Interindividual variability and intraindividual stability of oral fungal microbiota over time

Medical Mycology, 2014, 52, 496–503 doi: 10.1093/mmy/myu027 Advance Access Publication Date: 12 June 2014 Original Article Original Article Interindividual variability and intraindividual stability of oral fungal microbiota over time 1 Faculty of Dental Medicine, University of Porto, Portugal, 2 Nephrology Research and Development Unit, University of Porto, Portugal, 3 Institute of Molecular Pathology and Immunology, University of Porto, Portugal and and 4 Faculty of Sciences, University of Porto, Portugal *To whom correspondence should be addressed. Ricardo Araujo, IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr. Roberto Frias s/n, 4200-465 Porto. Tel: +351 225570700; Fax: +351 225570799; E-mail: Received 19 September 2013; Revised 7 March 2014; Accepted 16 March 2014 Abstract Oral microbiota is one of the most complex and diverse microbial communities in the human body. In the present study, we aimed to characterize oral fungi biodiversity and stability over time in a group of healthy participants with good oral health. Oral health and oral fungal microbiota were evaluated in 40 healthy individuals. A follow-up of 10 participants was carried out 28 weeks and 30 weeks after the first sampling. Oral rinse was collected and incubated in a fungal selective medium at 25o C and 37o C for 7 days. Fungi were identified based on macro- and microscopic morphology. API/ID32C was used for yeast identification, and molecular techniques were used to identify the most prevalent nonidentified moulds, mainly by sequencing 18S and internally transcribed spacer regions. Moulds were recovered from all participants and yeast from 92.5%. The most frequently isolated fungi were Candida spp., Rhodotorula spp., Penicillium spp., Aspergillus spp., and Cladosporium spp. The oral fungal community presented a high interindividual variability, but the frequency and quantification of each fungal taxon was constant over the 30-week observation period, showing a consistent intraindividual stability over time. The intraindividual stability opposed to interindividual variability may suggest a common and a variable group of fungi in the oral cavity. Key words: fungi, yeast, mould, oral health, mycobiome. Introduction The oral microbiome is one of the most complex and diverse microbial communities in the human body [1–3]. The microorganisms living in the oral cavity, as well as their interrelationships, are essential components in the balance between health and disease. Thus, it is crucial to study the microbiology of the human mouth, which is the portal of 496 entry for microbes to both the gastrointestinal and respiratory tracts, as this will allow us to understand what constitutes microbial communities in health and disease [4]. Fungi are ubiquitous organisms that colonize humans, animals, fruits, vegetables, and other plant material. Some fungi inhabit humans without any harmful effects, for example, the symptom-free oral carriage of Candida species  C The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: Filipa Monteiro-da-Silva1,2 , Ricardo Araujo3,4,∗ and Benedita Sampaio-Maia1,2 Monteiro-da-Silva et al. Methods Study participants Initially, 40 healthy students in their fifth year of the master’s degree program in the Dental Medicine Faculty, University of Porto, Portugal, were invited to voluntarily participate in the investigation. The ethics committee of the same faculty approved the consent form and research protocol. Exclusion criteria included parafunctional habits that could influence the oral microbiota (such as nail biting, finger sucking, and mouth breathing), probing depths >3 mm and/or retraction, and antibiotic or steroid therapy in the last 6 months. The medical and dental histories of each participant as well as information regarding oral hygiene habits, alcohol habits, and use of contraceptive drugs were obtained by interview. Oral clinical examination included assessment of probing depth, gingival retraction, and bleeding on probing at six sites/teeth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual) for all teeth. The prevalence of caries was assessed by DMFS (decayed, missing, and filled surface) index, and oral hygiene was assessed by plaque control record [11]. Sample collection and cultivation Oral samples were collected at least 1 h after a meal or tooth brushing, between 11 a.m. and 1 p.m., to avoid variation in salivary flow rates. Participants were asked not to use mouthwash during the week prior to sampling. To assess individual variation of oral fungi colonization over time, a follow-up of 10 participants was carried out 28 weeks and 30 weeks after the first sampling. This allowed for collection of samples within longer (6 months) and shorter (15 days) time periods. The selection criteria for the followup participants included continued participant availability and continued residence in the area (Porto, Portugal). Study participants rinsed their mouth with 15 ml sterile water for 15 s; then 10 ml of the collected samples was diluted in 250 ml of Sabouraud glucose agar supplemented with 50 mg/l of chloramphenicol in a vertical laminar flow hood. The culture media were immediately divided among 10 petri dishes using the pour plate method. Five dishes were incubated for 7 days at 25o C and the remaining five were incubated at 37o C. The cultures were examined every day, with the number of fungal colonies recorded in colony-forming units per milliliter (CFU/ml) and the growing fungi identified at a genus and species level (whenever possible) based on macro- and microscopic morphology [12]. We also used the API system (API/ID32C) for yeast identification, according to the manufacturer’s procedures (bioMérieux, Marcy L’Etoile, France). All fungi that were unable to be reliably identified based on traditional features were classified as nonidentified moulds or yeast. The most prevalent nonidentified moulds were subsequently identified using molecular techniques, mainly by sequencing 18S and internal transcribed spacer (ITS) regions of ribosomal RNA genes. The following criteria were established for Candida colony counts: patient whose sample values were <50 CFU/ml were considered low yeast carriers; patient whose sample values were between 50 and 400 CFU/ml were considered moderate yeast carriers; and patient whose sample values were >400 CFU/ml were considered high yeast carriers [13]. that has been recognized for many years [5]. On occasion, yeast can be problematic in immunosuppressed patients [6,7]. The shift from innocuous commensals to harmful pathogens may depend on factors other than the microorganism’s attributes. Local or systemic predisposing factors in the host may be of equal or greater importance in the pathogenesis of the disease [5,8]. Ou (...truncated)