Among the various factors, extracellular hydrolytic enzymes of which SAPs (secreted aspartyl proteinases) are considered to be one of the major virulence factors play a major role in over growth of the Candida, since these enzymes pave way to adhere, penetrate and for tissue invasion (Schaller et al., 2005).

Aspartyl proteinases are secreted by pathogenic species of Candida in vivo during infection. The enzymes are secreted in vitro when the organism is cultured in the presence of exogenous protein (usually bovine serum albumin) as the nitrogen source. Phospholipase enzymes, another important virulence factor, are associated with the function related to host cell damage, adherence and penetration (Kabir et al., 2012). They destroy phosphor-lipids in the host cell, hence results in the damage to the cell membrane, cell lysis and facilitating tissue invasion (Bhat et al., 2011). There are four secreted phosphorlipase, A to D (PLA, PLB, PLC and PLD). Their activity is very high during tissue damage because these enzymes carry out hydrolysis of one or more ester linkages of glycerophospholipids on the host cell membrane. Further-more, Candida albicans is able to acquire elemental iron from host tissues through haemolysin production, iron chelators (siderophores) and iron-transport proteins, which then is used by the fungus for metabolism, growth and establishment of infection in humans (Weinberg, 1978; Almeida et al., 2009). The ability of C. albicans to utilize hemoglobin as an iron source was first described by Moors et al. (1992). In humans, iron is found in some proteins, including hemoglobin (a component of erythrocytes). The first step of C. albicans infection in vivo involves binding to erythrocytes through receptors of the complement system. Next, C. albicans produces a hemolysis factor that induces lysis of the erythrocyte. This factor most likely corresponds to a mannoprotein bound to the cell surface of the fungus (Almeida et al., 2009; Watanabe et al., 1999). Almeida et al. (2008) observed that C. albicans caused greater damage to oral epithelial cells containing elevated concentrations of ferritin as compared to cells with lower iron levels.

Traditionally, antifungal drugs were developed either to inhibit or to kill the pathogenic organism. Because of the development of anti-fungal resistance to various anti-fungal drugs by the pathogen, there is a need to develop new antifungal strategy which specifically targets the virulence factors. The study of virulence factors provides a way to specifically target virulence of Candida sp. Therefore, the present study was conducted with an aim to determine in vitro phospholipase, proteinase, haemolysin, esterase activities and biofilm formation in oral Candida isolates.

Determination of haemolysin activity

To determine hemolytic activity, SDA (Oxoid) containing 7% sheep blood and 3% glucose with a final pH adjusted to 5.6 ± 0.2 was employed. Ten microliters of yeast suspension was inoculated onto plates; these were then incubated at 37°C for 48 h in aerobic condition. After incubation, a transparent/semitransparent zone around the inoculation site was considered as positive hemolytic activity (Manns et al., 1994). The ratio of the diameter of the colony to that of the translucent zone of haemolysis (mm) was used as the haemolytic index (Hz value).

Determination of esterase activity

To determine esterase activity, Tween-80 opacity test medium was used. The test medium with a pH adjusted to 6.8 consisted of 1% peptone, 0.5% NaCl, 0.01% CaCl₂ and 1.5% agar. After cooling the medium (50°C), 0.5% of Tween-80 was added. Ten microliters of previously prepared suspension was carefully deposited on the Tween-80 opacity test medium. This was then incubated at 30°C for 10 days in aerobic conditions. Esterase activity was considered as positive in the presence of a halo pervious to light around the inoculation site (Slifkin, 2000).

Determination of biofilm formation

Candida sp. was evaluated for biofilm formation using the method described by Melek et al. (2012). Sterile 96-well microplates were used to evaluate biofilm formation. Yeast culture was inoculated using a loop into a tube containing 2 ml of brain heart infusion broth (BHIB) medium with glucose (0.25%) and incubated at 37°C for 24 h. Then, all tubes were diluted at a ratio of 1:20 by using freshly prepared BHIB. From this final solution, 200 μL was placed into the microplate, which was then incubated at 37°C for 24 h. After incubation, the microplate was rinsed with PBS 3 times and then inverted to blot. Then 200 μL of 1% crystal violet was added to each well, followed by incubation for 15 min. After incubation, the microplate was again rinsed with PBS 3 times. Then 200 μL of ethanol: acetone mixture (80:20 w/v) was added to each well. They were read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader and the OD was recorded for each well. Three wells were used for biofilm formation and the arithmetical mean of 3 readings was used in analysis. Enterococcus faecalis ATCC 29212 was employed as the control strain. Sterile BHIB without microorganism was employed as the negative control. Samples with an OD higher than the cutoff value were considered positive, whereas those with lower value than cutoff were considered negative.

It has been reported that the enzymatic activity of Candida sp. may vary depending on the species and source of isolates (Mohandas and Ballal, 2002). In this study, out of 38 isolates, phospholipase activity was detected in 100% of the C. albicans. Tsang et al. (2007) also reported the same positivity rate of phospholipase activity, in samples from patients with oral Candida infection. Previous studies have reported phospholipase activity in 30 to 100% of Candidal isolates from various groups of patients and from various sites (Price et al., 1982; Wu et al., 1996). As shown in Table 1, 100% of C. albicans produced phospholipase, among the NAC species, C. tropicalis followed by C. krusei showed maximum phospholipase production. Phospholipase enzyme digests the host cell membrane phospholipid causing cell lysis and changes in the surface features that enhance adherence and consequent infection and hence phospholipase production may be used as one of the parameters to distinguish virulent invasive strains from non-invasive colonisers. About 90.9% of C. albicans showed proteolytic activity in the present study. C. tropicalis showed 100% proteolytic activity followed by C. krusei. This observation was similar to the reports given by previous workers (Marcos-arias et al., 2011).

It was noted that haemolysin activity was higher in C. tropicalis (77.7%) followed by C. albicans (63.63%). Manns et al. (1994) defined the condition under which C. albicans can display haemolytic activity and found that haemolysis is non-existent when no glucose is available in the culture medium. Rossoni et al. (2013) found that non-Candida species also produced same haemolytic activity as C. albicans. C. albicans secretes a haemolytic factor that causes the release of haemoglobin, which is then used as an iron source by the organisms. Watanabe et al. (1999) demonstrated that mannoprotein released from C. albicans bound to the band 3 protein on RBCs, thereby promoting their disruption. They detected the haemolytic activity in the culture supernatant of C. albicans in vitro. In the oral cavity, extracellular iron is bound mainly to lactoferrin, a protein present in saliva, while intracellular iron is stored as ferritin. Although, this element is bound to proteins and/or is present in the cytoplasm of cells, oral infections with C. albicans are frequent, suggesting that this yeast is able to take up different forms of iron from the oral cavity (Almeida et al., 2008).

About 59% of C. albicans expressed esterase activity (Pakshir et al., 2013; Aktas et al., 2002). It has been reported that both C. albicans and non-albicans Candida sp. express esterase activity. Rudek et al. (1978) demonstrated that esterase activity would appear to be a common feature of Candida species that are frequently isolated from clinical specimens. Kumar et al. (2006) reported that Tween 80 opacity test cannot be used as the sole phenotypic trait in the differentiation of C. albicans and C. dubiliancis though it appears to be simple, economical and easy method to perform for use in small clinical laboratories. Melak et al. (2012) detected that C. albicans showed esterase activity in aerobic conditions but not in anaerobic conditions.

Biofilm formation is one of the most important virulence factors of Candida sp. (Figure 1). Candida biofilms occur on tissue surfaces as well as the biomaterials of medical devices. As reported by Gultekin et al. (2011), no biofilm formation was detected in any C. albicans strains by microplate method, while it was found in 50% of non-albicans Candida sp. Demirbilek et al. (2007) also detected that the biofilm formation rate was higher in non-albicans Candida sp. than in C. albicans strains by the microplate method. In our study, the biofilm formation rates were found to be higher in C. tropicalis (88.8%) than C. albicans (86.3%). According to the above mentioned studies, the biofilm formation rate was higher in non-albicans Candida species as compared to C. albicans isolates.

It is necessary to understand the pathogenicity mechanisms of the Candida sp. for the development of new antifungal strategy. Developing anti-fungal therapies against selective target virulence factor is very crucial nowadays because of the multi-drug resistance developed by Candida sp. Hence, our study on virulence factors of Candida sp. pave way for the better understanding of the various virulence factors exhibited by Candida sp.

The authors did not declare any conflict of interest.