Mycobacterium tuberculosis resistance to novel anti-tuberculosis drugs in russia
Authors: Mokrousov Igor , Vyazovaya Anna , Akhmedova Gulnora , Fundovnaya Elena , Kozlova Elena , Maslennikova Natalia , Leontev Dmitrii , Polev Dmitrii , Ostankova Yulia , Shchemelev Alexander , Semenov Alexander , Vasilieva Neliya , Solovieva Natalia , Zhuravlev Viacheslav , Turkin Eugeni 
Published: January 2021

Aim and objectives:
Emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains requires use of novel compounds. New drugs require long-term treatment regimen and, consequently, long-term drug selective pressure gives more time to mycobacteria to develop resistance. At the same time, new compounds are expensive and, albeit efficient, they are not routinely used in the Russian Federation which situation with tuberculosis is characterized by high and increasing prevalence of multidrug resistant strains.

Methods:
M. tuberculosis isolates were recovered from sputum at the 1-3 months intervals. M. tuberculosis drug susceptibility testing for first- and second-line drugs was performed using modified proportion method on Middlebrook 7H9 liquid culture and Bactec MGIT 960 system (Becton Dickinson, Sparks, Md.) according to the manufacturer's instructions. The laboratory is externally quality assured by the System for External Quality Assessment “Center for External Quality Control of Clinical Laboratory Research” (Moscow, Russia). Whole genome sequencing was performed at the MiSeq platform (Illumina). The fastq files were aligned to the complete genome of the reference strain H37Rv (NC_00962.3) using Geneious 9 program (Biomatters, New Zealand) and additionally checked with PhyReSE online tool (https://bioinf.fz-borstel.de/mchips/phyresse/).

Results:
Mutations in Rv0678 (mmpR) were most frequent in BDQ resistant strains and different types of mutations were found (non-synonymous substitutions, frameshift, multi-codon indels). In some cases, different mutations coexisted and heteroresistance was observed in all cases, i.e., simultaneous presence of wild type and mutants alleles (which correlates with previous studies). Only in one patient, atpE mutation emerged in M. tuberculosis isolate during treatment. Analysis of genes associated with PCZ resistance (ethA, ethR, and hadABC) identified three mutations in ethA and one mutation in hadA. The ethA frameshift mutation (genome position 4327363 CT>C) was detected in isolates from patients who did not receive previous PCZ treatment but were all phenotypically resistant to PTH or ETH. Thus we assumed that this mutation emerged as PTH/ETH resistance mutation.

Conclusions:
To conclude, mutations in Rv0678 present a main mechanism of bedaquiline resistance in the Russian setting; development of resistance during treatment is not rare as bedaquiline resistance mutations emerged in 40% of patients. Furthermore, development of BDQ resistance 2-3 months after stop of treatment is possible, due to long half-life of the drug. With regard to PCZ resistance, presence of cross-resistance mutations to both PCZ and ETH/PTH suggests that these mutations should be assessed before inclusion of PCZ in the treatment regimen. A large prospective study of the more diverse M. tuberculosis collection is warranted to elucidate the development of resistance to these novel drugs in the Russian setting taking into consideration the population structure of M. tuberculosis population.