Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients dental prosthesis users in Brazil

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1 Mycopathologia (2007) 164: DOI /s Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients dental prosthesis users in Brazil Regina Helena Pires-Gonçalves Æ Elaine Toscano Miranda Æ Lilian Cristiane Baeza Æ Marcelo Teruyuki Matsumoto Æ José Eduardo Zaia Æ Maria José Soares Mendes-Giannini Received: 1 April 2007 / Accepted: 29 August 2007 / Published online: 29 September 2007 Ó Springer Science+Business Media B.V Abstract The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual s dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P \ 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P \ 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S AB for the entire collection R. H. Pires-Gonçalves E. T. Miranda L. C. Baeza M. T. Matsumoto M. J. S. Mendes-Giannini Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, SP, Brazil J. E. Zaia Universidade de Franca, Franca, SP, Brazil M. J. S. Mendes-Giannini (&) R. Expedicionários do Brasil, CEP , Araraquara, SP 1621, Brazil giannini@fcfar.unesp.br of 47 C. albicans isolates were ± Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host s clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others. Keywords Oral Candida species Randomly amplified polymorphic DNA (RAPD) C. albicans genotype Dental prostheses Oral carriage Introduction Candida albicans is a part of the normal microbial flora that colonizes mucocutaneous surfaces of the oral cavity, gastrointestinal tract, and vagina of the healthy human host. Although Candida does not normally cause disease, when immune defenses are compromised or the normal microflora balance is disrupted, C. albicans transforms itself into an opportunistic pathogenic, leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. On the other hand, dental prostheses provide a solid base favorable to yeast adherence and colonization [1], resulting in an inflammatory process in the oral soft tissue, especially in the subjacent area covered by complete or partial

2 256 Mycopathologia (2007) 164: dentures [2]. Current studies indicate that other factors within the oral environment such as saliva, ph, bacteria, hyphal formation, systemic diseases, and defects in the immune system have been showed to influence adhesion of Candida species to surface in the mouth [3, 4]. In addition, Diabetes mellitus and others high-risk patients could be considered as an additional variable that might influence not only oral Candida carriage, but also the ability of isolates to enhance the expression of virulence attributes [5]. Despite the high frequency of commensal carriage of C. albicans and its prominence as a major fungal pathogen, little is known about its genetic homogeneity, evolution, and persistence during commensalism or parasitism. The development of genotype techniques have helped to delineate subtypes of colonizing C. albicans strains and facilitated epidemiological analysis [6, 7]. For C. albicans, it has been demonstrated that RAPD analysis is highly effective in discriminating among completely unrelated strains, identifying the same strain, and discriminating microevolution in a single colonizing strain. RAPD is relatively cost-effective (for large numbers of isolates) and matches the resolving power of electrophoretic karyotyping. This, together with availability of computer-assisted software systems that generate dendrograms of genetic relatedness among C. albicans isolates and it has significantly advanced lineage studies over progressive infective episodes or during asymptomatic carriage [8, 9]. In addition, it has been found, by a variety of molecular methods that strains of C. albicans tend to be genetically similar if they are isolated from the same specific groups, such as HIV positive and negative patients [10], from geographically-related locations [10] or from several sites on the same or related patients [11 13]. Unfortunately, there have been very few studies of the genetic composition of commensal populations of infectious fungi. On the other hand, there are very little data in Brazil, on the incidence of yeast carriage and the identity of yeast species in the oral cavities of diabetic and hemodialysed patients and/or denture wearers. The main aim of this investigation was to assess the quantity, species and characterize the subtypes of oral C. albicans isolates from welldefined cohorts of diabetic and hemodialysed patients and healthy individual s dental prostheses users. Methods Patients Ethical approval for the project was granted by the Research Ethics Committee of the Post-Graduate Department and Research Department of the University of Franca, São Paulo State, Brazil (protocol 001/ 99) and it included: 175 insulin-using diabetes mellitus patients attending the diabetic clinics at the Endocrinology Department of SUS (Sistema Único de Saúde, the Public Health System) in the state of São Paulo, Brazil, and 77 patients with chronic renal failure who were receiving regular hemodialysis (the mean duration of hemodialysis therapy was three years, the range being two month to eight years) and 54 blood donors from the same region, who formed the healthy individuals group. All patients received a detailed oral examination to verify the integrity of the buccal mucosa at the time of sampling. Patients with clinical signs and symptoms of candidiasis were excluded from the study. The biological material was collected from November 13, 2000 to November 25, Patients were questioned about the use of partial or complete dentures, and these were noted. Collection of yeasts isolates and growth conditions A total of 91 yeast isolates was obtained from the cohort of 91 dental-prosthesis users. The microorganisms were recovered using the oral-rinse technique of Samaranayake et al. [14]. In brief, the patients were requested to rinse the mouth with 10 ml phosphate-buffered saline PBS for 60s, and the liquid was collected in plastic containers and centrifuged; pelleted material was re-suspended in 1 ml PBS and 50 ll was spread on Sabouraud Dextrose Agar (SDA, Sigma-Aldrich, Dorset, UK) plus chloramphenicol and incubated at C for h. The number of colony-forming units (CFU) for each sample was quantified (CP 600 Plus colony counter; Phoenix Luferco, Brazil), and one colony per sample was randomly selected and subcultured to obtain a pure growth. Morphologically distinct colonies from each culture was subcultured and stored on Sabouraud s dextrose slant for species differentiation, DNA fingerprinting and storage. All isolates

3 Mycopathologia (2007) 164: were identified by morphology, physiological, and biochemical characteristics [15]. The yeasts identified as C. albicans were screened for their ability to grow on SDA at 45 C for 48 h and xylose assimilation, to distinguish C. dubliniensis [16]. They were then evaluated with the API 20C Aux kit (BioMérieux SA, France). Isolates from a single sample yielding identical API 20C AUX profiles were considered the same yeast strain. Genotyping of C. albicans isolates Preparation of DNA for RAPD Only C. albicans isolates from patients using dental prostheses (removable and total superior and/or inferior) were subcultured on Sabouraud agar with chloramphenicol and incubated at 37 C for 48 h. One to three loops of cells from each colony were subcultured in 15 ml of YPG broth (1% of yeast extract, 2% of peptone, and 1% of glucose), constantly stirred for 18 h at 37 C, and the cultures used for the genomic DNA extraction. This was performed with the Puregene DNA Isolation Kit (Gentra Systems INC., Minneapolis, USA), following the maker s recommendations. DNA yield and integrity were verified by electrophoresis in 1% agarose gel. Randomly amplified polymorphic DNA analysis RAPD analysis was performed using Ready-To-Go RAPD Analysis Beads (Amersham Biosciences Corp., Piscataway, NJ, USA), as described by the manufacturer, carefully observing factors affecting reproducibility. Previous studies [17] were developed with the all ready to-go kit primers and the most discriminatory primer OPJ 6 (5 0 -d[cccgtcagca]- 3 0 ) was selected for use with all strains. The reproducibility of the method was verified by repeating all amplifications from two isolates of the same strain a minimum of three times with selected strains. One ll of the DNA template (containing approximately 25 ng) was mixed with 19 ll of distilled water, 5.0 ll (25 pmol) of OPJ 6, Ready-To-Go RAPD Analysis Bead, dntps (0.4 mm), 1 unit of AmpliTaq polymerase DNA, PCR-solution, 3 mm MgCl 2, 30 mm KCl, and 10 mm Tris (ph = 8.3) to give a final reaction volume of 25 ll. Thermocycling was performed in a GeneAmp 9700 machine (Perkin Elmer Corporation, Foster City, USA), as follows: an initial denaturation step at 95 C for 5 min, followed by 45 cycles consisting of denaturation at 95 C for 1 min, annealing at 36 C for 1 minute and with a final extension step at 72 C for 5 min. Control tubes without template DNA were included in each run and reproducibility was checked for each reaction [7, 18]. The PCR products were electrophoresed in agarose gels (2%) in TBE buffer (Tris base 54 g, boric acid 27.5 g, EDTA 0.5 M 20 ml; distilled water to 1,000 ml) 1 at 150 volts for 2 h at room temperature. Amplicons in the gel were stained with ethidium bromide (0.5 lg/ml) and visualized under ultraviolet transillumination. Gels were visually analyzed and, in order to standardize the quality of the images, recorded under UV light using the Image Master VDS System (Amersham Pharmacia Biotech, UK). All visible and well-defined bands obtained by visual analysis and confirmed by the software were included in the analysis. Dendrograms were used to determine the relationship among isolates on the basis of RAPD bands. Gels were normalized using Saccharomyces cerevisiae chromosomal DNA standards as a reference. All experiments were carried out in duplicate to assess reproducibility. Computer-assisted analysis of data and dendrogram generation RAPD profiles were analyzed by GelCompar software Version 2.0 (Applied Maths). The similarity coefficient (S AB ) between patterns for every pair of isolates A and B was computed with the formula S AB =2 E /(2 E + a + b), where E is the number of common bands in the patterns of A and B, a is the number of bands in pattern A with no correlates in pattern B, and b is the number of bands in pattern B with no correlates in pattern A. Dendrograms based on S AB values were generated by UPGMA, implemented in the GelCompar software. An S AB value of 1.00 indicates that the banding patterns for strain A are identical with that of strain B; S AB values of represent highly similar, but nonidentical, strains, and may suggest the occurrence of microevolution in a single strain; and S AB values below 0.80 represent unrelated strains [8, 19].

4 258 Mycopathologia (2007) 164: Statistical analysis The significance of differences among multiple groups was assessed using the chi-squared test and an analysis of variance. When the data sets failed the normality test, the Kruskal Wallis was used. Posthoc pair wise comparison between groups was tested for significance using Dunn s method with the level of significance set at P \ Data are reported as means ± SEM [18]. Results Out of the 306 patients screened for oral carriage of yeasts, 46% (141/306) were colonized. There were 68.4% carriers (91/133) among 133 dental prosthesis users (Table 1), and the rate of colonization varied significantly between these two subgroups (P \ 0.05). Only eight patients with prosthesis were not colonized. The total viable counts (CFU/ml of oral rinse) of yeast isolated are summarized in Table 2. By comparing the results it is noticeable that diabetic had higher viable counts, which was confirmed by the Dunn s multiple comparisons method (P \ 0.05). The frequency of yeasts among dental prosthesis users carriers related to age are displayed in Table 3, where it is seen that C. albicans predominated, and most isolates of C. parapsilosis are concentrated in group A 2 ( 51-years-old). C. albicans was the most commonly recovered species, isolated from 61.7% (87/141) of the overall individuals. Among the 91 dental prosthesis users who carried yeasts, the isolation frequency was: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). The group of diabetic patients was most frequently colonized (68.1%) and showed diversity of strains. A total of 47 C. albicans isolates recovered from the oral cavities of dental prosthesis users were submitted to RAPD analysis with OPJ 6. All the isolates were grouped into 11 RAPD profiles in four main clusters showing 50% of similarity (Fig. 1). The S AB ranged from 0.5 to 1 with an average ± Eleven genotypes and four major Table 1 Frequency of yeasts isolated from oral rinse samples of dental prosthesis users of three groups: diabetic; hemodialysed, and healthy group Individuals Oral carriers (Dental prosthesis users) Rate of colonization (%) Oral carriers (without a prosthesis) Rate of colonization (%) Diabetic a 62/ / Hemodialised b 24/ / Healthy c 05/ / Total d 91/ * 50/ a Diabetic group, n = 175 b Hemodialised group, n = 77 c Healthy individuals, n = 54 d Total, n = 306 *This was significantly (x 2 = 47.26, P \ 0.05) different when compared with patients without a prosthesis Table 2 Number of yeast colony-forming units per ml in oral rinse samples of three groups: diabetic; hemodialysed, and healthy group n Range Mean (Std error of mean) Median Healthy individuals ,078.9 ( 208.5) 800 Hemodialysed patients ,853.3 (2871.2) 1350 Diabetic patients ,321.8 (1587.4) 8500* *The Kruskal Wallis with a post-hoc pair wise comparison (Dunn s method) showed that the yeast CFU/ml in diabetic patients group was significantly (P \ 0.05) higher than other groups

5 Mycopathologia (2007) 164: Table 3 Proportions of yeast species as a function of age assessed by sugar assimilation patterns Age group Organism % of isolates A1 C. albicans 59.2 C. tropicalis 18.5 C. glabrata 7.4 C. krusei 7.4 C. parapsilosis 3.7 C. rugosa 3.7 A2 C. albicans 48.4 C. parapsilosis 28.1 C. tropicalis 12.5 C. glabrata 6.2 P. ohmeri 3.2 C. krusei 1.6 *Group A 1 50-years-old (n = 27 individuals) Group A 2 51-years-old (n = 64 individuals) genotypic clusters (I, II, III and IV) were derived at a threshold S AB of 80%. The cluster I isolates had approximately 80% similarity and were distributed into two groups IA and IB included 40 (85.0%) of the isolates. One of them (IB) had a coefficient of similarity of 90% containing 26 isolates, which comprised two subgroups with a high degree ([90%) of similarity between them. Both subgroups had a total of 26 isolates showed S AB values of 1.00, representing identical strains. The averages S AB of the hemodialysed and diabetic isolates were ± and ± 0.191, respectively. Clusters II, III and IV included only seven strains of diabetic patients, with S AB 0.70, suggesting unrelated isolates (Fig. 1). Discussion Yeast cells, especially Candida species, are common in oral cavities and in immunocompromised and immunocompetent individuals [20], with a predominance of C. albicans [1 3, 10, 21]. On the other hand, little is known about the microbiota of diabetic [22]. and hemodialysed individuals in Brazil. Published reports of the frequency of isolation of oral yeasts show considerable variation between similar sites and populations. A number of relevant factors have been associated with oral carriage of yeast in the population and certain groups of individuals, with varying degrees of immunodeficiency, have an increased risk of being infected by Candida. The methodology also influences in the obtained data. The concentrated rinse culture was simple to perform, equally sensitive and superior in quantifying yeast carriage than the imprint culture technique and, it is suggested that this technique could be preferentially employed in investigations to obtain comparable data from different centers [14]. Therefore, some controversy still remains regarding the role of diabetes in the colonization of the oral cavity by yeasts [23]. On the other hand, hemodialysed patients are especially group of individuals who had special needs and few studies have been developed to evaluate their oral microbiota. Specific factors (physiological, tegument continuity injury, nutritional factors, chemical and radiotherapy, surgical procedures and others, like poor oral hygiene) are associated with the increasing frequency of Candida species in the oral cavity [20, 24]. Previous studies [21, 22] have shown great variability in the number of patients whose oral cavities are colonized by yeasts. This may be due, at least in part, to the sampling method [20] and to the fact that the number of colony forming units per ml of patient saliva does not correlate with clinical evidence of oral candidiasis [20, 25]. This could be concluded from the present research, since the apparent amount of yeast (CFU/ml) among the diabetic (mean = 14,321.8 ± 1,587.4), hemodialysed (mean = 8,853.3 ± 2,871.2) and healthy patients (mean = 1,078.9 ± 208.5) has no relationship with oral mucosal disease. The high number of Candida colonies seen in diabetic and hemodialysed patients could reflect host-candida species interaction(s) favorable to colonization. It is important to note that diabetics, particularly those who are prosthesis wearers have an increased burden of asymptomatic colonization of the oral cavity in comparison to their controls. We have also demonstrated that the presence of C albicans and the cohabitation of different Candida species was more frequent in denture wearers. Barbeau et al. [2], have pointed out the presence of yeast on dentures was significantly associated with the extent of the inflammation. On the other hand, our findings suggest that the presence of dentures influenced the oral yeast carriage of diabetic and hemodialysed patients.

6 260 Mycopathologia (2007) 164: Fig. 1 UPGMA dendrogram generated from the similarity coefficient (S AB ) which shows interindividual similarity. S AB s were determined by DICE coefficients calculated for RAPD patterns of 47 isolates of C. albicans taken from 47 dental prosthesis users in three groups: diabetic (CD); hemodialysed (HD), and healthy group (HC). Vertical dashes line mark the position of S AB = 0.8, and subdivides the dendrogram into 2 large clusters. The cluster I isolates had approximately 80% similarity and were distributed into two subclusters IA (12 isolates) and IB (28 isolates). One of them (IB), containing 26 isolates, which comprised two subgroups with a high degree ([90%) of similarity between them. A high discriminatory power was found with primer 6, which produced up to 10 bands, as seen in RAPD patterns of isolates CD 131 and CD 130 Dice (Opt:1.50%) (Tol 3.0%-3.0%) (H>0.0%S>0.0%) [0.0%-100.0%] RAPD RAPD IA I IB II III IV CD104 CD116 HD49 HD40 HD31 CD56 CD103 HD38 CD158 CD61 CD79 HD68 CD33 CD43 CD23 CD85 CD163 CD166 HD39 HD63 HD69 HD13 CD22 CD40 CD119 CD142 CD50 HD03 HD30 HD23 HD32 HD75 HD73 CD154 CD156 HC10 HC39 HC50 CD86 HD59 CD131 CD130 CD45 CD94 CD108 CD24 CD30 In agreement with previous studies [2, 24, 25] but not with that of Al-Karaawi et al. [25], the presence of C. albicans on the oral mucosa in 66.9% of the studied patients must be considered as a representative high prevalence of this microorganism in persons wearing dental prosthesis. Patients with either partial or complete dentures were more often colonized, in some studies, as well as, in our work, twice or three times as often, than patients without dentures [24]. This may be explained by the fact that in denture

7 Mycopathologia (2007) 164: wearers, the fitting surface of the denture limits masticator movements, and is covered by a layer of microbial plaque that candidal species can readily colonize; also, when dentures are not properly adapted, they induce to tissue maceration, causing low-intensity and long-lasting local traumatisms, which can reduce tissue resistance against infections, increasing the permeability of the epithelium to soluble candidal antigens and toxins [26 29]. The oral lesions among elderly people are frequent and commonly related to the use of dentures [30] and, the principal risk factors for colonization were a dental prosthesis, poor oral hygiene and the use of antibiotics [31, 32]. There is clear evidence that C. albicans adheres to oral surfaces including acrylic dentures and mucosa [3] and, frequently colonize the oral mucous of patients wearing dental prosthesis, as well as the prosthesis [2, 24, 32]. C. albicans is the most adapted species as a commensal within the oral niche [2, 22, 33]. The present results suggest that for this test population, the C. albicans carriage (61.7%; 87/141) is more frequent than that of other species. Even so, the percentage of carriage non-albicans species was high (36.4%; 50/137), which could be explained by the larger percentage (68.1%) of diabetic patients than other groups, since previous studies associated diabetic patients with oral carriage of a range of Candida species [34, 35]. Another factor that has influenced this result was age, since 70.3% (64/91) were 51- years-old. The frequency of C. albicans in patients with increasing age decreases while non-c. albicans yeasts increases [36, 37]. Previous research [3, 12, 24, 36] suggests that intensity of carriers were greater among older individuals than in a lower age range (15 18 years-old). Besides that, 94.3% (133/141) were dental prosthesis users and in previous research, more diverse yeast species were isolated from the oral cavities of patients with dentures (either full or partial) than from dentate patients [27, 36 39] and, in the current study, the fact that selective media (that produce differential staining of candidal species and highlight morphological variations of colonies) were not used may have reduced the sensitivity of detection of that diversity. Understanding of species identification, commensalism and pathogenicity, person-to-person spread and the development of antifungal resistance within specific strains has been greatly enhanced by the utilization of molecular epidemiological methodology [6 8, 21]. Thus, the aim of this study was to analyze the genetic similarity of C. albicans isolates associated with dental prosthesis in diabetic, hemodialysed patients, and healthy individuals and the prevalence of C. albicans colonization in these individuals. RAPD is already recognized as a valuable tool for studying genetic epidemiology Candida infections [9, 20, 40 43]. Reproducibility in the patterns obtained by RAPD-PCR was assessed by carrying out the experiments in duplicate; no significant differences in profiles were observed. The Ready-To-Go RAPD analysis beads used, resulted in reproducible and stable banding patterns. As the RAPD technique is simple, rapid and rather cheap, it is already recognized as a valuable tool for studying genetic epidemiology. Interestingly, most of strains could be placed into major cluster (I) with a similarity index S AB 0.80 (mean S AB = ± 0.178), including diabetic, hemodialysed and control group. On the other hand, 88.9% of the isolates from the subgroup IB yielding RAPD profiles with high similarity 90%, demonstrating that these isolates are highly related. Among them, two types were found with strains of clonal origin. It has been shown that a single cluster of genetically related infection causing C. albicans isolates usually predominates in a given patient population in a given geographical locale [44, 45]. Alternatively it is possible that the predominant groups described in these studies are local representatives of one group, geographically widespread and causing infection in a variety of patient types. These strains, from different individuals, may perhaps adapt to the similar oral conditions in diverse individuals. These results are consistent with genetic relation studies of strains of C. albicans, in which different populations can have the same or similar genotypes [44, 45]. On the other hand, there is evidence that C. albicans is reproduced in a clonal way [46 48]. Organisms which reproduce clonally adapt to their environments if such niches are stable. Alternatively, when these microorganisms are exposed to frequent environmental changes they respond through microevolutionary selection based on their capacity to adapt to varied niches [48]. Thus, in our study, we noticed that 55.3% (26/47) of the individuals had highly similar but not identical strains in their oral

8 262 Mycopathologia (2007) 164: cavities, which suggests that these strains could have adapted to the oral environmental conditions and are suffering microevolution. According to Chong et al. [18], an S AB value of represents highly similar (but not identical) strains, suggesting that these infections were caused by a single strain that underwent microevolution. Some particular species present a selective advantage to specific locations in the human body [44, 49]. When analyzing the dendrogram, we noticed that over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses probably provides the necessary conditions for these strains to gain a growth-specific advantage over others; this was also reported by Hossain et al. [21] and Song et al. [50]. These findings may be attributed to oral conditions specific to these groups of patients. 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