Investigating Biofi lm Production , Coagulase and Hemolytic Activity in Candida Species Isolated From Denture Stomatitis Patients

Objective: Oral candidiasis, in the form of Candida-associated denture stomatitis, represents a common disease in a large percentage of denture wearers, and Candida albicans remains the most commonly isolated species. In this study, we aimed to evaluate biofi lm production, coagulase and hemolytic activity of Candida species isolated from denture stomatitis patients. Materials and Methods: This study included 70 patients (31 female, 39 male). Forty-eight of the patients were found to have a positive culture. A total of 48 Candida isolates representing fi ve species, C. albicans (n=17), C. glabrata (n=10), C. krusei (n=9), C. kefyr (n=7) and C. parapsilosis (n=5), were tested. Their coagulase activities were evaluated by a classical tube coagulase test with rabbit plasma. A blood plate assay on 3% enriched sheep blood Sabouraud-dextrose agar (SDA) was used to determine their in vitro hemolytic activities. Biofi lm production was determined by a visual tube method. Results: Twenty-one Candida isolates exhibited coagulase activity, and the coagulase activities of the C. albicans (64.7%) isolates were higher than other species. C. albicans, C. glabrata, C. kefyr and C. krusei species demonstrated beta hemolysis. C. parapsilosis strains failed to demonstrate any hemolytic activities. Fifteen (88.0%) of the C. albicans strains were biofi lm positive. Six (35.2%) of these strains were strongly positive, 8 (47.0%) C. albicans strains were moderately positive and 1 (5.8%) C. albicans strain was weakly positive. Sixteen (51.6%) of the non-albicans Candida strains were biofi lm positive while 15 (48.3%) did not produce biofi lms. Conclusion: The results of this present study indicate coagulase, hemolytic activity and biofi lm production by Candida spp. isolated from patients with denture stomatitis. Investigations of these virulence factors might be helpful in gaining information about the possible virulence of oral Candida species related to denture stomatitis.


Introduction
Candida is a commensal fungus that harmlessly inhabits various niches of the human body, including the oral cavity, gastrointestinal tract, vagina and skin of healthy individuals [1].Candida has been recognized as a part of the normal oral flora that has no harmful effects on the host.There are 300 to 400 species of microorganisms in the oral cavity, including 20 species of Candida.The fungus is frequently isolated from various oral sites, including the tongue, cheek, palatal mucosa, dentures, dental plaque, dental caries and subgingival flora [2].It is estimated that the oral cavity is colonized by Candida in 40 to 60% of the population.Changes in the oral environment, which can be affected by tooth loss or denture wearing, can cause changes in the oral microflora [3].When considering only those who wear dentures, the proportion of the population colonized by Candida increase to 60-100%.C. albicans is the most common species, and its various strains account for almost 70% of Candida isolates.In addition to C. albicans, other species including C. tropicalis, C. glabrata, C. kefyr, C. krusei and C. parapsilosis are also typically isolated from denture and non-denture wearers [4][5][6][7][8].
Denture stomatitis is a term used to describe certain pathological changes of the mucosa under complete or partially removable dentures.These changes are characterized by erythema and usually localized to both jaws, less frequently found in the mandible, and are commonly associated with angular cheilitis and median rhomboid glossitis [6,[9][10][11].It is more common in females than males.Deficient oral and denture hygiene is an important predisposing factor because poor hygiene facilitates both the presence of yeast and bacteria in saliva, and their colonization of the oral mucosa and denture surface.Other factors such as reduced salivary flow, roughness and micropores on the acrylic surface, trauma, pH alterations in the denture plaque, diabetes, long-term use of steroids and immunological impairment may also predispose an individual to the development of denture-induced stomatitis.Although the dominant etiological factor at present appears to be a fungal infection, other factors must also be considered.Candida species have been identified in most or all patients [4,8,[11][12][13][14], and the involvement of Candida as a potential causative agent in denture-induced stomatitis ("rubber sore mouth") was first described in 1936 [11].C. albicans remains the most frequently isolated yeast in the oral cavity.However, other species have been isolated and are involved in the disease [4][5][6][7][8]11].
An important co-factor associated with the pathogenesis of oral candidiasis appears to be virulence of the infecting organism.Several factors, such as adherence, persistence, dimorphism and/or germ tube formation, phenotypic switching, interference with host defense systems, synergism with bacteria and the production of hydrolases (lipases and proteases such as the secreted aspartyl proteinase, phospholipase, esterases, and phosphatases), have been proposed to be Candida spp.virulence factors [15][16][17][18][19].
One of the major factors contributing to the virulence of Candida is its versatility.It adapts to a variety of different habitats for growth and can form surface-attached microbial communities known as biofilms.Biofilm formation is thought to be one of Candida's most important growth adaptations because biofilms allow it the possibility to colonize oral surfaces.The possibility that these biofilms could serve as a reservoir for disseminated infections, such as aspiration pneumonia and gastrointestinal infection, has been pointed out earlier [1,2].
The capacity of Candida spp. to colonize host tissue and cause tissue invasion has been associated with its ability to produce extracellular enzymes.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 [17,[20][21][22].Hemolytic and coagulase activities in Candida spp.are virulence factors less well-studied, and their importance to pathogenicity require new and more rigorous studies.
The present study investigated biofilm production, coagulase and hemolytic activities of Candida spp.isolated from denture stomatitis patients.To our knowledge, this is the first study analyzing coagulase activity among Candida spp.isolated from denture stomatitis patients.

Subjects
This study included 70 (39 male and 31 female) complete denture wearers that were recruited from the Oral Diagnosis and Radiology Department of the Faculty of Dentistry.Denture stomatitis was assessed according to Newton's classification [6].

Mycological examination
Oral swab samples were obtained from the affected palatal mucosal site under the denture by passing a sterile cotton swab.Each swab was then transferred into 1 ml sterile phosphate-buffered saline and rinsed by vortexing to remove the yeast cells from the swab.From the diluted samples, 0.1 ml was used to inoculate Sabouraud dextrose agar (SDA) (Merck/Germany) supplemented with 1% chloramphenicol.Plates were incubated at 37°C for 48 h.After incubation, the isolates were identified by standard taxonomic procedures, which included germ tube production, typical microscopic appearances on cornmeal agar with Tween-80, production of chlamydospores, colony morphology and pigment production on chromogenic medium.Identifications were confirmed by the API 32 C AUX (bioMeriux, Marcy-l'Etoile, France) identification system for yeasts.

Determination of biofilm formation
Biofilm production was determined by visual methods.Colonies from the surface of SDA plate were inoculated into a polystyrene tube (Falcon conical tube with screw cap) containing 10 ml of Sabouraud-dextrose broth (SDB) supplemented with glucose (final concentration 8%).After incubation at 35°C for 48 h, the broth in the tubes was gently aspirated.The tubes were washed with distilled water twice and then stained with 2% safranin for 10 min.They were then examined for the presence of an adherent layer.Biofilm production was scored as negative (-), weak (+), moderate (++) or strong (+++).The biofilm producer Staphylococcus epidermidis ATCC 35984 was used as a positive control [23,24].

Assessment of hemolytic activity
Hemolytic activity was evaluated with a blood plate assay [20,25].Sheep blood SDA were prepared by adding 7 ml of fresh sheep blood to 100 ml of SDA supplemented with 3% glucose.Yeast strains were streaked onto SDA and incubated at 37°C for 18 h.Suspensions equal to McFarland 2 turbidity from the pure culture of the yeast colonies on SDA were prepared.Ten microliters of this suspension were spotted on sheep blood SDA (with 3% glucose).Plates were incubated at 37°C for 48 h.The presence of a distinctive translucent halo around the inoculum site indicated positive hemolytic activity.The ratio obtained by dividing the diameter of the colony by the total diameter of the colony plus the translucent halo was used as a hemolytic index (Hi) representing the intensity of the hemolysin production by the Candida species.

Statistical analysis
Statistical analyses were performed using SPSS version 13.0.To identify significant differences, the data were analyzed by analysis of variance using the general linear model, which permits the analysis of samples with different numbers of replicates.The mean values were separated by Duncan's multiple range tests.

Assessment of coagulase activity
A total of 48 Candida isolates were tested for coagulase activity using EDTA-rabbit plasma by a classical tube test.Approximately 0.1 ml of an overnight culture of each test strain in Sabouraud-dextrose broth was inoculated into a tube containing 500 ml of EDTA-rabbit plasma.The tubes were incubated at 35 o C and observed for clot formation at 2, 4, 6, and 24 h [12].The type strains Staphylococcus aureus ATCC 25923 and S. epidermidis ATCC 14990 were used as positive and negative controls, respectively, for coagulase expression [26].

Biofilm production by Candida spp.
Fifteen (88.0%) C. albicans strains were biofilm positive.Six (35.2%) of these strains were strongly positive, 8 (47.0%) strains were moderately positive and 1 (5.8%) strain was weakly positive (Table 1).Sixteen (51.6%) of the non-albicans Candida strains were biofilm positive while 15 (48.3%) did not produce biofilms.The distribution of strongly, moderately and weakly positive biofilm producers according to nonalbicans Candida spp.are shown in Table 1.2).The quantitative data indicated that the betahemolytic activities of C. albicans, C. glabrata and C. krusei showed significantly higher beta-hemolytic activities than C. kefyr (p<0.01)(Table 2).Further, there were no significant intraspecies differences in the beta-hemolytic activities among isolates belonging to C. albicans, C. glabrata and C. krusei (Table 2).3).

Discussion
The oral cavity provides a diverse number of surfaces including soft shedding non-keratinized buccal mucosal epithelia, the keratinized mucosa of the gums, the highly papil-lated mucosa of the tongue and the hard non-shedding surface of the teeth.In comparison with the dentate individual, the mouth of the denture wearer presents additional hard non-shedding areas and new environments (tissue-fitting surfaces) to support the growth of microorganisms and the development of plaque [9,27].
Oral candidiasis, in the form of Candida-associated denture stomatitis, represents a common disease in a large percentage of denture wearers, and C. albicans remains the most commonly isolated species in denture stomatitis [11].In this study, Candida spp.were isolated from the palatal mucosal surfaces of 48 (68.5%) cases.C. albicans 17 (35.4%)was the most commonly isolated species followed by C. glabrata 10 (20.8%), C. krusei 9 (18.7%), C. kefyr 7 (14.5%)and C. parapsilosis 5 (10.4%).Twenty-two (31.4%) patients had negative fungal cultures.In the present study, denture-induced stomatitis was more common among males (55.7%).The average male age was 62.8 (41-82), and the average female age was 55.6 .None of the patients had diabetes or an immune system-related illness, and none were using corticosteroid drugs for lengthy periods.
In the oral cavity, most colonizing and infecting microorganisms are not found as single-living cells but rather as complex structured microbial communities, which are often encapsulated within a matrix of exopolymeric material and attached to a biotic or abiotic surface.These communities are referred to as "biofilms".Yeasts cells adhere and colonize oral surfaces including mucosa and acrylic dentures and have the ability to co-aggregate with oral bacteria.The attachment of Candida to dental prostheses seems to be a critical event in the initiation of colonization and infection.This process may be especially important in the development of oral candidiasis and denture-related stomatitis, where Candida can adhere to the acrylic to form a reservoir for the chronic dissemination of fungal cells [9,[27][28][29].In addition, Candida cells in a biofilm exhibit distinct properties.The most significant property is resistance to several antimicrobial drugs, notably the popular azole drugs [7,10,[30][31][32].
In this study, we determined biofilm production in Candida strains isolated from denture stomatitis by tube adherence methods.The C. albicans strains were 88.2% positive for biofilm production.In contrast, C. glabrata, C. krusei, C. kefyr and C. parapsilosis were 60.0%, 44.4%, 57.1% and 40%, respectively, positive (Table 1).C. albicans exhibits a wide range of adhesion capabilities, and germ tube production may play a key role in the adhesion process [28,30].
Hemolysins are known to be putative virulence factors contributing to Candida pathogenesis, particularly facilitating hyphal invasion in disseminated candidiasis [36].The   [39].Tsang et al. [39] reported that the hemolytic activity of an oral C. albicans isolated from type 2 diabetes mellitus patients was significantly higher than those isolated from controls (a hemolysis index of 0.764±0.08 in the non-diabetic group vs. 0.673±0.06 in the diabetic group).In another study, Maia et al. [7] detected beta-hemolytic activities in C. albicans (2.08±0.389),C. glabrata (2.29±0.36)and C. krusei (1.97±0.33)strains isolated from the oral cavities of healthy elderly individuals.
Extensive research in Candida spp.has been focused on proteinases such as secreted aspartyl proteinases, phospholipases and hemolysins.Nevertheless, systematic studies on plasma coagulase are rare and, to the best of our knowledge, there are no studies on the coagulase activities of Candida spp.isolated from individuals with denture stomatitis.
In a previous study, Rodrigues et al. [17] detected coagulase activity in a large number of isolates from various clinical specimens, C. albicans (88.5%) and C. tropicalis (82.6%), and lower activities for other species using a rabbit plasma coagulase test after incubation for 24 h.Positive latex test results were obtained with 68.9% of the C. albicans strains, 56.5% of the C. tropicalis strains and 6.9% of the C. parapsilosis strains.None of the C. glabrata, C. guilliermondi or C. krusei strains had positive latex tests in the Rodrigues study [17].In another study, Becker et al. [40] observed that 40 (26.7%) of 150 yeast isolates had positive agglutination results from the Pastorex Staph-Plus LAT [24].In a previous study, we demonstrated coagulase activities for 125 Candida strains that represented 8 species isolated from different clinical specimens [21].
In the present study, the coagulase activities of 48 Candida isolates are shown in Table 3. Eleven (64.7%) C. albicans strains induced clot formation in a tube coagulase test with rabbit plasma.Coagulase activities were detected among other Candida species using a tube coagulase test with rabbit plasma.Three (30.0%) C. glabrata, 2 (22.2%) C. krusei, 3 (42.8%)C. kefyr and 2 (40.0%) C. parapsilosis strains were coagulase positive using a tube coagulase test with rabbit plasma (Table 3).
Our results also indicate the biofilm production of C. albicans was higher, and the beta hemolytic activities of C. albicans, C. glabrata and C. krusei were higher compared to C. kefyr.In addition, we report here for the first time the coagulase activity of Candida species isolated from denture stomatitis and that the coagulase activities of C. albicans strains were higher than those of non-albicans strains.Further studies on the simultaneous expression of candidal extracellular enzymes are urgently needed to understand the natural history and host-pathogen relationships associated with oral candidal infections.

Table 2 . Hemolytic activities of Candida species isolated from individuals with denture stomatitis
-: No activity.a : Within columns, the statistical difference between two means is given by the letters a and b.If the letters are different, the difference is significant (P < 0.01) by Duncan tests

Table 3 . Coagulase activities of Candida species isolated from individuals with denture stomatitis
[22]ida and Cryptococcus have been scarcely explored[37].A complement-mediated hemolysis induced by C. albicans has been reported by Manns et al.[25].Watanabe et al.[38]reported that C. albicans secretes a hemolytic factor that causes the release of hemoglobin, which is then used as an iron source by the organism.Luo et al.[20]studied 80 Candida isolates representing 14 species.These authors reported that C. albicans and others showed alpha and beta hemolysis.This study was the first to demonstrate variable hemolysin expression profiles by different Candida species[20].Recently, we reported that Candida species, including C. albicans, C. tropicalis, C. glabrata, C. kefyr and C. krusei, exhibit varying abilities to produce hemolysins on human, rabbit and sheep blood SDA supplemented with 3% glucose mediums[22].Studies on hemolysin activities from Candida spp.isolated from oral isolates are limited.The first study on a hemolytic activity from an oral cavity isolate of C. albicans was carried out byTsang et al.