Volume 19, Number 3—March 2013
Letter
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, 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.
References
- de Steenwinkel JEM, ten Kate MT, de Knegt GJ, Kremer K, Aarnoutse RE, Boeree MJ, Drug susceptibility of Mycobacterium tuberculosis Beijing genotype, association with MDR TB. Emerg Infect Dis. 2012;4:660–3. DOIPubMedGoogle Scholar
- David HL. Probability distribution of drug-resistant mutants in unselected populations of Mycobacterium tuberculosis. Appl Microbiol. 1970;20:810–4.PubMedGoogle Scholar
- Werngren J, Hoffner SE. Drug-susceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation-conferred resistance to rifampin at an elevated rate. J Clin Microbiol. 2003;41:1520–4. DOIPubMedGoogle Scholar
- Bergval I, Kwok B, Schuitema K, Kremer K, van Soolingen D, Klatser P, Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro. PLoS ONE. 2012;7:e29108. DOIPubMedGoogle Scholar
- Gillespie SH. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemother. 2002;46:267–74. DOIPubMedGoogle Scholar
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We performed no classical fluctuation assays. We compared the Beijing genotype with the East African/Indian genotype to learn how
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.
Acknowledgments
References
- Werngren J. Mycobacterium tuberculosis Beijing type mutation frequency [letter]. DOIGoogle Scholar
- Werngren J, Hoffner SE. Drug-susceptible Mycobacterium tuberculosis Beijing genotype does not develop mutation-conferred resistance to rifampin at an elevated rate. J Clin Microbiol. 2003;41:1520–4. DOIPubMedGoogle Scholar
- Bergval I, Kwok B, Schuitema K, Kremer K, van Soolingen D, Klatser P, Pre-existing isoniazid resistance, but not the genotype of Mycobacterium tuberculosis drives rifampicin resistance codon preference in vitro. PLoS ONE. 2012;7:e29108. DOIPubMedGoogle Scholar
- Clinical and Laboratory Standards Institute. Susceptibility testing of mycobacteria, nocardiae, and other aerobic actinomycetes. Approved standard M24–A2. 2nd ed. Wayne (PA): The Institute; 2011.
Table of Contents – Volume 19, Number 3—March 2013
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Please use the form below to submit correspondence to the authors or contact them at the following address:
Jim Werngren, Unit of Highly Pathogenic Microorganisms, Dept of Preparedness, Swedish Institute for Communicable Disease Control, Nobels väg 18 S-17182, Solna, Stockholm S 17182, Sweden
Address for correspondence: J.E.M. de Steenwinkel, Erasmus MC, Room L-327, PO Box 2040, 3000 CA, Rotterdam, the Netherlands; email: j.desteenwinkel@erasmusmc.nl
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