Mycobacterium tuberculosis resistance to novel anti-tuberculosis drugs in Russia: insight from whole genome sequencing of consecutive isolates

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. Bedaquiline is a novel and promising anti-TB drug. Substitutions in the M. tuberculosis atpE gene prevent interaction of the drug with its target, ATP synthase and were associated with bedaquiline resistance. Mutations in two other genes involved in intermediary metabolism and respiration were also linked to the bedaquiline resistance: pepQ coding for probable cytoplasmic peptidase and atpC coding for probable ATP synthase epsilon chain. Mutations in the repressor gene mmpR (Rv0678) lead to increased expression of the efflux genes mmpL5 and mmpS5, providing export of bedaquiline. Finally, mutations in Rv1979c coding for possible conserved permease involved in transport of amino acids across the membrane were also shown in bedaquiline-resistant isolates. Perchlozone is a new thiosemicarbazone that is similar to thiacetazone and approved for treatment of MDR-TB in Russia. It is a prodrug activated by monooxygenase EthA and its target is FASII dehydratase complex HadABC. A cross-resistance to TAC and PCZ was shown by in vitro experiments and was mediated by both ethA and hadA mutations. On the other hand, EthA is known to activate second-line drugs ethionamide (ETH) and prothionamide (PTH) and ethA or ethR mutations were described as ETH/PTH resistance mechanism. Linezolid is other important anti-TB drug used for treatment of MDR/XDR-TB. The drug binds to the ribosome at the peptidyl-transferase center, and in vitro and clinical studies described mutations in the 23S rRNA and L3 ribosomal protein genes in resistant strains. Russia is a recognized hot spot for MDR-TB. TB incidence is decreased in the European Russia and is very high in Siberia and Far East, however, MDR TB is high and increasing in all parts of the country. For example, in Northwest Russia, TB incidence is 31.3/100,000 and MDR rate in primary TB - 27.4%. Kaliningrad is the westernmost Russian region and has a unique geographic position, separated from the mainland Russia by Lithuania, Poland and the Baltic Sea. The region is characterized by high TB incidence (38.8/100,000) and very high rate of MDR among newly diagnosed TB patients (30.5%). The M. tuberculosis population structure is dominated by the Beijing genotype. In this study, we aimed to gain insight into molecular basis of M. tuberculosis resistance to bedaquiline, perchlozone and linezolid in the Kaliningrad region in Russia. We used whole-genome sequencing of multiple consecutive isolates from patients spanning long time period in order to gain comprehensive and quantitative view on the real-time mycobacterial adaptation to the human host. This study included patients admitted to the clinical tuberculosis dispensary in Kaliningrad whose treatment regimen included second-line, repurposed and new anti-TB drugs, including BDQ, PCZ and LZD. All samples used in this study were coded and lacked personal information about patients. All personal data were treated anonymously and, according to the regulations of the St. Petersburg Research Institute of Phthisiopulmonology, no additional ethical approval was required. 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. No mutations in rrl, rplC, rplD genes associated with LZD resistance were found. 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