The effectiveness of biofilm formation of daily cultures of clinically significant strains of opportunistic bacteria

   Background. The formation of biofilm structures by microorganisms living in the hospital environment is a serious medical problem. To conduct correct experimental studies, it is necessary to know the speed and efficiency of biofilm formation by clinically significant species of opportunistic bacteria.
   Aim: to study the kinetics of plankton culture growth and the rate of biofilm formation by clinically significant pathogens of infections associated with medical care to substantiate the methodology of further research.
   Materials and methods. The strains from the working collection of the Laboratory for Microbiome and Microecology of the Scientific Сentre for Family Health and Human Reproduction Problems were used. Experiments were carried out with conditionally pathogenic microorganisms of the Enterobacteriaceae family and non-fermenting gram-negative bacteria. The optical density was measured, the total microbial number of the cell suspension was determined, and the morphological structure of the biofilm was evaluated using a light microscope on sterile cover glasses for the
species Pseudomonas aeruginosa, Klebsiella pneumoniae and Serratia marcescens.
   Results. The duration of the lag phase of the kinetic curve of cell growth varied in isolates of S. marcescens, P. aeruginosa and K. pneumoniae from 1 to 4 and 6 hours of cultivation, respectively. Despite this, the exponential growth phase was the same for all tested isolates and amounted to 4 hours. Thus, isolates of clinically significant species entered the stationary growth phase after 5–10 hours of cultivation and were characterized as fast-growing. On abiotic surfaces, after 8 hours of incubation of the tested cultures, the initial stages of the formation of biofilm structures were observed, after 20 hours the formed multilayer biofilm was visualized, after 24 hours succession occurred, new single cells were attached to the place of the detached structures.
   Conclusion. The data obtained on the duration of the main stages of growth kinetics compared with the visualization of the formation of biofilm structures on abiotic surfaces should be taken into account when studying the effects of disinfectants, antiseptics and antibacterial drugs on planktonic cells and biofilm associations of clinically significant opportunistic microorganisms.

1. Verderosa A. D., Totsika M., Fairfull-Smith K. E. Bacterial biofilm eradication agents: A current review. Front Chem. 2019; 7: 824. doi: 10.3389/fchem.2019.00824

2. Jamal M., Tasneem U., Hussain T., Andleeb S. Bacterial biofilm: Its composition, formation and role in human infections. J Microbiol Biotechnol. 2015; 4: 1-14.

3. Pelling H., Nzakizwanayo J., Milo S., Denham E. L., MacFarlane W. M., Bock L. J., et al. Bacterial biofilm formation on indwelling urethral catheters. Lett Appl Microbiol. 2019; 68 (4): 277-293. doi: 10.1111/lam.13144

4. Patini R., Staderini E., Lajolo C., Lopetuso L., Mohammed H., Rimondini L., et al. Relationship between oral microbiota and periodontal disease: A systematic review. Eur Rev Med Pharmacol Sci. 2018; 22: 5775-5788. doi: 10.26355/eurrev_201809_15903

5. Talapko J., Škrlec I. The principles, mechanisms, and benefits of unconventional agents in the treatment of biofilm infection. Pharmaceuticals (Basel). 2020; 13 (10): 299. doi: 10.3390/ph13100299

6. Li Z., Knetsch M. Antibacterial strategies for wound dressing: Preventing infection and stimulating healing. Curr Pharm Des. 2018; 24 (8): 936-951. doi: 10.2174/1381612824666180213141109

7. Xie T., Wu Q., Zhang J., Xu X., Cheng J. Comparison of Vibrio parahaemolyticus isolates from aquatic products and clinical by antibiotic susceptibility, virulence, and molecular characterisation. Food Control. 2017; 71: 315-321. doi: 10.1016/j.foodcont.2016.06.046

8. 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. 2018; 284 (4): 332-345. doi: 10.1111/joim.12782

9. Pannanusorn S., Ramirez-Zavala B., Lünsdorf H., Agerberth B., Morschhäuser J., Römling U. Characterization of biofilm formation and the role of BCR1 in clinical isolates of Candida parapsilosis. Eukaryot Cell. 2014; 13 (4): 438-451. doi: 10.1128/EC.00181-13

10. Xu W., Dong S., Han Y., Li S., Liu Y. Hydrogels as antibacterial biomaterials. Curr Pharm Des. 2018; 24 (8): 843-854. doi: 10.2174/1381612824666180213122953

11. Linnes J. C., Ma H., Bryers J. D. Giant extracellular matrix binding protein expression in staphylococcus epidermidis is regulated by biofilm formation and osmotic pressure. Curr Microbiol. 2013; 66 (6): 627-633. doi: 10.1007/s00284-013-0316-7

12. Romaní A. M., Fund K., Artigas J., Schwartz T., Sabater S., Obst U. Relevance of polymeric matrix enzymes during biofilm formation. Microbial Ecology. 2008; 56 (3): 427-436. doi: 10.1007/s00248-007-9361-8

13. Kanwar I., Sah A. K., Suresh P. K. Biofilm-mediated antibiotic-resistant oral bacterial infections: Mechanism and combat strategies. Curr Pharm Des. 2017; 23 (14): 2084-2095. doi: 10.2174/1381612822666161124154549

14. Nemchenko U. M., Kungurceva E. A., Grigorova E. V., Bel’kova N. L., Markova Yu. A., Noskova O. A., et al. Simulation of bacterial biofilms and estimation of the sensitivity of healthcare-associated infection pathogens to bactericide Sekusept active. Russian Clinical Laboratory Diagnostics. 2020; 65 (10): 652-658. (In Russ.). doi: 10.18821/0869-2084-2020-65-10-652-658

15. Grigorova E. V., Nemchenko U. M., Voropaeva N. M., Bel’kova N. L., Noskova O. A., Savilov E. D. Biofilm formation exposed to disinfectants with different active ingredients in Pseudomonas aeruginosa. Bulletin of Experimental Biology and Medicine. 2021; 171 (6): 733-738. (In Russ.). doi: 10.47056/0365-9615-2021-171-6-733-738

16. Savilov E. D., Markova Yu. A., Nemchenko U. M., Noskova O. A., Chemezova N. N., Kungurtseva E. A., et al. Ability to biofilm formation in infectios agents isolated from patients of a large general children’s hospital. Pacific Medical Journal. 2020; 1 (79): 32-35. (In Russ.). doi: 10.34215/1609-1175-2020-1-32-35

17. Godovalov A. P., Stepanov M. C., Kobzarenko E. E. Surviving as a community: Antibiotic tolerance and persistence in bacterial biofilms: Patent No. 2719402 of the Russian Federation. 2020; (11). (In Russ.).

18. Osipova E. V., Shipicyna I. V., Naumenko Z. S. A comparative quantitative evaluation of the potential of biofilm formation by different bacterial clinical strains on polystirole and glass surface. International Journal of Applied and Fundamental Research. 2014; 8-1: 55-58. (In Russ.).

19. Leonov V. V. The quantitative evaluation of capacity of opportunistic pathogenic microorganisms to form biofilms in experiment. Russian Clinical Laboratory Diagnostics. 2012; 10: 57-59. (In Russ.).

20. Ignatenko A. V. Study of bacterial biofilm formation and assessment of their resistance to biocides. Trudy Belorusskogo Gosudarstvennogo Tekhnologicheskogo universiteta. Khimiya i tekhnologiya organicheskikh veshchestv. 2008; 1 (4): 173-176. (In Russ.).