A 24-hour Сandida sp. biofilm formation dynamically assesed with modified macrometric method

The aim of the study was to study the dynamics of 24-hour Candida spp. biofilm formation by using a modified macrometric method. The proposed macrometric method solves the problem of accelerating and simplifying the quantitative assessment of the biofilm formation process, increases sensitivity due to allowing to avoid mistakes related to applied polystyrene material. The ease of implementing such a technique makes it accessible to any laboratory. Reference strains from the American Type Culture Collection (ATCC) and clinical isolates of Candida spp. isolated from the female reproductive tract with candida dysbiosis were used for the study. Biofilm formation of Candida spp. studied according to the O’Toole G.A. et al. method modified by us. The biological activity of Сandida sp. biofilm formation was monitored for 48 hours with 4-hour intervals, in winter season, the IV phase of the moon. A 48-hour fungal culture corresponding to relevant maximum adhesion on glass surface was used. The study chrono-design implied obtaining 6 diurnal measurements for the function evaluated with a 3–5-repetits of the experimental conditions. Amplitude-phase characteristics of the studied biorhythms were graphically represented using cosinor analysis serving as the basic method to identify and model cyclic processes in biological systems. The study proved that the ability of micromycete cells to adhere is significantly higher in the stationary vs. logarithmic growth phase (p < 0.05). The chronobiological technique used here allowed to reveal the presence of diurnal fungal film-forming activity (p < 0.05) and reveal the general patterns of manifestated properties in representatives of all candida species examined. It has been experimentally established that the sequence and consistency of the biological properties of clinical Сandida sp. isolates over time were not fundamentally dependent on the type of fungus. During the study, rhythmometric markers of the strain-related pathogenicity was established reflecting contribution of rhythm and the amplitude-phase characteristic. It has been proven that the activity of biofilm formation increases along the “reference strains — clinical isolates” axis. For С. albicans the Mann–Whitney test data was 29, for C. tropicalis — 26, and for C. krusei — 30 (p < 0.05). We believe that chronobiological method opens up new perspectives in the studying physiology of Candida spp. because it allows to dynamically predict state of microorganism and take into account features of urgent and long-term adaptation to various environmental factors. Identifying diurnal rhythms in biofilm-forming activity of various Сandida sp. strains opens up an opportunity to control viability of bacterial-fungal associations and predict related resistance to diverse antimicrobial agents.