Development of Candida-associated denture stomatitis: new insights.

Despite therapeutic progress, opportunistic oral fungal infectious diseases have increased in prevalence, especially in denture wearers. The combination of entrapment of yeast cells in irregularities in denture-base and denture-relining materials, poor oral hygiene and several systemic factors is the most probable cause for the onset of this infectious disease. Hence colonization and growth on prostheses by Candida species are of clinical importance. The purpose of this review is to critically discuss several key factors controlling the adhesion of Candida species which are relevant to denture-associated stomatitis. Although there is some consensus on the role of surface properties, studies on several other factors, as the use of denture liners, salivary properties and yeast-bacterial interactions, have shown contradictory findings. A comprehensive fundamental understanding is hampered by conflicting findings due to the large variations in experimental protocols, while other factors have never been thoroughly studied. Surface free energy and surface roughness control the initial adherence, but temporal changes have not been reported. Neither have in vivo studies shown if the substratum type is critical in dictating biofilm accumulation during longer periods in the oral environment. The contribution of saliva is unclear due to factors like variations in its collection and handling. Initial findings have disclosed that also bacteria are crucial for the successful establishment of Candida in biofilms, but the clinical significance of this observation is yet to be confirmed. In conclusion, there is a need to standardize experimental procedures, to bridge the gap between laboratory and in vivo methodologies and findings and--in general--to thoroughly investigate the factors that modulate the initial attachment and subsequent colonization of denture-base materials and the oral mucosa of patients subjected to Candida infections. Information on how these factors can be controlled is required and this may help to prevent the disease. The societal impact of such information is significant given the magnitude of the candidosis problem worldwide.

INTRODUCTION

Candida infections receive increasing attention, presumably due to the increased prevalence worldwide. Numerous studies have shown that several Candida species possess a multitude of virulence mechanisms leading to successful colonization and infection of the host when suitable conditions occur. The recognition that Candida is an important pathogen has led to many laboratory studies evaluating these virulence attributes in an attempt to clarify the pathogenesis of the disease. The progress made in understanding some of these features, such as the mechanisms that result in adherence to surfaces79, cell surface hydrophobicity32, and saliva13 is very impressive though yet in many aspects inconclusive. Knowledge about how the adherence and biofilm formation process takes place and how to avoid or at least diminish Candida colonization are mandatory in clinical practice. This review aims to critically discuss several key factors controlling the adhesion of Candida species which are relevant to denture-associated stomatitis, to highlight areas of current controversy and to suggest future research.

Role of surface properties on Candida colonization

Fungi normally live as innocuous commensals and colonize various habitats in humans, notably skin and mucosa63,88. Commensal existence of oral Candida species varies from 20% to 50% in a healthy dentulous population79,88. As growth on surfaces is a natural part of the Candida lifestyle51, one can expect that Candida colonizes denture.

There is a large body of evidence indicating that Candida is able to adhere to acrylic resin dentures. This is the first step that may lead to the development of the infectious process and that may ultimately result in varying degrees of denture stomatitis of the adjacent mucosa13,15,84. Candida adheres directly or via a layer of denture plaque to denture base (polymethylmethacrylate – PMMA)7,23,86. Without this adherence, micro-organisms would be removed from the oral cavity when saliva or food is being swallowed.

It is well-known that innumerable factors are involved in the adhesion of Candida to the acrylic resin base, though contradictory results have been reported from in vitro studies68,78,93. Substrate surface properties, as surface charge, surface free energy, hydrophobicity, and roughness have all been reported to influence the initial adhesion of microorganisms8,104. Microbial adhesion on biomaterial surfaces depends on the surface structure and composition of biomaterials, and on the physicochemical properties of the microbial cell surface, again its surface charge and hydrophobicity4,11. Components of the resilient denture liners and acrylic resin may reduce the adhesion and inhibit the growth of Candida45,105,108.

(a) Surface free energy and surface roughness

Surface free energy is one of the main factors related to the development of denture related candidosis67. It is defined as the interaction between the forces of cohesion and adhesion and predicts whether or not wetting occurs113. A linear relationship between contact angle measurements on various types of substratum and Candida albicans adherence has been demonstrated, i.e. the higher the surface free energy, the higher will be the adhesion of microorganisms and alternatively, the more hydrophobic the surface, the less cell adherence is expected33,45,67.

Although the cited reports have found correlations between surface free energy and microbial' adhesion12, other factors should also be considered, such as cell surface factors, diet, salivary composition and secretion rates, and antibody titers, which are all controlling factors in plaque formation9 and could therefore influence yeast attachment. These many confounding factors might explain why recent studies have failed to show a direct correlation between surface free energy values and the adhesion of Candida species68,78,93,110.

Higher adherence of particular Candida species, e.g. C. tropicalis, C. glabrata and C.dubliniensis, when compared with C. albicans, might be attributed to their relative surface free energy values, since hydrophobic micro-organisms seem to be more adherent to acrylic surfaces. While there are no studies regarding hydrophobicity of C. tropicalis and C. dubliniensis, Luo and Samaranayake55 (2002) stated that C. glabrata is more hydrophobic than C. albicans.

Commonly used biomaterials exhibit significant differences in surface free energy. Heat-polymerized acrylic resin was reported to be more wettable than microwave-polymerized acrylic resin, due to acid-base interactions68,94.

Surface roughness is calculated as the arithmetic average deviation of the surface valleys and peaks of a given surface1. It directly influences micro-organisms initial adherence to surfaces, biofilm development, and Candida species colonization. Materials with the roughest surface usually exhibit higher yeast counts70,78,83,105. This happens because surfaces may serve as a reservoir, with surface irregularities providing an increased chance of microorganism retention and protection from shear forces, even during denture cleaning. In addition, these irregularities sometimes allow the entrapped microbial cells time to attach irreversibly to a surface98.

Quirynen, et al.79 (1990) postulated a threshold roughness value (0.2 μm) below which no effect on the adhesion should be expected. Smooth and highly polished surfaces are of utmost importance not only for patient's comfort but also for denture/restoration longevity, good aesthetical results, oral hygiene and low plaque retention101.

The presence of saliva is known to change this scenario. The nature of the substratum may influence the formation and the composition of the salivary pellicle, which layer may then become more relevant than the surface properties of the dental material itself30. It has been shown that saliva immersion decreases the surface roughness83 and surface free energy94 of acrylic resins. This might explain the general decrease of Candida species in those studies where specimens were coated with saliva. Saliva, its components and properties on Candida adherence and colonization is thoroughly discussed in the following paragraph Role of the salivary properties on Candida colonization.

The available studies on surface properties raise questions regarding the role of surface free energy and surface roughness. There is general agreement that the hydrophobicity of the cell surface and substratum is an important predictor in the adhesion process, i.e. surface free energy indicates the ease with which saliva spreads over a surface67,94. There is also consensus on the role of surface roughness and the initial adherence process, i.e. surface roughness is positively correlated with the rate of bacterial/fungal colonization of biomaterials. If such rougher surfaces become exposed to the oral environment, they may be more susceptible to micro-organisms adhesion and biofilm formation and lead to infections. However, no studies on the application of certain treatments on different substratum types have been reported (i.e. application of different treatments diminishes the number of yeasts but may lead to detrimental changes of the substratum). In vivo studies may lead to different outcomes when compared with in vitro studies.

(b) Denture liners surface and characteristics

New materials have been developed in order to reduce and redistribute occlusal forces from dentures that might damage the underlying mucosal tissues60,97. In recent years, the use of denture liners, either hard or soft, has increased.

Liners are needed in many clinical situations in which patients have thin, sharp, or badly resorbed residual alveolar ridges or chronic tissue irritation from dentures57,60. Even though these materials exhibit excellent tissue tolerance, one of the problems is the colonization of Candida spp. on and within the material. Fungal growth is known to destroy the surface properties of the liner and this may lead to irritation of the oral tissues. This is due to a combination of increased surface roughness and high concentrations of exotoxins and metabolic products produced by the fungal colonies57. This observation is the rationale why attempts have been undertaken to incorporate antifungal agents or antiseptics in these materials.

Unfortunately, conflicting adherence/colonization results are reported on these lining materials. Some in vitro studies reported significant inhibitory effects on C. albicans 21,112. More recent studies, however, showed only limited antifungal properties and no significant reduction on Candida adherence and colonization17,21,24,31,49,50,53,58,75,78.

As can be seen in Figure 1A and B and as was also reported previously105, denture liners, especially the soft ones, introduce a higher surface roughness. The porous surface texture of the material will entrap yeast cells (Figure 2A and B), leading to an increased (re)colonization in spite of the antifungals. Concomitantly, the nutrient-rich environment of the oral cavity might overrule any inhibitory effect induced by antifungals released from the denture liners31.

FIGURE 1A and B

Scanning electron microscopy of a soft denture reliner showing the extents of defect; it is notable to observe that the material not only exhibits porosities, but also show surface irregularities, which may turn into adhesion sites (A: x 40; B: x 100). Sample analyzed was prepared according to the manufacturer's directions (CoeSoft, GC America, Alsip IL, USA). It was subsequently mounted on a stub, air-dried, sputtercoated with gold (Balzers Union MED 010 evaporator), and examined with a Zeiss (Thornwood, NY) DSM940A scanning electron microscope at an accelerating voltage of 20.0 kV for surface characterization

FIGURE 2A and B

Adherence of Candida albicans and bacteria on a soft denture liner coated with saliva A – Note that bacteria and fungi are united. B – The sample was not coated with saliva; note that bacteria and fungi do not seem connected when compared to the coated sample

Even though some in vitro studies have shown limited inhibitory effects, a reasonable explanation on why lining materials do not keep their antifungal characteristics could be the constant bathing in saliva in the mouth. Saliva extracts the antifungal ingredients, possibly even within a short time after the denture is placed in the oral environment, or dilutes the concentration near the denture surface to below fungicidal concentrations. Moreover, the antifungal included might not be effective against the particular Candida species (or mixture of micro-organisms, see below) that is causing the infection. Judging the literature the need emerges to systematically evaluate liners against various Candida species in relevant assays, e.g. involving various Candida and bacterial mixtures and saliva.

Role of salivary properties on Candida colonization

The role of human saliva in the Candida adhesion process is still controversial68,73. Saliva shows a physical cleaning effect and innate defence molecules, including lysozyme, histatin, lactoferrin, calprotectin and IgA20,96, interact with Candida species, thereby decreasing adherence to and colonization of oral surfaces. Other components in whole saliva, including mucins20,25, statherin42 and proline-rich-proteins13,96 have been reported to adsorb to C. albicans, thereby facilitating adherence to saliva-coated acrylic resins2.

However, studies regarding the influence of whole saliva on Candida adherence are mutuality contradictory and no consensus can be found in the literature (Table 1). Several investigators reported that a saliva coating reduces the adherence of C. albicans in acrylic resin based materials6,59,65,66,68,72,78,86,110 Others showed increased adherence rates with saliva coating23,65,71,102. Three other research groups found no effect at all of a saliva coating41,72,97. A dynamic effect, depending on the morphological phase of C. albicans was also found84,91, where initially adherence was increased, but subsequently decreased after 24 hours.

TABLE 1

The effect of saliva on Candida species adherence/biofilm formation on acrylic surfaces, according to published data

Authors	Saliva Collection	Saliva Type	Candida Species	Effect on Candida spp.
Samaranayake, et al.86, 1980	Unstimulated	Whole	C. albicans	Reduction
	Stimulated	Parotid	C. albicans	No effect
MacCourtie, et al.61, 1986	Unstimulated	Whole	C. albicans	Reduction
Nikawa, et al.72, 1992	Unstimulated	Whole	C. albicans	No effect
Vasilas, et al.102, 1992	Stimulated	Whole	C. albicans	Increase
		Parotid	C. albicans	Increase
		Submandibular-
		Sublingual	C. albicans	Increased/reduced[1]
Edgerton, et al.23, 1993	Stimulated	Submandibular-
		Sublingual	C. albicans	Increase
		Mucin-free	C. albicans	No effect
Nikawa, et al.71, 1993	Unstimulated	Whole	C. albicans	Increase
Waters, et al.110, 1997	Unstimulated	Whole	C. albicans	Reduction
Radford, et al.81, 1999			C. albicans
Millsap, et al.65, 1999	Stimulated	Whole	C. albicans	Reduction/Increase[2]
San Millán, et al.91, 2000	Unstimulated	Whole	C. albicans	Increased/reduction[3]
Millsap, et al.66, 2001	Stimulated	Whole	C. albicans	Reduction
			C. krusei	Reduction
			C. tropicalis	Reduction
Ramage, et al.85, 2001	Stimulated	Whole	C. dubliniensis	Increase
Maza, et al.59, 2002	Unstimulated	Whole	C. albicans	Reduction
Bosch, et al.6, 2003	Unstimulated	Whole	C. albicans	Reduction
Jin, et al.41, 2004	Unstimulated	Whole	C. albicans	No effect
Ramage, et al.84, 2004	Stimulated	Whole	C. albicans	Increase[4]
Moura, et al.68, 2006	Stimulated	Whole	C. albicans	Reduction
			C. glabrata	No effect
			C. dubliniensis	Reduction/no effect[5]
			C. tropicalis	Reduction
Pereira-Cenci, et al.78, 2007	Stimulated	Whole	C. albicans	Reduction
			C. glabrata	Reduction
Tari, et al.97, 2007	Stimulated	Whole	C. albicans	No effect

dependent upon the donor

dependent upon the co-existence with other bacteria

dependent on Candida morphological phase

but decreased over time

dependent upon the substratum

Several reasons might explain these divergent results. The most important are probably differences in the use of stimulated versus unstimulated saliva, resulting in different protein composition and viscosity, hence protection103. Furthermore, different incubation periods, use of filtered or whole saliva, different saliva temperatures when performing the study, and the presence or absence of nutrients in the different studies may have interfered with cell viability and adherence capacity20,41,83,86. Obviously inter-individual variations in the composition of saliva affect the outcome of three component adherence system studies of substratum, saliva and yeast19,25,68,73,78.

In the oral cavity a denture is coated with a salivary pellicle, which provides receptor sites for the adherence of micro-organism28. Again surface roughness and surface free energy are confounding factors in the coating. Although surface characteristics are important in determining the final composition of an acquired pellicle and hence can dictate colonization of Candida species, there are only few studies where the effects of different types of acrylic resins on this process are compared67,83.

Studies dealing with the effect of saliva on adherence of Candida species, other than C. albicans, to acrylic resins in vitro and in vivo, indicate variable adherence levels66,68,78. C. dubliniensis counts have been shown to decrease25, increase85 or show no effect68 in the presence of saliva, while C. glabrata counts were not influenced by saliva in one study68 but decreased in another report78.

Thus there is contradicting evidence with regard to the relationship in vitro between saliva and Candida adhesion. In general it may be concluded that low molecular weight proteins are related to the adherence levels of Candida 10. This is in agreement with clinical studies20,74,80,96, where patients with low or impaired salivary flow and/or composition presented higher Candida species counts when compared with saliva from patients with normal salivary flow. Collectively this confirms the regulating role of saliva in inhibiting Candida species adherence.

Candida species' shift

The Candida species most often reported to be associated with oral mucosal lesions is Candida albicans. But C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, and C. dubliniensis have also been isolated from diseased tissues18,56,89,90. Recently a shift in disease-associated Candida species from Candida albicans towards these non-albicans species was observed48,87,107. While C. albicans is still by far the predominant isolate under inflammatory conditions34, C. glabrata emerges as the second most prevalent species, frequently isolated from acrylic denture surfaces and the palatal mucosa89. Candida glabrata used to be considered a non-pathogenic Candida species, but the increased use of immunosuppressive drugs, as a cure of the immunosuppressive syndrome, have now led to increasing C. glabrata infections with high mortality rates47. The explanation for this trend towards morbidity due to ''less pathogenic'' yeasts remains to be established, but it has already been suggested that the increased worldwide use of antifungals has contributed to this phenomenon92,95. Besides the shift from C. albicans to C. glabrata, there is increasing evidence that more than one Candida species may simultaneously colonize mucosal habitats, as reported for the oral mucosa22, tongue and palate92, both in healthy and diseased subjects.

Bacteria and Candida interactions

Microbial cell to cell communication plays an important role in the colonization process. Micro-organisms present in the oral environment interact with each other in many ways, such as by using each other's metabolic end-products, or by communicating more directly through signalling molecules5. Understanding the complex interactions between surfaces, saliva, eukaryotic and prokaryotic microorganisms during infections is crucial in developing prevention and treatment strategies. In studies on Candida biofilm formation and Candida susceptibility, the characteristics of the oral environment in which the biofilms are naturally formed should be mimicked as closely as feasible52.

The multicellular lifestyle of bacterial and yeast biofilms44,69 is induced by environmental stress and/or restricted nutrient supplies76. These cooperation lead to adaptation to natural stress responses and result in a balanced microflora62,64,76,77. In addition to various forms of metabolic dependence micro-organisms may co-aggregate, with two or more genetically distinct strains interacting through specific cell to cell recognition38. Such coaggregation has been observed between C. albicans and several other oral micro-organisms36,37,39 and is an important factor in the microbial colonization and progression of infections in the oral cavity.

Bacteria and yeasts also interact via quorum sensing (QS). Quorum sensing is a polymicrobial coordination within a microbial community, based on excreted small molecules triggering a genetic response when present in sufficiently high concentrations. QS occurs both in single species bacterial communities and in complex mixed bacterial-yeast communities16,43. A recent study35 showed that Candida hyphal formation can be modulated by Gram negative bacterial quorum sensing molecules. Particularly in the multispecies biofilm communities QS molecules may accumulate to high concentrations and hence are important in controlling physiology and homeostasis46.

Although studies on biofilm development and species interactions have, so far, focused largely on bacterial species it has become clear that synergistic interactions among micro-organisms increase the efficiency of the impropagation29,54. Oral biofilm are not random mixtures of micro-organisms; but organized structures though varying in space and time while modulating adherence and metabolic properties99. Immediately after brushing or prophylaxis, the surface will be recoated with salivary pellicle and the first pioneer bacteria will colonize. These "early colonizers" are followed by the "late colonizers", if the conditions of/in the biofilm become amenable for other species to survive40.

Although there is variability in composition of an oral biofilm community depending on patient dependent characteristics, the mere presence of a specific microorganism does not induce pathology. Typically this depends on a complex of micro-organisms-host interactions that modulate the host's response leading to inflammation. Depending on the local conditions, bacteria may provide fungi with compounds that activate virulence determinants of fungi109. This is not only important for Candida infections but also why Candida may be responsible for non-Candida infections induced by the patient's indigenous microflora27.

Several researchers have studied interactions among Candida and bacteria in an attempt to determine how oral bacteria may modulate Candida adherence and colonization. The influence of Streptococcus salivarius has been reported to decrease Candida adherence86, while cooperation between several Streptococci and Candida albicans has also been reported7,106. Other research groups assessed in vivo biofilms, with various plaque collection methods generally destructive to the biofilm structure14,26,82,84,111. In contrast, the new confocal scanning laser microscopy using molecular biological staining techniques may elucidate unsolved issues or even identify artefacts arising from traditional methodologies. A recent study using acrylic resin samples of denture wearers in vivo has shown that different subjects present different biofilm formation rates, architecture and densities3. Unfortunately, the only substratum tested was acrylic resin and there was no attempt to characterize the surface properties, which might have resulted in a better understanding of the process. Clearly, understanding the biofilm behaviour of Candida species under various environmental conditions is the key to the development of effective preventive measures for Candida infections100. Further studies are needed to establish whether or not these interactions are strain-specific and on which other parameters they depend. As a result it may be possible to identify the stages when C. albicans and other emerging pathogenic species can be targeted in treatment and prevention.

Future research and final remarks

From the literature the picture emerges that many factors determine Candida harbouring biofilms. These factors include surface properties, micro-organisms interactions, biofilm architecture, and saliva. Obviously it is tempting to study the individual parameters in simple mechanistic studies. However, the level of contradictions in the pertaining literature should be interpreted by assuming multiple interactions between the various factors. A meaningful study of Candida biofilms thus only seems possible when the various factors are studied in a comprehensive experimental design.

As recent studies are pointing to the role of multi-species biofilms on the onset of the disease, studies that may explain how such biofilms interact with surfaces and how to prevent their growth are important. Fungal adhesion may be greater in materials presenting higher surface roughness. Consequently, the rehabilitation material chosen in clinical situations has to be carefully considered. When the oral cavity is re-colonized after antimycotic treatment withdrawal in patients with oral candidiasis, the yeasts may be harboured in more remote sites of the material.

While the initial adhesion of Candida species is influenced by surface roughness, and may be influenced by the materials' surface free energy (question still under discussion), these characteristics should be evaluated in in vivo-like conditions. Indeed, the presence of a rehabilitation material that could favour health and avoid the oral cavity re-colonization is mandatory. Therefore, studies that could explore the factors related to initial re-colonization by Candida in different materials are of utmost importance. The relationship of denture base materials and their effect on fungal growth requires further investigation through epidemiologic, clinical, and basic research. These new studies may include surface characteristics, but other important matters discussed on this review are fundamental to facilitate treatment protocols. New research should be on multispecies biofilm, as close as possible to the in vivo situation. Furthermore, other emerging fungal pathogens, such as Candida glabrata, should be under investigation, as the results found for one Candida species (mainly Candida albicans) may not generally hold, again in experimental setups where other organisms and saliva are present.