The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3–V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome.

Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized.

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Head and neck squamous cell carcinoma (HNSCC) patients often present with poor oral health, making it difficult to assess the relationship between oral microbes, inflammation, and carcinoma. This study investigates salivary microbes and inflammatory cytokines as biomarkers for HNSCC, with consideration of oral health. Saliva was collected from 30 participants, including 14 HNSCC patients and 16 participants representing both dentally compromised and healthy individuals. Bacterial and fungal communities were analyzed based on 16S rRNA gene and ITS1 amplicon sequencing, respectively, and concentrations of inflammatory cytokines were quantified using a cytometric bead array, with flow cytometry. Diversity‐based analyses revealed that the bacterial communities of HNSCC patients were significantly different to those of the healthy control group but not the dentally compromised patients. Fungal communities were dominated by Candida, irrespective of cohort, with Candida albicans comprising ≥96% of fungal sequences in most HNSCC patients. Significantly higher concentrations of interleukin (IL)‐1β and IL‐8 were detected in HNSCC and dentally compromised patients, when independently compared with healthy controls. IL‐1β and IL‐8 concentrations were significantly positively correlated with the abundance of C. albicans. Our findings suggest that salivary microbial and inflammatory biomarkers of HNSCC are influenced by oral health.