We developed a multiplexed DNA microarray-based assay allowing identification of 12 causative agents of reproductive tract infections with the simultaneous detection of 47 genetic determinants of resistance to antimicrobial substances. The microarray was tested on 93 isolates of Neisseria gonorrhoeae, 32 isolates of Treponema pallidum and 29 samples of Ureaplasma spp./Mycoplasma spp. The N. gonorrhoeae isolates had multiple mutations in the penA, ponA, rpsJ, gyrA, parC, and mtrR genes; their prognostic value significantly increased when combinations of mutations were detected. In the analyzed T. pallidum isolates, single A2058G substitution in the 23S rRNA gene responsible for macrolide resistance was found. DNA sequences of Ureaplasma spp./Mycoplasma spp. were determined as wild type, which was not fully consistent with the results of analysis of their antimicrobial susceptibility.

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

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Мониторинг возбудителей инфекционных заболеваний, развивающих множественную устойчивость к антимикробным препаратам, является важной и актуальной задачей. Целью работы являлось генотипирование современных российских клинических изолятов N. gonorrhoeae по протоколу NG-MAST (Neisseria gonorrhoeae multi-antigen sequence types) и сравнительный филогенетический анализ возбудителя гонококковой инфекции в России, странах ЕС и Японии. Всего исследовано 822 изолята, собранных в РФ в период с 2013 по 2018 гг. Использовали также данные NG-MAST-типирования из баз данных «PathogenWatch» (страны ЕС, 1071 образец) и «PubMLST» (Япония, 206 образцов). Изоляты РФ принадлежали к 301 различному NG-MAST типу, наиболее распространенными являлись 807, 228, 1993, 5714, 9476 (8,3%; 3,3%; 3,2%; 3,2%; 2,7% соответственно). В РФ обнаружено только 3 изолята (0,4%) пандемически значимого NG-MAST 1407, характеризующегося множественными детерминантами резистентности к антимикробным препаратам и доминирующего во многих странах мира. Построено филогенетическое древо NG-MAST типов, найденных в России и в европейских странах. Кластерный анализ данных по доле изолятов с уникальными сиквенс-типами и численности населения показал существование двух кластеров (уровень значимости 0,01): первый составили Россия и Япония, второй -европейские страны. Показана тенденция в распределении уникальных сиквенс-типов -их доля тем выше, чем больше численность населения страны. Филогенетический анализ показал генетическую отдаленность наиболее распространенных российских, европейских и японских сиквенс-типов, что указывает на локальный характер формирования и эволюции российской популяции N. gonorrhoeae.Surveillance of multidrug-resistant infections is a priority task for contemporary epidemiology. The aim of this study was to genotype modern clinical isolates of N. gonorrhoeae using the NG-MAST technique (Neisseria gonorrhoeae multi-antigen sequence typing) and to compare the phylogeny of the gonococcal pathogens coming from Russia, European Union and Japan. We studied a total of 822 isolates collected in Russia from 2013 through 2018. We also used NG-MAST data from the following databases: PathogenWatch (European Union, 1,071 isolates) and PubMLST (Japan, 206 isolates). Russian isolates represented 301 different NG-MAST types. The most common were types 807, 228, 1993, 5714, and 9476 (8.3%, 3.3%, 3.2%, 3.2%, and 2.7%, respectively). There were only 3 isolates (0.4%) from Russia that represented the epidemiologically significant sequence type 1407 prevailing in many countries and characterized by multiple determinants of antimicrobial resistance. A phylogenetic tree for the NG-MAST types found in Russia and European countries was constructed. The cluster analysis of the proportion of isolates belonging to unique sequence types and the country population size allowed us to identify 2 clusters (significance level -0.01): the first cluster included Russia and Japan, the second, European countries. A distribution pattern was identified for unique sequence types: the gr...

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