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, rifampin-resistant mutants of the Beijing 1585 strain did not emerge in low-density cultures (5 × 10⁵ CFU/mL) used for time-kill kinetics experiments, although 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 high-density culture of 10¹⁰ 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.