Oral Mycobiome Characterization Via Cultivation and Molecular Approaches

Objectives: Fungi are important but understudied components of the oral microbiome. A diverse mycobiome has been reported via molecular methods but it is not clear which of these taxa are cultivable. In particular, Malassezia is very abundant in molecular mycobiome studies but it has never been cultivated. The aim of this study is to compare the prevalence of oral fungi via cultivation and molecular detection.
Methods: 24 subjects were enrolled from the general population (n=12) or from subjects undergoing treatment for cancer (n=12). An oral examination was performed and unstimulated saliva was collected. The mycobiome was characterized via high throughput sequencing of the ITS1 region and also via cultivation on Chromagar Malassezia™, a medium containing lipids to encourage growth of Malassezia. Isolates were identified via Sanger sequencing of ITS1.
Results: Subjects enrolled were 50+/-19 years old, 58% males, 21% wore a removable denture and 33% had evidence of mucosal lesions in the form of denture stomatitis, mucositis or oral candidiasis. Fungi were detected in all subjects via molecular methods, while only 75% of subjects were positive via cultivation. Average salivary cultivable fungal load was 2.4x10³ colony forming units per mL [range 0 to 4.6x10⁴]. Two hundred and six genera were detected by sequencing, while only 6 genera via cultivation. The molecular and cultivation prevalence of these genera was 100% and 17% for Malassezia, 96% and 50% for Candida, 75% and 17% for Rhodotorula, 29% and 8% for Pichia, 21% and 4% for Exophiala and 17% and 4% for Clavispora. Apart from these, Apergillus (71%), Saccharomyces (63%), Cryptococcus (63%), Cladosporium (58%), Penicillum (54%) and Wallemia (50%) were also very prevalent but only detected molecularly.
Conclusions: We report the first cultivation of Malassezia from the oral cavity confirming it is an oral resident. Fungal prevalence by molecular methods appears to be much higher compared to cultivation.