Oral Distribution of Genera, Species and Biotypes of Yeasts in Patients with Marginal Periodontitis

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1 æoriginal ARTICLE æ Oral Distribution of Genera, Species and Biotypes of Yeasts in Patients with Marginal Periodontitis Xiaobo Song 1,2, Jinglu Sun 2, B. Frode Hansen 1 and Ingar Olsen 2 From the 1 Department of Periodontology and the 2 Institute of Oral Biology, Dental Faculty, University of Oslo, Oslo, Norway Correspondence to: Xiaobo Song, Department of Periodontology, Dental Faculty, University of Oslo, P. B Blindern, NO-0317 Oslo, Norway. xiaobos@odont.uio.no Microbial Ecology in Health and Disease 2003; 15: 114/119 Yeasts can be members of the commensal flora in the healthy human mouth. They have also been found in periodontal pockets of patients with marginal periodontitis, but little is known about their distribution in other oral sites of such patients. The aim of the present study was to examine the distribution of genera, species and biotypes of yeasts in various sites of oral cavities of patients with chronic marginal periodontitis. Samples were taken from the periodontal pockets, buccal mucosa and palate of 70 marginal periodontitis patients and 45 healthy individuals. After cultivation yeast colonies were selected according to morphology and the biotype was determined by a commercial kit (ID 32C, biomérieux, France). Oral yeasts were detected in 34.3% of the marginal periodontitis patients and 42.2% of the healthy subjects. Among the sites examined, the buccal mucosa was the major site for yeasts, being colonized in 31.4% of the marginal periodontitis patients and in 35.6% of the healthy subjects. Two genera (Candida and Saccharomyces), four species (C. albicans, C. dubliniensis, C. parapsilosis and S. cerevisiae) and 19 biotypes were identified from the marginal periodontitis patients, while one genus (Candida), two species (C. albicans and C. dubliniensis) and 11 biotypes were obtained from the healthy subjects. C. albicans was the most dominant species in all the oral sites. Among the other Candida species, C. dubliniensis dominated, being most prominent in the periodontal pockets. Two biotypes ( and ) of C. albicans and one biotype ( ) of C. dubliniensis were predominant in the marginal periodontitis patients, whereas three biotypes ( , and ) of C. albicans dominated in the healthy subjects. The differences in the distribution of yeast species and biotypes may suggest cell, tissue or site tropism in oral yeast colonization. Key words: yeasts, biotypes, periodontitis. INTRODUCTION During the last 10 years there has been an increase in human yeast infections all over the world (1). Yeasts infect skin and mucosa locally, but can also cause life-threatening systemic infection. These organisms are frequent members of the commensal flora of the mouth with a prevalence of 2 /70%, the average being 34.4% in healthy humans (2). Oral yeast infections occur particularly among patients with HIV infection; patients on broad-spectrum antibiotics, corticosteroids or cytotoxic drugs; patients receiving irradiation therapy or subjects wearing intra-oral removable appliances. A number of other local and general factors can predispose to oral yeast infections (2, 3). In the mouth, Candida albicans is the most commonly isolated species, accounting for 70/75% of the isolates; C. glabrata and C. tropicalis, which are the second commonest species, each constitute about 7% (2). The prevalence of yeasts in periodontal pockets has been reported to be 14/19% (4/ 6) and it has been speculated, but not shown yet, that yeasts may contribute to breakdown of the tooth-supporting tissues in marginal periodontitis. There is little information on the distribution of yeasts in oral sites besides the periodontal pocket in periodontitis patients. Furthermore, it is often assumed that most cases of candidosis originate from the commensal strains inhabiting the oral cavity, vaginal canal or gastrointestinal tract prior to infection (7/10). Some studies have suggested that indigenous yeasts are replaced by unusual phenotypic or genotypic Candida strains in oral candidosis (11/13). However, no consistent associations between pathogenicity and biotypes or genotypes of C. albicans have been reported so far (14/16). The aim of the present study was to assess the distribution of genera, species and biotypes of yeasts in the buccal mucosa, palate, periodontal pockets and gingival crevices of patients with chronic marginal periodontitis (MP) and of healthy subjects. MATERIALS AND METHODS Participants The patient group comprised 70 patients (35 males and 35 females) with MP, chosen from patients who came for periodontal treatment at the Institute of Clinical Dentistry, Dental Faculty, University of Oslo. The criterion for patient inclusion was at least two teeth with periodontal pockets # Taylor & Francis ISSN X Microbial Ecology in Health and Disease DOI: /

2 Yeast-colonized oral sites in periodontitis 115 deeper than 5 mm. The exclusion criteria were full or partial maxillar or mandibular denture; oral mucosal lesions; usage of antibiotics, corticosteroids or immunosuppressive agents, cytotoxic chemotherapy, irradiation therapy, in the last 3 months; pregnancy; endocrine disorders; malignancies; immunodeficiencies and malnutrition. The age of the patients varied from 14 to 84 years (51.29/12.8 years). The healthy group comprised 45 individuals (19 males, 26 females) chosen from people attending dental examination and staff members of the Dental Faculty, University of Oslo. The criterion for healthy subject inclusion was no oral infection. The exclusion criteria were the same as for the MP patients. The age of the healthy subjects varied from 16 to 74 (32.99/14.1) years. Sampling and culture Yeast samples were taken from periodontal pockets ]/5 mm in the MP patients and from the gingival crevices in the healthy subjects, as well as from the buccal and palatal mucosa of each participant. Pooled subgingival plaque samples were collected by inserting two to five sterile paper points (Roeko, Germany) into two to four of the deepest pockets or gingival crevices for 15 seconds after removing supragingival plaque. Palatal and buccal mucosa samples were taken by streaking the sites with sterile cotton-tipped swabs (Selefatrade, Sweden). The samples were streaked at the chairside directly onto sterile Sabouraud dextrose agar plates. These were incubated in air at 378C for 3 days. Colonies exhibiting distinct morphologies from each oral site were picked, subcultured, transferred to liquid Todd Hewitt medium with DMSO, and stored at /808C until further use. MP and healthy groups. A significance level of 5% was chosen and a program package, SRSS 11.0, was used for statistical analysis. RESULTS Occurrence of yeasts Oral yeasts were detected in 34.3% of the MP patients and in 42.2% of the healthy subjects. The frequency of yeasts in the buccal mucosa, palate and periodontal pockets/gingival crevices is shown in Fig. 1. Morphology of yeast colonies The total number of isolates selected was 110 from the MP patients and 58 from the healthy subjects, with a mean of 4.6 isolates per MP patient and 3.1 isolates per healthy subject. The yeast colonies of the MP patients, especially those obtained from the periodontal pockets, exhibited multiple morphologies, with rough, fuzzy or smooth surfaces under the stereomicroscope, whereas the colonies from the healthy subjects showed identical morphology of the smooth type. Genera and species of yeasts Two genera (Candida, Saccharomyces) and four species (C. albicans, C. dubliniensis, C. parapsilosis and S. cerevisiae) were identified from the oral cavity of the MP patients. The occurrence of the four species is shown in Table I. C. Biotyping The isolates from different oral sites of each participant were identified by using a commercial kit, ID 32C (biomérieux, France), which is based on assimilation tests. The inoculation and incubation of the kit were done according to the manufacturer s instructions, using a Vitek System ATB 1574 (biomérieux) robot. Automatic reading of the kits was done by the mini API instrument (bio- Mérieux). The results of the reactions, transferred into a numerical code, were treated in a database (API, bio- Mérieux) for identification of each yeast isolate. The numerical code was also used as the biotype profile of each isolate. Statistical analyses A two-sided Chi-square test, and when appropriate a Fisher s exact test, was used for statistical analyses of differences in the frequency of yeast species and biotypes between the MP and healthy group. A two-sided sign test was applied for statistical analyses of differences within the Fig. 1. Frequency (%) of yeasts from patients (n/70) with marginal periodontitis (MP) and healthy subjects (n/45) in different oral sites.

3 116 X. Song et al. Table I Frequency of oral yeast species found in patients with marginal periodontitis (MP) and healthy subjects Yeast species Percentage of all subjects Percentage of yeast-positive subjects Percentage of all isolates MP (n/70) Healthy (n/45) MP (n/24) Healthy (n/19) MP (n/110) Healthy (n/ 58) C. albicans C. dubliniensis C. parapsilosis 1.4 / 4.2 / 0.9 / S. cerevisiae 1.4 / 4.2 / 1.8 / albicans was detected in all the three oral sites, most often in the buccal mucosa; C. dubliniensis in all three oral sites, mostly in the periodontal pockets; S. cerevisiae was detected in the palate and pockets; while C. parapsilosis was isolated exclusively from pockets (Fig. 2). This figure indicates that C. albicans was the most dominant species in all the oral sites. Among the other Candida species, C. dubliniensis dominated, being most prominent in the periodontal pockets. The ratio of the isolation frequency between C. albicans and C. dubliniensis was 6.7 in the buccal mucosa, 2.0 in the palate and 1.3 in the periodontal pockets. The occurrence of C. albicans was significantly higher than that of C. dubliniensis in the buccal mucosa (pb/0.001), but not in the palate and the periodontal pockets of the MP patients. One genus (Candida) and two species (C. albicans, C. dubliniensis) were found in the healthy subjects (Table I). The frequency ratio between C. albicans and C. dubliniensis was quite similar in the oral sites of the healthy subjects, i.e. 4.3 in the buccal mucosa, 4.5 in the palate and 5.5 in the gingival crevices (Fig. 2). The occurrence of C. albicans was significantly higher than that of C. dubliniensis in the buccal mucosa (p/ 0.021) and the gingival crevices (p/ 0.022), but not in the palate. Biotypes of yeasts Table II shows that 19 biotypes (ID 32C profiles) were identified from the isolates selected by distinct colony morphology in the MP patients. Biotypes and of C. albicans and of C. dubliniensis were the most dominant biotypes in both patients and isolates. Fig. 3 shows that biotype was most frequently recovered from the periodontal pockets, while biotype was most often found in the buccal mucosa. The occurrence of biotype was significantly higher than that of biotype in the buccal mucosa (p/0.021), but not in the palate and the periodontal pockets of the MP patients. Eleven biotypes were identified from the healthy subjects. Biotypes , and of C. albicans were most commonly detected among healthy subjects and isolates (Table II). There were no significant differences in the occurrence of the predominant biotypes between the two subject groups, except that biotype was significantly higher in the healthy subjects than in the MP patients (p/ 0.013), being higher in the gingival crevices than in the periodontal pockets (p/ 0.022). There was a dissimilar distribution of Fig. 2. Distribution (%) of oral yeast species isolated from patients (n/70) with marginal periodontitis (MP) and healthy subjects (n/45) in different oral sites.

4 Yeast-colonized oral sites in periodontitis 117 Table II Frequency of oral yeast biotypes* found in patients with marginal periodontitis (MP) and healthy subjects Biotype Percentage of all subjects Percentage of yeast-positive subjects Percentage of all the isolates MP (n/70) Healthy (n/45) MP (n/24) Healthy (n/19) MP (n/110) Healthy (n/58) / 4.2 / 0.9 / / 4.2 / 0.9 / / 4.2 / 0.9 / / 12.5 / 6.4 / / 2.2 / / / $ / 4.2 / 1.8 / / 4.2 / 0.9 / $% % / 4.2 / 0.9 / / 4.2 / 0.9 / $, % / 2.2 / 5.3 / / 4.2 / 0.9 / / 2.2 / 5.3 / / 4.2 / 0.9 / *According to the API ID 32C system, biomérieux. $Predominant biotypes of MP patients. %Predominant biotypes of healthy subjects. the predominant biotypes between or within the two subject groups (Fig. 3). DISCUSSION ID 32C is a commercially available identification system for yeasts based on standardized and miniaturized assimilation tests. The system has proven to be accurate and reproducible in the identification of the majority of yeasts isolated in clinical microbiology laboratories (17 /19). None of the MP patients had any clinical signs and symptoms of oral yeast infection, or local or general factors predisposing to candidosis (3). Of the mouths with MP 34.3% harboured yeasts. This corresponded with the average prevalence of yeasts, i.e. 34.4%, reported from healthy volunteers (2) but was lower than the frequency of Fig. 3. Distribution (%) of the predominant yeast biotypes isolated from patients (n/70) with marginal periodontitis (MP) and healthy subjects (n/45) in different oral sites.

5 118 X. Song et al. yeasts in the healthy group, 42.2%. This was unexpected, as the average age of the MP group was higher than that of the healthy group and the prevalence of yeasts seems to increase with age (20). Predilection sites for various types of oral candidosis are the palate and the buccal mucosa. These sites were therefore selected for sampling together with the periodontal pockets. The presence of yeasts in the buccal mucosa was more frequent than in the other sites of both groups. This emphasizes the buccal mucosa as a predilection site for yeasts in the oral cavity and suggests that it could be a reservoir for yeasts in the periodontal pocket rather than vice versa. Yeasts were found in the periodontal pockets of 21.4% of the MP patients. This is somewhat higher than has been reported in other MP studies: 14 /19.6% (4/6). Yeasts from the MP patients exhibited more colony morphologies than those from the healthy subjects. This agreed with the diversity of species and biotypes in these patients, and with the observations of Hellstein et al. (21) who found significant differences in colony phenotype between isolates recovered from diseased and healthy mouths. These colony phenotype differences were related to carbon source utilization. C. albicans appeared in all the oral locales at the highest frequency, especially in the healthy subjects. The high prevalence corresponds with findings (70/75%) in a previous report (2), emphasizing this species as the most dominant in the oral cavity. The high occurrence of C. dubliniensis in the periodontal pockets indicates that infected pockets could be a predilection site for C. dubliniensis. This species has been recovered mainly from the oral cavity, especially in immunocompromised individuals (22, 23). C. dubliniensis differs from C. albicans with respect to potency of virulence factors such as higher tendency to phenotype switching (24), and enhanced adherence to buccal epithelial cells (25) and to Fusobacterium nucleatum (26). F. nucleatum is a major anaerobic bacterium in periodontal pockets, and may play an important role in microbial colonization of the oral cavity (27). The ability of C. dubliniensis to adhere to F. nucleatum may aid in the microbial colonization of deep pockets (26, 28). C. parapsilosis was isolated only from the periodontal pockets and S. cerevisiae from the pockets and palate at a low frequency in the MP patients. Hannula et al. (29) recovered only C. albicans from periodontal pockets, whereas Rams and Slots (30) detected C. albicans, C. parapsilosis, C. tropicalis, C. krusei and an unidentified Candida species. The ratio of C. albicans and C. dubliniensis might be associated with the local health status, since it varied greatly in the MP sites but was fairly constant in the healthy sites. Three biotypes ( and of C. albicans and of C. dubliniensis) dominated in the MP group while two biotypes ( and of C. albicans) dominated in the periodontal pockets. Rams and Slots (30) reported a single biotype accounting for 57.8% of subgingival C. albicans strains in adult periodontitis. Three biotypes of C. albicans ( , and ) were predominant in the healthy group. Biotypes and were predominant in both groups, which corresponds with a number of previous studies that found one or two major biotypes in individuals with and without oral candidosis (10, 16, 31). The high occurrence of biotype in the periodontal pockets suggests a possible association with periodontal infection, whereas the great compatibility of biotype with the healthy oral locales, especially the gingival crevices, implicates a harmless role of this biotype in the oral cavity. However, there was no obvious correlation between biotypes and clinical infections. The dissimilar distribution of the predominant biotypes between or within the diseased and healthy group agrees with the results of Hellstein et al. (21), who demonstrated significant differences in sugar assimilation patterns of candidal isolates recovered from diseased and non-diseased patients, but disagrees with a number of studies showing similarity in distribution of biotypes regardless of their pathological sources (8, 16, 30). The difference in distribution of yeast species and biotypes detected may suggest cell, tissue or site tropism in oral yeast colonization. CONCLUSIONS MP did not increase the carrier rate of yeasts in the oral cavity, but the variation in yeast colony morphology, species and biotype was higher in the MP patients than in the healthy subjects. Among the oral sites examined in the MP patients, i.e. the buccal mucosa, periodontal pocket and palate, the buccal mucosa was the major site for yeast colonization. Species and biotypes differed between the MP patients and the healthy subjects, as well as among the oral sites of the MP patients. C. albicans was most dominant in all the oral sites of the MP patients and the healthy subjects. Among the other Candida species, C. dubliniensis was the most prominent in the periodontal pockets. The high occurrence of C. dubliniensis and its biotype from the periodontal pockets suggested that they may be associated with periodontal infection. The great compatibility of biotype of C. albicans with the healthy oral locales, especially the gingival crevices, indicates a harmless role of this biotype in the oral cavity. The difference in distribution of yeast species and biotypes may suggest cell, tissue or site tropism in oral yeast colonization.

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