ANALYSIS OF SINGLE ACCUMULATED NUCLEOTIDE VARIANTS AND MUTATION RATE IN M. TUBERCULOSIS ISOLATE PAIRS USING THE WHOLE GENOME SEQUENCING APPROACH

Objectives. Whole genome sequencing (WGS) has become an irreplaceable tool in studying different aspects of Mycobacterium tuberculosis (Mtb) infection, such as microevolution, drug resistance and recurrence. However, data about acquired genomic mutations during human infection are still limited. The aim of this research was to explore mutation rate and accumulated single nucleotide variants (SNVs) in Mtb isolate pairs obtained during either a single or two different tuberculosis (TB) episodes.
Materials and Methods. WGS was performed on 52 Mtb isolate pairs of identical genotypes acquired from pulmonary TB patients (2002-2019). Gene-based annotation and functional effect prediction were performed on differing SNVs detected in isolate pairs with pairwise SNV-distance ≤ 10. Mycobacterial
mutation rate was calculated as SNVs/genome/year for isolate pairs where differing SNVs were found only in subsequent isolates.
Results. 77 differing SNVs among 29 Mtb isolate pairs were detected, and the majority was located in protein-coding genes (68/77). Missense (33/68) and synonymous (32/68) mutations were more common than stop mutations (3/68). No SNV accumulation pattern could be determined since new variants were
mostly randomly distributed throughout the whole Mtb genome. In contrast, four patients infected with multidrug-resistant Mtb strains of Beijing (n = 3) and LAM (n = 1) genotypes developed fluoroquinolone resistance due to accumulation of additional SNVs in gyrA gene. The mutation rate was calculated for 37 Mtb isolate pairs. In 18 cases it was equal to zero, in 17 isolate pairs the mean mutation rate was 0.36 ± 0.16 SNVs/genome/year, and two outlier values of 2 and 2.47 SNV/genome/year were obtained for drugresistant single-episode isolates of Beijing genotype.
Conclusions. This study highlighted the overall tendencies of SNV accumulation in Mtb genome. Further investigation is needed to decipher factors triggering selective fluoroquinolone resistance development and high mycobacterial mutation rate in different Mtb genotypes. This study was supported by the ERDF grant No 1.1.1.1/20/A/0 46.