Candida - associated denture stomatitis . Aetiology and management : A review. Part 1. Factors influencing distribution of candida species in the Oral Cavity

Abstract Candida species are yeasts and within the oral cavity, Candida albicans is the most frequently isolated. There is clear evidence that C. albicans adheres to oral surfaces including acrylic dentures and mucosa. The mechanisms of attachment differ, with candidal adhesion to inert surfaces under the control of hydrophobic and electrostatic forces and adhesion to mucosa dependent on a number of complex ligand-recognition systems. Other factors within the oral environment such as saliva, pH, bacteria and hyphal formation have been shown to influence adhesion of candida species to surfaces in the mouth. Key words: Candida albicans, adhesion, hyphae, denture. (Received for publication January 1997. Revised April 1997. Accepted April 1997.) Introduction In recent years there has been an increasing interest in candidal infections in immunosuppressed and otherwise medically compromised individuals. This has resulted in a large volume of research data which has focused attention on Candida albicans as the primary aetiologic agent of candidosis, a disease which can vary from superficial mucosal lesions to a life-threatening systemic form. Oral candidosis in the form of candida-associated denture stomatitis is a common disease in some 65 per cent of denture we a r e rs, and C. albicans, Candida glabrata and Candida tropicalis have been isolated from such cases.1 This review focuses on the ecology of candida species in the mouth. Australian Dental Journal 1998;43:(1):45-50 Candida - associated denture stomatitis . Aetiology and management : A review. Part 1. Factors influencing distribution of candida species in the Oral Cavity B. C. Webb*† C. J. Thomas† M. D. P. Willcox‡ D. W. S. Harty* K. W. Knox* Australian Dental Journal 1998;43:1. 45 *Institute of Dental Research, Sydney. †Faculty of Dentistry, The University of Sydney. ‡Cooperative Research Centre for Eye Research Technology, The University of New South Wales. Factors influencing distribution of candida species in the oral cavity Yeast cells or blastospores are unicellular, eukaryo t i c organisms which multiply by a specific process of mitotic cell division known as budding. A nomenclature for the different morphological stages of candida development has been clearly defined and budding involves growth of new cellular material from a particular site on the blastospore surface.2When the bud has grown to optimal size, nuclear division occurs and a septum is formed between the two cell units. A hypha has been defined as a microscopic tube which contains multiple cell units divided by septa and which may arise from existing hyphae or from blastospores. The hyphae, which develop from blastospores, are known as germ tubes and they grow continuously by apical extension.2When blastospores are produced one from another in linear fashion without separating, a structure t e rmed pseudohypha is form e d .3 True septat e hyphae are produced by some candida species, such as C. tropicalis, under certain circumstances, but true hyphae are mainly associated with C. albicans. The entire candidal cellular aggregate including hyphae, branches and lateral buds is referred to as a mycelium.2 Macroscopic characteristics Candida species form soft cream-coloured colonies (Fig. 1) with a yeasty odour when grown under aerobic conditions on medium to assimilate lactose.3 Certain rare strains of C.tropicalis may assimilate cellobiose weakly and show an assimilation pattern similar to that of Candida parapsilosis. The inclusion of arabinose is useful, since C. parapsilosis readily assimilates this carbohydrate whereas most strains of C. tropicalis do not.3 The most frequently used yeast identification system is the API 32 C§ and this utilizes the carbohydrate assimilation tests described above. Microscopic characteristics The gross microscopic appearance of all the species is similar; all yeasts are Gram-positive but sometimes the shapes of the blastospores can vary from ovoid to elongated or spheri c a l .2M i c r o s c o p i c a l l y, C. albicans exhibits dimorphism (Fig. 2) in which there is a transition from ovoid budding blastospores ( yeast cells) to parallel-sided hy p h a e .2 Size also va ri e s , with measurements for C. albicans and C. krusei blastospores being given as 2.9-7.2_2.9-14.4 µm and 2.2-5.6_4.3-15.2 µm respectively.2 The cells of C. krusei appear elongated and have the appearance of ‘long grain rice’, and Candida kefyr (Candida pseudotropicalis) another clinically important species, has a similar microscopic appearance.4 Identification The production of pseudohyphae is one of the major differences between C. glabrata (a species that cannot form pseudohyphae) and other medically important candida species. Observation of germ tubes and chlamydospores (large thick-walled cells which develop at the tips of pseudohyphae) are also helpful in identifying C. albicans.3 All pathogenic candida species assimilate and ferment glucose as a carbon source, none assimilates nitrate as a nitrogen source, but they vary in their abilities to utilize other carbon and nitrogen s o u r c e s.2 Carbon assimilation and occasionally f e rm e n t ation studies are needed to differentiate candida species, for example, the clinically import a n t candida species, Candida guilliermondii is the only one to assimilate dulcitol and C. kefyr the only one pH in the range of 2.5-7.5, and a temperature in the range of 20°-38°C. Growth is usually detected in 48-72 hours, and sub-cultures may grow more rapidly. The ability of yeasts to grow at 37°C is an important characteristic to be considered in their identification from clinical specimens as most pathogenic species grow readily at 25°C and 37°C, whereas saprophytes usually fail to grow at the higher temperature.3 In contrast to the convex colonies of other candida species, Candida krusei grows as spreading colonies with a matt or rough whitish yellow surface.4 Fig. 1.–Colonies of Candida albicans grown on Sabouraud’s medium for 48 hours at 37°C. Fig. 2.–Scanning electron micrograph showing dimorphism in Candida albicans grown on denture acrylic for 48 hours at 37°C; budding blastospores and hyphae forming mycelium are clearly visible (bar=10 µm). Oral microbial ecology and distribution of candida species The opportunistic fungus C. albicans is the commonest of the candida species found within the oral cavity while other species which have been isolated include C. glabrata, C. tropicalis, C. kefyr, C. krusei and C. guilliermondii.5 Candida organisms, as commensal members of the normal oral microbiota, are present on average in 40 per cent (20-60 per cent range) of the human population.5 The primary oral source of C. albicans is considered to be the dorsum of the tongue and other oral sites such as mucosa and plaque-covered tooth surfaces are colonized secondarily.6 Candida species are also found as harmless commensals of the digestive and vaginal tracts. However, when the host’s defence system is impaired, as in medically compromised and immunosuppressed individuals, C. albicans infection can lead to the establishment of candidosis, which is manifested as superficial, involving the mucosa, or disseminated, which is the more serious invasive form.7 Oral candidosis is one of the most common fungal infections associated with HIV infection and C. albicans is the species most often isolated, with C.glabrata, C. tropicalis and C. krusei occasionally detected.8,9 C. kru s e i has been recently identified as an emergi n g nosocomial pathogen because of its pathogenic and clinical manifestations in hospitalized patients and immunosuppressed and otherwise medically compromised persons.4 Specific factors affecting distribution of oral candida Saliva Studies have shown that saliva reduces the adhesion of C. albicans to acrylic whereas serum, which may enter the oral cavity as a result of trauma to the palatal mucosa, enhances adhesion.10,11 In another study it was suggested that it was the salivary proteins or mucin that act as receptors for mannoproteins on the surface of C. albicans.12 This was confirmed by the work of Edgerton et al.13 and Hoffman and Haidari s ,1 4 who reported that C . albicans selectively adsorbs salivary mucins and that mucincontaining saliva enhances adhesion of yeast cells to acrylic. The reduction or complete absence of salivary secretion in individuals with xerostomia due to Sj¨ogren’s syndrome (a group of symptoms including enlargement of the parotid gland) has a profound effect on the normal oral microbiota. The reduced moisture level favours the growth of bacteria such as Staphylococcus aureus, which is resistant to dryness, and inhibits oral commensals adapted to high moisture levels.5 The study also showed that a low salivary pH and a high oxygen tension alter the oral e nvironment, and, as a result the numbers of veillonella, commensal neisseria and micrococcus species are reduced, while the growth of candida species, Streptococcus mutans and lactobacillus species is favoured. pH It has been suggested that an acidic environment favours the colonization of candida species,15 and low pH levels have been observed in denture plaque obtained from upper dentures of denture stomatitis patients on sucrose or glucose-rich diets.16 The effect of pH on the adhesion of C. albicans strains to mucosal surfaces has been shown to vary with the source of the mucosal cell and the strain.17 A recent study by Verran et al.18 has shown that C. albicans strains appeared to behave differently in response to a change of pH and that all strains were capable of adhering to buccal epithelial cells (BEC) at pH 7.3, 6.0 and 2.6, although adhesion was low. However, adhesion to BEC was increased when stationary phase cells were used instead of early exponential phase cells, with one strain producing hyphae at pH 2.6. In the same study pH did not significantly influence adhesion to acrylic except in one instance where the strain that adhered best did so at pH 7.3, while two strains isolated from cases of denture stomatitis produced hyphae at lower pH values. Adhesion The interactions between C. albicans and the host are complex, and several inve s t i g at o rs have suggested that the mechanism of attachment involves interactions between candida cell ligands and host cell receptors.19,20 The ligand receptors of C. albicans are thought to be mannoproteins, and it has been demonstrated that mammalian cell proteins iC3b, fibrinogen, fibronectin and laminin will bind to C. albicans as a result of candida CR3-like recognition.20 C. albicans produces extra-cellular polymeri c material which contains a mannoprotein adhesin. The interaction between candida and epithelial cells is thought to be one involving the protein portion of the mannoprotein adhesin and the fucose or Nacetylglucosamine- containing surface glycoproteins of epithelial cells.21 Recent inve s t i g ations indicate that the adhesion of C. albicans to host cells is dependent on the type of host cell and strain of the organism, and at least four different candida-host cell recognition systems have been descri b e d .2 2 , 2 3 The first invo l ves blastoconidium mannoprotein with lectin-like properties (mentioned a b ove), which recognizes the terminal fucose or Nacetylglucosaminecontaining glycosides of host epithelial cells. The second system invo l ves the CR2/CR3 complement receptor of C. albicans, which recognizes host endothelial cells. A 60 kDa mannoprotein extracted from hyphae appears to bind both the amino acid sequence argi n i n e - g l y c i n e - a s p a rt i c acid (RGD peptides) and non-RGD-containing ligands; the RGD ligands are important constituents of the extracellular mat rix of endothelial cells with fibronectin being one of the proteins containing the RGD sequence. Calderone2 2 also mentions two other systems which recognize receptors of epithelial cells, n a m e l y, one involving a mannan oligosaccharide and the other concerned with the chitin content of the candida cell wall. It was suggested that the existence of these ligand-recognition systems may explain the wide range of sites susceptible to invasion by candida species throughout the body.2 2 O d d s2 4 has indicated that the production of v i rulence fa c t o rs in candida species may va ry a c c o r d i n g to site, stage of invasion and nature of host response. Most investigators agree that stationary phase blastospores adhere better to tissue cells, mucosal cells and acrylic than those at the exponential (log) phase.11,25,26 C. albicans undergoes a change, producing an outer fibrillar-floccular layer from the cell surface when it is grown to the stationary phase in media containing high concentrations of specific sugars. This material together with electrostatic forces, is thought to be responsible for enhanced candidal adhesion to acrylic in vitro. In the oral cavity, colonization of the denture requires attachment to the salivary pellicle (consisting of adsorbed proteins and glycoproteins) covering the denture. Mannoprotein (cell surface polysaccharide) The cell wall of C. albicans is composed primarily of the polysaccharides mannan, glucan and chitin. The wall is of variable thickness and consists of several layers with differing electron density. The number of layers and their morphology varies and this is related to such factors as stage of growth, yeast form or germ tube, and growth medium but usually there are five layers within the cell wall. The outer fibrillar layer is composed of mannan or mannoprotein which is also found in different locations throughout the cell wall. Mannans represent about 40 per cent of the total cell wall polysaccharide or 15.2-22.9 per cent of the yeast cell wall (dry mass), while ß-1, 3-D-glucans and ß-1, 6-D-glucans form 47-60 per cent by mass of the cell wall. Proteins, lipids and chitin account for 6-25 per cent, 1-7 per cent and 0.6-9 per cent by mass of the cell wall respectively.20 The role of mannoproteins as candida cell wall adhesins has been discussed above. Cell surface hydrophobicity (CSH) Studies have shown that CSH is involved in the adhesion of blastospores to human epithelial cells and plastics. External cell wall protein changes in C. albicans are thought to be responsible for changes in hydrophobicity and hy d r o p h i l i c i t y.2 7 The hy d r o p h o b i c cells of C. albicans have been shown to bind diffusely and plentifully to host tissues, whereas the hydrophilic cells’ attachment is restricted to specific sites. Hydrophilic cells bind to regions with macrophages in contrast to hydrophobic cells which bind to tissue areas free of macrophages,28 earlier studies agreeing with these findings.29-31 This would indicate that hydrophilic cells are more easily removed from the body by phagocytosis than hydrophobic cells, which can colonize epithelial surfaces. Hydrophobic interactions which can operate over relat i vely long separation distances could possibly facilitate specific adhesin-receptor interactions, by bri n ging the surfaces closer together.28,32 It has been shown that the adhesion of candida species to plastic surfaces is controlled by attractive London-van der Waals forces (hydrophobic forces) and electrostatic forces. The ability of candida species to adhere to inert polymeric surfaces may give the organisms direct access to the human host.33 Oral bacteria Bacteria may contribute to the colonization and proliferation of candida strains in the oral cavity.34 A study has shown that the coaggregation of Strepto - coccus sanguis, Streptococcus gordonii, Streptococcus oralis and Streptococcus anginosus with C. albicans was enhanced by subjecting the blastospores to glucose starvation.35 It was suggested that the coaggregations involved proteincarbohydrate interactions, and it was shown that heat or protease treatment of starved candida cells eliminated their coaggregation with S. sanguis and S. gordonii.35 Another study demonstrated that C. albicans would not readily adhere to acrylic that had not been preincubated with streptococci, but did adhere to the adherent S. sanguis and Streptococcus salivarius.36 Branting et al. showed that adhesion of C. albicans to acrylic surfaces was enhanced when the yeast was incubated simultaneously with S. mutans.37 Hyphae There is agreement amongst most investigators that the hyphal form of C. albicans is associated with its invasiveness.20,26,38 It has been demonstrated that there is a strong correlation between germ tube formation and increased adhesion of C. albicans to BEC.38 The study showed that germ tubes of C. albicans exhibited enhanced adhesion to human mucosal cells, which it was suggested, could be one of the mechanisms related to virulence in candida species.38 It is thought that proteinases are produced during hyphal formation which help to disrupt the integrity of the oral mucosa.39-41 A number of factors regulate the transition of C.albicans from blastospores to hyphae. These include temperature and pH of the growth medium, the medium containing an inducer such as serum, NacetylD-glucosamine, L-proline, ethanol or a mixture of amino acids, as in Lee’s medium and va rious tissue culture media.2 , 4 2 - 4 6 It has been demonstrated in vitro that a temperature of 37-40°C, a pH of 6.5- 7.0 and an initial blastospore concentration not exceeding 106/mL are essential for growth of C. albicans hyphae within several hours.2 The same study also showed that at temperatures below 30°C and pH less than 6.0 most C. albicans strains on nutrient-poor media can form very long hyphae over a period of several days.2 Verran et al.18 demonstrated that at pH 2.6, adherent C. albicans were able to produce hyphae but suspended candida cells did not have this ability. A calcium-calmodulin interaction was shown to induce blastospore-hyphae transition, whereas u n r e s t ricted calcium uptake resulted in specific inhibition of C. albicans hyphal growth47,48 while another study demonstrated that carbon dioxide alone can induce germ tube form ation in C. albicans.4 9 Holmes and Shepherd5 0 found that exponential phase blastospores of C. albicans formed germ tubes after a period of glucose starvation followed by transfer to glucose ammonium ion medium at 37°C and pH 6.5. It was shown that the presence of both a carbohydrate and a nitrogen source was essential for blastospore-hyphae transition. Other studies have also demonstrated that the nutritional status of the blastospore is an important factor in regulating C. albicans morphogenesis.51,52 Odds2 considers that both morphological forms of C. albicans, blastospore and hyphae, appear to i n i t i ate and sustain pat h o l o gical responses in mammalian hosts. However, it seems that one form may be better adapted than the other to survive under specific ecological conditions. Enzymes Studies of phospholipase A and lysophospholipase activities have shown that C. albicans isolates that adhered most strongly to BEC and were the most pathogenic to mice, had the highest phospholipase activities.53 Some investigators have suggested that hyphae produced by C. albicans m ay possess phospholipase which allows entry to the host epithelial cells39 while Samaranayake16 reported that extracellular phospholipases and acid proteases of C. albicans were activated by low pH conditions. Proteinase has been shown to have an important influence on candidal adhesion to and invasion of oral epithelium.40 In another study it was demons t r ated that blastospores, after ingestion by macrophages, quickly express proteinase which is followed by increasing proteolytic activity with the f o rm ation of germ tubes by the ingested blastospores causing macrophage destruction.54 This paper has reviewed data relating to factors that influence the colonization of candida species within the oral environment and candidal adhesion to denture and mucosal surfaces. Part 2 of this review will be concerned with oral diseases caused by candida species. Acknowledgements This study was supported by a research grant from the Faculty of Dentistry, University of Sydney. The assistance of the Photographic Department, Electron Microscopy Unit, University of Sydney is gratefully acknowledged. References 1. Budtz-Jorgensen E, Stenderup A, Grabowski M. 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Demonstration of fungal proteinase during phagocytosis of Candida albicans and Candida tropicalis. J Med Vet Mycol 1990;28:3-14. Address for correspondence/reprints: Dr B. C. Webb, Institute of Dental Research, 2 Chalmers Street, Surry Hills, New South Wales 2010.