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25VETERINARY SCIENCES
VIRULENCE POTENTIAL AND RESISTANCE TO ANTIFUNGAL DRUGS OF CANDIDA GENUS
YEAST FUNGI
Manoyan M.,
Phd, assistant professor, Head of the Department of Mycological Expertise and Standardization of Medicines Against Mycoses and Mycotoxicosis, The Russian State Center for Quality and Standardization of Veterinary Drugs and Feed (VGNKI).1, Moscow
Sokolov V.,
Researcher, Department of Mycological Expertise and Standardization of Medicines Against Mycoses and Mycotoxicosis, The Russian State Center for Quality and Standardization of Veterinary Drugs and Feed
(VGNKI), Moscow Gursheva A.,
Researcher, Department of Mycological Expertise and Standardization of Medicines Against Mycoses and Mycotoxicosis, The Russian State Center for Quality and Standardization of Veterinary Drugs and Feed
(VGNKI), Moscow Gabuzyan N.
Junior Researcher, Department of Mycological Expertise and Standardization of Medicines Against Mycoses and Mycotoxicosis, The Russian State Center for Quality and Standardization of Veterinary Drugs and
Feed (VGNKI), Moscow
Abstract
Sixteen Candida catenulata isolates and twelve Candida lipolytica isolates isolated from cattle udder skin and raw milk were studied. The growth rate of the isolates at +37°C, the activity of proteolytic enzymes and phospholipases, the intensity of mycelial structures' formation, and the sensitivity to fluconazole and voriconazole were studied.
As a result of the studies, the potential virulence of the studied isolates was demonstrated; more than half of Candida lipolytica isolates were resistant to fluconazole or voriconazole, cases of cross-resistance to both drugs were shown.
Keywords: virulence potential, resistance, antifungal drugs, genus Candida, yeast fungi, cattle, udder skin, raw milk
Introduction
The spectrum of infections caused by yeast fungi is extremely wide: candidiasis of the skin [1] and mucous membranes [2, 3], invasive intestinal candidiasis [4], candidemia [candida septicemia]. Importantly that yeast fungi of the genus Candida are not species-specific pathogens, the same pathogen can infect many species of domestic, farm and wild animals, as well as humans [5].
Among the great variety of yeast infections, the infections of mucous membranes and skin, such as cattle mastitis, are of utmost importance.
The proportion of yeast and yeast-like fungi in the etiology of cattle mastitis in China (Heilongjiang Province) was 35.6% [6]. Of these, 79.4% were yeast-like fungi of the genus Candida (C. krusei 37%, C. tropicals 10.4%), followed by yeast fungi of the genus Tricho-sporon (5.9%). At the same time, the incidence of mastitis caused by fungi was up to 53%.
In a study conducted between 1996 and 2000 in the Lublin region (Poland), Candida fungi were isolated from milk of cows with clinically pronounced mastitis in 6.9% of cases [7].
In Brazil, yeast-like fungi were found in the cow milk with clinically confirmed mastitis in 43.3% of cases, while in the milk of healthy animals such fungi were detected in 23.4% of cases [8].
A study conducted in 2010-2014 in 11 breeding farms of the Sverdlovsk region also found a connection between Candida yeast-like fungi found in milk and mastitis [9].
Yeast fungi have a rich arsenal of agents that determine their pathogenicity. Among these mechanisms, or virulence factors, are the following: growth at high temperature (up to 37 °C and above) [10], adhesion ability, formation of mycelial structures causing thigmotro-pism, proteolytic and lipolytic enzymes including phos-pholipases, biofilm formation ability, low antigenicity and mechanisms that reduce the intensity of the immune response [11, 12].
Yeast fungi can also be resistant to antifungal drugs. It is not only about pathogens of nosocomial infections, but also about fungi isolated from the external environment. Recently, isolates resistant to antifungal drugs have been found in milk of cows both clinically healthy and with signs of mastitis [13], in droppings of wild birds [14], at poultry farms [15] and in the external environment [16].
Candida catenulata and Candida lipolytica are often found in food, especially in milk and milk products [17]. A number of researchers associate Candida catenulata and Candida lipolytica with mastitis [18, 19].
Goal of research
The aim of this work was to study some virulence factors of Candida catenulata and Candida lipolytica
yeast fungi isolated from different sources associated with farm animals: growth rate at +37°C, activity of proteolytic enzymes and phospholipases, intensity of mycelial structure formation and antifungal drugs sensitivity - fluconazole and voriconazole.
Materials and methods
The yeast fungi studied were isolated from raw milk samples and from cattle udder skin using standard methods. Identification was performed using the Can-didaTest21 test system (Erba LaChema). A total of 16
Candida catenulata isolates, 12 Candida lipolytica isolates and 10 Candida albicans strains were used as controls.
The growth rate at a temperature of +37°C.
Each isolate was cultured in Sabouraud liquid medium for 24 hours. The optical density (OD)of the culture at the beginning of the experiment was 0.5 units. McF. At the end of cultivation, the OD was determined and calculated how many times it increased in relation to the initial OD. This value was taken as an index of growth rate.
Activity of proteolytic enzymes and phospho-lipases.
Each isolate was cultivated in YCB nutrient medium (Yeast carbon base, HiMedia laboratories) with the addition of bovine serum albumin at 5% (weight/volume) to determine proteolytic activity; egg yolk emulsion at 5% (volume/volume) was added to the nutrient medium to determine phospholipase activity. Five sterile discs of filter paper were placed on the surface of the nutrient medium, on which 10 mcL of culture suspension with a density of 0.5 units McF was then applied, incubated for 24 hours at +37°C. After cultivation, the diameter of the lysis zone around the disk was measured, and then the ratio of the colony (disk)diameter to the diameter of the lysis zone was determined. These values were taken as indicators of the activity of proteolytic enzymes and phospholipases.
Formation intensity of mycelial structures.
Each isolate was cultivated in liquid nutrient medium RPMI-1640 with the addition of 20% (volume/volume) of cattle fetal serum (experimental group) and without it (control group) for 60 minutes at +37°C with constant stirring. After cultivation, the total number of cells, the number of mycelial structures, and their ratio in each group were counted. Then it was calculated how many times the relative number of mycelial structures in the experimental group exceeds this value in the control group. This value was taken as the intensity of formation of mycelial structures.
Sensitivity to antifungal drugs.
The sensitivity of isolated isolates to antifungal drugs was determined by agar diffusion according to the procedure described in CLSI M44. The results obtained were interpreted according to CLSI M44s-3.
In the experiment, we used HiMedia discs containing 25 ^g of fluconazole and 1 ^g of voriconazole and Muller-Hinton agar modified for fungi (HiMedia).
Results
Growth rate at a temperature of + 37 ° C, Figure 1 (Annex).
Candida catenulata: 4.63±0.18;
Candida lipolytica: 4.31±0.16;
Candida albicans: 4.66±0.26.
The optical density of cultures of Candida lipolytica isolates grown at 37°C is significantly lower than the optical density of cultures of Candida albicans strains grown under the same conditions. This difference may indicate a lower growth rate of C. lipolytica under the conditions in which the experiment was conducted. No significant differences in OD between Candida catenulata and Candida albicans isolates were found.
Activity of proteolytic enzymes and phospho-lipases, Figure 2 (Annex).
Candida catenulata: 0.92±0.04 (proteinases), 0.94±0.04 (phospholipases);
Candida lipolytica: 0.96±0.02 (proteinases), 0.95±0.04 (phospholipases);
Candida albicans: 0.8±0.04 (proteinases), 0.84±0.04 (phospholipases).
All the differences obtained by comparing the studied isolates with the control strains in terms of enzyme activity are statistically reliable. Based on these data, it was concluded that proteolytic enzymes and phospholipases of Candida catenulata and Candida lipolytica isolates have less activity in comparison with the control Candida albicans strains under the experimental conditions.
Intensity of formation of mycelial structures, Figure 3, Figure 4 (Annex).
Candida catenulata: 3.37±0.28;
Candida lipolytica: 2.66±0.2;
Candida albicans: 4.08±0.18.
All the differences obtained by comparing the studied isolates with the control strains in terms of the intensity of mycelial structures' formation are statistically reliable. Based on these data, it was concluded that Candida catenulata and Candida lipolytica isolates less intensively form mycelial structures - true and pseudomycelium in comparison with the control Candida albicans strains under the experimental conditions.
Sensitivity to antifungal drugs, Figure 5, Figure 6 (Annex).
3 Candida catenulata isolates and 8 Candida lipolytica isolates were determined to be resistant to fluconazole (19 and 67%, respectively), with a growth suppression zone diameter >14 mm.
3 Candida catenulata isolates and 7 Candida lip-olytica isolates were found to be resistant to voriconazole (19 and 58%, respectively), with a growth suppression zone diameter >13 mm.
Findings
The virulence potential of the studied Candida ca-tenulata and Candida lipolytica isolates was characterized as lower in comparison with Candida albicans. Indicators of growth rate at +37°C, activity of proteolytic enzymes and phospholipases, intensity of formation of mycelial structures in the studied isolates were significantly lower than in the control virulent strains of Candida albicans.
More than half of the studied Candida lipolytica isolates were resistant to fluconazole or voriconazole, and cross-resistance to both drugs was observed in 7 of
8 isolates; among Candida catenulata isolates the proportion resistant to fluconazole or voriconazole was 19%.
Discussions
A number of authors used similar methods to determine the virulence of Candida yeast fungi. In the first case, clinical isolates of Candida parapsilosis were studied and their virulence was confirmed in the laboratory conditions [20]. In another study [21], the diversity of yeast fungi inhabiting the gastrointestinal tract of broilers [Gallus gallus d.], the phospholipase and proteolytic activity of isolated yeast fungi, as well as their sensitivity to antifungal drugs were studied.
Cross-resistance to several drugs has been repeatedly described in the literature [22, 23], and such data correlate well with the known mechanisms of resistance to azole antifungal drugs [24].
The number of drugs used to treat yeast infections is small, especially in comparison with antibacterial antibiotics. These are mainly azoles: fluconazole and voriconazole, which are the first-line drugs, ketocona-zole, clotrimazole, miconazole, itraconazole and some others. The cases of cross-resistance, reducing the already small list of available drugs, undoubtedly pose a danger to public health.
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Already as of 2011, the proportion of resistant Candida fungi isolates was quite high [25], and continues to grow steadily [26]. Most of these data were obtained for pathogens of nosocomial infections, as well as isolates isolated from humans [27, 28, 29]; antifun-gal drugs were highly likely to be used by such patients.
Recently, pathogens of fungal infections that are resistant to most of the available drugs have appeared, Candida auris is an excellent example of such a pathogen [30, 31, 32]. According to the available data, this yeast fungi, that initially was a soil saprophyte, acquired both virulence [33] and resistance to antifungal drugs; a potential mechanism of such changes was described using the example of other yeast species [34].
Conclusions
Our results raise some concerns because the isolates studied were isolated from animals in which azole antimycotics are not used in therapy, or from environmental objects, and these isolates already have a high level of resistance, including cross-resistance. The isolates studied are also virulent.
We will continue studies of fungi, including yeasts isolated from animals and their habitats, for their potential virulence and pathogenicity, resistance to antifun-gal drugs and their potential for further spread.
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