mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2021.027mes-142Research ArticleОРИГИНАЛЬНОЕ ИССЛЕДОВАНИЕORIGINAL RESEARCHВыделение и характеристика бактериофагов Pseudomonas aeruginosa — потенциальных агентов для фаговой терапииIsolation and characterization of Pseudomonas aeruginosa bacteriophages — potential agents for phage therapyКорниенкоМ. А.KornienkoM. A.

Мария Андреевна Корниенко

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Maria A. Kornienko

Malaya Pirogovskaya, 1a, Moscow, 119435

kornienkomariya@gmail.comКупцовН. С.KuptsovN. S.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ДаниловД. И.DanilovD. I.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ГородничевР. Б.GorodnichevR. B.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

МалаховаМ. В.MalakhovaM. V.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

БеспятыхД. А.BespiatykhD. A.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ВеселовскийВ. А.VeselovskyV. A.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ШитиковЕ. А.ShitikovE. A.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

ИльинаЕ. Н.IlinaE. N.

ул. Малая Пироговская, д. 1а, г. Москва, 119435

Malaya Pirogovskaya, 1a, Moscow, 119435

Федеральный научно-клинический центр физико-химической медицины Федерального медико-биологического агентстваРоссияFederal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological AgencyRussian Federation2021261020242331623Copyright © Корниенко М.А., Купцов Н.С., Данилов Д.И., Городничев Р.Б., Малахова М.В., Беспятых Д.А., Веселовский В.А., Шитиков Е.А., Ильина Е.Н., 20242024Корниенко М.А., Купцов Н.С., Данилов Д.И., Городничев Р.Б., Малахова М.В., Беспятых Д.А., Веселовский В.А., Шитиков Е.А., Ильина Е.Н.Kornienko M.A., Kuptsov N.S., Danilov D.I., Gorodnichev R.B., Malakhova M.V., Bespiatykh D.A., Veselovsky V.A., Shitikov E.A., Ilina E.N.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/142

Одним из патогенов, характеризующихся критическим показателем доли штаммов с множественной лекарственной устойчивостью (МЛУ), является Pseudomonas aeruginosa. В качестве альтернативы антибиотикам при терапии инфекций, вызванных штаммами с МЛУ, рассматривают фаготерапию. Целью исследования было выделить и охарактеризовать бактериофаг P. aeruginosa, потенциально пригодный для терапии инфекционных заболеваний. Выделение проводили методом накопительных культур. Спектр литической активности устанавливали спот-тестированием на коллекции из 40 штаммов P. aeruginosa. Полногеномное секвенирование выполняли на платформе MiSeq (Illumina). Филогенетический анализ геномов проводили с помощью VICTOR. Выделенные бактериофаги vB_PaeA-55-1w и vB_PaeM-198, принадлежащие к семействам Autographiviridae и Myoviridae соответственно, обладали широким спектром литической активности (около 50% каждый), в том числе вызывали лизис штаммов с МЛУ. Геномы vB_PaeA-55-1w и vB_PaeM-198 представлены двухцепочечной ДНК длиной 42,5 и 66,3 т.п.н. соответственно. В составе геномов аннотировано 52 (vB_PaeA-55-1w) и 95 (vB_PaeM-198) открытых рамок считывания, среди них гены интеграз и токсинов не обнаружены. На филогенетическом древе vB_PaeA-55-1w располагался в кластере совместно с бактериофагами рода Phikmvvirus семейства Autographiviridae, в том числе с используемыми в фаготерапии, а vB_ PaeM-198 входил в кластер, включающий бактериофаги рода Pbunavirus семейства Myoviridae. Бактериофаги vB_PaeA-55-1w и vB_PaeM-198 можно рассматривать в качестве кандидатов для применения в фаготерапии, в том числе и для лечения инфекций, вызванных штаммами P. aeruginosa с МЛУ.

Pseudomonas aeruginosa — is one of the pathogens characterized by the critical number of multidrug-resistant (MDR) strains. Phage therapy is considered an alternative to antibiotics, especially in treatment of infections caused by MDR strains. The aim of this study was to isolate and characterize P. aeruginosa phages that could potentially be suitable for treating infectious diseases. To isolate the P. aeruginosa phages, enrichment cultures were used. The lytic activity spectrum was confirmed by spot testing on 40 P. aeruginosa strains. Whole-genome sequencing was performed using Illumina MiSeq instrument. Phylogenetic analysis was done using VICTOR tool. Isolated phages vB_PaeA-55-1w and vB_PaeM-198 from Autographiviridae and Myoviridae families, respectively, had a broad spectrum of lytic activity (about 50% each), including lysis of MDR strains. The genomes vB_PaeA-55-1w and vB_PaeM-198 comprise double-stranded DNA of 42.5 and 66.3 kbp in length, respectively. Open reading frames were annotated for both phages (52 for vB_PaeA-55-1w, and 95 for vB_PaeM-198), no integrases and toxins were detected. On a phylogenetic tree, vB_PaeA-55-1w phage was clustered with phages from the Phikmvvirus genus (Autographiviridae family), which are also used in phage therapy. vB_PaeM-198 phage was clustered with phages from the Pbunavirus genus (Myoviridae family). vB_PaeA-55-1w and vB_PaeM-198 phages could be considered as candidates for phage therapy and may be used to treat infections caused by MDR P. aeruginosa.

Pseudomonas aeruginosaвирулентные бактериофагифаготерапияAutographiviridaeMyoviridaeполногеномное секвенированиефилогенетический анализPseudomonas aeruginosavirulent bacteriophagesphage therapyAutographiviridaeMyoviridaewhole genome sequencingphylogenetic analysisИсследование выполнено за счет средств, предоставленных для выполнения государственного задания «Разработка персонализированного подхода терапии инфекционных процессов с применением вирулентных бактериофагов» (ШИФР: Бактериофаг).The authors thank the Center for Precision Genome Editing and Genetic Technologies for Biomedicine, the Federal Research and Clinical Center of Physical-Chemical Medicine of the Federal Medical Biological Agency for their help with sequencing the genomes of bacteriophages.ReferencesTacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnetet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018; 18 (3): 318–27.Tacconelli E, Carrara E, Savoldi A, Harbarth S, Mendelson M, Monnetet DL, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018; 18 (3): 318–27.Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, Gómez-Zorrilla S, et al. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev. 2019; 32 (4).Horcajada JP, Montero M, Oliver A, Sorlí L, Luque S, Gómez-Zorrilla S, et al. Epidemiology and treatment of multidrug-resistant and extensively drug-resistant Pseudomonas aeruginosa infections. Clin Microbiol Rev. 2019; 32 (4).Kuzmenkov AY, Trushin IV, Vinogradova AG, Avramenko AA, Sukhorukova MV, Malhotra-Kumar S, et al. AMRmap: an interactive web platform for analysis of antimicrobial resistance surveillance data in Russia. Front Microbiol. 2021; 12: 620002.Kuzmenkov AY, Trushin IV, Vinogradova AG, Avramenko AA, Sukhorukova MV, Malhotra-Kumar S, et al. AMRmap: an interactive web platform for analysis of antimicrobial resistance surveillance data in Russia. Front Microbiol. 2021; 12: 620002.Pena C, Cabot G, Gómez-Zorrilla S, Zamorano L, Ocampo-Sosa A, Murillas J, et al. Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections. Clin Infect Dis. 2015; 60 (4): 539–48.Pena C, Cabot G, Gómez-Zorrilla S, Zamorano L, Ocampo-Sosa A, Murillas J, et al. Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections. Clin Infect Dis. 2015; 60 (4): 539–48.2020 Antibacterial agents in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization, 2021. Available from: https://www.who.int/publications/i/item/9789240021303.2020 Antibacterial agents in clinical and preclinical development: an overview and analysis. Geneva: World Health Organization, 2021. Available from: https://www.who.int/publications/i/item/9789240021303.Gordillo Altamirano FL, Barr JJ. Phage therapy in the postantibiotic era. Clinical Microbiology Reviews. 2019; 32 (2): e00066-18. DOI: 10.1128/CMR.00066-18.Gordillo Altamirano FL, Barr JJ. Phage therapy in the postantibiotic era. Clinical Microbiology Reviews. 2019; 32 (2): e00066-18. DOI: 10.1128/CMR.00066-18.Акимкин В. Г., Дарбеева О. С., Колков В.Ф. Бактериофаги: исторические и современные аспекты их применения: опыт и перспективы. Клиническая практика. 2010; 1 (4): 48–54.Akimkin VG, Darbeeva OS, Kolkov VF. Bakteriofagi: istoricheskie i sovremennye aspekty ih primenenija: opyt i perspektivy. Klinicheskaja praktika. 2010; 1 (4): 48–54. Russian.Chen F, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Novel lytic phages protect cells and mice against Pseudomonas aeruginosa infection. J Virol. 2021; 95 (8): e01832-20. DOI: 10.1128/JVI.01832-20.Chen F, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Novel lytic phages protect cells and mice against Pseudomonas aeruginosa infection. J Virol. 2021; 95 (8): e01832-20. DOI: 10.1128/JVI.01832-20.Jault P, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial. Lancet Infect Dis. 2019; 19 (1): 35–45.Jault P, Cheng X, Li J, Yuan X, Huang X, Lian M, et al. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial. Lancet Infect Dis. 2019; 19 (1): 35–45.Oechslin F. Resistance development to bacteriophages occurring during bacteriophage therapy. Viruses. 2018; 10 (7): 351. DOI: 10.3390/v10070351.Oechslin F. Resistance development to bacteriophages occurring during bacteriophage therapy. Viruses. 2018; 10 (7): 351. DOI: 10.3390/v10070351.Купцов Н. С. и др. Эффективность препаратов бактериофагов против патогенов группы ESKAPE. Вестник РГМУ. 2020; (3): 19–26.Kuptsov NS, Kornienko MA, Gorodnichev RB, Danilov DI, Malakhova MV, Parfenova TV, et al. Efficacy of commercial bacteriophage products against ESKAPE pathogens. Bulletin of RSMU. 2020; (3): 18–24.Mazzocco A, et al. Enumeration of bacteriophages using the small drop plaque assay system. Methods Mol Biol. 2009; 501: 81–85.Mazzocco A, et al. Enumeration of bacteriophages using the small drop plaque assay system. Methods Mol Biol. 2009; 501: 81–85.Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Pr. 1989, 2200 p.Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Pr. 1989, 2200 p.Seemann T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics. Oxford University Press. 2014; 30 (14): 2068–9.Seemann T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics. Oxford University Press. 2014; 30 (14): 2068–9.Aziz RK, et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genomics. BioMed Central. 2008; 9: 75.Aziz RK, et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genomics. BioMed Central. 2008; 9: 75.Laslett D. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004; 32 (1): 11–16.Laslett D. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004; 32 (1): 11–16.Lefkowitz EJ, et al. Virus taxonomy: The database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res. 2018: 46 (1): 708–17.Lefkowitz EJ, et al. Virus taxonomy: The database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res. 2018: 46 (1): 708–17.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.Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol. 2015; 32 (10): 2798–800.Lefort V, Desper R, Gascuel O. FastME 2.0: A comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol. 2015; 32 (10): 2798–800.FigTree. Available from: http://tree.bio.ed.ac.uk/software/figtree/. (Дата обращения: 16.07.2021).FigTree. Available from: http://tree.bio.ed.ac.uk/software/figtree/. (Data obrashhenija: 16.07.2021).Enright MC, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 2000; 38 (3): 1008–15.Enright MC, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 2000; 38 (3): 1008–15.Alvi IA, et al. RLP, a bacteriophage of the family Podoviridae, rescues mice from bacteremia caused by multi-drug-resistant Pseudomonas aeruginosa. Arch Virol. 2020. 165 (6): 1289–97.Alvi IA, et al. RLP, a bacteriophage of the family Podoviridae, rescues mice from bacteremia caused by multi-drug-resistant Pseudomonas aeruginosa. Arch Virol. 2020. 165 (6): 1289–97.Farlow J, et al. Complete Genome Sequences of 10 Phages Lytic against Multidrug-Resistant Pseudomonas aeruginosa. Microbiol Resour. 2020. 9: 29.Farlow J, et al. Complete Genome Sequences of 10 Phages Lytic against Multidrug-Resistant Pseudomonas aeruginosa. Microbiol Resour. 2020. 9: 29.Alvi IA, Asif M, Rehman S. A single dose of a virulent bacteriophage vB PaeP-SaPL, rescues bacteremic mice infected with multi drug resistant Pseudomonas aeruginosa. Virus Res. 2021; 292: 198250.Alvi IA, Asif M, Rehman S. A single dose of a virulent bacteriophage vB PaeP-SaPL, rescues bacteremic mice infected with multi drug resistant Pseudomonas aeruginosa. Virus Res. 2021; 292: 198250.Adnan M, et al. Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm. Biologicals. 2020; 63: 89–96.Adnan M, et al. Isolation and characterization of bacteriophage to control multidrug-resistant Pseudomonas aeruginosa planktonic cells and biofilm. Biologicals. 2020; 63: 89–96.Treepong P, et al. Global emergence of the widespread Pseudomonas aeruginosa ST235 clone. Clin Microbiol Infect. 2018; 24 (3): 258–66.Treepong P, et al. Global emergence of the widespread Pseudomonas aeruginosa ST235 clone. Clin Microbiol Infect. 2018; 24 (3): 258–66.Principi N, Silvestri E, Esposito S. Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections. Front Pharmacol. 2019; 10: 513.Principi N, Silvestri E, Esposito S. Advantages and Limitations of Bacteriophages for the Treatment of Bacterial Infections. Front Pharmacol. 2019; 10: 513.Guo Y, Chen P, Lin Z, Wang T. Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2. Viruses. 2019; 11 (4): 318. DOI: 10.3390/v11040318.Guo Y, Chen P, Lin Z, Wang T. Characterization of Two Pseudomonas aeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2. Viruses. 2019; 11 (4): 318. DOI: 10.3390/v11040318.Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, et al. Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. MBio. 2012; 3 (2): e00029-12. DOI: 10.1128/mBio.00029-12.Alemayehu D, Casey PG, McAuliffe O, Guinane CM, Martin JG, Shanahan F, et al. Bacteriophages φMR299-2 and φNH-4 can eliminate Pseudomonas aeruginosa in the murine lung and on cystic fibrosis lung airway cells. MBio. 2012; 3 (2): e00029-12. DOI: 10.1128/mBio.00029-12.Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis. 2010; 201 (7): 1096–104.Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis. 2010; 201 (7): 1096–104.

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