Genotypic analysis of Candida albicans isolates obtained from removable prosthesis wearers

Aims: To assess of the genotypic diversity of Candida albicans isolated from removable prosthesis wearers, with and without denture‐related stomatitis (DRS). The occurrence of different genotypes in pathological and control cases was investigated. Methods and Results: One hundred and sixty‐four isolates of C. albicans obtained from different oral cavity locations were compared by randomly amplified polymorphic DNA (RAPD). The coherence of this analysis was confirmed by genotyping a selected group of isolates with pulsed field gel electrophoresis (PFGE). Among the 164 isolates, 150 were grouped into seven groups on the basis of their RAPD patterns. Three of these groups (comprising 54 isolates) had significant (α < 0·10) predominance of clinical or control cases. For the other isolates, no significant differences were observed between control and DRS cases. Occasionally, more than one genotype was found in the same person. These findings were sustained by PFGE analysis. No relevant associations between the genotypic patterns and pathology level were found. Conclusions: This study evidenced that C. albicans with similar genotypes may be found in individuals with DRS and in control cases. Significance and Impact of the Study: This conclusion hints the involvement of other aetiological factors that alone or in association with C. albicans may trigger the emergence of DRS.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

Denture contamination by yeasts in the elderly

Objectives: The aim of this study was to investigate yeast carriage in healthy denture wearers by swabbing and to evaluate the effect of denture hygiene habits. Materials and methods: Denture wearers (n = 87) without evidence of denture stomatitis or any other oral disease were investigated by separately swabbing the fitting surface of the upper denture and the corresponding palatal mucosa in contact with the appliance. In a group of volunteers, a gel without any active compound was spread on the palatal side of the denture once in every morning for 2 weeks. Results: Screening showed Candida colonisation of upper prosthesis in 75.9% of individuals. The most frequent species isolated were Candida albicans (77.9% of the positive cultures), Candida glabrata (44.1%) and Candida tropicalis (19.1%). Carriage of more than one yeast species was found in 48.5% of the contaminated dentures. There was a statistically significant association between denture contamination and palatal mucosa colonisation (chi‐squared test: p < 0.0001). Repeated swabbings after 1 week as well as during a weekly follow‐up for 1 month confirmed the denture contamination and its degree of severity. A daily gel application produced a yeast‐count decrease to 10% of the initial value after 2 weeks (chi‐squared test: p = 0.0134 and p = 0.2841 for prosthesis and palatal mucosa, respectively). Conclusion: This study documented the reliability of oral swabbing when investigating yeast carriage in healthy denture wearers. Moreover, just a diagnostic tool, sampling upper dentures for Candida could be the opportunity to verify the patient’s compliance to hygiene advice.     

Virulence Factors and Antifungal Susceptibility of Candida albicans Isolates from Oral Candidosis Patients and Control Individuals

Sixty isolates of Candida albicans, 30 obtained from the oral cavity of denture wearers presenting signs of candidosis and 30 obtained from the oral cavity of denture wearers with normal palatal mucosa were assayed for phospholipase and proteinase production, as well as for adherence to buccal epithelial cells. Likewise, susceptibility of the isolates to antifungals was determined by the NCCLS reference method and the E-test method. Proteinase activity was increased among the strains obtained from oral candidosis patients. In contrast, no significant differences between the two groups of isolates were observed in their adherence ability in vitro, in phospholipase production, and susceptibility to antifungal drugs.     

Investigation of extracellular phospholipase and proteinase activities of Candida species isolated from individuals denture wearers and genotypic distribution of Candida albicans strains

In order to determine the relationship between the development of denture related stomatitis (DRS) and the production of phospholipase and proteinase by Candida species, 156 Candida isolates isolated from the individuals in the control group and from the individuals different denture wearers were included in this study. According to the results of the study, C. albicans strains were determined to produce high levels of phospholipase and proteinase. It was also determined that the prevalence of phospholipase and proteinase activities in C. albicans strains isolated from individuals with DRS and from the individuals without DRS was not different. In order to determine genotypic variation of 109 C. albicans strains isolated, CA-INT-L and CA-INT-R primers specific to the site of the transposable group I intron of the 25S rRNA gene (rDNA) region were used. As a result, it was considered that, there were several other virulence factors belonging to the microorganism which played a role in the development mechanisms of the infection caused by C. albicans. In addition, according to the results of microbial genotyping, it was determined that there were no C. albicans strains specifically responsible for the development of DRS.     

Enhancement of <Emphasis Type="Italic">Candida albicans</Emphasis> Virulence After Exposition to Cigarette Mainstream Smoke

The habit of cigarette smoking is associated with higher oral candidal carriage and possible predisposition to oral candidosis. The effects of exposure to smoke on the virulence properties of oral yeasts remain obscure. Hence, we showed in vitro the effect of cigarette smoke condensate (CSC) on ten clinical isolates of Candida albicans obtained from nonsmoking volunteers, as well the type-strain CBS562. CSC was generated by complete burn of five commercial cigarettes in an in-house smoking machine and used to prepare the culture broth in which the strains were grown. In 24-h intervals (T₂₄, T₄₈, and T₇₂), the cells were harvested, washed, subcultured, and the resultant growth were evaluated for possible variations for secreted aspartyl protease, phospholipase, chondroitinase, and hemolysins, adhesion to acrylic and cell surface hydrophobicity (CSH). The results indicated a temporal increase in the secretion rates of enzymes, particularly when yeast cells were exposed to CSC for 48–72 h (P < 0.05). Similarly, adhesion to acrylic and CSH increased with exposure period (P < 0.05). Based on foregoing, we concluded that CSC may promote significant enhance in the secretion of candidal histolytic enzymes and adherence to denture surfaces, thereby promoting oral yeast carriage and possible infection.     

Relationship between salivary histatin 5 levels and Candida CFU counts in healthy elderly

Objectives: Few epidemiological studies have confirmed the antifungal activity of histatin 5 in saliva against Candida species. The purpose of this study was to examine the relationship between concentrations of histatin 5 and the number of cultivable Candida in saliva samples from elderly. Methods: Whole saliva samples were obtained from 124 elderly people, 65 years or older, living in a rural community. The concentrations of histatin 5 in saliva samples were determined by the enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody. Total colony‐forming units (CFUs) were counted on a selective medium for Candida. Multiple linear regression analysis was performed to determine the independent contribution of explanatory variables to Candida CFUs using age, sex, histatin 5 concentration and type of denture prosthesis as independent variables. Results: Saliva samples from 104 subjects (84%) were candidal colony‐positive. The youngest group (65–69 years old) showed significantly smaller Candida CFU counts than those in the older group. The mean Candida CFU count of denture wearers was significantly higher than that of non‐denture wearers. Significantly negative associations were found between Candida CFU counts and histatin 5 level in the oldest group (p < 0.05) and in the full‐denture wearers (p < 0.01). Multiple linear regression analysis revealed that Candida CFU counts were mostly associated with type of dentures, followed by histatin 5 concentration. Conclusion: Possible activity of histatin 5 against Candida in whole saliva of elderly people was epidemiologically confirmed. The area covered by the prostheses was a strong factor associated with the Candida CFU count.     

The Oral Microbiome of Denture Wearers Is Influenced by Levels of Natural Dentition

Objectives

The composition of dental plaque has been well defined, whereas currently there is limited understanding of the composition of denture plaque and how it directly influences denture related stomatitis (DS). The aims of this study were to compare the microbiomes of denture wearers, and to understand the implications of these towards inter-kingdom and host-pathogen interactions within the oral cavity.

Methods

Swab samples were obtained from 123 participants wearing either a complete or partial denture; the bacterial composition of each sample was determined using bar-coded illumina MiSeq sequencing of the bacterial hypervariable V4 region of 16S rDNA. Sequencing data processing was undertaken using QIIME, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. The dentures were sonicated to remove the microbial flora residing on the prosthesis, sonicate was then cultured using diagnostic colorex Candida media. Samples of unstimulated saliva were obtained and antimicrobial peptides (AMP) levels were measured by ELISA.

Results

We have shown that dental and denture plaques are significantly distinct both in composition and diversity and that the oral microbiome composition of a denture wearer is variable and is influenced by the location within the mouth. Dentures and mucosa were predominantly made up of Bacilli and Actinobacteria. Moreover, the presence of natural teeth has a significant impact on the overall microbial composition, when compared to the fully edentulous. Furthermore, increasing levels of Candida spp. positively correlate with Lactobacillus spp. AMPs were quantified, though showed no specific correlations.

Conclusions

This is the first study to provide a detailed understanding of the oral microbiome of denture wearers and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS, though we were unable to show a defined role for AMPs.     

Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva

doi: 10.1111/j.1741‐2358.2010.00390.x
Correlation between factors associated with the removable partial dentures use and Candida spp. in saliva Objectives: To correlate the presence and number of Candida spp. in the saliva of wearers of removable partial dentures retained with precision attachments with the proportion of metal/acrylic resin present in the dentures. Methods: Saliva samples from 40 removable partial denture wearers (test) and one paired sample of individuals, non‐wearers of any type of removable denture (control) were collected, seeded, and the colony forming units of Candida counted and identified. The metal/acrylic resin proportion of each denture was quantified, using silicone plates pressed over each denture. Results: Candida spp. was found in the saliva of 80% of the individuals in the test group and 65% of the control, with C. albicans being the most prevalent species. The test group presented with the highest number of colony forming units of Candida per ml of saliva, and there was weak correlation between this number and the metal and resin area of the denture (Pearson’s coefficient of correlation). Greater prevalence and a higher number of colony forming units of Candida per ml of saliva occurred in removable partial denture wearers (p = 0.04) with a weak positive correlation between the metal and resin area and the number of colony forming units of Candida per ml of saliva. However, this correlation was more significant for the area of resin.     

Effect of ill-fitting dentures on the swallowing duration in patients using polygraphy

doi: 10.1111/j.1741‐2358.2011.00536.x Effect of ill‐fitting dentures on the swallowing duration in patients using polygraphy Background: Surface electromyography (SEMG) has been widely used in the recent years to study swallowing physiology, offering a valid and reliable tool for identifying normal swallowing. The goal of our study was to assess the contribution of denture fitness in the age‐related increase of swallowing duration. Methods: Twenty denture wearers and 20 dentate individuals were analysed using SEMG and a computerised kinesiography of mandibular movement. Three spontaneous saliva swallowings were recorded for each patient with both their old and new prostheses. Three spontaneous saliva swallowings were recorded for each dentate person in two different recording sessions. Results: Old prosthesis mean swallowing time was 1.84 (SD ± 0.85) seconds while the new well‐fitting prostheses needed a 1.28 (SD ± 0.55) (p = 0.0009) swallowing time. The difference in swallowing time was significant (p = 0.01) between dentate subjects and individuals wearing an old prosthesis. No significant difference was found between dentate subjects and the same prosthesis wearers when a new well‐fitting prosthesis was worn. Conclusion: Data presented in this work suggest that part of the increased duration of swallowing showed by elderly and healthy people is because of incorrect an dental prosthesis. Prolongation of swallowing duration in the elderly population could be reconsidered in the light of the quality of dental device worn by the aged population.     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00506.x Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers Objective: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. Materials and methods: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher’s exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. Results: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue‐coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. Conclusion: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.     

The role of candidal histolytic enzymes on denture-induced stomatitis in patients living in retirement homes

Material and methods: Fifty nine elders wearing complete dentures and living in retirement homes in Curitiba (southern Brazil), were divided into two groups: group #1, 26 patients with denture‐induced stomatitis and group #2, 33 patients without denture‐induced stomatitis. The two groups were evaluated in relation to the degree of denture‐induced stomatitis, salivary fungal loads, and secretion of some histolytic enzymes. Results: Patients from group #1 showed higher degrees of colonisation by Candida albicans (p = 0.031). Candida krusei, Candida tropicalis, and Candida parapsilosis were also isolated, but there were no differences between the groups (p > 0.05). Secretory aspartyl protease (Sap) and chondroitinase did not show significant differences among the isolated Candida spp. in the two groups. Phospholipase secretion rates were higher among the strains of C. albicans from group #2 (p = 0.036). The same behaviour was not detected for non‐albicans Candida species. Conclusions: The results could infer that differences in the secretion rates of candidal histolytic enzymes should not be imputed as imperative for the progress of denture‐induced stomatitis.     

Ageing exacerbates damage of systemic and salivary neutrophils from patients presenting Candida-related denture stomatitis

Background  

Ageing leads to a decline in the function of the immune system, increasing the body's susceptibility to infections through the impairment of T-cells, macrophages, neutrophils and dendritic cells Denture stomatitis is a primary oral disease affecting elderly denture wearers. The major etiologic factor involved in this pathology is the infection by Candida albicans, an opportunistic pathogen that causes local and disseminated diseases in immunosuppressed humans. Neutrophils play a critical role in the immune response against C. albicans and are continually present in the salivary fluid and in the blood. The aim of this study was to determine ageing-related changes in salivary and blood neutrophils and their potential implications in Candida-related denture stomatitis.     

Oral mucosal lesions in denture wearers

doi:10.1111/j.1741‐2358.2009.00289.x
Oral mucosal lesions in denture wearers Objective: To determine the prevalence of oral mucosal lesions (OMLs) and denture‐related mucosal lesions (DMLs) in denture wearers and to co‐relate the prevalence with age, gender, type of denture and any systemic conditions. Materials and methods: Dental records of 380 denture wearers were retrospectively reviewed for OMLs and DMLs. Results: We found 45% of the denture wearers had DMLs and 60.8% had OMLs not related to denture wearing. Although the prevalence of DMLs was higher in complete denture wearers than in partial denture wearers (49% vs. 42.2%), this difference was not significant. The most common DMLs were traumatic ulcer (19.5%) and denture‐induced stomatitis (18.1%). When analysed by type, traumatic ulcer, denture hyperplasia, frictional keratosis and candidiasis were more common in complete denture wearers, whereas denture‐induced stomatitis was more common in partial denture wearers. Frictional keratosis was more common in men than in women. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers, and the most common OML was fissured tongue (27.6%). No association between DMLs and systemic conditions or xerostomic drugs was noted. Conclusion: No differences in the prevalence of DMLs in association with denture type were found. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers. This difference was affected by age, and the data were similar to the findings observed in the elderly.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Effect of denture adhesive on the micro-organisms in vivo

doi: 10.1111/j.1741‐2358.2010.00381.x Effect of denture adhesive on the micro‐organisms in vivo Background: Denture adhesives increase the retention and stability of dentures in edentulous patients, especially in cases where salivary flow is impaired or in the management of traumatised oral mucosa. Objectives: The effect of a denture adhesive on the oral flora at different time intervals. Method: Thirty denture‐wearing patients were involved in this study. While half of the group received a denture adhesive, the other half did not. At baseline, 1 and 2 months after delivering the dentures, smear samples were obtained from the saliva, palate and the dentures. Candida albicans, Candida krusei, Candida glabrata, Candida spp., Staphylococcus aureus, Moraxella catarrhalis, α‐haemolytic streptococci, β‐haemolytic streptococci, Pneumococcus aureus, S. anginosus, S. intermedius, S. constellatus, S. sanguis, S. gordonii, S. mitis, S. mutans, S. salivarius, and yeasts were investigated. The data were statistically analysed using anova and repeated measures. Results: Most types of the micro‐organisms were not seen and could not be analysed statistically except α‐haemolytic streptococci and C. albicans. No statistically significant difference was found for α‐haemolytic streptococci and C. albicans in saliva, palate and the denture at all time intervals. Conclusions: Prolonged use of the denture adhesive tested up to 2 months did not yield to increase in micro‐organisms of the oral flora.     

The effect of denture adhesives on Candida albicans growth in vitro

doi: 10.1111/j.1741‐2358.2011.00478.x
The effect of denture adhesives on Candida albicans growth in vitro Objective: Denture‐wearing favours the growth of Candida. In view of the fact that many denture wearers regularly use adhesives to enhance denture retention, stability and function, the aim of this work was to study the effect of denture adhesives on Candida albicans growth in vitro. Materials and methods: The denture adhesives tested were Corega^® cream, Kukident^® cream, Novafix^® cream, Polident^® cream, Protefix^® cream, Steradent^® cream, Aderyn^® powder, Corega^® ultra powder, Protefix^® powder and Corega^® strip. C. albicans growth curves were obtained in the presence or absence of a 1% solution of the denture adhesive diluted in Sabouraud broth. Macro‐ and microscopic morphological changes in C. albicans were analysed, as was microbial contamination of the denture adhesive. Results: Most of the denture adhesives studied induced morphological changes in C. albicans cells and colonies, but only two had any significant inhibitory effect on yeast growth. Kukident^® cream markedly inhibited C. albicans growth in a concentration‐dependent way, reducing the growth rate by 95%, whereas Corega^® cream also inhibited C. albicans growth but in a non‐concentration‐dependent way, reducing the growth rate by 37%. In addition, denture adhesives available as powders had detectable microbial contamination. Conclusion: Some commercially available denture adhesives showed microbial contamination and some had significant inhibitory effect on C. albicans growth.     

Candida-associated denture stomatitis

Candida-associated denture stomatitis was demonstrated by its cultivation in 171 out of 240 patients examined with partial or total dentures. After taking smears from lesions of the oral mucosa (tongue, cheeks, palate) and the contiguous denture surface by cotton wool swabs and inoculating them onto Sabouraud glucose agar and CHROMagarCandida, individual yeast species were identified by a germ tube. filamentous, and assimilation tests employing the commercial kit AuxaColor. SevenCandida species were identified in smears from the oral mucosa lesions and the contiguous denture surface:C. albicans (95 patients),C. tropicalis (26).C. parapsilosis (20),C. krusei (14),C. guilliermondii (12),C. lusitaniae (1) andC. freyschusii (1). Diabetes mellitus, neoplastic diseases, chemotherapy, radiotherapy, and broad-spectrum antibiotic therapy were identified as some of the large number of factors predisposing patients to stomatitis prothetica.     

Prevalence and Antifungal Susceptibility of Yeasts Obtained from the Oral Cavity of Elderly Individuals

Infections caused by Candida yeasts are common in elderly individuals. Seventy-five isolates of Candida spp. were obtained from saliva samples of 136 institutionalized elderly individuals resident in six retirement homes of Belo Horizonte, Brazil. Forty-seven isolates (62.66%) were identified as Candida albicans, 15 (20%) as C. tropicalis, 7 (9.33%) as C. glabrata, 4 (5.33) as C. parapsilosis, and 2 (2.67%) as C. guilliermondii. Among the 136 elderly individuals studied, 49 (36%) were male and 87 (64%) were female. Ages ranged from 60 to 90 years old. Sixty-three (46.3%) of the institutionalized individuals were denture wearers and, among them, 53 (84.1%) carried Candida yeasts in the oral cavity. Forty-four subjects presented lesions in the oral mucosa and among these, 36 (82%), had positive culture for Candida spp. The samples were tested for the in vitro susceptibility to amphotericin B, itraconazole, fluconazole and 5-flucytosin, and great variations were observed in the minimum inhibitory concentrations (MIC) of these drugs according to the species.