Browsing by Author "Mokrousov, Igor"
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- Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain clusterPublication . Mokrousov, Igor; Vyazovaya, Anna; Shitikov, Egor; Badleeva, Maria; Belopolskaya, Olesya; Bespiatykh, Dmitry; Gerasimova, Alena; Perdigão, JoãoBackground The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern. Results The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes). Conclusions We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted.
- Molecular Insight into Mycobacterium tuberculosis Resistance to Nitrofuranyl Amides Gained through Metagenomics-like Analysis of Spontaneous MutantsPublication . Mokrousov, Igor; Slavchev, Ivaylo; Solovieva, Natalia; Dogonadze, Marine; Vyazovaya, Anna; Valcheva, Violeta; Masharsky, Aleksey; Belopolskaya, Olesya; Dimitrov, Simeon; Zhuravlev, Viacheslav; Portugal, Isabel; Perdigão, João; Dobrikov, Georgi M.We performed synthesis of new nitrofuranyl amides and investigated their anti-TB activity and primary genetic response of mycobacteria through whole-genome sequencing (WGS) of spontaneous resistant mutants. The in vitro activity was assessed on reference strain Mycobacterium tuberculosis H37Rv. The most active compound 11 was used for in vitro selection of spontaneous resistant mutants. The same mutations in six genes were detected in bacterial cultures grown under increased concentrations of 11 (2×, 4×, 8× MIC). The mutant positions were presented as mixed wild type and mutant alleles while increasing the concentration of the compound led to the semi-proportional and significant increase in mutant alleles. The identified genes belong to different categories and pathways. Some of them were previously reported as mediating drug resistance or drug tolerance, and counteracting oxidative and nitrosative stress, in particular: Rv0224c, fbiC, iniA, and Rv1592c. Gene-set interaction analysis revealed a certain weak interaction for gene pairs Rv1592–Rv1639c and Rv1592–Rv0224c. To conclude, this study experimentally demonstrated a multifaceted primary genetic response of M. tuberculosis to the action of nitrofurans. All three 11-treated subcultures independently presented the same six SNPs, which suggests their non-random occurrence and likely causative relationship between compound action and possible resistance mechanism.
- Practical approach to detection and surveillance of emerging highly resistant Mycobacterium tuberculosis Beijing 1071-32-clusterPublication . Mokrousov, Igor; Vyazovaya, Anna; Sinkov, Viacheslav; Gerasimova, Alena; Ioannidis, Panayotis; Jiao, Weiwei; Khromova, Polina; Papaventsis, Dimitrios; Pasechnik, Oksana; Perdigão, João; Rastogi, Nalin; Shen, Adong; Skiba, Yuriy; Solovieva, Natalia; Suffys, Philip; Tafaj, Silva; Umpeleva, Tatiana; Vakhrusheva, Diana; Yarusova, Irina; Zhdanova, Svetlana; Zhuravlev, Viacheslav; Ogarkov, OlegAncient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster.
- Synthesis, Characterization and Complex Evaluation of Antibacterial Activity and Cytotoxicity of New Arylmethylidene Ketones and Pyrimidines with Camphane SkeletonsPublication . Slavchev, Ivaylo M.; Mitrev, Yavor; Shivachev, Boris; Valcheva, Violeta; Dogonadze, Marine; Solovieva, Natalia; Vyazovaya, Anna; Mokrousov, Igor; Link, Wolfgang; Jiménez, Lucía; Cautain, Bastien; Mackenzie, Thomas A.; Portugal, Isabel; Lopes, Francisca; Capela, Rita; Perdigão, João; Dobrikov, Georgi M.The synthesis of 20 arylidenecamphors and 15 pyrimidines with camphane skeleton is described in the current report. A modified method for preparation of sterically hindered 2-aminopyrimidines in two steps was demonstrated. The evaluation of their in vitro activity against Mycobacterium tuberculosis H37Rv showed different MIC values (up to 0.91 μM for ketone 39). Compound 35 demonstrated moderate (8.23 μM), but sustainable activity toward a collection of drug-resistant M. tuberculosis strains. Many of the compounds (especially among 2-aminopyridines 42–56) exhibited good to excellent activity against different strains of pathogenic bacteria and fungi (MIC up to 0.60 μM for compound 50), compared with reference antibiotics. Many of the newly designed compounds possess also in vitro cytotoxicity.