Method for the estimation of antibacterial activity of the polyfunctional protein from transferrin family in the experimental model of the kinetics of Staphylococcus aureus development
Authors: Tsarev V. N. , Makeeva I. M. , Sadchikova El. R. , Podporin M. S. , Trefilova Yu. A. , Arzukanyan A. V. , Goldman I. L. 
Published: January 2022

Introduction. Lactoferrin is a cationic monomeric glycoprotein produced by acinar cells and glands, present in different places of the mucous membrane in different concentrations. In connection with the development of various variants of hygienic and medicinal products for the treatment of inflammatory diseases of the oral cavity based on lactoferrin, there was a need for an objective assessment of its antibacterial and antibiofilms properties, followed by an analysis of the preservation of activity in various variants of the isolation of this protein from the substrate and storage.Aim - to improve the effectiveness of evaluating the antibacterial activity of lactoferrin and the duration of its preservation in various biological substrates containing the active substance and individual experimental batches of the manufactured drug using automatic cultivation.Materials and methods. As part of the experiment, a microbiological diagnostic technique employing a system for the automatic cultivation of microbial populations was used. A pre-prepared bacterial suspension was inoculated into the nutrient broth and the studied lactoferrin samples were added, followed by cultivation and analysis of the possible antibacterial effects of transferrin protein. To determine the sensitivity of the isolated strains, we used our own modification of the serial dilution method developed at the Department of microbiology, virology, immunology of the A.I. Yevdokimov Moscow State University of Medicine and Dentistry. The experiment was based on the programmed automatic cultivation using the RTS-1 bioreactor. The interpretation of the results was carried out by changing the optical density at a wavelength of λ = 850 nm. The study of the growth dynamics of microorganisms was carried out in several repetitions, which was reflected in the graphs of the development of bacterial populations. The assessment of the growth control of the corresponding bacterial species was reflected in the change in the optical density values, on the basis of which the curve was built. Results and discussion. According to the results of an experimental study of the growth curves of bacterial populations, statistically significant differences in the number of viable cells in different phases of the growth curves were noted, when using different lactoferrin samples. Higher activity of human recombinant lactoferrin samples was established. An analysis of growth dynamics revealed differences in the onset of the maximum reproduction and its inhibition under the influence of various aggravating factors during cultivation. The bacteriostatic effect of lactoferrin is realized through the binding of iron ions, depriving the bacteria of this microelement, causing inhibition of their development. Along with this, lactoferrin is active against certain virulence factors of microorganisms, splitting them like serine proteases, and thus prevents their penetration into human cells.Conclusion. The method used for automatic cultivation of microorganisms in the bioreactor used allows one to obtain reproducible results, is available for wide use, and can be recommended for obtaining objective, comparable, reliable information about the antimicrobial properties of various samples of the bactericidal protein lactoferrin produced by the domestic pharmaceutical industry. The studied substrate containing recombinant human lactoferrin of Russian production is characterized by high antibacterial activity that persists for 3 years as minimum.