Candida parapsilosis culture extracts: in vitro , antagonistic action against Candida albicans , Candida auris and Candida para-psilosis

: Fungal infections caused by Candida species has increased significantly in recent years. Additionally, resistance to conventional therapy is an aggravating factor causing high morbidity and mortality, especially in immunocompromised patients or those undergoing treatment with another antimicrobials. During the infection process, Candida species produces metabolites which can self-regulate population density, in addition to controlling several virulence factors and exerting action on other microorganisms. In view of this fact, the present study evaluated the in vitro anti-fungal activity of the pure culture extract of Candida parapsilosis against Candida albicans , Candida auris and Candida parapsilosis . Fungal culture extracts of C. parapsilosis were prepared in Sabouraud Dextrose Broth, extracted with ethyl acetate and dried in a rotary evaporator. Subsequently, tests were performed to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). The antifungal activity of the extracts against Candida species showed values of MIC ranging between 500 - 2000 µg/mL and a MFC range of 1414 µg/mL - 2000 µg/mL. Future investigations for the identification and composition of this fungal culture extract will provide insights about metabolites are present and involved in the antifungal activity shown here, contributing for the future to new therapeutic approaches in the control of infections caused by Candida.


Introduction
Candida species are the most common causal agents of infections in hospitalized patients with high rates of morbidity and mortality. The main etiologic agent of these infections is the Candida albicans yeast, however, in recent years, there has been an increasing incidence of infections caused by non-albicans species such as Candida parapsilosis [1]. The yeast Candida auris has gained prominence, as a pathogen of great concern for public health services, responsible for invasive infections with a high mortality rate. In addition, its rapid dispersion in healthcare environments, and its profile of resistance to multiple classes of conventional antifungals [2,3], impact all health area. The multidrug resistance associated with virulence and prolonged survival on surfaces makes this microorganism gain considerable attention from the scientific community [4]. The treatment for infections fungal agents consists of the use of topical or systemic antifungal agents or a combination of both, whose mechanism of action involves the main structure as cell wall or membrane constituents [5]. In recent years, the incidence of recurrent infections, with the emergence of strains resistant to the drugs available for treatment, has increased and become a challenge for the control of these infections [6,7]. Therefore, the search for new compounds with antifungal action has gained considerable scientific interest over the years, essential for new therapeutic practices in the control of these infections. The present study evaluated the antifungal effect of the fungal culture extract of Candida parapsilosis against Candida albicans, Candida auris and Candida parapsilosis.

Preparation of Extracts
To obtain the C. parapsilosis (CP) culture extract, colonies were subcultured on Sabouraud agar (DIFCO®) incubated at 35°C for 24 hours. After growing, new inoculation was performed into 500 mL of Sabouraud Dextrose broth (DIFCO®), following Mac Farland scale turbidity adjustment 10, and incubation at 35°C for 72 hours.
Subsequently, filtration was performed on a millipore 0.2 μm membrane and liquidliquid extraction, using ethyl acetate as counter-phase. The procedure was repeated three times to allow the total extraction of fungal metabolites from the culture media. The ethyl acetate phase was submitted to a rotary evaporator for drying. The resulting bulk compound was solubilized in sterile distilled water solution with 10% dimethyl sufoxide (Synth®).
Sterile control (containing 0.2 mL of RPMI) and growth control (containing only inoculum and RPMI) were prepared. The plates were incubated at 35°C for 24 hours. The minimum inhibitory concentration (MIC) of fluconazole was determined as the lowest concentration capable to inhibiting 70% of fungal growth (MIC70), and for the extract, the concentration capable to inhibiting 100% (MIC100). To determine the minimum fungicide concentration (MFC), an aliquot from each well was plated in Sabouraud Agar medium. The CFM was defined as the lowest concentration of the extract to inhibit visible growth in solid medium. All tests were performed in triplicate.

Results
MIC values for fluconazole of Candida species are described in table 1. C. albicans and C. parapsilosis strains showed values of inhibition between 0.25 and 2 µg/mL. For C. auris strains, the higher MIC value was observed for the clinical strain (> 128 µg/mL) when compared to the reference strain (8 µg/mL).
The antifungal activity of the extracts against Candida species showed MIC values between 500 -2000 µg/mL ( Table 2). C. parapsilosis strains showed greater sensitivity to the extract when compared to C. albicans strains. Regarding to CFM, for C. albicans and C. parapsilosis strains MIC value was 2000 µg/mL. The highest CFM value was observed for clinical C. auris (4000 µg/mL) and the lowest for reference C. auris (500 µg/mL) (Figure 1). Considering geometric mean of MIC values, the higher inhibition values were observed for C. albicans (2000 µg/mL), followed by C. parapsilosis (1260 µg/mL) and C. auris (1000 µg/mL). Regarding CFM, both C. albicans and C. parapsilosis had a higher geometric mean (2000 µg/mL), when compared to C. auris strains (1414 µg/mL) ( Table 3).

Discussions
The incidence of fungal infections increases worldwide, especially in immunocompromised people or those undergoing treatment with antimicrobials. Yeasts of the Candida genus are the most common microorganisms involved in this process, with high rates of resistance to antifungal therapy [9]. The C. albicans species is a commensal pathogen of the oropharyngeal cavity, skin, and human gastrointestinal tract, but in homeostasis disorders they evolve to clinical manifestations. which comprise superficial to systemic infections [10]. In this context, a wide variety of virulence factors such as polymorphism, protease lipases and biofilm formation are produced for colonization and invasion of the host tissue [11]. Despite C. albicans being the main etiologic agent in this category, the growing increases in species such as C. parapsilosis with strains resistant to antifungal drugs has changed this epidemiological profile [12]. The particular importance of the increased incidence of C. parapsislosis is due to its ability to form biofilms on intravascular devices and prosthetic materials, gastrointestinal colonization and transmission by colonized hands of health professionals, making it difficult to control [13].
Epidemiological data reported by Dizbay et al., showed a high incidence of candidemia by C. parapsilosis in non-neutropenic critical patients from a tertiary hospital in Turkey. At this reported data, C. parapsilosis corresponded to 77% of the cases against 23% of C. albicans with a high mortality rate (65%) [14]. Corroborating with these data, Mesini et al. presented a study about epidemiology changes of candidemia, reporting the increases cases of fluconazole-resistant by C. parapsilosis strains with mortality rates of 33% [15]. In contrast to the Mesini study, here the C. albicans, C. parapsilosis strains showed fluconazole-sensitive susceptibility profiles.
The MIC of extracts slightly lower for C. parapsilosis (1260 µg/mL) when compared to C. albicans (2000 µg/mL). In addition to the rise of C. parapsilosis, the emergence of C. auris and its resistance profile to available antifungal agents is a concern for global public health. Since 2009 infections caused by C. auris have been reported in more than 40 countries with strains isolated predominantly resistant to one or more classes of antifungal drugs [16]. Studies by Maphanga and colleagues revealed that up to 90% of strains isolated in South Africa were resistant to fluconazole [17].
In the present study the reference strain of C. auris presented a profile sensitive to fluconazole and, additionally to the extract (500 µg/mL) compared to the same clinical strain (2000 µg/mL). The differences in MICs values can be explained by individual biological characteristics of these strains, with previous exposure to environments that demand physiological changes as a defense mechanism for tolerance and survival of these microorganisms.
During the process of invasion and infection, Candida species generate metabolites in competitive environments. Such metabolites can self-regulate population density and controlling several virulence factors [18]. Oliver et al. showed in study the metabolic profiles of Candida spp. with 66 metabolites identified and quantified, associated with glycolysis or gluconeogenesis, tricarboxylic acid cycle, nucleic acid synthesis, amino acid and lipid metabolism [19]. The antifungal action of C. parapsilosis extract against strains of C. parapsilosis itself species when administered exogenously, are observed in other situations, such as farnesol, a molecule produced by C. albicans that regulates population density and several virulence factor [20]. However, in exogenous administration against strains of C. albicans present a high antimicrobial and antibiofilm potential [21,22].
Although the composition of the C. parapsilosis extract obtained is unknown, its antifungal potential against Candida spp is evident. Future investigations to identify the composition of this extract will allow further clarification of which metabolites involved in the antifungal activity shown here, contributing to new therapeutic approaches in infections control of yeasts, mostly the genus Candida.

Research Ethics Committee Approval: None.
Acknowledgments: Authors would like to the financial support of the National Council for the Improvement of Higher Education (CAPES) and the National Council for Scientific and Technological Development (CNPq), for the scholarships granted. We acknowledge the Multi-user Laboratory (LMU) at São Jose do Rio Preto (Famerp).

Conflicts of Interest:
The authors declare no conflict of interest.