Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy Individuals

The oral microbiome–organisms residing in the oral cavity and their collective genome–are critical components of health and disease. The fungal component of the oral microbiota has not been characterized. In this study, we used a novel multitag pyrosequencing approach to characterize fungi present in the oral cavity of 20 healthy individuals, using the pan-fungal internal transcribed spacer (ITS) primers. Our results revealed the “basal” oral mycobiome profile of the enrolled individuals, and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. Among these genera, 39 were present in only one person, 16 genera were present in two participants, and 5 genera were present in three people, while 15 genera (including non-culturable organisms) were present in ≥4 (20%) participants. Candida species were the most frequent (isolated from 75% of participants), followed by Cladosporium (65%), Aureobasidium, Saccharomycetales (50% for both), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). Four of these predominant genera are known to be pathogenic in humans. The low-abundance genera may represent environmental fungi present in the oral cavity and could simply be spores inhaled from the air or material ingested with food. Among the culturable genera, 61 were represented by one species each, while 13 genera comprised between 2 and 6 different species; the total number of species identified were 101. The number of species in the oral cavity of each individual ranged between 9 and 23. Principal component (PCO) analysis of the obtained data set followed by sample clustering and UniFrac analysis revealed that White males and Asian males clustered differently from each other, whereas both Asian and White females clustered together. This is the first study that identified the “basal mycobiome” of healthy individuals, and provides the basis for a detailed characterization of the oral mycobiome in health and disease.

[1]  G. McVean.  A Genealogical Interpretation of Principal Components Analysis , 2009, PLoS genetics.

[2]  D. Ojcius,et al.  The oral microbiota: living with a permanent guest. , 2009, DNA and cell biology.

[3]  K. Marr,et al.  Candida glabrata sepsis secondary to oral colonization in bone marrow transplantation. , 2004, Medical mycology.

[4]  E. Sonnhammer,et al.  Kalign2: high-performance multiple alignment of protein and nucleotide sequences allowing external features , 2008, Nucleic acids research.

[5]  T. Lion,et al.  Species-Specific Identification of a Wide Range of Clinically Relevant Fungal Pathogens by Use of Luminex xMAP Technology , 2009, Journal of Clinical Microbiology.

[6]  D. Reich,et al.  Principal components analysis corrects for stratification in genome-wide association studies , 2006, Nature Genetics.

[7]  J. Lopez-Ribot,et al.  Replacement of Candida albicans with C. dubliniensis in Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis Treated with Fluconazole , 2002, Journal of Clinical Microbiology.

[8]  R. Lamont,et al.  Oral microbial communities in sickness and in health. , 2005, Trends in microbiology.

[9]  M. Hirata,et al.  Molecular identification of Candida dubliniensis isolated from oral lesions of HIV-positive and HIV-negative patients in São Paulo, Brazil. , 2006, Revista do Instituto de Medicina Tropical de Sao Paulo.

[10]  J. A. Aas,et al.  Defining the Normal Bacterial Flora of the Oral Cavity , 2005, Journal of Clinical Microbiology.

[11]  F. Dewhirst,et al.  Bacterial Diversity in Human Subgingival Plaque , 2001, Journal of bacteriology.

[12]  Samuel A. Lee,et al.  Candida krusei sepsis secondary to oral colonization in a hemopoietic stem cell transplant recipient. , 2007, Medical mycology.

[13]  T. Lion,et al.  Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region , 2009, European Journal of Clinical Microbiology & Infectious Diseases.

[14]  J. Versalovic,et al.  DNA pyrosequencing-based identification of pathogenic Candida species by using the internal transcribed spacer 2 region. , 2008, Archives of pathology & laboratory medicine.

[15]  M. Ghannoum,et al.  Single-Step PCR Using (GACA)4 Primer: Utility for Rapid Identification of Dermatophyte Species and Strains , 2008, Journal of Clinical Microbiology.

[16]  H. Helenius,et al.  Fungal colonization of haematological patients receiving cytotoxic chemotherapy: emergence of azole-resistant Saccharomyces cerevisiae. , 2000, The Journal of hospital infection.

[17]  R. Knight,et al.  UniFrac: a New Phylogenetic Method for Comparing Microbial Communities , 2005, Applied and Environmental Microbiology.

[18]  Zeng-Tong Zhou,et al.  A metabonomic approach to the diagnosis of oral squamous cell carcinoma, oral lichen planus and oral leukoplakia. , 2008, Oral oncology.

[19]  B. Roe,et al.  A core gut microbiome in obese and lean twins , 2008, Nature.

[20]  Kun Tang,et al.  Global diversity in the human salivary microbiome. , 2009, Genome research.

[21]  Adam Godzik,et al.  Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences , 2006, Bioinform..

[22]  N. Rao,et al.  Abundant and Diverse Fungal Microbiota in the Murine Intestine , 2006, Applied and Environmental Microbiology.

[23]  C. Linton,et al.  Molecular identification of pathogenic fungi. , 2008, The Journal of antimicrobial chemotherapy.

[24]  David W Williams,et al.  Detection of Candida in Concentrated Oral Rinse Cultures by Real-Time PCR , 2004, Journal of Clinical Microbiology.

[25]  A. De Luca,et al.  Rapid identification of Candida species in oral rinse solutions by PCR , 2006, Journal of Clinical Pathology.

[26]  Andreas Günther,et al.  Relatedness of medically important strains of Saccharomyces cerevisiae as revealed by phylogenetics and metabolomics , 2008, Yeast.

[27]  Chi-Hong Tseng,et al.  Molecular Analysis of Fungal Microbiota in Samples from Healthy Human Skin and Psoriatic Lesions , 2006, Journal of Clinical Microbiology.

[28]  Rob Knight,et al.  UniFrac – An online tool for comparing microbial community diversity in a phylogenetic context , 2006, BMC Bioinformatics.

[29]  J. A. Aas,et al.  Subgingival plaque microbiota in HIV positive patients. , 2007, Journal of clinical periodontology.

[30]  J. Elborn,et al.  Development of a novel PCR assay for the identification of the black yeast, Exophiala (Wangiella) dermatitidis from adult patients with cystic fibrosis (CF). , 2008, Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society.

[31]  J. A. Aas,et al.  The breadth of bacterial diversity in the human periodontal pocket and other oral sites. , 2006, Periodontology 2000.

[32]  W. Merz,et al.  Recovery of Candida dubliniensis and Other Yeasts from Human Immunodeficiency Virus-Associated Periodontal Lesions , 2001, Journal of Clinical Microbiology.

[33]  L. Samaranayake,et al.  The oral prevalence of aerobic and facultatively anaerobic gram-negative rods and yeasts in Hong Kong Chinese. , 1994, Archives of oral biology.

[34]  F. Bushman,et al.  DNA bar coding and pyrosequencing to identify rare HIV drug resistance mutations , 2007, Nucleic acids research.

[35]  M. Ronaghi,et al.  A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing , 2007, Nucleic acids research.

[36]  R. Newcombe,et al.  Oral carriage of yeasts, coliforms and staphylococci in patients with advanced malignant disease. , 1992, Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology.

[37]  Jonathan P. Bollback,et al.  The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing , 2007, PloS one.

[38]  E. Alfonso.  Genotypic identification of Fusarium species from ocular sources: comparison to morphologic classification and antifungal sensitivity testing (an AOS thesis). , 2008, Transactions of the American Ophthalmological Society.

[39]  G. Schuster.  Oral flora and pathogenic organisms. , 1999, Infectious disease clinics of North America.