Dynamic population changes in Mycobacterium tuberculosis during acquisition and fixation of drug resistance in patients

Abstract

Background: Drug-resistant tuberculosis poses a growing challenge to global public health. However, the diversity and dynamics of the bacterial population during acquisition of drug resistance have yet to be carefully examined.

Methods: Whole-genome sequencing was performed on 7 serial Mycobacterium tuberculosis (M. tuberculosis) populations from 3 patients during different stages in the development of drug resistance. The population diversity was assessed by the number and frequencies of unfixed mutations in each sample.

Results: For each bacterial population, 8-41 unfixed mutations were monitored by the fraction of single-nucleotide polymorphisms at specific loci. Among them, as many as 4 to 5 resistance-conferring mutations were transiently detected in the same single sputum, but ultimately only a single type of mutant was fixed. In addition, we identified 14 potential compensatory mutations that occurred during or after the emergence of resistance-conferring mutations.

Conclusions: M. tuberculosis population within patients exhibited considerable genetic diversity, which underwent selections for most fit resistant mutant. These findings have important implications and emphasize the need for early diagnosis of tuberculosis to decrease the chance of evolving highly fit drug-resistant strains.

Figure 1.

Patients and samples. The treatment durations are indicated as solid lines, which start with a hollow square on the 0th day and end with a triangle (default) or circle (death). The treatment schedules are presented in grayish shadows, and the poor compliance of patient A is indicated by the dark gray shadow. Sample information such as collection time points, designations, and resistance profiles are presented in boxes.

Figure 2.

Characteristic SNPs. A, Venn diagrams showing the number of characteristic SNPs in each sample. B–D, Genomic distribution and frequencies of characteristic SNPs identified in each patient. Different colors represent the serially collected samples. Frequencies are estimated from the mapping result on M. tuberculosis H37Rv, using a previously described maximum likelihood approach [17].

Figure 3.

The dynamic heteroresistance in 3 patients. Resistance-conferring mutations are labeled as M1–M6. A, Dynamic heteroresistance to isoniazid in patient A. In sample a3, the 4 minor mutations (M2, M3, M5, and M6) are below the frequency threshold of 5% but still detectable. Therefore, all of them are presented for a comprehensive view. B and C, Unfixed drug resistant mutations in patients B and C, respectively.