculture concentration standard, we applied rifampin at 1 mg/L (4) because bacteria growing at this concentration are considered resistant to rifampin. Our mutation frequency and time-kill kinetics assay results are not contradictory because in the time-kill kinetics assays, the subculture rifampin concentration was 4 mg/L.

Jim Werngren
We performed no classical fluctuation assays. We compared the Beijing genotype with the East African/Indian genotype to learn how M. tuberculosis strains differed in their capacity to withstand antituberculosis drug treatment. For reference strain H37Rv, mutation frequency was 1.5 × 10-6, higher than that found with higher subculture concentrations.
With regard to the 3 other issues, our drug-susceptibility testing of mutants showed a stable rifampin-resistant phenotype. We agree that these bacteria might represent preexisting mutants selected during drug exposure in a certain drug concentration window. By using different concentrations in subculture plates in our mutation frequency assay, we detected such preexisting mutants. Heteroresistance probably does not explain our observations because in our time-kill kinetics experiments, the whole mycobacterial population decreased over time in a drug concentration-dependent way, and regrowth of a drug-resistant subpopulation was not observed.
By not sticking to the fixed test conditions as used in the classical drug-susceptibility assays, research leads to highly interesting findings. One can conclude that serendipity flourishes with variation.