mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2023.060mes-117Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕORIGINAL RESEARCHВыделение и характеристика вирулентных бактериофагов против Klebsiella pneumoniae значимых капсульных типовIsolation and characterization of virulent bacteriophages against Klebsiella pneumoniae of significant capsular typesГородничевР. Б.GorodnichevR. B.

Москва

Moscow

КорниенкоМ. А.KornienkoM. A.

Москва

Moscow

БеспятыхД. А.BespiatykhD. A.

Москва

Moscow

МалаховаМ. В.MalakhovaM. V.

Москва

Moscow

КривуляА. О.KrivuliaA. O.

Москва

Moscow

ВеселовскийВ. А.VeselovskyV. A.

Москва

Moscow

БеспятыхЮ. А.BespyatykhYu. A.

Москва

Moscow

ГолощаповО. В.GoloshchapovO. V.

Санкт-Петербург

Saint Petersburg

ЧерненькаяТ. В.ChernenkayaT. V.

Москва

Moscow

ШитиковЕ. А.ShitikovE. A.

Москва

Moscow

Федеральный научно-клинический центр физико-химической медицины имени Ю. М. Лопухина Федерального медико-биологического агентстваРоссияLopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological AgencyRussian FederationПервый Санкт-Петербургский государственный медицинский университет имени И. П. ПавловаРоссияPavlov First Saint Petersburg State Medical UniversityRussian FederationНаучно-исследовательский институт скорой помощи имени Н. В. Склифосовского Департамента здравоохранения города МосквыРоссияSklifosovsky Research Institute for Emergency MedicineRussian Federation202325102024254159167Copyright © Городничев Р.Б., Корниенко М.А., Беспятых Д.А., Малахова М.В., Кривуля А.О., Веселовский В.А., Беспятых Ю.А., Голощапов О.В., Черненькая Т.В., Шитиков Е.А., 20242024Городничев Р.Б., Корниенко М.А., Беспятых Д.А., Малахова М.В., Кривуля А.О., Веселовский В.А., Беспятых Ю.А., Голощапов О.В., Черненькая Т.В., Шитиков Е.А.Gorodnichev R.B., Kornienko M.A., Bespiatykh D.A., Malakhova M.V., Krivulia A.O., Veselovsky V.A., Bespyatykh Y.A., Goloshchapov O.V., Chernenkaya T.V., Shitikov E.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/117

В контексте растущей устойчивости к антибиотикам бактериофаги — альтернатива традиционной антимикробной терапии. Терапия бактериофагами — одна из таких альтернатив. Целью исследования были выделение и характеристика бактериофагов, эффективных против штаммов Klebsiella pneumoniae клинически значимых капсульных типов. Из проб сточных и речных вод методом накопительных культур было выделено восемь фагов. Определение спектра литической активности фагов проводили на коллекции клинических изолятов K. pneumoniae (n = 279). Бактериофаги лизировали 52,8–100% изолятов K. pneumoniae соответствующих капсульных типов: фаг VKV295 — 100% изолятов с капсульным типом KL1, SAA231 — 52,8% с KL2, NNK-G4 — 100% с KL39, VSG32 — 66,7% с KL41, NKA196 — 87,5% с KL47, Rappa3 — 87,5% с KL57, PEA128 — 95,5% с KL64 и ChM-G5 — 69,6% с KL102.

Их геномы были секвенированы и проанализированы биоинформатически. Фаги принадлежали к семейству Autographiviridae и относились к трем родам. Литический спектр фагов был ограничен конкретными капсульными типами вследствие наличия специфичных рецептор-связывающих белков — полисахариддеполимераз. Выделенные бактериофаги были строго вирулентными, не несли вредных генетических детерминант, что позволяет их применять в терапевтической практике для борьбы с антибиотикорезистентными инфекциями, вызванными K. pneumoniae.

The growing proportion of antibiotic-resistant Klebsiella pneumoniae strains raises challenges to the healthcare system and requires the development of alternative treatment options. Bacteriophage therapy is one of such options. The study was aimed to isolate and describe bacteriophages effective against K. pneumoniae strains of clinically significant capsular types. The bacteriophages were isolated from the sewage and river water samples using the enrichment culture technique. The spectrum of lytic activity of the phages was tested on the collection of K. pneumoniae clinical isolates (n = 279). The studied bacteriophages lysed 52.8–100% of K. pneumoniae strains of respective capsular types: phage VKV295 lysed 100% of strains with the capsular type KL1, SAA231 — 52.8 of strains with KL2, NNK-G4 — 100% of strains with KL39, VSG32 — 66.7% of strains with KL41, NKA196 — 87.5% of strains with KL47, Rappa3 — 87.5% of strains with KL57, PEA128 — 95.5% of strains with KL64, and ChM-G5 — 69.6% of strains with KL102. Whole-genome sequencing and subsequent bioinformatic analysis revealed that the phages belong to the Autographiviridae family and are classified into three genera.The lytic spectrum of phages was limited to specific capsular types due to the presence of specific receptor-binding proteins, polysaccharide depolymerases. The isolated bacteriophages were strictly virulent, did not carry harmful genetic determinants, and had a specific host range, making them applicable in therapeutic practice for combating antibiotic-resistant infections caused by K. pneumoniae.

вирулентные бактериофагиKlebsiella pneumoniaeантибиотикорезистентностьполисахарид-деполимеразыvirulent bacteriophagesKlebsiella pneumoniaeantibiotic resistancepolysaccharide depolymerasesReferencesHe Y, Li W, Wang Z, Chen H, Tian L, et al. Nosocomial infection among patients with COVID-19: A retrospective data analysis of 918 cases from a single center in Wuhan, China. Infect Control Hosp Epidemiol. 2020; 41 (8): 982–3.He Y, Li W, Wang Z, Chen H, Tian L, et al. Nosocomial infection among patients with COVID-19: A retrospective data analysis of 918 cases from a single center in Wuhan, China. Infect Control Hosp Epidemiol. 2020; 41 (8): 982–3.European Centre for Disease Prevention and Control. Antimicrobial resistance in the EU/EEA (EARS-Net). AER for 2021. Surveillance report, 2022; p. 20.European Centre for Disease Prevention and Control. Antimicrobial resistance in the EU/EEA (EARS-Net). AER for 2021. Surveillance report. 2022; p. 20.Сухорукова М. В., Эйдельштейн М. В., Иванчик Н. В., Склеенова Е. Ю., Шайдуллина Э. Р., Азизов И. С. и др. Антибиотикорезистентность нозокомиальных штаммов Enterobacterales в стационарах России: результаты многоцентрового эпидемиологического исследования «МАРАФОН 2015–2016». Клиническая микробиология и антимикробная химиотерапия. 2019; 21 (2): 147–59.Sukhorukova MV, Edelstein MV, Ivanchik NV, Skleenova EYu, Shajdullina ER, Azyzov IS, et al. Antimicrobial resistance of nosocomial Enterobacterales isolates in Russia: Results of multicenter epidemiological study «MARATHON 2015–2016». Clinical Microbiology and Antimicrobial Chemotherapy. 2019; 21 (2): 147–59. Russian.Li D, Huang X, Rao H, Yu H, Long S, Li Y, et al. Klebsiella pneumoniae bacteremia mortality: a systematic review and meta- analysis. Front Cell Infect Microbiol. 2023; 13 (April): 1–9.Li D, Huang X, Rao H, Yu H, Long S, Li Y, et al. Klebsiella pneumoniae bacteremia mortality: a systematic review and meta-analysis. Front Cell Infect Microbiol. 2023; 13: 1–9.Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399 (10325): 629–55.Murray CJ, Ikuta KS, Sharara F, Swetschinski L, Aguilar GR, Gray A, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399 (10325): 629–55.Górski A, Międzybrodzki R, Węgrzyn G, Jończyk‐Matysiak E, Borysowski J, Weber–Dąbrowska B. Phage therapy: Current status and perspectives. Med Res Rev. 2020; 40 (1): 459–63.Górski A, Międzybrodzki R, Węgrzyn G, Jończyk‐Matysiak E, Borysowski J, Weber–Dąbrowska B. Phage therapy: Current status and perspectives. Med Res Rev. 2020; 40 (1): 459–63.Aslam S, Lampley E, Wooten D, Karris M, Benson C, Strathdee S, et al. Lessons learned from the first 10 consecutive cases of intravenous bacteriophage therapy to treat multidrug-resistant bacterial infections at a single center in the United States. Open Forum Infect Dis. 2020; 7 (9): ofaa389.Aslam S, Lampley E, Wooten D, Karris M, Benson C, Strathdee S, et al. Lessons learned from the first 10 consecutive cases of intravenous bacteriophage therapy to treat multidrug-resistant bacterial infections at a single center in the United States. Open Forum Infect Dis. 2020; 7 (9): ofaa389.Dedrick RM, Smith BE, Cristinziano M, Freeman KG, Jacobs- Sera D, Belessis Y, et al. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease. Clin Infect Dis. 2023; 76 (1): 103–12.Dedrick RM, Smith BE, Cristinziano M, Freeman KG, Jacobs- Sera D, Belessis Y, et al. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease. Clin Infect Dis. 2023; 76 (1): 103–12.Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, et al. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob. Agents Chemother. 2017; 61 (10): 1–15.Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, et al. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob. Agents Chemother. 2017; 61 (10): 1–15.Купцов Н. С., Корниенко М. А., Городничев Р. Б., Данилов Д. И., Парфенова Т. В., Макаренко Г. И. и др. Эффективность препаратов бактериофагов против патогенов группы ESKAPE. Вестник РГМУ. 2020; 3 (2020): 19–26.Kuptsov NS, Kornienko MA, Gorodnichev RB, Danilov DI, Parfenova TV, Makarenko GI, et al. Efficacy of commercial bacteriophage products against ESKAPE pathogens. Bulletin of RSMU. 2020; 3 (2020): 19–26. Russian.Pires DP, Oliveira H, Melo LD, Sillankorva S, Azeredo J. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications. Appl Microbiol Biotechnol. 2016; 100 (5): 2141–51.Pires DP, Oliveira H, Melo LD, Sillankorva S, Azeredo J. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications. Appl Microbiol Biotechnol. 2016; 100 (5): 2141–51.Follador R, Heinz E, Wyres KL, Ellington MJ, Kowarik M, Holt KE, et al. The diversity of Klebsiella pneumoniae surface polysaccharides. Microb genomics. 2016; 2 (8): e000073.Follador R, Heinz E, Wyres KL, Ellington MJ, Kowarik M, Holt KE, et al. The diversity of Klebsiella pneumoniae surface polysaccharides. Microb genomics. 2016; 2 (8): e000073.Liao CH, Huang YT, Hsueh PR. Multicenter surveillance of capsular serotypes, virulence genes, and antimicrobial susceptibilities of Klebsiella pneumoniae causing bacteremia in Taiwan, 2017– 2019. Front Microbiol. 2022; 13: 783523.Liao CH, Huang YT, Hsueh PR. Multicenter surveillance of capsular serotypes, virulence genes, and antimicrobial susceptibilities of Klebsiella pneumoniae causing bacteremia in Taiwan, 2017– 2019. Front Microbiol. 2022; 13: 783523.Jin Y, Dong C, Shao C, Wang Y, Liu Y. Molecular epidemiology of clonally related Metallo-β-Lactamase-Producing Klebsiella pneumoniae isolated from newborns in a hospital in Shandong, China. Jundishapur Journal of Microbiology. 2017; 10 (9): 14046.Jin Y, Dong C, Shao C, Wang Y, Liu Y. Molecular epidemiology of clonally related Metallo-β-Lactamase-Producing Klebsiella pneumoniae isolated from newborns in a hospital in Shandong, China. Jundishapur Journal of Microbiology. 2017; 10 (9): 14046.Rojas LJ, Weinstock GM, De La Cadena E, Diaz L, Rios R, Hanson BM, et al. An analysis of the epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: convergence of two evolutionary mechanisms creates the “Perfect Storm”. J Infect Dis. 2018: 217 (1): 82–92.Rojas LJ, Weinstock GM, De La Cadena E, Diaz L, Rios R, Hanson BM, et al. An analysis of the epidemic of Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae: convergence of two evolutionary mechanisms creates the “Perfect Storm”. J Infect Dis. 2018: 217 (1): 82–92.Shaidullina ER, Schwabe M, Rohde T, Shapovalova VV, Dyachkova MS, Matsvay AD, et al. Genomic analysis of the international high- risk clonal lineage Klebsiella pneumoniae sequence type 395. Genome Med. 2023; 15 (1): 17.Shaidullina ER, Schwabe M, Rohde T, Shapovalova VV, Dyachkova MS, Matsvay AD, et al. Genomic analysis of the international high-risk clonal lineage Klebsiella pneumoniae sequence type 395. Genome Med. 2023; 15 (1): 17.Егоров С. А., Семёнов В. М., Дмитраченко Т. И. Анализ изолятов Klebsiella pneumoniae, обладающих широкой резистентностью к антибиотикам. Педиатрия. Восточная Европа. 2022; 10 (3): 325–33.Egorov SK, Semenov VM, Dmitrachenko TI. Analysis of Klebsiella pneumoniae isolates with extremely high antibiotic resistance. Paediatrics. Eastern Europe. 2022; 10 (3): 325–33. Russian.Ryzhov V, Fenselau C. Characterization of the protein subset desorbed by MALDI from whole bacterial cells. Anal Chem. 2001; 73 (4): 746–50.Ryzhov V, Fenselau C. Characterization of the protein subset desorbed by MALDI from whole bacterial cells. Anal Chem 2001; 73 (4): 746–50.Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, et al. Wzi gene sequencing, a rapid method for determination of capsulartype for klebsiella strains. J Clin Microbiol. 2013; 51 (12): 4073–8.Brisse S, Passet V, Haugaard AB, Babosan A, Kassis-Chikhani N, Struve C, et al. Wzi gene sequencing, a rapid method for determination of capsulartype for klebsiella strains. J Clin Microbiol. 2013; 51 (12): 4073–8.Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by the Direct Plating Plaque Assay. Methods Mol Biol. 2009; 501: 77–80.Mazzocco A, Waddell TE, Lingohr E, Johnson RP. Enumeration of bacteriophages by the Direct Plating Plaque Assay. Methods Mol Biol. 2009; 501: 77–80.Городничев Р. Б., Корниенко М. А., Купцов Н. С., Малахова М. В., Беспятых Д. А., Веселовский В. А. и др. Молекулярно- генетическая характеристика трех новых бактериофагов Klebsiella pneumoniae, перспективных для применения в фаговой терапии. Медицина экстремальных ситуаций. 2021; 23 (3): 90–7.Gorodnichev RB, Kornienko MA, Kuptsov NS., Malakhova MV, Bespiatykh DA, Veselovsky VA, et al. Molecular genetic characterization of three new Klebsiella pneumoniae bacteriophages suitable for phage therapy. Extreme medicine. 2021; 23 (3): 90–7. Russian.Green MR, Sambrook J. Molecular cloning. A Laboratory Manual 4th. Cold Spring Harbor Laboratory, 2012; p. 1936.Green MR, Sambrook J. Molecular cloning. A Laboratory Manual 4th. Cold Spring Harbor Laboratory, 2012; p. 1936.Liu B, Zheng D, Jin Q, Chen L, Yang J. VFDB. 2019: A comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res. 2019; 47 (D1): D687–D692.Liu B, Zheng D, Jin Q, Chen L, Yang J. VFDB. 2019: A comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res. 2019; 47 (D1): D687–D692.Liu B, Pop M. ARDB — Antibiotic resistance genes database. Nucleic Acids Res. 2009; 37 (SUPPL 1): 443–7.Liu B, Pop M. ARDB — Antibiotic resistance genes database. Nucleic Acids Res. 2009; 37 (SUPPL 1): 443–7.Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017; 33 (21): 3396–404.Meier-Kolthoff JP, Göker M. VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics. 2017; 33 (21): 3396–404.Darzentas N. Circoletto: visualizing sequence similarity with Circos. Bioinformatics. 2010; 26 (20): 2620–1.Darzentas N. Circoletto: visualizing sequence similarity with Circos. Bioinformatics. 2010; 26 (20): 2620–1.Fursova NK, Astashkin EI, Ershova ON, Aleksandrova IA, Savin IA, Novikova TS, et al. Multidrug-resistant Klebsiella pneumoniae causing severe infections in the neuro-ICU. Antibiotics. 2021; 10 (8): 1–17.Fursova NK, Astashkin EI, Ershova ON, Aleksandrova IA, Savin IA, Novikova TS, et al. Multidrug-resistant Klebsiella pneumoniae causing severe infections in the neuro-ICU. Antibiotics. 2021; 10 (8): 1–17.Beamud B, García-González N, Gómez-Ortega M, González- Candelas F, Domingo-Calap P, Sanjuan R. Genetic determinants of host tropism in Klebsiella phages. Cell Rep. 2023; 42 (2): 112048.Beamud B, García-González N, Gómez-Ortega M, González- Candelas F, Domingo-Calap P, Sanjuan R. Genetic determinants of host tropism in Klebsiella phages. Cell Rep. 2023; 42 (2): 112048.Turner D, Kropinski AM, Adriaenssens EM. A roadmap for genome-based phage taxonomy. Viruses. 2021; 13 (3): 506.Turner D, Kropinski AM, Adriaenssens EM. A roadmap for genome-based phage taxonomy. Viruses. 2021; 13 (3): 506.

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