microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2021-1-95-102microbe-1443Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESМультилокусное сиквенс-типирование штаммов сибиреязвенного микроба, выделенных на территории России и сопредельных государствMultilocus Sequence-Typing of Anthrax Microbe Strains Isolated in Russia and Neighboring Countrieshttps://orcid.org/0000-0003-1682-8788ГончароваЮ. О.GoncharovaYu. O.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

info@obolensk.orghttps://orcid.org/0000-0002-6897-3613БахтееваИ. В.BakhteevaI. V.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

https://orcid.org/0000-0002-5710-4746МироноваР. И.MironovaR. I.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

БогунА. Г.BogunA. G.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

https://orcid.org/0000-0001-7611-3608ХлоповаК. В.KhlopovaK. V.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

https://orcid.org/0000-0002-9501-1380ТимофеевВ. С.TimofeevV. S.

142279, Московская обл., п. Оболенск

Obolensk, Moscow Region, 142279

ФБУН «Государственный научный центр прикладной микробиологии и биотехнологии»РоссияState Research Center of Applied Microbiology and BiotechnologyRussian Federation2021160420210195102Copyright © Гончарова Ю.О., Бахтеева И.В., Миронова Р.И., Богун А.Г., Хлопова К.В., Тимофеев В.С., 20212021Гончарова Ю.О., Бахтеева И.В., Миронова Р.И., Богун А.Г., Хлопова К.В., Тимофеев В.С.Goncharova Y.O., Bakhteeva I.V., Mironova R.I., Bogun A.G., Khlopova K.V., Timofeev V.S.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.microbe.ru/jour/article/view/1443

Цель – генотипирование методом мультилокусного сиквенс-типирования (MLST) и проведение филогенетического анализа выборки из 40 штаммов Bacillus anthracis, выделенных на территории России и сопредельных государств.

Материалы и методы

Материалы и методы. В работе осуществлена сборка последовательностей семи генов домашнего хозяйства штаммов B. anthracis на основе данных полногеномного секвенирования нового поколения, после чего описаны выявленные мутации и их координаты. Полученные последовательности использованы для генотипирования исследуемой выборки методом MLST. Результаты сравнены с данными, представленными в базе данных PubMLST. Филогенетический анализ проведен для слитых in silico последовательностей семи локусов выявленных сиквенс-типов. Для построения дендрограмм использован программный пакет MEGA 7.0.

Результаты и обсуждение

Результаты и обсуждение. В исследованной выборке обнаружено два сиквенс-типа (ST): 35 штаммов относились к ST-1, а пять штаммов, отличающихся одной общей мутацией в локусе glpF, – к ST-3 (согласно кодировке PubMLST), что подчеркивает генетическую обособленность данной группы штаммов. У одного штамма обнаружена уникальная мутация в гене gmk, находящаяся за пределами участка локуса, используемого для MLST. 

Objective – genotyping by multilocus sequence-typing (MLST) and phylogenetic analysis of 40 Bacillus anthracis strains isolated in Russia and neighboring countries.

Materials and methods

Materials and methods. In this study, the sequences of seven housekeeping genes of B. anthracis strains were assembled based on the data of a whole genome new generation sequencing, after which the identified mutations and their coordinates were described. The obtained sequences were used for genotyping of the investigated sample using the MLST method. The results are compared with the data presented in PubMLST database. A phylogenetic analysis was performed for the in silico fused sequences of the seven loci of the identified sequence types. The MEGA 7.0 software package was used to build the dendrograms.

Results and discussion

Results and discussion. Two sequence types (ST) have been found in the examined sample: 35 strains belong to ST-1, and five strains that differed by one common mutation at the glpF locus – to ST-3 (according to PubMLST coding), which emphasizes the genetic separation of this group of strains. One strain has a unique mutation in the gmk gene located outside the region used for MLST. 

Bacillus anthracisBacillus cereusMLSTгенотипированиеBacillus anthracisBacillus cereusMLSTgenotypingReferencesCarroll L.M., Kovac J., Miller R.A., Wiedmann M. Rapid, high-throughput identification of anthrax-causing and emetic Bacillus cereus group genome assemblies via BTyper, a computational tool for virulence-based classification of Bacillus cereus group isolates by using nucleotide sequencing data. Appl. Environ. Microbiol. 2017; 83(17):e01096-17. DOI: 10.1128/AEM.01096-17.Carroll L.M., Kovac J., Miller R.A., Wiedmann M. Rapid, high-throughput identification of anthrax-causing and emetic Bacillus cereus group genome assemblies via BTyper, a computational tool for virulence-based classification of Bacillus cereus group isolates by using nucleotide sequencing data. Appl. Environ. Microbiol. 2017; 83(17):e01096-17. DOI: 10.1128/AEM.01096-17.Маринин Л.И., Дятлов И.А., Шишкова Н.А., Герасимов В.Н. Сибиреязвенные скотомогильники: проблемы и решения. М.: Династия; 2017. 215 с.Marinin L.I., Dyatlov I.A., Shishkova N.A., Gerasimov V.N. [Anthrax Cattle Burial Grounds: Problems and Solutions]. Moscow: “Dynasty”; 2017. 215 p.Hoffmann C., Zimmermann F., Biek R., Kuehl H., Nowak K., Mundry R., Agbor A., Angedakin S., Arandjelovic M., Blankenburg A., Brazolla G., Corogenes K., Couacy-Hymann E., Deschner T., Dieguez P., Dierks K., Düx A., Dupke S., Eshuis H., Formenty P., Yuh Y.G., Goedmakers A., Gogarten J.F., Granjon A.C., McGraw S., Grunow R., Hart J., Jones S., Junker J., Kiang J., Langergraber K., Lapuente J., Lee K., Leendertz S.A., Léguillon F., Leinert V., Löhrich T., Marrocoli S., Mätz-Rensing K., Meier A., Merkel K., Metzger S., Murai M., Niedorf S., De Nys H., Sachse A., van Schijndel J., Thiesen U., Ton E., Wu D., Wieler L.H., Boesch C., Klee S.R., Wittig R.M., Calvignac-Spencer S., Leendertz F.H. Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest. Nature. 2017; 548(7665):82–6. DOI: 10.1038/nature23309.Hoffmann C., Zimmermann F., Biek R., Kuehl H., Nowak K., Mundry R., Agbor A., Angedakin S., Arandjelovic M., Blankenburg A., Brazolla G., Corogenes K., Couacy-Hymann E., Deschner T., Dieguez P., Dierks K., Düx A., Dupke S., Eshuis H., Formenty P., Yuh Y.G., Goedmakers A., Gogarten J.F., Granjon A.C., McGraw S., Grunow R., Hart J., Jones S., Junker J., Kiang J., Langergraber K., Lapuente J., Lee K., Leendertz S.A., Léguillon F., Leinert V., Löhrich T., Marrocoli S., Mätz-Rensing K., Meier A., Merkel K., Metzger S., Murai M., Niedorf S., De Nys H., Sachse A., van Schijndel J., Thiesen U., Ton E., Wu D., Wieler L.H., Boesch C., Klee S.R., Wittig R.M., Calvignac-Spencer S., Leendertz F.H. Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest. Nature. 2017; 548(7665):82–6. DOI: 10.1038/nature23309.Helgason E., Tourasse N.J., Meisal R., Caugant D.A., Kolsto A.B. Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl. Environ. Microbiol. 2004; 70(1):191–201. DOI: 10.1128/aem.70.1.191-201.2004.Helgason E., Tourasse N.J., Meisal R., Caugant D.A., Kolsto A.B. Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl. Environ. Microbiol. 2004; 70(1):191–201. DOI: 10.1128/aem.70.1.191-201.2004.Agren J., Hamidjaja R.A., Hansen T., Ruuls R., Thierry S., Vigre H., Janse I., Sundstrom A., Segerman B., Koene M., Löfström C., Van Rotterdam B., Derzelle S. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. Virulence. 2013; 4(8):671–85. DOI: 10.4161/viru.26288.Agren J., Hamidjaja R.A., Hansen T., Ruuls R., Thierry S., Vigre H., Janse I., Sundstrom A., Segerman B., Koene M., Löfström C., Van Rotterdam B., Derzelle S. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. Virulence. 2013; 4(8):671–85. DOI: 10.4161/viru.26288.Maiden M.C., Bygraves J.A., Feil E., Morelli G., Russell J.E., Urwin R., Zhang Q., Zhou J., Zurth K., Caugant D.A., Feavers I.M., Achtman M., Spratt B.G. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA. 1998; 95(6):3140–5. DOI: 10.1073/pnas.95.6.3140.Maiden M.C., Bygraves J.A., Feil E., Morelli G., Russell J.E., Urwin R., Zhang Q., Zhou J., Zurth K., Caugant D.A., Feavers I.M., Achtman M., Spratt B.G. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA. 1998; 95(6):3140–5. DOI: 10.1073/pnas.95.6.3140.Enright M.C., Spratt B.G., Kalia A., Cross J.H., Bessen D.E. Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect. Immun. 2001; 69(4):2416–27. DOI: 10.1128/iai.69.4.2416-2427.2001.Enright M.C., Spratt B.G., Kalia A., Cross J.H., Bessen D.E. Multilocus sequence typing of Streptococcus pyogenes and the relationships between emm type and clone. Infect. Immun. 2001; 69(4):2416–27. DOI: 10.1128/iai.69.4.2416-2427.2001.Van Loo I.H.M., Heuvelman K.J., King A.J., Mooi F.R. Multilocus sequence typing of Bordetella pertussis based on surface protein genes. J. Clin. Microbiol. 2002; 40(6):1994–2001. DOI: 10.1128/jcm.40.6.1994-2001.2002.Van Loo I.H.M., Heuvelman K.J., King A.J., Mooi F.R. Multilocus sequence typing of Bordetella pertussis based on surface protein genes. J. Clin. Microbiol. 2002; 40(6):1994–2001. DOI: 10.1128/jcm.40.6.1994-2001.2002.Noller A.C., McEllistrem M.C., Stine O.C., Morris J.G.Jr., Boxrud D.J., Dixon B., Harrison L.H. Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis. J. Clin. Microbiol. 2003; 41(2):675–9. DOI: 10.1128/jcm.41.2.675-679.2003.Noller A.C., McEllistrem M.C., Stine O.C., Morris J.G.Jr., Boxrud D.J., Dixon B., Harrison L.H. Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis. J. Clin. Microbiol. 2003; 41(2):675–9. DOI: 10.1128/jcm.41.2.675-679.2003.Kotetishvili M., Stine O.C., Kreger A., Morris J.G., Sulakvelidze A. Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. J. Clin. Microbiol. 2002; 40(5):1626–35. DOI: 10.1128/jcm.40.5.1626-1635.2002.Kotetishvili M., Stine O.C., Kreger A., Morris J.G., Sulakvelidze A. Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. J. Clin. Microbiol. 2002; 40(5):1626–35. DOI: 10.1128/jcm.40.5.1626-1635.2002.Priest F.G., Barker M., Baillie L.W.J., Holmes E.C., Maiden M.C.J. Population structure and evolution of the Bacillus cereus group. J. Bacteriol. 2004; 186(23):7959–70. DOI: 10.1128/jb.186.23.7959-7970.2004.Priest F.G., Barker M., Baillie L.W.J., Holmes E.C., Maiden M.C.J. Population structure and evolution of the Bacillus cereus group. J. Bacteriol. 2004; 186(23):7959–70. DOI: 10.1128/jb.186.23.7959-7970.2004.Sorokin A., Candelon B., Guilloux K., Galleron N., Wackerow-Kouzova N., Ehrlich S.D., Bourguet D., Sanchis V. Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Appl. Environ. Microbiol. 2006; 72(2):1569–78. DOI: 10.1128/aem.72.2.1569-1578.2006.Sorokin A., Candelon B., Guilloux K., Galleron N., Wackerow-Kouzova N., Ehrlich S.D., Bourguet D., Sanchis V. Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Appl. Environ. Microbiol. 2006; 72(2):1569–78. DOI: 10.1128/aem.72.2.1569-1578.2006.Tourasse N.J., Helgason E., Okstad O.A., Hegna I.K., Kolsto A.B. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J. Appl. Microbiol. 2006; 101(3):579–93. DOI: 10.1111/j.1365-2672.2006.03087.x.Tourasse N.J., Helgason E., Okstad O.A., Hegna I.K., Kolsto A.B. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J. Appl. Microbiol. 2006; 101(3):579–93. DOI: 10.1111/j.1365-2672.2006.03087.x.PubMLST – Public databases for molecular typing and microbial genome diversity. [Электронный ресурс]. URL: https://pubmlst.org/bcereus/(дата обращения 20.11.2020).PubMLST – Public databases for molecular typing and microbial genome diversity. (Cited: November 20, 2020). [Internet]. Available from: https://pubmlst.org/bcereus/.Ogawa H., Fujikura D., Ohnuma M., Ohnishi N., Hang'ombe B.M., Mimuro H., Ezaki T., Mweene A.S., Higashi H. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species. PLoS One. 2015; 10(3):e0122004. DOI: 10.1371/journal.pone.0122004.Ogawa H., Fujikura D., Ohnuma M., Ohnishi N., Hang’ombe B.M., Mimuro H., Ezaki T., Mweene A.S., Higashi H. A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species. PLoS One. 2015; 10(3):e0122004. DOI: 10.1371/journal.pone.0122004.Venkateswaran K, Singh N.K, Checinska Sielaff A., Pope R.K., Bergman N.H., van Tongeren S.P., Patel N.B., Lawson P.A., Satomi M., Williamson C., Sahl J.W., Keim P., Pierson D., Perry J. Non-toxin-producing Bacillus cereus strains belonging to the B. anthracis clade isolated from the international space station. mSystems. 2017; 2(3):e00021-17. DOI: 10.1128/mSystems.00021-17.Venkateswaran K, Singh N.K, Checinska Sielaff A., Pope R.K., Bergman N.H., van Tongeren S.P., Patel N.B., Lawson P.A., Satomi M., Williamson C., Sahl J.W., Keim P., Pierson D., Perry J. Non-toxin-producing Bacillus cereus strains belonging to the B. anthracis clade isolated from the international space station. mSystems. 2017; 2(3):e00021-17. DOI: 10.1128/mSystems.00021-17.Hunter P.R., Gaston M.A. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J. Clin. Microbiol. 1988; 26(11):2465–6. DOI: 10.1128/JCM.26.11.2465-2466.1988.Hunter P.R., Gaston M.A. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J. Clin. Microbiol. 1988; 26(11):2465–6. DOI: 10.1128/JCM.26.11.2465-2466.1988.Sue D., Marston C.K., Hoffmaster A.R., Wilkins P.P. Genetic diversity in a Bacillus anthracis historical collection (1954 to 1988). J. Clin. Microbiol. 2007; 45(6):1777–82. DOI:10.1128/JCM.02488-06.Sue D., Marston C.K., Hoffmaster A.R., Wilkins P.P. Genetic diversity in a Bacillus anthracis historical collection (1954 to 1988). J. Clin. Microbiol. 2007; 45(6):1777–82. DOI:10.1128/JCM.02488-06.Timofeev V., Bahtejeva I., Mironova R., Titareva G., Lev I., Christiany D., Borzilov A., Bogun A., Vergnaud G. Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia. PLoS One. 2019; 4(5):e0209140. DOI: 10.1371/journal. pone.0209140.Timofeev V., Bahtejeva I., Mironova R., Titareva G., Lev I., Christiany D., Borzilov A., Bogun A., Vergnaud G. Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia. PLoS One. 2019; 4(5):e0209140. DOI: 10.1371/journal. pone.0209140.Гончарова Ю.О., Бахтеева И.В., Титарева Г.М., Миронова Р.И., Кисличкина А.А., Майская Н.В., Мокриевич А.Н., Тимофеев В.С. Аллельный полиморфизм гена lef у штаммов возбудителя сибирской язвы из Государственной коллекции патогенных микроорганизмов («ГКПМ-Оболенск»). Бактериология. 2019; 4(2):7–12. DOI: 10.20953/2500-1027-2019-2-7-12.Goncharova Yu.O., Bakhteeva I.V., Titareva G.M., Mironova R.I., Kislichkina A.A., Mayskaya N.V., Mokrievich A.N., Timofeev V.S. [Allelic polymorphism of the lef gene in anthrax pathogen strains from the State collection of pathogenic microorganisms (“GKPM-Obolensk”)]. Bakteriologiya [Bacteriology]. 2019; 4(2):7–12. (In Russian). DOI: 10.20953/2500-1027-2019-2-7-12.BLAST. [Электронный ресурс]. URL: https://blast.ncbi. nlm.nih.gov/Blast.cgi (дата обращения 01.11.2020).BLAST. (Cited: November 01, 2020). [Internet]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi.Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Y., Evchenko A.Y., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Tchmerenko D.K., Kulichenko A.N., Morozov V.Y. Genotyping and phylogenetic location of one clinical isolate of Bacillus anthracis isolated from a human in Russia. BMC Microbiol. 2019; 19(1):165. DOI: 10.1186/s12866-019-1542-3.Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Y., Evchenko A.Y., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Tchmerenko D.K., Kulichenko A.N., Morozov V.Y. Genotyping and phylogenetic location of one clinical isolate of Bacillus anthracis isolated from a human in Russia. BMC Microbiol. 2019; 19(1):165. DOI: 10.1186/s12866-019-1542-3.Braun P., Grass G., Aceti A., Serrecchia L., Affuso A., Marino L., Grimaldi S., Pagano S., Hanczaruk M., Georgi E., Northoff B., Schöler A., Schloter M., Antwerpen M., Fasanella A. Microevolution of anthrax from a young ancestor (M.A.Y.A.) suggests a soil-borne life cycle of Bacillus anthracis. PLoS One. 2015; 10(8):e0135346. DOI: 10.1371/journal.pone.0135346.Braun P., Grass G., Aceti A., Serrecchia L., Affuso A., Marino L., Grimaldi S., Pagano S., Hanczaruk M., Georgi E., Northoff B., Schöler A., Schloter M., Antwerpen M., Fasanella A. Microevolution of anthrax from a young ancestor (M.A.Y.A.) suggests a soil-borne life cycle of Bacillus anthracis. PLoS One. 2015; 10(8):e0135346. DOI: 10.1371/journal.pone.0135346.Vergnaud G. Bacillus anthracis evolution: taking advantage of the topology of the phylogenetic tree and human history to propose dating points. Erciyes Med. J. 2020; 42(4):362–9. DOI: 10.14744/etd.2020.64920.Vergnaud G. Bacillus anthracis evolution: taking advantage of the topology of the phylogenetic tree and human history to propose dating points. Erciyes Med. J. 2020; 42(4):362–9. DOI: 10.14744/etd.2020.64920.Keim P., Van Ert M.N., Pearson T., Vogler A.J., Huynh L.Y., Wagner D.M. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect. Genet. Evol. 2004; 4(3):205–13. DOI: 10.1016/j.meegid.2004.02.005.Keim P., Van Ert M.N., Pearson T., Vogler A.J., Huynh L.Y., Wagner D.M. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect. Genet. Evol. 2004; 4(3):205–13. DOI: 10.1016/j.meegid.2004.02.005.

The authors declare that there are no conflicts of interest present.