ЕКСПЕРИМЕНТАЛЬНА МЕДИЦИНА https://doi.org/10.31718/mep.2019.23.1-2.06 SUSCEPTIBILITY OF PLANKTONIC AND FILM FORMS OF CANDIDA GLABRATA AND CANDIDA ALBICANS TO CATIONIC SURFACTANT

біоплівки The aim of the study was to investigate the sensitivity of planktonic and film forms of C. albicans and C. glabrata to cati- onic surfactant antiseptics. Materials and methods. The study was based on investigating 20 clinical strains of C. albicans and 15 C. glabrata isolated from surgical inpatientsю. The sensitivity of planktonic forms of investigated strains to anti- septic agents was quantitatively evaluated by two-fold serial dilutions (macrodilution) in Sabouraud liquid nutrient medium. Biofilm-forming properties of clinical strains C. albicans and C. glabrata were assessed by using the Christensen’s spectrophotometric method (MtP-test “microtiter plate test”). The influence of the antiseptics on C. albicans and C. glabrata film forms was assessed by the reproduction of the biofilms according to the above-described procedure with adding antiseptics in sub-bacteriostatic concentrations and the subsequent spectrophotometric ODU assessment. In the study we used antiseptics based on cationic surfactants, chlorhexidine digluconate 0.05 (Chlorhexidine-KR, manufactured by PJSC “Khimfarmzavod “Chervona zirka"”, Kharkiv, Ukraine (CHH)) and decamethoxin 0.2 (Decasan, produced by Yu-ria-Farm LLC ", Kyiv, Ukraine (DCM)). Results. According to the research results, lower sensitivity of C. glabrata strains to CHH was found, compared to the sensitivity of C. albicans strains. In addition, the activity of DCM in the investigated representatives of Candida spp. did not differ significantly. Clinical strains of C. glabrata were more susceptible to DCM compared to their susceptibility to CHH. C. albicans strains showed medium film-forming properties, while C. glabrata high. The investigated cationic surfactant antiseptics possessed the same degree of activity on the film-forming proper- ties of clinical strains of Candida spp. Conclusions. Cationic surfactant antiseptics (CHH and DCM) possess antifungal activity against planktonic and film forms of C. albicans and C.


Introduction
Yeast-like fungi that belong to the genus Candida are known to be an inseparable component of human microbiota. However, immune deficiency conditions or / and impaired homeostasis can contribute to the development of candidiases [1].
According to the relevant literature, until the beginning of the 21st century yeast fungi caused about 3% of postoperative infectious complications [2]. Lately, however, Candida spp. has been reported to as a rapidly growing cause of infectious post-operative complications. Fungal infections have been found out to result in high mortality rate, which sometimes reaches the rates of deaths due to tuberculosis and malaria [3]. Fungi of the genus Candida rank the sixth place among the main nosocomial pathogens and the fourth place among the dominant causative agents of nosocomial bacteria. This is due to the decline in community immunity among the population, alterations in the biological properties of pathogens and the rapid development of microbial resistance to antimicrobial agents [2,4]. In order to improve the situation it is of great importance to reconsider and update existing treatment standards.
In the past, C. albicans was known as the most prevalent species of Candida spp. throughout the world and described as a causative agent of about two thirds of all cases of invasive candidiasis [5]. However, today there are some changes within the spectrum of etiological factors of candidiasis infection that indicate a growing shift towards the so-called non-albicans species (C. glabrata, C. krusei, C. tropicalis) [5][6][7].
While C. albicans, C. krusei, C. tropicalis are genetically related, C. glabrata does not have a close phylogenetic relationship with other members of the genus. This determines the existence of significant differences in their morphology, pathogenicity factors, sensitivity to medications and thus the pathogenesis of mycoses caused by this causative agent [8]. This requires a comprehensive in-depth study of the biological properties of Candida spp. within the boundaries of the genus and the search for medicines that would have the same equal antifungal effect on different representatives of the genus.
The aim of the study was to investigate the sensitivity of planktonic and film forms of C. albicans and C. glabrata to cationic surfactant antiseptics.

Materials and methods
The study was based on investigating 20 clinical strains of C. albicans and 15 C. glabrata isolated from surgical inpatients staying at M. Sklifosovskiy Poltava Regional Clinical Hospital and M. Pyrogov Vinnytsia Regional Clinical Hospital. The research has been carried out according to Helsinki Declaration on the ethical principles for medical research involving human subjects and approved by the Bioethics Committee of M. Pyrogov Vinnytsia National Medical University and the Commission on Ethical Issues and Bioethics of Ukrainian Medical Stomatological Academy.
To investigate the microflora, the material was taken from the surface of the infected surgical wound, followed by cultivation on Sabouraud culture medium for 48 hours. The final identification was carried out in accordance with the standard methodology for morphological, tinctorial and biochemical properties and by applying automatic bacteriological analyzer Vitec -2compact bioMérieux (France).
The sensitivity of planktonic forms of investigated strains to antiseptic agents was quantitatively evaluated by two-fold serial dilutions (macrodilution) in Sabouraud liquid nutrient medium in accordance with the Order of the Ministry of Public Health of Ukraine No. 167, April 5, 2007 "On Approval of Methodological Instructions on "Assessment of Sensitivity of Microorganisms to Antibacterial Drugs " [9]. For this purpose, a microbial suspension (inoculum) equivalent to 1.0 was prepared according to the McFarland standards, and diluted 100-fold in a saline, after that the microbial concentration obtained was 3x1010 CFU / cm 3 . Then, inoculums prepared ex tempore and taken in a dose of 1.0 ml were applied into each test tube with a two-fold dilution of antiseptic solution and into one test tube containing 1.0 ml of nutrient broth ("negative" control). All test tubes with the exception of "negative" control were incubated in the standard atmosphere at 35 ° C for a day. The "negative" control test tube was kept at a temperature 4 ° C until the results were recorded.
Biofilm-forming properties of clinical strains C. albicans and C. glabrata were assessed by using the Christensen's spectrophotometric method (MtP-test "microtiter plate test"). Biofilms were modelled in Sabouraud liquid nutrient medium in wells of a sterile, flat-bottom 96-well polystyrol tablet (Corning, USA) and stained with 1% solution of crystalline violet. The properties of microorganisms to form a biofilm were measured by absorbance of the dye in units of optical density (ODU) using a spectrophotometer with a wavelength of 570 nm. The ability of microorganisms to form biofilms was evaluated as low (ODU <0.120), average (ODU = 0.121-0.239) and high (ODU > 0.240) [10].
The influence of the antiseptics on C. albicans and C. glabrata film forms was assessed by the reproduction of the biofilms according to the above-described procedure with adding antiseptics in sub-bacteriostatic concentrations and the subsequent spectrophotometric ODU assessment.
The statistical analysis of the obtained findings was performed by using the standard SPSS Statistics 23 and Microsoft Excel 2010 software packages. We calculated the arithmetic mean (M), the mean error of the arithmetic mean (± m), and the criterion of the reliability of the differences (p). The differences between the indicators studied were evaluated according to Student's t-test.

Results and discussion
The obtained findings demonstrated that CHH and DCM were found to produce varying antifungal effect on investigated clinical strains of yeast fungi ( Table 1). The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFcC) of CHH for C. glabrata significantly exceeded the MIC and MFcC showed by DCM for C. albicans in 1.6 times (p <0.05). This indicated a lower sensitivity of C. glabrata strains to CHH when compared with the C. albicans strain sensitivity. In addition, the activity of DCM against the investigated representatives of Candida spp. did not differ significantly (p <0.05). However, it should be noted that MFcC demonstrated by DCM against clinical strains of C. glabrata was 1.7 times lower than MFcC of CHH against the relevant strain (p <0.05). This pointed out a significantly higher sensitivity of C. glabrata to DCM than to CHH. The study showed the representatives of the genus Candida differed in their ability to biofilm formation (Fig.  1). The ability to absorb the dye by C. glabrata (0,251 ± 0,04 ODU) exceeded the C. albicans values (0,203 ± 0,05 ODU) in 1,2 times (p = 0,004), i.e. the clinical strains of C. albicans demonstrated average film-forming properties, while those of C. glabrata strains were higher.
It should be noted that the cationic surfactants antiseptics studied had a strong activity against the film for- The biofilm formation by C. albicans is known to start by the microorganism adhesion to the artificial surface with the subsequent production of the extracellular matrix. Cells embedded into this matrix multiply, forming a biofilm. In turn, the ability of C. glabrata to form a similar extracellular matrix has not been experimentally proven. Accordingly, the powerful film-forming properties of the latter are genetically determined and associated with the presence of a number of surface adhesins involved in the process of biofilm formation. Recently, the genome of representatives of the Candida species is being actively studied, establishing a connection between their strains' properties of biofilm formation and the presence of surperficial Epa6, Epa7 and other proteins [11].
Cationic surfactant antiseptics used in the study have been found out to reliably suppress the biofilm-forming properties of both C. albicans clinical strains and C. glabrata strains, which are different from C. albicans by their biological properties. The mechanism of action of the drugs of this group consists in the ability to change the surface tension of the cell of the microorganism. It impacts the surface structure of the cell and stimulates the disruption of its osmotic balance, resulting in "osmotic shock" and the death of the microorganism [12].
The obtained results point out the high efficacy of cationic surfactant antiseptics on planktonic and film forms of Candida spp. clinical strains. The equally potent anti-fungal action of CHH and DCM against both C. albicans and C. glabrata strains makes the medicines studied appropriate in the treatment of candidiasis in view of the significant differences in their antimycotic effects to microbes within the genus.

Conclusions
Cationic surfactants antiseptics (CHH and DCM) possess antifungal activity against clinical strains of Candida spp. Clinical strains of C. albicans demonstrate higher susceptibility to CHH, compared with the clinical strains of C. glabrata. In turn, DCM has a significantly higher anti-fungal effect against C. glabrata, compared with CHH. C. albicans clinical strains show average biofilm properties, while C. glabrata strains have high biofilmforming properties. CHH and DCM significantly inhibit the ability to form biofilms by clinical strains of Candida spp.