Аннотация
Приведены результаты исследования биоплёнок Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, выделенных от пациентов с хронической ЛОР-инфекцией. Для изучения биоплёнок применены фотометрические методы и люминесцентная микроскопия в сочетании с компьютерным 3D-моделированием. Проведено измерение массы биоплёночного матрикса и массы бактериальных клеток в динамике развития биоплёнки. Биоплёнкобразование у всех культур бактерий исследовано в течение семи дней. Показано, что все исследуемые штаммы бактерий способны образовывать биоплёнки. Последовательная визуализация с помощью 3D моделей выявила стадийную организацию при формировании биоплёнок. Данные 3D моделирования подтверждены результатами фотометрического анализа изменений массы матрикса биоплёнки и клеточной составляющей. Образование зрелой биоплёнки у всех культур бактерий происходило на четвёртые сутки. Процесс биоплёнкообразования у штаммов бактерий имел различия в динамике изменения массы клеток и межклеточного матрикса. Наиболее быстрое нарастание бактериальной массы наблюдалось у S. aureus и P. aeruginosa, тогда как наиболее интенсивное образование межклеточного матрикса характерно для P. aeruginosa. Люминесцентная микроскопия с компьютерным 3D моделированием дополняют фотометрический метод исследования биоплёнкообразующих свойств бактерий, что расширяет представление о развитии микробной популяции внутри биоплёнки, роли матрикса в поддержании структуры биоплёнки. Комбинация нескольких методов позволяет определить оптимальное время и мишени для антимикробного воздействия на биоплёнку, оценить различные способы регулирования процесса биоплёнкообразования у бактерий при некоторых инфекциях.
Annotation
The article presents the results of a study of biofilms of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae isolated from patients with chronic ENT infection. Various photometric methods and luminescence microscopy combined with 3D computer modeling were used for biofilm studying. The mass of the biofilm matrix and the mass of bacterial cells in dynamics of biofilm development were measured. Biofilm formation in all bacterial cultures was studied for seven days. It was shown all investigated bacterial strains are capable to biofilm forming. Sequential visualization using 3D models revealed a staged organization of biofilm formation. The 3D modeling data were confirmed by the results of photometric analysis the change of biofilm matrix and the cellular component. The formation of a mature biofilm in all bacterial cultures occurred on the fourth day. However, the process of biofilm formation of bacterial strains had differences in the dynamics of changes in cell mass and extracellular matrix. The most rapid increase in the bacterial mass was observed in S. aureus and P. aeruginosa, while the most intensive formation of the extracellular matrix was typical for P. aeruginosa. Luminescent microscopy with 3D computer modeling complements the photometric method investigation of bacteria biofilm-forming properties, which expands the understanding of microbial population development inside the biofilm, as well as the role of matrix in maintaining the biofilm structure. Using the combination of several methods makes it possible to determine the optimal time and targets for the antimicrobial effect on the biofilm, as well as to evaluate the different ways of regulation of bacterial biofilm formation in some infections.
Key words: bacterial biofilm; biofilm formation; matrix; luminescent microscopy; 3D modeling; ENT infections.
Список литературы
1.Plekhanov N.A., Zadnova S.P., Kritskiy A.A. Vibrio cholerae biofilm: mechanisms that regulate formation and environmental signals that promote its formation. Problemy osobo opasnykh infektsiy [Internet]. 2019; (3):19-25. Available at: https://journal.microbe.ru/jour/article/view/1170. (in Russian)
2.Lade H., Park J.H., Chung S.H., Kim I.H., Kim J.M., Joo H.S. et al. Biofilm Formation by Staphylococcus aureus Clinical Isolates is Differentially Affected by Glucose and Sodium Chloride Supplemented Culture Media. J. Clin. Med. [Internet]. 2019; 8(11). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912320/.
3.Cavalheiro M., Pereira D., Formosa-Dague C., Leitão C., Pais P., Ndlovu E.et al. From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view. Commun. Biol. [Internet]. 2021; 4(1). Available at: https://pubmed. ncbi.nlm.nih.gov/34285314/.
4.Sharipova M.R., Mardanova A.M., Rudakova N.L., Pudova D.S. Bistability and biofilm matrix formation as adaptation mechanisms of Bacillus subtilis in the stationary phase. Mikrobiologiya [Internet]. 2021; 90(1):24-42. Available at: https://elibrary.ru/item. asp?doi=10.31857/S0026365620060178. (in Russian)
5.Bjarnsholt T., Buhlin K., Dufrêne Y.F., Gomelsky M., Moroni A., Ramstedt M. et al. Biofilm formation – what we can learn from recent developments. J. Intern. Med. [Internet]. 2018; 284(4):332-45. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927207/.
6.Kropotov V.S., Zaslavskaya M.I., Ignatova N.I., Kryazhev, D.V. Modern methods for examining the ultrastructure of bacterial biofilms (literature review). Problemy meditsinskoy mikologii. 2022; 24(4):10-20. (in Russian)
7.Schilcher K., Horswill A.R. Staphylococcal Biofilm Development: Structure, Regulation, and Treatment Strategies. Microbiol. mol. biol. rev. MMBR [Internet]. 2020; 84(3). Available at: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC7430342/.
8.Ilyina T.S., Romanova Y. Bacterial biofilms: their role in chronical infection processes and the means to combat them. Mol. genet. microbiol. virol. (Russ. Version). 2021; 39:14. (in Russian)
9.Abu Khweek A., Amer A.O. Factors mediating environmental biofilm formation by Legionella pneumophila [Internet]. Frontiers in cellular and infection microbiology. Frontiers Media S.A. 2018; 8:38. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835138/.
10.Jamal M., Ahmad W., Andleeb S., Jalil F., Imran M., Nawaz M.A. et al. Bacterial biofilm and associated infections. J. Chin. med. assoc. JCMA. 2018; 81(1):7-11.
11.Arciola C.R., Campoccia D., Montanaro L. Implant infections: Adhesion, biofilm formation and immune evasion [Internet]. Nature Reviews Microbiology. Nature Publishing Group; 2018; 16: 397-409. Available at: https://pubmed.ncbi.nlm.nih.gov/29720707/.
12.Bell T., O’Grady N.P. Prevention of Central Line–Associated Bloodstream Infections [Internet]. Infectious Disease Clinics of North America. 2017; 31:551-9. Available at: https://www.ncbi.nlm.nih. gov/pmc/articles/PMC5666696/.
13.Mirzaei R., Mohammadzadeh R., Alikhani M.Y., Shokri Moghadam M., Karampoor S., Kazemi S. et al. The biofilm-associated bacterial infections unrelated to indwelling devices. IUBMB Life [Internet]. 2020; 72(7):1271-85. Available at: https://onlinelibrary.wiley.com/doi/abs/10.1002/iub.2266.
14.Mesrati I., Saidani M., Jemili M., Ferjeni S., Slim A., Boubaker I.B.B. Virulence determinants, biofilm production and antimicrobial susceptibility in Staphylococcus aureus causing device-associated infections in a Tunisian hospital. Int. J. antimicrob. agents [Internet]. 2018; 52(6):922-9. Available at: https://pubmed.ncbi.nlm.nih. gov/29775684/.
15.Ding L., Wang J., Cai S., Smyth H., Cui Z. Pulmonary biofilm-based chronic infections and inhaled treatment strategies. Int. J. Pharm. 2021; 604:120768.
16.Kinnari T.J. The role of biofilm in chronic laryngitis and in head and neck cancer. Curr. Opin. Otolaryngol. Head Neck. Surg. 2015; 23(6):448-53.
17.Kania R., Vironneau P., Dang H., Bercot B., Cambau E., Verillaud B. et al. Bacterial biofilm in adenoids of children with chronic otitis media. Part I: a case control study of prevalence of biofilms in adenoids, risk factors and middle ear biofilms. Acta оtolaryngol. (Stockh.). 2019; 139(4):345-50.
18.Vestby L.K., Grønseth T., Simm R., Nesse L.L. Bacterial Biofilm and its Role in the Pathogenesis of Disease. Antibiotics [Internet]. 2020; 9(2):59. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167820/.
19.Grande R., Puca V., Muraro R. Antibiotic resistance and bacterial biofilm. Expert. Opin. Ther. Pat. 2020; 30(12):897-900.
20.Vuotto C., Pascolini C., Balice M., Libori M., Tiracchia V., Salvia A. et al. Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains. J. Appl. Microbiol. [Internet]. 2017; 123(4):1003-18. Available at: https://pubmed.ncbi.nlm.nih. gov/28731269/.
21.Chebotar` I.V., Bocharova Y.A., Gur’ev A.S., Mayansky N.A. Bacteria survival strategies in contact with antibiotics. Klinicheskaya Laboratornaya Diagnostika. 2020; (2):116-21. (in Russian)
22.Badar W., Ullah Khan M.A. Analytical study of biosynthesised silver nanoparticles against multi‐drug resistant biofilm‐forming pathogens. IET Nanobiotechnol. [Internet]. 2020; 14(4):331–40. Available at: https://onlinelibrary.wiley.com/doi/10.1049/iet-nbt.2019.0287.
23.Hall C.W., Mah T.F. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiology Reviews. Oxford University Press; 2017; 41: 276-301.
24.O’Toole G.A. Microtiter Dish Biofilm Formation Assay. J. Vis. Exp. JoVE [Internet]. 2011; (47):2437. Available at: https://www.ncbi. nlm.nih.gov/pmc/articles/PMC3182663/.
25.Kryazhev D.V., Ermolina G.B., Kropotov V.S. Method of evaluation of bacterial biofilm. Patent RF № 2770413 C1; 2022. (in Russian)
26.Relucenti M., Familiari G., Donfrancesco O., Taurino M., Li X., Chen R. et al. Microscopy methods for biofilm imaging: Focus on sem and VP-SEM pros and cons [Internet]. Biology (Basel); 2021; 10: 1-17. Available at: https://pubmed.ncbi.nlm.nih.gov/33445707/.
27.Caizán-Juanarena L., Krug J.R., Vergeldt F.J., Kleijn J.M., Velders A.H., Van As H. et al. 3D biofilm visualization and quantification on granular bioanodes with magnetic resonance imaging. Water Res. 2019; 167:115059.