vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/vjgb-25-62vavilov-4684Research ArticleГЕНЕТИКА МИКРООРГАНИЗМОВMICROBIAL GENETICSГенетический потенциал к образованию биопленок клинических штаммов Pseudomonas aeruginosaGenetic potential for biofilm formation of clinical strains of Pseudomonas aeruginosaНемченкоУ. М.NemchenkoU. M.

Иркутск

Irkutsk

БельковаН. Л.BelkovaN. L.

Иркутск

Irkutsk

nlbelkova@gmail.comКлименкоЕ. С.KlimenkoE. S.

Иркутск

Irkutsk

СмуроваН. Е.SmurovaN. E.

Иркутск

Irkutsk

ЗугееваР. Е.ZugeevaR. E.

Иркутск

Irkutsk

СиньковВ. В.SinkovV. V.

Иркутск

Irkutsk

СавиловЕ. Д.SavilovE. D.

Иркутск

Irkutsk

Институт эпидемиологии и микро6иологии, Научный центр про6лем здоровья семьи и репродукции человекаРоссияInstitute of Epidemiology and Microbiology, Scientific Center for Family Health and Human Reproduction ProblemsRussian Federation202520072025294594599Copyright © Немченко У.М., Белькова Н.Л., Клименко Е.С., Смурова Н.Е., Зугеева Р.Е., Синьков В.В., Савилов Е.Д., 20252025Немченко У.М., Белькова Н.Л., Клименко Е.С., Смурова Н.Е., Зугеева Р.Е., Синьков В.В., Савилов Е.Д.Nemchenko U.M., Belkova N.L., Klimenko E.S., Smurova N.E., Zugeeva R.E., Sinkov V.V., Savilov E.D.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/4684

. Бактерии вида Pseudomonas aeruginosa являются одной из ведущих причин нозокомиальных инфекций дыхательного тракта и играют важную роль в инфицировании нижних дыхательных путей у 6ольных муковисцидозом (MB). Биопленки, представляя со6ой организованные скопления клеток, о6еспечивают выживаемость микроорганизмов в не6лагоприятных условиях окружающей среды и спосо6ствуют хронизации инфекции и формированию персистирующих форм. Целью ра6оты стало определение фенотипической спосо6ности и генетического потенциала к 6иопленкоо6разованию у клинических штаммов P. aeruginosa, персистирующих у пациентов с MB на фоне постоянного приема антимикро6ных препаратов. Для характеристики пяти штаммов P. aeruginosa, полученных от пациентов с MB, в ра6оте применены 6актериологические, молекулярногенетические и 6иоинформатические методы. Фенотипически все штаммы отнесены к умеренно-о6разующим 6иопленку, при этом коэффициент 6иопленкоо6разования варьировал от 2.10 до 3.15. Анализ драфт-геномов выявил различия в представленности некоторых генов или отдельных локусов трех из четырех известных сигнальных путей, цАMФ/Vfr, Gac/Rsm и c-di-GMP, которые описаны в геномах P. aeruginosa и имеют отношение к регуляции о6разования 6иопленок. Дополнительно показаны отличия в представленности таких генов, как frzE, tcpE и rcsC. Несомненно интересен анализ генов pppA, icmF, clpV1, trpE, trpG и stp1, которые используются для расширенного мультилокусного типирования PubMLST и различаются по структуре локусов у всех проанализированных штаммов. Эти гены потенциально могут 6ыть применены для типирования клинических штаммов Р. aeruginosa с целью характеристики их 6иопленкоо6разующих свойств. Таким о6разом, в геномах клинических штаммов P. aeruginosa, длительно персистирующих у пациентов на фоне постоянного получения анти6иотикотерапии, охарактеризованы гены, которые потенциально могут участвовать как в процессе 6иопленкоо6разования, так и в его регуляции. Характеристика генетического потенциала к о6разованию 6иопленок дает возможность поиска надежных генетических маркеров этого процесса для мониторинга эволюции воз6удителя в результате длительной персистенции в организме хозяина.

Pseudomonas aeruginosa is one of the leading causes of nosocomial respiratory tract infections and plays an important role in lower respiratory tract infection in patients with cystic fibrosis (CF). Biofilms, which are organized cell clusters, ensure the survival of microorganisms in unfavorable environmental conditions and contribute to the chronicity of infection and the formation of persistent forms. The aim of this study was to determine the phenotypic ability and genetic potential for biofilm formation in clinical strains of P. aeruginosa persisting in patients with CF against the background of constant intake of antimicrobial drugs. Bacteriological, genetic, and bioinformatic methods were used to characterize five P. aeruginosa strains obtained from patients with CF. Phenotypically, all strains were classified as moderately biofilm-forming, while the biofilm formation coefficient varied from 2.10 to 3.15. Analysis of draft genomes revealed differences in the representation of some genes or individual loci of three of the four known signaling pathways (cAMP/Vfr, Gac/Rsm, and c-di-GMP) that have been described in P. aeruginosa genomes and are related to the regulation of biofilm formation. In addition, differences in the representation of genes such as frzE, tcpE, and rcsC are shown. Of undoubted interest is the analysis of genes such as pppA, icmF, clpV1, trpE, trpG, and stp1, which are used for extended multilocus typing PubMLST and differed in the structure of loci in all analyzed strains. These genes can be used to identify clinical strains of P. aeruginosa and to characterize their biofilm-forming properties. Thus, genes potentially participating in both biofilm formation and regulation have been characterized in the genomes of clinical P. aeruginosa strains that persist for a long time in patients receiving continuous antibiotic therapy. Characterization of the genetic potential for biofilm formation makes it possible to search for reliable genetic markers of this process in order to monitor the evolution of the pathogen as a result of long-term persistence in the host organism.

Pseudomonas aeruginosaмуковисцидоз6иопленкиполногеномное секвенированиесигнальные путиPseudomonas aeruginosacystic fibrosisbiofilmswhole genome sequencingsignaling pathwaysThis work was carried out within the framework of technology No. 123051600027-1ReferencesBankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., … Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-477. doi 10.1089/cmb.2012.0021Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., … Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455-477. doi 10.1089/cmb.2012.0021Bjarnsholt T., Jensen P.Ø., Fiandaca M.J., Pedersen J., Hansen C.R., Andersen C.B., Pressler T., Givskov M., Høiby N. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol. 2009;44(6):547-558. doi 10.1002/ppul.21011Bjarnsholt T., Jensen P.Ø., Fiandaca M.J., Pedersen J., Hansen C.R., Andersen C.B., Pressler T., Givskov M., Høiby N. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol. 2009;44(6):547-558. doi 10.1002/ppul.21011Brencic A., Lory S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Mol Mi crobiol. 2009;72(3):612-632. doi 10.1111/j.1365-2958.2009.06670.xBrencic A., Lory S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Mol Mi crobiol. 2009;72(3):612-632. doi 10.1111/j.1365-2958.2009.06670.xCao P., Fleming D., Moustafa D.A., Dolan S.K., Szymanik K.H., Redman W.K., Ramos A., Diggle F.L., Sullivan C.S., Goldberg J.B., Rumbaugh K.P., Whiteley M. A Pseudomonas aeruginosa small RNA regulates chronic and acute infection. Nature. 2023;618(7964): 358-364. doi 10.1038/s41586-023-06111-7Cao P., Fleming D., Moustafa D.A., Dolan S.K., Szymanik K.H., Redman W.K., Ramos A., Diggle F.L., Sullivan C.S., Goldberg J.B., Rumbaugh K.P., Whiteley M. A Pseudomonas aeruginosa small RNA regulates chronic and acute infection. Nature. 2023;618(7964): 358-364. doi 10.1038/s41586-023-06111-7Coggan K.A., Wolfgang M.C. Global regulatory pathways and crosstalk control Pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol. 2012;14(2):47-70. doi 10.21775/cimb.014.047Coggan K.A., Wolfgang M.C. Global regulatory pathways and crosstalk control Pseudomonas aeruginosa environmental lifestyle and virulence phenotype. Curr Issues Mol Biol. 2012;14(2):47-70. doi 10.21775/cimb.014.047Deretic V., Schurr M.J., Yu H. Pseudomonas aeruginosa, mucoidy and the chronic infection phenotype in cystic fibrosis. Trends Microbiol. 1995;3(9):351-356. doi 10.1016/s0966-842x(00)88974-xDeretic V., Schurr M.J., Yu H. Pseudomonas aeruginosa, mucoidy and the chronic infection phenotype in cystic fibrosis. Trends Microbiol. 1995;3(9):351-356. doi 10.1016/s0966-842x(00)88974-xElfadadny A., Ragab R.F., AlHarbi M., Badshah F., Ibáñez-Arancibia E., Farag A., Hendawy A.O., De Los Ríos-Escalante P.R., Aboubakr M., Zakai S.A., Nageeb W.M. Antimicrobial resistance of Pseudomonas aeruginosa: navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front Microbiol. 2024;15:1374466. doi 10.3389/fmicb.2024.1374466Elfadadny A., Ragab R.F., AlHarbi M., Badshah F., Ibáñez-Arancibia E., Farag A., Hendawy A.O., De Los Ríos-Escalante P.R., Aboubakr M., Zakai S.A., Nageeb W.M. Antimicrobial resistance of Pseudomonas aeruginosa: navigating clinical impacts, current resistance trends, and innovations in breaking therapies. Front Microbiol. 2024;15:1374466. doi 10.3389/fmicb.2024.1374466Fernández-Billón M., Llambías-Cabot A.E., Jordana-Lluch E., Oliver A., Macià M.D. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa biofilms. Biofilm. 2023;5:100129. doi 10.1016/j.bioflm.2023.100129Fernández-Billón M., Llambías-Cabot A.E., Jordana-Lluch E., Oliver A., Macià M.D. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa biofilms. Biofilm. 2023;5:100129. doi 10.1016/j.bioflm.2023.100129Grigorova E.V., Nemchenko U.M., Voropaeva N.M., Belkova N.L., Noskova O.A., Savilov E.D. Effect of disinfectants with different active ingredients on biofilm formation in Pseudomonas aeruginosa. Bull Exp Biol Med. 2021;171(6):745-749. doi 10.1007/s10517-021-05308-yGrigorova E.V., Nemchenko U.M., Voropaeva N.M., Belkova N.L., Noskova O.A., Savilov E.D. Effect of disinfectants with different active ingredients on biofilm formation in Pseudomonas aeruginosa. Bull Exp Biol Med. 2021;171(6):745-749. doi 10.1007/s10517-021-05308-yJolley K.A., Bray J.E., Maiden M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3:124. doi 10.12688/wellcomeopenres.14826.1Jolley K.A., Bray J.E., Maiden M.C.J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018;3:124. doi 10.12688/wellcomeopenres.14826.1Jurado-Martín I., Sainz-Mejías M., McClean S. Pseudomonas aeruginosa: an audacious pathogen with an adaptable arsenal of virulence factors. Int J Mol Sci. 2021;22(6):3128. doi 10.3390/ijms22063128Jurado-Martín I., Sainz-Mejías M., McClean S. Pseudomonas aeruginosa: an audacious pathogen with an adaptable arsenal of virulence factors. Int J Mol Sci. 2021;22(6):3128. doi 10.3390/ijms22063128Kanehisa M., Sato Y., Kawashima M. KEGG mapping tools for uncovering hidden features in biological data. Protein Sci. 2022;31(1): 47-53. doi 10.1002/pro.4172Kanehisa M., Sato Y., Kawashima M. KEGG mapping tools for uncovering hidden features in biological data. Protein Sci. 2022;31(1): 47-53. doi 10.1002/pro.4172Kearns D.B., Shimkets L.J. Chemotaxis in a gliding bacterium. Proc Natl Acad Sci USA. 1998;95(20):11957-11962. doi 10.1073/pnas.95.20.11957Kearns D.B., Shimkets L.J. Chemotaxis in a gliding bacterium. Proc Natl Acad Sci USA. 1998;95(20):11957-11962. doi 10.1073/pnas.95.20.11957Nemchenko U.M., Kungurtsevа E.A., Grigorova E.V., Belkova N.L., Markova Yu.A., Noskova O.A., Chemezova N.N., Savi lov E.D. Simulation of bacterial biofilms and estimation of the sensitivity of healthcare-associated infection pathogens to bactericide Sekusept active. Klinicheskaya Laboratornaya Diagnostika = Russian Clini cal Laboratory Diagnostics. 2020;65(10):652-658. doi 10.18821/0869-2084-2020-65-10-652-658 (in Russian)Nemchenko U.M., Kungurtsevа E.A., Grigorova E.V., Belkova N.L., Markova Yu.A., Noskova O.A., Chemezova N.N., Savi lov E.D. Simulation of bacterial biofilms and estimation of the sensitivity of healthcare-associated infection pathogens to bactericide Sekusept active. Klinicheskaya Laboratornaya Diagnostika = Russian Clini cal Laboratory Diagnostics. 2020;65(10):652-658. doi 10.18821/0869-2084-2020-65-10-652-658 (in Russian)Nemchenko U.M., Sitnikova K.O., Belkova N.L., Grigorova E.V., Voropaeva N.M., Sukhоreva M.V., Sukhareva E.S., Savilov E.D. Effects of antimicrobials on Pseudomonas aeruginosa biofilm formation. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J. Genet. Breed. 2022;26(5):495-501. doi 10.18699/VJGB-22-60Nemchenko U.M., Sitnikova K.O., Belkova N.L., Grigorova E.V., Voropaeva N.M., Sukhоreva M.V., Sukhareva E.S., Savilov E.D. Effects of antimicrobials on Pseudomonas aeruginosa biofilm formation. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J. Genet. Breed. 2022;26(5):495-501. doi 10.18699/VJGB-22-60Nemchenko U.M., Belkova N.L., Klimenko E.S., Smurova N.E., Savilov E.D. Phenotypic and genetic variability of clinical isolates of Pseudomonas aeruginosa persisting in patients with cystic fibrosis. Kliniceskaa Mikrobiologia i Antimikrobnaa Himioterapia = Clinical Microbiology and Antimicrobial Chemotherapy. 2024;26(S1):42 (in Russian)Nemchenko U.M., Belkova N.L., Klimenko E.S., Smurova N.E., Savilov E.D. Phenotypic and genetic variability of clinical isolates of Pseudomonas aeruginosa persisting in patients with cystic fibrosis. Kliniceskaa Mikrobiologia i Antimikrobnaa Himioterapia = Clinical Microbiology and Antimicrobial Chemotherapy. 2024;26(S1):42 (in Russian)O’Toole G.A. Microtiter dish biofilm formation assay. J Vis Exp. 2011; 30(47):2437. doi 10.3791/2437O’Toole G.A. Microtiter dish biofilm formation assay. J Vis Exp. 2011; 30(47):2437. doi 10.3791/2437Parkins M.D., Somayaji R., Waters V.J. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev. 2018;31(4):e00019-18. doi 10.1128/cmr.00019-18Parkins M.D., Somayaji R., Waters V.J. Epidemiology, biology, and impact of clonal Pseudomonas aeruginosa infections in cystic fibrosis. Clin Microbiol Rev. 2018;31(4):e00019-18. doi 10.1128/cmr.00019-18Penesyan A., Paulsen I.T., Kjelleberg S. Three faces of biofilms: a microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. NPJ Biofilms Microbiomes. 2021;7(1):80. doi 10.1038/s41522-021-00251-2Penesyan A., Paulsen I.T., Kjelleberg S. Three faces of biofilms: a microbial lifestyle, a nascent multicellular organism, and an incubator for diversity. NPJ Biofilms Microbiomes. 2021;7(1):80. doi 10.1038/s41522-021-00251-2Rissman A.I., Mau B., Biehl B.S., Darling A.E., Glasner J.D., Perna N.T. Reordering contigs of draft genomes using the Mauve Aligner. Bioinformatics. 2009;25(16):2071-2073. doi 10.1093/bioinformatics/btp356Rissman A.I., Mau B., Biehl B.S., Darling A.E., Glasner J.D., Perna N.T. Reordering contigs of draft genomes using the Mauve Aligner. Bioinformatics. 2009;25(16):2071-2073. doi 10.1093/bioinformatics/btp356Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-2069. doi 10.1093/bioinformatics/btu153Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-2069. doi 10.1093/bioinformatics/btu153Shaginyan I.A., Avetisyan L.R., Chernukha M.Yu., Siyanova E.A., Burmistrov E.M., Voronkova A.Yu., Kondratieva E.I., Chuchalin A.G., Gintzburg A.L. Epidemiological significance of genome variations in Pseudomonas aeruginosa causing chronic lung infection in patients with cystic fibrosis. Kliniceskaya Mikrobiologiya i Antimikrobnaya Himioterapiya = Clinical Microbiology and Antimicrobial Chemotherapy. 2019;21(4):340-351. doi 10.36488/cmac.2019.4.340-351 (in Russian)Shaginyan I.A., Avetisyan L.R., Chernukha M.Yu., Siyanova E.A., Burmistrov E.M., Voronkova A.Yu., Kondratieva E.I., Chuchalin A.G., Gintzburg A.L. Epidemiological significance of genome variations in Pseudomonas aeruginosa causing chronic lung infection in patients with cystic fibrosis. Kliniceskaya Mikrobiologiya i Antimikrobnaya Himioterapiya = Clinical Microbiology and Antimicrobial Chemotherapy. 2019;21(4):340-351. doi 10.36488/cmac.2019.4.340-351 (in Russian)Sitnikova K.O., Nemchenko U.M., Voropaeva N.M., Grigorova E.V., Savilov E.D., Markova Yu.A., Belkova N.L. The effectiveness of biofilm formation of daily cultures of clinically significant strains of opportunistic bacteria. Acta Biomedica Scientifica. 2022;7(5-1): 119-128. doi 10.29413/ABS.2022-7.5-1.13 (in Russian)Sitnikova K.O., Nemchenko U.M., Voropaeva N.M., Grigorova E.V., Savilov E.D., Markova Yu.A., Belkova N.L. The effectiveness of biofilm formation of daily cultures of clinically significant strains of opportunistic bacteria. Acta Biomedica Scientifica. 2022;7(5-1): 119-128. doi 10.29413/ABS.2022-7.5-1.13 (in Russian)Thelin K.H., Taylor R.K. Toxin-coregulated pilus, but not mannosesensitive hemagglutinin, is required for colonization by Vibrio cho lerae O1 El Tor biotype and O139 strains. Infect Immun. 1996; 64(7):2853-2856. doi 10.1128/iai.64.7.2853-2856.1996Thelin K.H., Taylor R.K. Toxin-coregulated pilus, but not mannosesensitive hemagglutinin, is required for colonization by Vibrio cho lerae O1 El Tor biotype and O139 strains. Infect Immun. 1996; 64(7):2853-2856. doi 10.1128/iai.64.7.2853-2856.1996Thöming J.G., Tomasch J., Preusse M., Koska M., Grahl N., Pohl S., Willger S.D., Kaever V., Müsken M., Häussler S. Parallel evolutionary paths to produce more than one Pseudomonas aeruginosa biofilm phenotype. NPJ Biofilms Microbiomes. 2020;6:2. doi 10.1038/s41522-019-0113-6Thöming J.G., Tomasch J., Preusse M., Koska M., Grahl N., Pohl S., Willger S.D., Kaever V., Müsken M., Häussler S. Parallel evolutionary paths to produce more than one Pseudomonas aeruginosa biofilm phenotype. NPJ Biofilms Microbiomes. 2020;6:2. doi 10.1038/s41522-019-0113-6Wall E., Majdalani N., Gottesman S. The complex Rcs regulatory cascade. Annu Rev Microbiol. 2018;72:111-139. doi 10.1146/annurevmicro-090817-062640Wall E., Majdalani N., Gottesman S. The complex Rcs regulatory cascade. Annu Rev Microbiol. 2018;72:111-139. doi 10.1146/annurevmicro-090817-062640Winstanley C., O’Brien S., Brockhurst M.A. Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections. Trends Microbiol. 2016;24:327-337. doi 10.1016/j.tim.2016.01.008Winstanley C., O’Brien S., Brockhurst M.A. Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections. Trends Microbiol. 2016;24:327-337. doi 10.1016/j.tim.2016.01.008

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