In vitro evaluation of virulence factors associated with clinical isolates of Candida species from patient in tertiary care hospital in Central India

Background: Adherence to host tissues, morphological changes and secretion of extracellular hydrolytic enzymes are important virulence attributes of Candida species. These enzymes play pivotal roles in pathogenicity of Candida infection. Aim: The present study was conducted with an aim to determine in vitro phospholipase, haemolysin, Coagulase activities and biofilm production of Candida species isolated from various Clinical samples. Material and Method: A total of 156 Candida species isolated from various clinical specimens were identified up to species level by standard mycological techniques and was tested for extracellular hydrolytic enzyme activity and Biofilm formation. Results: 156 Candida species were isolated from 915 clinical specimens. Biofilm production was shown by 77 candidal strains (49.35%), phospholipase activity was observed by 74 candidal strains (47.43%). haemolysin activity was observed by 66 candidal strains (42.30%) and Coagulase production in 38 of Candida (24.35%) isolates. Conclusion: Both C. albicans and Non-albicans Candida (NAC) species produce extracellular hydrolytic enzymes. Since these enzymes are important to understand the co-relation between the species and infection, their detection is extremely important in the management of infections caused by various Candida species.


Introduction
The incidence of invasive fungal infection has increased steadily over the past two decades. With recent advances in medical and surgical intervention and increasing population of immunocompromised patients, the diversity and list of fungal pathogens continue to grow [1]. The virulence attributes of Candida includes hyphal switching, germination, surface recognition and production of extracellular hydrolytic enzyme [2]. Among these, extracellular hydrolytic enzymes play an important role in adherence and infection of Candida [3]. Recently, microbial biofilms have gained prominence because of the increase in infections related to indwelling medical devices. The advantages of forming a biofilm for the organism include protection from the environment, nutrient availability, metabolic cooperation, and acquisition of new genetic traits [4]. Research on Candida pathogenicity has been focused on hydrolytic enzymes, such as proteinases, phospholipases and lipases. Little information concerning Candida coagulase and hemolytic activities was found in a recent literature search.
Hemolytic and coagulase activities in Candida spp. are virulence factors less well-studied and their importance to pathogenicity require new and more rigorous studies [5].
The present study was conducted with an aim to determine in vitro phospholipase, haemolysin, Coagulase activities and biofilm production of Candida species isolated from various Clinical samples.

Aim:
To determine the virulence factors of Candida species isolated from various clinically suspected samples of Candidiasis. The study was approved by the Institutional research and ethics committee. Speciation of Candida isolates were done by evaluating the germ tube formation, assimilation, and sugar fermentation test. Culture on cornmeal agar was for demonstration of chlamydospores while culture on candid chromagar was for the identification of the species. 156 isolates were screened for extracellular enzymes production and Biofilm formation.

Determination of virulence factor
1) Phospholipase enzyme production 2) Haemolytic enzyme production 3) Coagulase production 4) Production of biofilm 1) Phospholipase enzyme production: The phospholipase activity of Candida isolates was detected by the method of Samaranayakeet al. [6] (color plate-1)

Preparation of media-
The egg-yolk medium consisted of 13.0g Sabouraud's dextrose agar (SDA), 11.7g sodium chloride, 0.11g Calcium chloride and 10% sterile egg yolk in 184ml distilled water. First, the components without the egg yolk were mixed and sterilized (autoclave). After autoclaving the egg yolk was centrifuged at 500 rpm for 10 minute at room temperature and 20ml of the supernatant was added to the sterilized medium.

Yeast suspension preparation-10 8 yeast cells/ml was prepared in sterile saline
Procedure-Extracellular phospholipase activity was detected by inoculating 5 µl of yeast suspension onto the surface of the egg-yolk medium and left to dry at room temperature. Candida albicans 90028 was used as Positive control. Culture plate was incubated at 37˚C for 2 days. The lower the Pz value, higher the phospholipase activity [7,8].
Haemolytic enzyme production: Haemolysin activity was assessed by the method of Manns et al. [9] on blood agar plate. (color plate-2) Media preparation-SDA supplemented 7% sheep blood and 3% glucose with a final pH adjusted to 5.6 ± 0.2 was used.
Procedure-10µl of previously prepared yeast suspension was inoculated onto plates and incubated at 37 °C in 5%CO 2 for 48 h.
Interpretation-Beta (β)-haemolysis was indicated by a clear, colourless zone surrounding the colony of the organism and alpha (α)-haemolysis showed a greenish zone. Gamma (γ) haemolysis was indicated by an absence of any apparent haemolysis or discolouration.
To represent the extent of haemolysin activity by different Candida isolates [Haemolysin activity (Hz) was determined by calculating the ratio of the diameter of the colony to that of the translucent zone of haemolysis (in mm). The assay was conducted on three In present study C.tropicalis shows the maximum Phospholipase activity. Phospholipase activity was seen in 54.08% NAC species and 36.20% C.albicans species. II) Coagulase production-Maximum Coagulase production was seen in C.tropicalis (28.76%) isolates in sheep plasma C.tropicalis (57.53%) was the most common biofilm producer

Discussion
We have done study on selective virulence factors such as phospholipase, hemolysin, coagulase enzyme production and biofilm.
Out of 156 Candida species studied, phospholipase activity was seen in 74 (47.43%) isolates. Previous studies have reported phospholipase activity in 30 to 100% of Candidal isolates. Similar observations were found in other Indian studies [14]. Vinita et al study indicates that even though all the isolated strains were pathogenic, not all strains of Candida produced phospholipase as virulent factors. The virulence of Candida species is attributed not to a single factor but to a combination of several factors, like proteinase, phospholipase, biofilm production etc. Phospholipase positivity among non-albicans Candida strains was reported by Vinita et al [14] and Sachin et al [11]. In present study among the NAC species C. tropicalis showed maximum phospholipase production (58.90%), so it means it is more invasive among various isolates. Similar observations were found in other Indian studies [15]. In our study Phospholipase was the major virulent factor expressed by C. tropicalis isolates. Researchers have reported contradictory findings regarding phospholipase activity in C. tropicalis. Investigators like Thangam et al [16] reported high phospholipase activity in C. tropicalis isolates among NAC spp. while others like Samaranayake et al [17] reported no activity. These inconsistencies in observations may be due to biological differences among the isolates tested [15].
Most of the studies on exoenzymes are focused on phospholipases and secreted aspartyl proteinases (Sap). Coagulase production and haemolytic activity of are the least studied [12].
In present study haemolysin activity was detected in 42.30% isolates of Candida species. Study by Sachin et al [1] in 2012 noted (51.8%) haemolytic activity and 48% in C. tropicalis. In our study all isolates showed ß type of haemolysis and C. tropicalis showed maximum haemolytic activity (50.68%). Haemolysin is another putative virulence factor thought to contribute to candidal pathogenesis. In particular, the secretion of haemolysin, followed by iron acquisition, facilitates hyphal invasion in disseminated candidiasis [5].

Research Article
Tropical Journal of Pathology & Microbiology Available online at: www.pathologyreview.in 192 | P a g e In the present study 24.35% isolates were coagulase test positive when tested with sheep plasma. None of the these Candida isolates reacted positively in human plasma. In the study by Sachin et al [11] in 2013 reported 30.1% isolates were reported coagulase positive. Coagulase binds plasma fibrinogen and activates a cascade of reactions that induce clotting of plasma. Rodrigues et al. [18] reported high coagulase activity in C.tropicalis (82.6%). Variations in coagulase production by Candida spp may be related to their pathogenicity. It is concluded that the laboratory detection of coagulase activity in clinical isolates of Candida spp may help the diagnosis of Candida-related infections.
Among the three virulence factors, Phospholipase (47.43%) was the most common virulent factor followed by Haemolysin production (42.30%) and Coagulase production (24.35%) in the present study.
NAC species 53/98 (54.08%) produced biofilm in comparison to C. albicans 24/58 (41.37%) showing relatively lower slime production. Also strong biofilm production was seen in cases of C. tropicalis compared to weak biofilm production that was seen in C. kefyr, C. krusei, and C.guillermondii.
Similar observation was seen in study by Yigit et al. [5] Studies, investigating biofilm production among Candida species detected higher levels of positivity among non-albicans Candida strains (51-60%) than that of C.albicans strains (8-12%) [8]. Biofilms are surface associated microbial communities which are firmly fixed within an extracellular matrix [19]. It limits the penetration of an antifungal agent through the matrix and protects the yeast cells from host immune response.
Virulence of Candida depends on the type of species and the clinical sample from which it has been isolated. Hence, significance of virulence determination in rational antifungal therapy cannot be ignored and should be adopted as a routine procedure in the laboratory.

Conclusion
In conclusion, it was seen that NAC spp. had greater biofilm-forming ability when compared to C. albicans species especially C. tropicalis showed the highest ability to produce phospholipase, haemolysin, Coagulase and biofilms. Species of Candida other than C. albicans which were previously considered as less or non virulent are now implicated in human disease. In our study extracellular hydrolytic enzyme activities was shown by C. albicans and NAC species. Detection of virulence factors like phospholipase, haemolysin, Coagulase and Biofilm formation will help to provide sufficient data in the establishment of a relationship between the infection and Candida species isolated.
This study will provide data about distribution of various Candida species and their Virulence markers among patients visiting our tertiary care hospital and our geographical area specially in and around Ujjain district (M.P.).

Detection of Virulence factor
Color plate-1 Color plate -2 Phospholipase enzyme Production Haemolysin enzyme production