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Molecular Identification and Virulence Factors Determination in Candida Species Isolated from Egyptian Patients

Nehal Ahmed, Dina E Rizk, Manal El Said*, Rasha MF Barwa, Mohammed Adel Elsokary, Ramadan HI Hassan

Microbiology Department, Theodor Bilharz Research Institute, Giza, Egypt

Abstract: Candida-related infections are becoming a universal threat to the health of human who undergo immunosuppressive therapy or aggressive medical intervention. Objectives: The aim was to study the distribution of Candida species among winter and summer seasons and to determine the expression of their virulence factors. Methods: A total of 164 Candida isolates were collected from clinical specimens at Mansoura University Hospitals. Candida species were identified by polymerase chain reaction (PCR). Extracellular phospholipase, secretory aspartyl proteinase (SAP) and coagulase enzymes and biofilm formation were determined. SAP 9 and 10 genes were detected by PCR. Results: Non-albicans (NAC) isolates were more dominant than C. albicans isolates (P value < 0.0001). C. tropicalis was the most prevalent (59.2%) followed by C. albicans (31.1%), then C. glabrata, C. krusie, unidentified NAC and C. kefyr in 3.7%, 2.4%, 2.4% and 1.2% respectively. Extracellular phospholipase activity was detected in 31.7% of Candida isolates. All C. albicans had phospholipase activity (100%) and one isolate of C. tropicalis was positive while other species were negative. SAPs activities were determined in 61.6% of Candida isolates and were detected in 70.1% and 62.7% among C. tropicalis and C. albicans isolates respectively. SAP9 and SAP 10 genes were detected in 27.7% and 12.9% of Candida isolates showed positive SAPs activity respectively and they were all C. albicans strains. Other species did not harbor either SAP9 or SAP10. Coagulase activity was detected in 80.4% of Candida isolates with higher activity in C. albicans (88.2%), followed by C. tropicalis (81.4%), then other NAC isolates. Biofilm formation was determined in 69.5% of Candida isolates and was more prevalent in C. tropicalis (82.5%) followed by C. albicans (19.6%), C. krusie (100%), unidentified NAC (75%), C. glabrata (33.3%) and C. kefyr (50%). Conclusion: NAC with a preponderance of C. tropicalis was the most common isolated Candida species. Biofilm production, proteinase, phospholipases and coagulase enzymes were observed in both C. albicans and NAC. SAP9 and SAP 10 genes were detected only in C. albicans strains.

Keywords: candida, polymerase chain reaction, phospholipases, proteinase, coagulase, biofilm, SAP 9, 10 genes

Received August 10, 2019; Revised September 22, 2019; Accepted October 24, 2019

Citation: Nehal Ahmed, Dina E Rizk, Manal El Said, Rasha MF Barwa, Mohammed Adel Elsokary, Ramadan HI Hassan, Molecular Identification and Virulence Factors Determination in Candida Species Isolated from Egyptian Patients. American Journal of Microbiological Research, 7 (4). 108-117. January 2019. doi:10.12691/ajmr-7-4-2

1. Introduction

Candida species are member of the human normal microbiota, colonizing the oral cavity, the gastrointestinal and genitourinary tracts in most of healthy individuals [1]. However, these species can become pathogenic in case the host’s normal flora is disrupted or the immunity is impaired [2]. Candida species are the most common cause of fungal infections in humans [3] with Candida albicans (C. albicans) being the most prevalent pathogen [4,5]. When host defenses become compromised due to hospitalization, treatment with antibiotics, surgery and the use of catheters and prosthetic devices, C. albicans can cause symptomatic infections, ranging from superficial skin or mucosal infections to life-threatening systemic ones [1,6,7].

The expanding spread of antifungal agents and the frequent use of broad-spectrum immune-inhibitors, changes the epidemiology of candidiasis with a shift in the prevalence of Candida species so that a reduced proportion of C. albicans and an increase in non-albicans Candida (NAC) species can be seen [2]. Emerging NAC species are causing increasingly high morbidity and mortality [5,7]. The rise in the incidence of Candida infections is complicated by the antimicrobial resistance and the limited number of available anti-fungal drugs [8].

The rapid and correct identification of Candida species can narrow therapy options by preventing treatment with potentially toxic antifungal agents, thus reducing costs of hospitalization and improving negative patient outcomes [2,9,10]. The precise identification of the strains at species and sub-species levels is highly demanded to perform epidemiological investigations and control the outbreaks [2]. Phenotypic and biochemical assays are frequently used for species identification. However, these assays are time-consuming, prone to error and delay appropriate antifungal treatment. Nowadays, polymerase chain reaction (PCR) is regarded as a standard platform in many clinical laboratories due to its affordability and reproducibility [9,11,12].

Candida species pathogenicity and its ability to cause infection is attributed to a set of virulence factors, including the ability to escape host defense, phenotyping switching, adhesion, biofilm formation and secretion of coagulase and hydrolytic enzymes [7,8,13,14,15]. Hydrolytic enzyme secretion during infection increases the ability of organisms for adhesion, invasion, as well as destruction of immune factors in the host, in addition to acquisition of nutrients. These enzymes include secreted aspartyl proteinases (Saps), phospholipases and hemolysins [6,16].

Secreted aspartic proteases (Saps) are encoded by a family of 10 SAP genes (SAP1- SAP 10) which have a vital role in virulence of Candida by degrading host tissue proteins as well as adhere to epithelial host tissue [5,8]. They degrade proteins related to immunological defense such as antibodies, complement and cytokines, allowing the opportunistic fungus to escape from the first line of host defenses [1]. Sap1 to Sap8 are fully secreted to the extracellular environment while Sap9 and Sap10 remain attached to the cell wall via a glycosylphosphatidylinositol anchor [17]. Sap9 and Sap10 enzymes maintain cell surface integrity of the Candida cell wall, and promote biofilm formation [8]. Sap9 is the most highly expressed Sap in strains isolated from patients with both oral and vaginal Candida infections [17]. All Candida species secrete proteinases, but non-C. albicans appear to do so at a lower level. These genes exhibit differential expression profiles at different stages and sites of infection [6].

Extracellular phospholipases are responsible for lipids digestion for nutrient acquisition, adhesion to host tissues, synergistic interactions with other enzymes, nonspecific hydrolysis and initiation of inflammatory processes by provoking cells of the immune system and self-defense [1]. The phospholipases catalyze the hydrolysis of phospholipids, the main part of cell membranes in humans, causing cell lysis that facilitate adhesion and penetration [6,18].

Compared with other enzymes, coagulase activity of Candida is the least studied with very few researches enumerated its activity. Coagulase is a protein enzyme which enables conversion of plasma fibrinogen to fibrin [19]. Candidal coagulase activity was first reported by Rodrigues et al [20] and previous studies have demonstrated that Candida species show varied coagulase activities against different animal plasma [21]. The exact mechanism of candidal coagulase enzyme in disease pathogenesis is not clearly elucidated [22].

Biofilms formed by Candida are a complicated three-dimensional structure, with yeast, hyphal, and pseudohyphal cells embedded in extracellular matrix [5,23]. Living within a biofilm has many advantages, which include protection against the environment, resistance to physical and chemical stresses, metabolic cooperation and joint regulation of gene expression for the associated microorganisms [1]. Candida presents in biofilm structure show a decrease in susceptibility to some anti-fungals and a reduction in killing by the host immune system [8]. Therefore, Candida infections associated with biofilms are often refractory and recurrent [23].

Expression of Candida virulence factors may vary depending on the infecting species, geographical origin, type of infection, the site and stage of infection, and host reaction [24]. Therefore, understanding the virulence factors will be a main tool to determination pathogenesis of candidiasis and also will help discovery new antifungal drug targets for improved therapeutic regimens [18].

Seasonality in pathogen dynamics influences the impact of disease on populations and can enhance pathogen spread [25]. Seasonal differences in host habitat can affect the transmission and persistence of pathogens by changing the contact with infective stages in the environment. Seasonal diversities in host immune function can also modify the growth of the pathogens within hosts [26]. Sociality fluctuates seasonally in many species and varies the transmission by increasing or decreasing contact rate [27]. Understanding the patterns and drivers of seasonality increases our understanding of disease effect on inhabitants and the rate of spread of invading pathogens [26].

The aim of this work was to study the prevalence of different Candida species isolates and their distribution among seasons using PCR for identification and to determine the expression of virulence factors by different species of Candida.

2. Methods

2.1. Candida Isolates

In this study, 164 isolates of different Candida species were isolated from Mansoura University Hospitals from different specimens including urine, sputum, bed sores, oral and throat swabs. Standard strain C. albicans (ATCC 10231) was used as control. The isolates were collected in two seasons; group I collected in May-August representing summer season isolates and group II collected from October-January representing winter isolates. All samples were inoculated on Sabouraud dextrose agar. Candida species were inoculated in yeast peptone dextrose broth with 20% glycerol and stored at -20°C [28].

2.2. Identification of Candida Species

Candida isolates were identified by their colony morphology, Gram staining, germ tube test and subculture on morphologic media (brilliance Candida agar) [29]. Molecular identification of Candida species were performed by the PCR using specific oligonucleotide primers (Table 1). For Candida DNA extraction, a colony was collected and inoculated into 20 μl of TE buffer. The mixture was heated for 10 minutes in water bath at 100ºC and, then heated in microwaves at high power for 2 minute [28]. Candida species were detected by the PCR using specific oligonucleotide primers (Table 1).

DNA samples were amplified in a reaction mixture that contained (2.5 μl) genomic DNA samples, 5 μl 5X Green buffer, 0.75 μl (10mM) dNTP Mix, 1 μl forward primer (10μM), 1μl reverse primer (10μM) (Table 1), 0.125μl Taq Polymerase, 1.5 μl MgCl₂ solution (25Mm), and distilled water (13.125 μl). For C. albicans, C. dubliniensis, C. glabrata, C. parapsilosis and C. tropicalis DNA was amplified in a PCR thermal cycler (Eppendorf PCR Master cycler) by running one cycle at 95°C for 3 minutes, then 40 cycles as follows: 60 s of denaturation at 94°C, 30s of annealing at 55°C and 45s of primer extension at 72°C. Following the last cycle, additional 10 minutes incubation at 72°C was carried out to ensure the complete polymerization of any remaining PCR products [30]. While for C. krusie and C. kefyr the PCR cycle parameters were as follows; preheating at 96°C for 2 minutes; then 30 cycles of 96°C for 30s, 57°C for 3s and 74°C for 60s [31]. The amplified genes were visualized by 1.5% agarose gel electrophoresis and ethidium bromide staining under UV transilluminator. The standard strain C. albicans (ATCC 10231) was used as control.

2.3. Virulence Factors of Candida Species

2.3.1. Detection of Extracellular Phospholipase Activity

An aliquot (10μl) of the yeasts suspension was inoculated onto Sabouraud egg yolk agar and incubated at 37°C for four days. Colony diameter and precipitation zone plus colony diameter were measured and interpreted for each isolate according to Mahmoudabadi et al [32]; negative (Pz value = 1), weak (+) (Pz value <0.90- 0.99), poor (++) (Pz value =0.80-0.89), moderate (+++) (Pz value =0.70-0.79) and strong (++++) (Pz value <0.70). The standard strain C. albicans (ATCC 10231) was used as control.

2.3.2. Detection of Aspartic Proteinase Activity

Candida isolates were suspended in saline to produce turbidity equivalent to a 0.5 McFarland standard. A 6 mm sterile filter paper discs were impregnated with 10 μl of the suspension and placed on the surface of the bovine serum albumin agar. The plates were incubated at 30°C up to 7 days. Enzyme activities were scored according to the criteria by Patil et al [33]; (1+) when the zone of agar clarification around the margin of the colony was 1-2 mm in diameter and (2+) when the zone was >2 mm (3 to 5 mm). Standard strain C. albicans (ATCC 10231) was used as control.

2.3.3. Molecular Detection of Sap9 and Sap10 Genes by PCR

Sap9 and Sap10 genes were detected by the PCR using specific oligonucleotide primers (Table 2).

DNA samples were amplified in a reaction mixture (12.5 μl) that contained (1.25 - 2.5 μl) genomic DNA, 2.5 μl 5X Green buffer, 0.375 μl dNTP Mix (10 mM), 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM) and (1.25- 2.5μl) template DNA and 0.0625 μl Taq Polymerase (500 μl), 0.75 μl MgCl2 solution (25 Mm), and distilled water (6.563 - 5.3125 μl). DNA was amplified in a PCR thermal cycler by running one cycle at 95°C for 5 minutes, and then 40 cycles as follows: 5s of denaturation at 95°C, 10s of annealing at 59°C and 30 s of primer extension at 72°C. Following the last cycle, additional 3 minutes incubation at 72°C was carried out to ensure the complete polymerization of any remaining PCR products.

The amplified genes were visualized by 1% agarose gel electrophoresis and ethidium bromide staining under UV transilluminator [34]. Standard strain C. albicans (ATCC 10231) was used as control.

Table 1. Primers for PCR amplification used for identification of Candida species and their oligonucleotide sequences

Table 2. Specific amplification and sequencing primer sets for SAP9 and SAP10 genes

2.3.4. Detection of Coagulase Enzyme Activity

Rabbit plasma was filter-sterilized and aliquots of the filtrates were placed in screw-cap tubes and frozen at -20°C until used [21]. Candida strains were cultured overnight in Sabouraud dextrose broth. Approximately 0.1 ml of each culture was inoculated into tubes containing 500 μl of rabbit plasma. The tubes were incubated at 25, 37 or 45°C and observed for clot formation after 2, 4, 6 and 24 hours. The standard strain C. albicans (ATCC 10231) was used as control.

2.3.5. Quantification of Biofilm Formation by Crystal Violet

Quantitative analysis of biofilm production was based on method described by Jin et al [35]. Briefly, a 20 ml aliquot of Candida cell suspension containing 3x10⁷ CFU/ml was inoculated into wells of microtiter plate containing 180 ml Sabouraud glucose broth and incubated at 35°C for 24 hours without agitation. The biofilm coated wells were washed twice with 200 μl of sterile distilled water and stained with 110 μl of 0.4% aqueous crystal violet solution for 45 minutes. Afterwards, the wells were washed 4 times with 350 μl of sterile distilled water and destained with 200 μl of 95% ethanol. After 45 minutes of destaining, 100 μl of destaining solution was transferred to a new plate. The amount of the crystal violet stain in the destaining solution was measured with a microtiter plate reader at 595 nm. According to Stepanovic et al [36], the classification of the results obtained has four categories; non-adherent (0), weakly (+), moderately (++), or strongly (+++) adherent, based upon the optical density (ODs) of bacterial films. The standard strain C. albicans (ATCC 10231) was used as control.

3. Results

3.1. Identification of Candida Species by Polymerase Chain Reaction (PCR)

In this study, 164 of Candida isolates were collected in two seasons; group I isolates (80/164; 48.8%) in summer season and group II isolates (84/164; 51.2%) representing winter season. The most prevalent Candida species was C. tropicalis with isolation rate of 59.2% (97/164) followed by C. albicans in 31.1% (51/164), then C. glabrata, C. krusie and C. kefyr isolates in 3.7% (6/164), 2.4% (4/164) and 1.2% (2/164) respectively (Figure 1). Four (4/164; 2.4%) isolates could not be identified (unidentified NAC) by PCR. C. tropicalis was statistically significant prevalent than C. albicans (P value = 0.000004). Also, the total number of NAC isolates was significantly higher than that of C. albicans isolates (P value < 0.0001).

Distribution of isolated Candida species among the clinical specimens is illustrated in Table 3. Candida isolates were mostly recovered from urine specimens (77.4%; 127/164) followed by sputum (12.8%; 21/164) then bed sore, oral and throat swabs in 4.3% (7/164), 3.7% (6/164) and 1.8% (3/164) respectively. The distribution of Candida species among different season’s groups is illustrated in Table 4. It was found that C. albicans and C. kefyr have a higher significance occurrence among summer group (P value = 0.01 and 0.04 respectively), while C. glabrata was higher among winter group (P value = 0.0005).

Among the 164 studied isolates, 86 (52.4%) isolates were collected from males and 78 (47.6%) isolates from females. For male’s isolates, 62 (63.9%) were C. tropicalis followed by C. albicans (19; 37.3%), then C. glabrata (1; 16.6%), C. kefyr (1; 50%) and unidentified NAC (2; 50%). For female’s isolates, C. tropicalis was detected in 35 (36.08%), followed by C. albicans (32; 62.7%), then C. glabrata (5; 83.3%), C. krusie (4; 5.1%), C. kefyr (1; 50%) and unidentified NAC (2; 2.7%). There was significant difference between female and male group concerning the number of C. tropicalis (P value = 0.0001) as it was more prevalent among male group. C. glabrata, C. albicans and C. krusie were more prevalent among females (P value = 0.02, 0.01 and 0.0046 respectively).

3.2. Virulence Factors Determination in Candida Species

3.2.1. Detection of Extracellular Phospholipase Activity

Extracellular phospholipase activity was demonstrated in 31.7% (52/164) of Candida isolates. All C. albicans had phospholipase activity (51/51; 100%) and only one (1/97; 1%) isolate of C. tropicalis. Other species showed negative phospholipase activity. The level of phospholipase activity among Candida species and according to different seasons is illustrated in Table 4. There was significant difference between total positive phospholipase activity between summer and winter groups as summer group showed higher prevalence (P value = 0.006), while no significant difference were detected between summer and winter seasons among Candida isolates expressing strong, moderate or week levels of phospholipase activity.

3.2.2. Detection of Secretory Aspartyl Proteinases Activity

Secretory aspartic proteinase activities was determined in 61.6% (101/164) of Candida isolates and was detected among 70.1% of C. tropicalis (68/97), 62.7% of C. albicans isolates (32/51) and 25% of unidentifiable NAC isolates (1/4). No activity was detected among other species. Table 4 showed the level of secretory aspartic proteinase activity among Candida in different seasons. There was no significant difference between summer and winter groups concerning secretory aspartyl protease activity and their levels (P value >0.05).

3.2.3. Sap 9 and 10 genes Detection by Polymerase Chain Reaction

SAP9 and SAP 10 genes were detected in 27.7% (28/101) and 12.9% (13/101) of Candida isolates showed positive aspartyl protinase activity respectively (Table 4, Figure 2). All of them were C. albicans isolates. SAP9 or SAP10 were determined simultaneously in 4.9% (5/101). Other species did not harbor either SAP9 or SAP10. There was no significant difference between summer and winter groups concerning SAP9 and 10 genes (P value > 0.05).

Table 3. Distribution of isolated Candida species among the clinical specimens

Table 4. The distribution of Candida species and virulence factors among different season’s groups

Figure 1. Agarose gel electrophoresis of polymerase chain reaction. M: DNA marker (100 bp), lanes (1-7) were identified as C. tropicalis, lanes (8-9) were identified as C. albicans and lanes (10-13) were negative for C. parapsilosis identification

Figure 2. Agarose gel electrophoresis of polymerase chain reaction for SAP9 gene detection. M: DNA marker (50 bp), Lanes 2, 3, 6 and 7 were positive for SAP9 gene and lanes 1, 4, 5 and 8 were negative

3.2.4. Detection of Plasma Coagulase

Coagulase activity was detected in 80.4% (132/164) of Candida isolates. C. albicans isolates showed coagulase activity in 88.2% (45/51) followed by C. tropicalis (81.4%; 79/97), C. krusie (75%3/4), C. glabrata 50% (3/6), C. kefyr (50%; 1/2) and unidentified NAC in 33.3% (1/3). There was no significant difference in coagulase producing Candida species in winter group versus summer group among C. tropicalis, C. kefyr and unidentified NAC. There was significant difference in coagulase activity of C. albicans and C. krusei (P value= 0.001 and 0.01 respectively) with higher predominance in summer season while C. glabrata showed higher prevalence in winter (P value = 0.01) (Table 4).

3.2.5. Detection of Biofilm Formation

Biofilm formation was determined in 69.5% (114/164) of Candida isolates and were significantly more prevalent in NAC (91.2%; 104/114) species than in C. albicans isolates (8.8%; 10/114) (P value = 0.00001). Among the 114 biofilm producer Candida species, biofilm formation was more frequent in C. tropicalis of (82.5%; 94/97) followed by C. albicans (19.6%; 10/51), C. krusie (100%; 4/4), unidentified NAC (75%; 3/4), C. glabrata (33.3%; 2/6) and C. kefyr (50%; 1/2). Biofilm production among Candida species with their categories and statistically significant values in different seasons is illustrated in Table 4. Biofilm formation were more dominant among the total Candida species, C. tropicalis and C. glabrata in winter season than in the summer (P value = 0.017, 0.04, 0.04 respectively) whereas weakly adherence C. krusie isolates have more occurrence in summer season than the winter (P value = 0.04).

4. Discussion

Candida species are prevalent opportunistic fungi that become pathogenic in patients with reduced immune competence or in individuals with an imbalance of competing bacterial microflora [13]. Candida-related infections are becoming a universal threat to the health of human who usually undergo immunosuppressive therapy or aggressive medical intervention [9].

C. albicans is the most prevalent pathogen in mucosal and systemic fungal infections. However, NAC species, are now emerging as important contributors in series of disturbs ranging from mildly superficial mucosal discomforts to deadly disseminated bloodstream and deep-seated tissue infections [5].

Considering the variation of Candida species in susceptibility to antifungal agents, the rapid and accurate identification of the species may assist in finding an appropriate therapy for candidiasis [2]. Polymerase chain reaction (PCR) has been developed to help establish an early diagnosis of infection with the aim of allowing prompt initiation of antifungal therapy and improving patient outcomes [12].

The virulence of the Candida species is attributed to a wide variety of mechanisms including adherence, biofilm formation, extracellular enzymes secretion, and dimorphism [18]. Hydrolytic enzyme include secreted aspartyl proteinases (SPAs) and phospholipases [6]; SPAs have a potential role in pathogenicity through facilitating the invasion and counteracting the host defense system [8] and extracellular phospholipase lyses host cells to facilitate adhesion and penetration [18]. Biofilms protect Candida from the harmful effects of the host and the natural environment and increase their chance of survival [15].

In the present study, the prevalence of different Candida species isolates and their distribution among seasons was detected using PCR for identification and the expression of virulence factors by different species of Candida were determined.

In this study, 164 isolates of Candida species were collected in two seasons as group I (80/164; 48.8%) summer season isolates and group II (84/164; 51.2%) representing winter isolates. The isolation rate of Candida species was higher in the winter which was consistent with the results of other studies [37,38]. Edi-Osagie and Emmerson [39] evaluated the season of birth as a risk factor for the development of invasive Candida in preterm low-birth weight infants and found that 73% of Candida infections occurred during the months from September to February and they recommended the consideration of seasonal associations when targeting selective antifungal chemoprophylaxis. Our results was different from other reports who stated that the isolation rate of Candida species was highest in April and lowest in February [40,41]. Fungal infections by Candida occur easily in a body with a weakened immune system. Based on this fact, it is thought that the immune system is significantly weakened at the change of seasons leading to a susceptible condition of infection.

In our study, C. tropicalis (59.2%) was the most prevalent Candida species followed by C. albicans (31.1%). Less frequently isolated Candida species were C. glabrata, C. krusie and C. kefyr (3.7%, 2.4% and 1.2%) respectively. This result is in accordance with many studies that identified C. tropicalis as the predominant widespread pathogenic yeast species of the non-albicans (NAC) group [16,42,43]. The incidence of infection attributed to C. tropicalis species were reported to be varying in a range of 20-45% depending on infection site and geography [44]. In India, epidemiological data showed that 67-90 % of nosocomial candidaemia were as a result of NAC species, of which C. tropicalis was the most predominant [45]. Formerly, C. albicans was thought to be the predominant species that causes candidiasis in immunocompetent and immunocompromised individuals. However, infections due to NAC including C. tropicalis have elevated dramatically on a universal scale. Therefore, C. tropicalis is proclaimed to be emerging pathogenic yeast [5,44]. On contrary, other studies reported C. albicans as the most frequently identified species [46,47].

In our study, the virulence activity of different Candida species was determined with the detection of seasonality effect on the virulence expression. Extracellular phospholipase activity was demonstrated in 31.7% of Candida isolates. All C. albicans had phospholipase activity (100%) and only one isolate of C. tropicalis was positive while other species were negative. Our results were in agreement with many studies who stated that 100% of C. albicans demonstrated phospholipase activity [24,32,48,49]. Pinto et al [50] reported that phospholipase activity was detected only in C. albicans strains in a rate of 99.4%. Vidotto et al [51], believed that the correlation between phospholipase activity and germ tube formation in C. albicans facilitate the mucosa penetration. Several studies have shown that clinical isolates of C. albicans have higher levels of expression of extracellular phospholipase activity that allows C. albicans to acquired nutrients in host nutrient-poor niches and contributes to invasion [3,16,18]. Moreover, few studies reported phospholipase positivity among NAC isolates with low enzymatic activity [3,48,52] while Gokce et al [53] found that all NAC strains were phospholipase negative.

In the present study, secretory aspartic proteinase activities (Saps) was determined in 61.6% of Candida isolates with detection rates of 70.1% and 62.7% among C. tropicalis and C. albicans isolates respectively. Our results were in agreement with Kaur et al [54] who reported that the SAPs activity was determined in 69.23% of Candida isolates where C. tropicalis and C. albicans exhibited SAPs activity in 77% and 61.1% respectively. Another study by Sachin et al. [55] declared that SAPs activity was detected in 52% of C. tropicalis isolates. Nawaz et al [56] reported that NAC strains have higher protease activity than C. albicans where C. tropicalis showed the highest proteolytic activity. Controversy, another study stated that the highest proteinase expression was seen in C. albicans, followed by the NAC species [18]. The absence of SAPs activity in our study among other identified NAC species was in accordance with the previous reports [3,57]. The production of SAPs is a critical virulence trait in C. albicans, which destruct the surface proteins (albumin, keratin) and degrade the locally protective IgA and C3 component. This enables tissue invasion and resistance to the antimicrobial attack by the host [3,18].

In this study, SAP9 and SAP 10 genes were detected in 27.7% and 12.9% of Candida isolates showed positive aspartyl protinase activity respectively and all of them were C. albicans strains. Other species did not harbor either SAP9 or SAP10. These results are in agreement with Ikonomova et al [17] who stated that Sap 9 is the most highly expressed Sap in strains isolated from patients with both oral and vaginal Candida infections. Another study reported that Sap5 and Sap9 are the major SAPs expressed in vivo in mucosal biofilms [58]. Other Candida species in our results did not harbor either SAP9 or SAP10 which agreed with other reports who documented that C. glabrata and C. krusei do not possess any SAP genes [8,57]. While Sap1 to Sap8 are fully secreted to the extracellular environment, Sap9 and Sap10 remain attached to the cell wall via a glycosylphosphatidylinositol (GPI) anchor [17,58]. Sap9 seems to be predominantly located in the cell membrane, and Sap10 is located in the cell wall and membrane [5]. In contrast to all other members of the Saps family, the Saps 9-10 proteases monitored under in vitro and in vivo conditions are independent of pH and morphotype [59]. Sap 9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins, which mediate biofilm formation and promote adherence to host cells and invasion into epithelial cell layers [8,58]. The effects of SAPs on C. albicans virulence can be supported by the activation of other genes such as HWP1 that encodes the hyphal cell wall protein promoting C. albicans adhesion to different surfaces [5]. Kadry et al [8] declared that non-pathogenic Candida species usually has fewer genes encoding SAP than pathogenic species and this fact was confirmed by gene sequencing.

In this study, coagulase activity was detected in 80.4% of Candida isolates with higher activity in C. albicans (88.2%), followed by C. tropicalis (81.4%), then other NAC isolates. Our results is agreed with Rodrigues et al [20] who detected high coagulase activity in C. albicans (88.5%) and C. tropicalis (82.6%), but lower activities in other species using the coagulase tube test with rabbit plasma after incubation for 24 hours. Yigit et al [60] reported that coagulase activity of C. albicans (64.7%) isolates were higher than other species. In previous study, Padmajakshi et al [22] detected higher coagulase activity among C albicans isolates (68%) followed by C tropicalis (59%). In our study, the rabbit plasma were used in the coagulase tube test for the detection of coagulase activity as many studies [19,21,22] stated that the rabbit plasma appeared to give the best indication of coagulase activity, whereas sheep plasma was less sensitive and human plasma expressed no activity for any of the Candida species. These data indicate that rabbit plasma is the most appropriate medium for coagulase testing of Candida strains). Variations in coagulase production by Candida species may be related to their pathogenicity. Thus, laboratory detection of coagulase activity in clinical isolates of Candida species may help in the diagnosis of Candida-related infections [21].

In the current study, biofilm formation was determined in 69.5% of Candida isolates and was more prevalent in NAC species (91.2%) species than in C. albicans isolates (8.8%). These results were in accordance with Lahkar et al [18] who reported that of a total 33 (62.3%) biofilm positive isolates, significant production was observed in the NAC species. In a study by Mohandas and Ballal [61], a total of 73% of Candida species produced biofilm where only 51% of C. albicans isolates formed biofilm, which was significantly lower than the percentage of all NAC species. The later authors found that strong biofilm production was seen in C. krusei and C. tropicalis while weak biofilm production was detected in C. albicans. Several previous reports were in accordance with these results [62,63]. On the other hand, another study declared that C. albicans biofilms were bulkier than the ones formed by C. glabrata [3]. Candida biofilms formed both in vitro on abiotic surfaces and in vivo on biotic surfaces such as the oral and vaginal mucosa [8]. Because biofilms are associated with a protective extracellular matrix, the cells in biofilms are more resistant to conventional antifungal drugs and host immune factors [64]. Further, cross contamination through medical devices via biofilm is a major source of infection in hospitals [65]. It was found that SAP 9 and SAP 10 enzymes maintain cell surface integrity of the Candida cell wall, and promote biofilm formation [8].

5. Conclusion

The present study showed predominance of NAC in different clinical samples. Biofilm production, proteinase and phospholipases were observed in both C. albicans and NAC. The number of NAC isolates producing biofilm is more than the number of C. albicans producing this virulence factor. This result suggests that the biofilm production is more important for NAC strains and that C. albicans possess mechanisms other than biofilm production to establish infections. Our study showed that the percentage of NAC producing proteinase is higher than C. albicans, whereas C. albicans are higher producers of phospholipase than NAC. The knowledge on how the pathogen regulates the production of different virulence factors contributes to better understanding of the pathogenesis. Thus, innovative strategies to develop newer and safer antifungal agents are needed to suppress and eradicate the virulence factors involved in the pathogenicity of life threatening Candida species.

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