In vitro ACTIVITY OF THE ANTIBIOTIC BATUMIN AGAINST Candida albicans BIOFILM

The aim of this work was to study action of batumin on the strains of Candida albicans and Candida krusei in planktonic and biofilm form and also to obtain more detailed insights into the influence of batumin on biofilm formation by using atomic-force microscopy. The Minimum Inhibitory Concentration (MIC) of batumin was studied according to CLSI standards. Formation of a biofilm was studied by the photometric O’Toole method by means of a plate photometer ELx808 (BioTek, USA) at wavelength of 630 nanometers. The batumin has a high selective activity against staphylococci (MIC  0,25 μg/ml), at the same time, antibiotic, being not active concerning yeast of the genus Candida (MIC  512 μg/ml) showed the inhibiting action on biofilm formation of these microorganisms. Batumin influence on biofilm formation was studied in type, collection strains С. albicans , C. krusei and clinical isolates. Presence 0,125 μg/ml of batumin in the broth (1/2 MIC for staphylococci) reduced the biofilm formation at 55.6% of the studied strains. Their biofilm formation values varied for C. albicans from 1.5–3.9 CU (conventional unit: OD 630 in experimental samples/OD 630 in control samples), for C. krusei of 2.3–3.0. Batumin was more effective against Candida strains with strong biofilm formation.Atomic force microscopy revealed qualitative changes in the exopolymeric matrix due to batumin treatment, as well as a significant reduction in the number of cells adhered to the coverslip, preventing formation of C. albicans 127 biofilm, However, C. albicans ATCC 24433 a significant reduction in the number of cells adhered to the coverslip weren’t observed. The data obtained by an Аtomic force microscopy confirm ability of a batumin to prevent formation of a biofilm at the studied strains that allows to consider it as the preventive agent at treatment of the infections caused

The aim of this work was to study action of batumin on the strains of Candida albicans and Candida krusei in planktonic and biofilm form and also to obtain more detailed insights into the influence of batumin on biofilm formation by using atomic-force microscopy. The Minimum Inhibitory Concentration (MIC) of batumin was studied according to CLSI standards. Formation of a biofilm was studied by the photometric O'Toole method by means of a plate photometer ELx808 (BioTek, USA) at wavelength of 630 nanometers.
The batumin has a high selective activity against staphylococci (MIC  0,25 μg/ml), at the same time, antibiotic, being not active concerning yeast of the genus Candida (MIC  512 μg/ml) showed the inhibiting action on biofilm formation of these microorganisms. Batumin influence on biofilm formation was studied in type, collection strains С. albicans, C. krusei and clinical isolates. Presence 0,125 μg/ml of batumin in the broth (1/2 MIC for staphylococci) reduced the biofilm formation at 55.6% of the studied strains. Their biofilm formation values varied for C. albicans from 1.5-3.9 CU (conventional unit: OD 630 in experimental samples/OD 630 in control samples), for C. krusei of 2.3-3.0. Batumin was more effective against Candida strains with strong biofilm formation.
Atomic force microscopy revealed qualitative changes in the exopolymeric matrix due to batumin treatment, as well as a significant reduction in the number of cells adhered to the coverslip, preventing formation of C. albicans 127 biofilm, However, C. albicans ATCC 24433 a significant reduction in the number of cells adhered to the coverslip weren't observed.
The data obtained by an Аtomic force microscopy confirm ability of a batumin to prevent formation of a biofilm at the studied strains that allows to consider it as the preventive agent at treatment of the infections caused by yeast-like fungi of the genus Candida.
The objective of this work was to study action of batumin on the strains of C. albicans and C. krusei in planktonic and biofilm form and also to obtain more detailed insights into the influence of batumin on biofilm formation by using atomic-force microscopy.

Materials and Methods
Batumin, obtained by fermentation of Pseudomonas batumici, was purified by silica gel preparative chromatography to 85% of purity.
Batumin is commercially available from Santa Cruz Biotechnology (Santa Cruz, CA) or Enzo Life Sciences Antwerp, Belgium).
The object of the study were type and collection strains of С. albicans (n = 33) and C. krusei (n = 12) (Table 1), isolated from patients after examination for intestinal dysbiosis, from skin microbial dysbiosis (microbial collection of the Institute of Cellular and Intracellular Symbiosis, Ural Branch of Russian Academy of Sciences, Orenburg, Russia).
Identification of С. albicans and C. krusei was carried out on the basis of morphological, cultural and biochemical properties with the use of the commercial test system API20CAUX (bioMerieux, France).
The Minimal inhibitory concentration (MIC) of batumin was studied according to CLSI Standards in Mueller-Hinton agar [8]. The microbial load of C. albicans was 0.5  10 7 cfu/ml and the Petri dishes were incubated at 37 С for 24 hours.
Concentration of batumin (0.125 μg/ml) was used to study its effect upon biofilm formation in Candida. Biofilm formation was studied by a photometric method determining the bacterial capacity to adhere to the 96well polystyrole plane-table surface (Thermo Scientific, USA) with subsequent crystal violet staining [9]. An antibiotic was added into culture medium simultaneous with the culture of fungi and cultivated 24 hours. At the study of influence of batumin (0.125 g /ml) on the different stages of biofilm formation by C. albicans strains the antibiotic was added into culture medium simultaneously with yeast-like fungi in 90 minutes, 24 and 48 hours from the beginning of incubation.
Optical density measurement was done using a photometer ELx808 (BioTek, USA) at a wavelength of 630 nm. Degree of biofilm formation was presented in conditional units (CU) which was the optical density of the broth after growth of the strain relative to the nutrient broth optical density.
For the study of batumin effect on biofilm formation by atomic force microscopy, we used C. albicans ATCC 24433 as test-culture and C. albicans 127 (clinical isolate). For testing the influence of batumin on biofilm production, glass coverslips were immersed into Luria-Bertani broth with 0.125 μg/ml of batumin and incubated for 48 h at 37 C.
Visualization of the biofilms was done by atomic force microscopy using the SMM-2000 microscope (Proton-MIET Closed JOINT Stock Company, Russia), in contact mode in an air environment [10] [11].
Statistic analysis was performed by nonparametric method using Mann Whitney U-test [12].

Results and Discussion
All the studied 45 strains of C. albicans and C. krusei were highly resistant to batumin (MIC of 512 μg/ml), in correspondence with earlier data [6]. The resistance of strains to batumin was studied according to CLSI Standards in Mueller-Hinton agar [8]. At the same time, our preliminary research showed effectiveness of the antibiotic on inhibition of formation of a biofilm in the cultures of the genus Candida [7,13].
In experiments on batumin effect on biofilm formation by strains of Candida we used concentration 0.125 μg/ml, which causes only modification of biological properties, including biofilm formation and did not influence on grows properties of cultures.
The obtained results showed that the effect of batumin on the formed biofilm in the representatives of the genus Candida was variable for different strains and species ( Table 1).
The biofilm formation values varied for C. albicans from 1.5 to 3.9 CU, for C. krusei from 2.3 to 3.0. Presence of batumin (0.125 μg/ ml) in the broth reduced the biofilm formation for 55.6% in the studied strains of fungi, whereas for staphylococci this was 85% [14].
Of special interest is the fact that the change in biofilm in the presence of batumin differs for different strains of Candida, and that these differences are caused at a concentration of only 0.125 μg/ml, for strains resistant to the studied antibiotic (Table 1).
Apparently, the changes we have detected in the biofilms of yeast-like fungi under the influence of batumin made them unstable, incapable of persistence and dissemination in the human body.
It should be noted that batumin is more effective against С. albicans strains with The analysis of the experimental data on batumin at the stage of biofilm formation for C. albicans showed dependence of batumin effectiveness at the stage of biofilm formation ( Table 2).
Addition of batumin to the cultivation medium simultaneously with C. albicans did not influence biofilm formation of fungi in 40% of cases, and promoted reduction of biofilm formation values in 60% of cases for 63.1 ± 3.4% of the initial level (Р < 0.05).
Addition of batumin in 90 min after incubation of C. albicans (an initial stage of biofilm formation) reduced values of biofilm formation in 70% of cases on average by 48.8 ± 4.4% versus control (Р < 0.05). Addition after 24 hours of growth, the antibiotic reduced formation of biofilm in culture in 50% of cases versus 24.8% of control (Р < 0.05), and in 50% of cases -stimulated this parameter by 32.4%.
After incubation of C. albicans with the antibiotic during the 48 hours in 70% of cases the lack of the preparation influence was noted and only in 30% of strains decrease in biofilm formation was noted only by 5.6%.
Thus, the biofilm of the studied strains of C. albicans is sensitive to batumin at early stages of its formation whereas the wellestablished biofilm was more resistant to studied preparation.
On the contrary, batumin in 22.2% of cases stimulated low level of biofilm formation in yeast-like fungi. Possibly, the obtained data reflect the developed relationship between species of microorganisms in a microbiocenosis, as it is known that in interaction of bacteria of the genus Pseudomonas and fungi of the genus Candida there is a mutual depression of biofilm formation of microorganisms at all stages of development [15,16].
By its action on biofilms of cultures of Candida batumin is similar to action on biofilms of Staphylococci [13]. Sidrim et al. (2015) reported a similar effect when studying -lactam antibiotics and vancomycin effects on formation of biofilms of Candida spp.
A more detailed study of batumin action upon C. albicans biofilm formation was carried out using atomic force microscopy for C. albicans ATCC 24433 T and C. albicans 127 (clinical isolate).
On the coverslip incubated with the cells C. albicans ATCC 24433 T without batumin the set of cells adhered to its surface (Figure, A). The glass coverslips were immersed into Luria-Bertani broth with 0.125 μg/ml of batumin and incubated for 48 h at 37 C. At visualization of the surface of microorganisms, the spherical formations which are presumably gemmating daughter cells (Figure, B) are clearly visible. However, a significant decrease of the number of adherent cells in the presence of the antibiotic was not observed (Figure, C, D).
A detailed study of the surface of bacterial cells treated with batumin allows establishing significant reduction of their roughness values ( Table 3).
The cells of C. albicans 127 were observed in the form of a monolayer of separate islets (Figure, F). This strain formed biofilm on the surface of the glass, as can be seen. Surface biofilm was formed by an exopolymeric matrix with cells of round shape immersed in it (Figure, G). The addition in the medium of batumin caused, on the one hand, the lack of signs of an exopolymeric matrix (Figure,  H, I), and on the other hand, the change of cell morphology. So the mean diameter of cells was equal to 1.88 ± 0.43 μm, which was significantly less than control values 2.29 ± 0.20 (Table 3).
Atomic force microscopy revealed qualitative changes in the exopolymeric matrix due to batumin treatment, as well as a significant reduction in the number of cells adhered to the coverslip, preventing formation of C. albicans 127 biofilm. In C. albicans ATCC 24433 T , a significant reduction in the number of cells adhered to the coverslip was not observed. In this case a nonspecific interaction of batumin and a surface ligand of C. albicans ATCC 24433 T is probable. The results presented in this work showed that all studied С. albicans and C. krusei strains were highly resistant against batumin (MIC  512 μg/ml). However, the antibiotic showed inhibition of biofilm formation of these microorganisms.
The biofilm of the studied strains of C. albicans and C. krusei was sensitive to batumin at early stages of its formation, whereas the well-established biofilm was more resistant to studied preparation.
The data obtained by an atomic-force microscopy confirm the ability of batumin to prevent formation of biofilm in the studied strains that allows to consider it as the preventive agent for treatment of yeast-like fungi of the genus Candida.

Acknowledgement
We thank prof, Mario Vaneechoutte (University of Gent, Belgium) for critical reading of the manuscript and Vasilchenko Alexey for provided pictures by AFM.   Note: * -P < 0.05 (Mann-Whitney U-test).