Journal of microbiology, epidemiology and immunobiologyJournal of microbiology, epidemiology and immunobiologyЖурнал микробиологии, эпидемиологии и иммунобиологии0372-93112686-7613Central Research Institute for Epidemiology108110.36233/0372-9311-192ORIGINAL RESEARCHESОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯResearch ArticleCompetitive ability of Escherichia coli strains in the intestinal microbiota of patients with Crohn's disease and healthy volunteers: physiological, biochemical and genetic characteristicsФизиолого-биохимическая и генетическая характеристика конкурентных свойств штаммов Escherichia coli в кишечной микрофлоре пациентов с болезнью Крона и здоровых добровольцевhttps://orcid.org/0000-0002-8138-9235SiniaginaMaria N.СинягинаМария Николаевна
Russian Federation

researcher, Research laboratory “Omics technologies”

н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0002-4250-2645LaikovAlexander V.ЛайковАлександр Владимирович
Russian Federation

researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0001-7445-2091MarkelovaMaria I.МаркеловаМария Ивановна
Russian Federation

researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0003-3458-1176BoulyginaEugenia A.БулыгинаЕвгения Александровна
Russian Federation

researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0002-9982-9059KhusnutdinovaDilyara R.ХуснутдиноваДиляра Рашидовна
Russian Federation

researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0001-9542-3580AbdulkhakovSayar R.АбдулхаковСайяр Рустамович
Russian Federation

Cand. Sci. (Med.), Head, Department of fundamentals of clinical medicine, senior researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

к.м.н., зав. каф. фундаментальных основ клинической медицины, с.н.с. НИЛ «Омиксные технологии Института фундаментальной медицины и биологии

marias25@mail.ruhttps://orcid.org/0000-0001-5314-7012GrigoryevaTatyana V.ГригорьеваТатьяна Владимировна
Russian Federation

Cand. Sci. (Biol.), senior researcher, Research laboratory “Omics technologies”, Institute of Fundamental Medicine and Biology

к.б.н., с.н.с. НИЛ «Омиксные технологии» Института фундаментальной медицины и биологии

marias25@mail.ruKazan Federal UniversityКазанский (Приволжский) федеральный университет1512202299666968126082021Copyright ©; 2022, Siniagina M.N., Laikov A.V., Markelova M.I., Boulygina E.A., Khusnutdinova D.R., Abdulkhakov S.R., Grigoryeva T.V.Copyright ©; 2022, Синягина М.Н., Лайков А.В., Маркелова М.И., Булыгина Е.А., Хуснутдинова Д.Р., Абдулхаков С.Р., Григорьева Т.В.2022Siniagina M.N., Laikov A.V., Markelova M.I., Boulygina E.A., Khusnutdinova D.R., Abdulkhakov S.R., Grigoryeva T.V.Синягина М.Н., Лайков А.В., Маркелова М.И., Булыгина Е.А., Хуснутдинова Д.Р., Абдулхаков С.Р., Григорьева Т.В.https://creativecommons.org/licenses/by/4.0https://microbiol.crie.ru/jour/article/view/1081

Introduction. Crohn's disease (CD) is a chronic inflammation of various parts of the gastrointestinal tract with an increased proportion of Escherichia coli. However, the role of E. coli in disease remains unclear.

This study aims to evaluate the competitive abilities of E. coli strains from CD patients and healthy volunteers, and to identify the biochemical and genetic determinants underlying these features.

Materials and methods. The antagonistic activity was assessed by co-cultivation of 11 clinical E. coli strains inhibiting the growth of the K-12, with Enterobacter cloacae, Klebsiella pneumonia and Salmonella enterica. To elucidate the mechanism of antagonistic activity, the evaluation of biochemical properties and a comparative genomic analysis were used.

Results and discussion. Genes of bacteriocin production systems were identified in genomes of 11 strains from CD patients and healthy volunteers active against the E. coli K-12 strain. Three strains from healthy individuals demonstrated activity against several Enterobacteriaceae bacteria. The strains’ biochemical properties were typical of representatives of E. coli. Strains 1_34_12, active against E. cloacae, and 1_45_11, inhibiting all tested enterobacteria, are phylogenetically related to the laboratory strain K-12. Strain 1_39_1, active against K. pneumonia and S. enterica, is phylogenetically close to the Nissle1917, contains the genes for colibactin biosynthesis and a variant of the fimH gene that increases the adhesive ability of bacteria.

Conclusion. The identified E. coli strains are able to displace Enterobacteriaceae bacteria and can be used to study the bacteria-bacteria and host-bacteria interactions, to understand their role in gut homeostasis and intestinal inflammation.

Введение. Болезнь Крона (БК) характеризуется хроническим воспалением различных отделов желудочно-кишечного тракта и сопровождается увеличением доли Escherichia coli. Однако роль E. coli в развитии заболевания остаётся невыясненной.

Цель работы — оценить конкурентные свойства штаммов E. coli, выделенных из кишечника пациентов с БК и здоровых добровольцев, и выявить биохимические и генетические детерминанты, лежащие в их основе.

Материалы и методы. Антагонистическую активность оценивали путём совместного культивирования 11 клинических штаммов E. coli, способных ингибировать рост штамма K-12, с Enterobacter cloacae, Klebsiella pneumonia и Salmonella enterica. Для выяснения механизма антагонистической активности проводили оценку биохимических свойств штаммов и сравнительный геномный анализ.

Результаты и обсуждение. В геномах 11 штаммов от пациентов с БК и здоровых добровольцев, проявивших антагонистическую активность в отношении штамма E. coli K-12, выявлены гены систем продукции бактериоцинов. Выявлены 3 штамма от здоровых добровольцев, ингибирующие рост нескольких представителей семейства Enterobacteriaceae. Биохимические возможности данных штаммов характерны для типичных представителей вида E. coli. Штаммы 1_34_12, активный в отношении E. cloacae, и 1_45_11, способный подавлять рост всех протестированных энтеробактерий, филогенетически родственны лабораторному штамму K-12. Штамм 1_39_1, активный в отношении K. pneumonia и S. enterica, филогенетически близок пробиотическому штамму Nissle 1917 и содержит кластер генов биосинтеза колибактина и вариант гена fimH, повышающий адгезивную способность бактерий.

Заключение. Выявленные штаммы E. coli способны вытеснять энтеробактерии в экспериментах in vitro и могут быть использованы для дальнейшего изучения особенностей взаимодействия комменсалов друг с другом и с организмом хозяина, а также для понимания их роли в поддержании гомеостаза и развитии воспалительных реакций кишечника.

Escherichia coliCrohn’s diseasemicrobiotaantagonistic activityEscherichia coliболезнь Кронамикробиотаантагонистическая активностьПравительство Российской ФедерацииGovernment of the Russian Federation0671-2020-0058This research was partially funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (project #0671-2020-0058)Работа выполнена за счет средств субсидии, выделенной Казанскому федеральному университету для выполнения Государственного задания в сфере научной деятельности (проект № 0671-2020-0058).Das B., Nair G.B. Homeostasis and dysbiosis of the gut microbiome in health and disease. J. Biosci. 2019; 44(5): 1–8. https://doi.org/10.1007/s12038-019-9926-yPessione E. The Russian doll model: how bacteria shape successful and sustainable inter-kingdom relationships. Front. Microbiol. 2020; 11: 2325. https://doi.org/10.3389/fmicb.2020.573759Freter R. Factors affecting the microecology of the gut. In: Probiotics. Dordrecht: Springer; 1992: 111–44. https://doi.org/10.1007/978-94-011-2364-8_6Stubbendieck R.M., Straight P.D. Multifaceted interfaces of bacterial competition. J. Bacteriol. 2016; 198(6): 2145–55. https://doi.org/10.1128/JB.00275-16Massip C., Oswald E. Siderophore-microcins in Escherichia coli: determinants of digestive colonization, the first step toward virulence. Front. Cell. Infect. Microbiol. 2020; 10: 381. https://doi.org/10.3389/fcimb.2020.00381Hayes C.S., Koskiniemi S., Ruhe Z.C., Poole S J., Low D.A. Mechanisms and biological roles of contact-dependent growth inhibition systems. Cold Spring Harb. Perspect. Med. 2014; 4(2): a010025. https://doi.org/10.1101/cshperspect.a010025Aoki S.K., Pamma R., Hernday A.D., Bickham J.E., Braaten B.A., Low D.A. Contact-dependent inhibition of growth in Escherichia coli. Science. 2005; 309(5738): 1245–8. https://doi.org/10.1101/cshperspect.a010025Russell A.B., Peterson S.B., Mougous J.D. Type VI secretion system effectors: poisons with a purpose. Nat. Rev. Microbiol. 2014; 12(2): 137–48. https://doi.org/10.1038/nrmicro3185Anderson M.C., Vonaesch P., Saffarian A., Marteyn B.S., Sansonetti P.J. Shigella sonnei encodes a functional T6SS used for interbacterial competition and niche occupancy. Cell Host Microbe. 2017; 21(6):769–76. https://doi.org/10.1016/j.chom.2017.05.004Endt K., Stecher B., Chaffron S., Slack E., Tchitchek N., Benecke A., et al. The microbiota mediates pathogen clearance from the gut lumen after non-typhoidal salmonella diarrhea PLoS Pathog. 2010; 6(9): e1001097. https://doi.org/10.1371/journal.ppat.1001097Kamada N., Seo S.U., Chen G.Y., Nunez G. Role of the gut microbiota in immunity and inflammatory disease Nat. Rev. Immunol. 2013; 13(5): 321–35. https://doi.org/10.1038/nri3430Abt M.C., Pamer E.G. Commensal bacteria mediated defenses against pathogens. Curr. Opin. Immunol. 2014; 29: 16–22. https://doi.org/10.1016/j.coi.2014.03.003Manichanh C., Rigottier-Gois L., Bonnaud E., Gloux K., Pelletier E., Frangeul L., et al. Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach. Gut. 2006; 55(2): 205–11. https://doi.org/10.1136/gut.2005.073817Dicksved J., Halfvarson J., Rosenquist M., Järnerot G., Tysk C., Apajalahti J., et al. Molecular analysis of the gut microbiota of identical twins with Crohn's disease. ISME J. 2008; 2(7): 716–27. https://doi.org/10.1038/ismej.2008.37Frank D.N., Amand A.L., Feldman R.A., Boedeker E.C., Harpaz N., Pace N.R. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc. Nat. Acad. Sci. 2007; 104(34): 13780–5. https://doi.org/10.1073/pnas.0706625104Danilova N.A., Abdulkhakov S.R., Grigor'eva T.V., Markelova M.I., Vasil'ev I.Yu., Bulygina E.A., et al. Markers of dysbiosis in patients with ulcerative colitis and Crohn's disease. Terapevticheskiy arkhiv. 2019; (4): 13–20. https://doi.org/10.26442/00403660.2019.04.000211 (in Russian)Данилова Н.А., Абдулхаков С.Р., Григорьева Т.В., Маркелова М.И., Васильев И.Ю., Булыгина Е.А. и др. Маркеры дисбиоза у пациентов с язвенным колитом и болезнью Крона. Терапевтический архив. 2019; (4): 13–20. https://doi.org/10.26442/00403660.2019.04.000211Hewitt J.H., Rigby J. Effect of various milk feeds on numbers of Escherichia coli and Bifidobacterium in the stools of new-born infants. J. Hyg. 1976; 77(1): 129–39. https://doi.org/10.1017/S0022172400055601Bel'skiy V.V., Moskovtseva A.I. Colonization of Escherichia coli of the intestine and the frequency of detection of plasmid factors of multidrug resistance and colicinogenicity in children during the first year of life. Pediatriya. Zhurnal im. G.N. Speranskogo. 2007; 86(1): 20–4. (in Russian)Бельский В.В., Московцева А.И. Колонизация Escherichia coli кишечника и частота выявления плазмидных факторов множественной лекарственной устойчивости и колициногенности у детей на протяжении первого года жизни. Педиатрия. Журнал им. Г.Н. Сперанского. 2007; 86(1): 20–4.Kamada N., Seo S.U., Chen G.Y., Nunez G. Role of the gut microbiota in immunity and inflammatory disease Nat. Rev. Immunol. 2013; 13(5): 321–35. https://doi.org/10.1038/nri3430Leimbach A., Hacker J., Dobrindt U. Escherichia coli as an all-rounder: the thin line between commensalism and pathogenicity. Curr. Top. Microbiol. Immunol. 2013; 358: 332. https://doi.org/10.1007/82_2012_303Lapaquette P., Glasser A.L., Huett A., Xavier R.J., Darfeuille‐Michaud A. Crohn's disease‐associated adherent‐invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cell. Microbiol. 2010; 12(1): 99–113. https://doi.org/10.1111/j.1462-5822.2009.01381.xLapaquette P., Glasser A.L., Huett A., Xavier R.J., Darfeuille‐Mi- chaud A. Crohn's disease‐associated adherent‐invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly. Cell. Microbiol. 2010; 12(1): 99–113. https://doi.org/10.1111/j.1462-5822.2009.01381.xSiniagina M.N., Markelova M.I., Boulygina E.A., Laikov A.V., Khusnutdinova D.R., Abdulkhakov S.R., et al. Diversity and adaptations of Escherichia coli strains: exploring the intestinal community in Crohn’s disease patients and healthy individuals. Microorganisms. 2021; 9(6): 1299. https://doi.org/10.3390/microorganisms9061299Siniagina M., Markelova M., Laikov A., Boulygina E., Khusnutdinova D., Kharchenko A., et al. Cultivated Escherichia coli diversity in intestinal microbiota of Crohn's disease patients and healthy individuals: whole genome data. Data Brief. 2020; 28: 104948. https://doi.org/10.1016/j.dib.2019.104948Ohno M., Hasegawa M., Hayashi A., Caballero-Flores G., Alteri C.J., Lawley T.D., et al. Lipopolysaccharide O structure of adherent and invasive Escherichia coli regulates intestinal inflammation via complement C3. PLoS Pathog. 2020; 16(10): e1008928. https://doi.org/10.1371/journal.ppat.1008928Meier-Kolthoff J.P., Göker M. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat. Commun. 2019; 10(1): 2182. https://doi.org/10.1038/s41467-019-10210-3Meier-Kolthoff J.P., Auch A.F., Klenk H-P., Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform. 2013; 14: 60. https://doi.org/10.1186/1471-2105-14-60Lefort V., Desper R., Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. Mol. Biol. Evol. 2015; 32(10): 2798–800. https://doi.org/10.1093/molbev/msv150Kreft L., Botzki A., Coppens F., Vandepoele K., Van Bel M. PhyD3: A phylogenetic tree viewer with extended phyloXML support for functional genomics data visualization. Bioinform. 2017; 33(18): 2946–7. https://doi.org/10.1093/bioinformatics/btx324David L.A., Maurice C.F., Carmody R.N., Gootenberg D.B., Button J.E., Wolfe B.E., et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014; 505(7484): 559–63. https://doi.org/10.1038/nature12820De Filippo C., Cavalieri D., Di Paola M., Ramazzotti M., Poullet J.B., Massart S., et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl Acad. Sci. USA. 2010; 107(33): 14691. https://doi.org/10.1073/pnas.1005963107Tyakht A.V., Alexeev D.G., Popenko A.S., Kostryukova E.S., Govorun V.M. Rural and urban microbiota: to be or not to be? Gut Microbes. 2014; 5(3): 351–6. https://doi.org/10.4161/gmic.28685Donaldson G.P., Lee S.M., Mazmanian S.K. Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 2016; 14(1): 20–32. https://doi.org/10.1038/nrmicro3552Pereira F.C., Berry D. Microbial nutrient niches in the gut. Environ. Microbiol. 2017; 19(4): 1366–78. https://doi.org/10.1111/1462-2920.13659Garrity G. Bergey's Manual of Systematic Bacteriology: Volu- me 2: The Proteobacteria, Part B: The Gammaproteobacteria. Springer Science & Business Media; 2007.Garrity G. Bergey's Manual of Systematic Bacteriology: Volume 2: The Proteobacteria, Part B: The Gammaproteobacteria. Springer Science & Business Media; 2007.Bingle L.E., Bailey C.M., Pallen M.J. Type VI secretion: a beginner’s guide. Curr. Opin. Microbiol. 2008; 11(1): 3–8. https://doi.org/10.1016/j.mib.2008.01.006Pomares M.F., Delgado M.A., Corbalán N.S., Farías R.N., Vincent P.A. Sensitization of microcin J25-resistant strains by a membrane-permeabilizing peptide. App. Environ. Microbiol. 2010; 76(20): 6837–42. https://doi.org/10.1128/AEM.00307-10Laviña M.A., Gaggero C., Moreno F. Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli. J. Bacteriol. 1990; 172(11): 6585–8. https://doi.org/10.1128/jb.172.11.6585-6588.1990Chiuchiolo M.J., Delgado M.A., Farı́as R.N., Salomón R.A. Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25. J. Bacteriol. 2001; 183(5): 1755–64. https://doi.org/10.1128/JB.183.5.1755-1764.2001Connell N., Han Z., Moreno F., Kolter R. An E. coli promoter induced by the cessation of growth. Mol. Microbiol. 1987; 1(2): 195–201. https://doi.org/10.1111/j.1365-2958.1987.tb00512.xKurepina N.E., Basyuk E.I., Metlitskaya A.Z., Zaitsev D.A., Khmel I.A. Cloning and mapping of the genetic determinants for microcin C51 production and immunity. Mol. Gen. Genet. 1993; 241(5-6): 700–6. https://doi.org/10.1007/BF00279914Schneider T., Hahn-Löbmann S., Stephan A., Schulz S., Giritch A., Naumann M., et al. Plant-made Salmonella bacteriocins salmocins for control of Salmonella pathovars. Sci. Rep. 2018; 8(1): 1–0. https://doi.org/10.1038/s41598-018-22465-9Mushtaq H., Bakht J., Khan I., Ahmad B. Antimicrobial efficacy and prevalence of colicinogenic E. coli in faecal matter of human, cow and sheep. Int. J. Antimicrob. Agents. 2021; 57(1): 106221. https://doi.org/10.1016/j.ijantimicag.2020.106221Sassone-Corsi M., Nuccio S.P., Liu H., Hernandez D., Vu C.T., Takahashi A.A., et al. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature. 2016; 540(7632): 280–3. https://doi.org/10.1038/nature20557Johnson J.R., Johnston B., Kuskowski M.A., Nougayrede J.P., Oswald E. Molecular epidemiology and phylogenetic distribution of the Escherichia coli pks genomic island. J. Clin. Microbiol. 2008; 46(12): 3906–11. https://doi.org/10.1128/JCM.00949-08Putze J., Hennequin C., Nougayrède J.P., Zhang W., Homburg S., Karch H., et al. Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae. Infect. Immun. 2009; 77(11): 4696–703. https://doi.org/10.1128/IAI.00522-09Suresh A., Ranjan A., Jadhav S., Hussain A., Shaik S., Alam M., et al. Molecular genetic and functional analysis of pks-harboring, extra-intestinal pathogenic Escherichia coli from India. Front. Microbiol. 2018; 9: 2631. https://doi.org/10.3389/fmicb.2018.02631Nougayrède J.P., Homburg S., Taieb F., Boury M., Brzuszkiewicz E., Gottschalk G., et al. Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science. 2006; 313(5788): 848–51. https://doi.org/10.1126/science.1127059Olier M., Marcq I., Salvador-Cartier C., Secher T., Dobrindt U., Boury M., et al. Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity. Gut Microb. 2012; 3(6): 501–9. https://doi.org/10.4161/gmic.21737Dreux N., Denizot J., Martinez-Medina M., Mellmann A., Billig M., Kisiela D., et al. Point mutations in FimH adhesin of Crohn's disease-associated adherent-invasive Escherichia coli enhance intestinal inflammatory response. PLoS Pathog. 2013; 9(1): e1003141. https://doi.org/10.1371/journal.ppat.1003141Drider D. Gut microbiota is an important source of bacteriocins and their in situ expression can be explored for treatment of bacterial infections. Prob. Antimicrob. Proteins. 2021; 13(6): 1759–65. https://doi.org/10.1007/s12602-021-09843-yŠmajs D., Micenková L., Šmarda J., Vrba M., Ševčíková A., Vališová Z., et al. Bacteriocin synthesis in uropathogenic and commensal Escherichia coli: colicin E1 is a potential virulence factor. BMC Microbiol. 2010; 10: 288. https://doi.org/10.1186/1471-2180-10-288Micenková L., Frankovičová L., Jaborníková I., Bosák J., Dítě P., Šmarda J., et al. Escherichia coli isolates from patients with inflammatory bowel disease: ExPEC virulence-and colicin-determinants are more frequent compared to healthy controls. Int. J. Med. Microbiol. 2018; 308(5): 498–504. https://doi.org/10.1016/j.ijmm.2018.04.008