With this review, we aim to increase the quality standards for clinical studies with microbiome as an output parameter. We critically address the existing body of evidence for good quality practices in oral microbiome studies based on 16S rRNA gene amplicon sequencing. First, we discuss the usefulness of microbiome profile analyses. Is a microbiome study actually the best approach for answering the research question? This is followed by addressing the criteria for the most appropriate study design, sample size, and the necessary data (study metadata) that should be collected. Next, we evaluate the available evidence for best practices in sample collection, transport, storage, and DNA isolation. Finally, an overview of possible sequencing options (eg, 16S rRNA gene hypervariable regions, sequencing platforms), processing and data interpretation approaches, as well as requirements for meaningful data storage, sharing, and reporting are provided.

Aim: To assess the microbial effects of mechanical debridement in conjunction with a mouthrinse on sites with peri-implant mucositis and gingivitis.Materials and methods: Eighty-nine patients with peri-implant mucositis were included in a double-blinded, randomized, placebo-controlled trial with mechanical debridement and 1-month use of either delmopinol, chlorhexidine (CHX), or a placebo mouthrinse. Submucosal and subgingival plaque samples of implants and teeth were collected at baseline and after 1 and 3 months, processed for 16S V4 rRNA gene amplicon sequencing, and analysed bioinformatically. Results:The sites with peri-implant mucositis presented with a less diverse and less anaerobic microbiome. Exposure to delmopinol or CHX, but not to the placebo mouthrinse resulted in microbial changes after 1 month. The healthy sites around the teeth harboured a more diverse and more anaerobe-rich microbiome than the healthy sites around the implants.Conclusions: Peri-implant sites with mucositis harbour ecologically less complex and less anaerobic biofilms with lower biomass than patient-matched dental sites with gingivitis while eliciting an equal inflammatory response. Adjunctive antimicrobial therapy in addition to mechanical debridement does affect both dental and peri-implant biofilm composition in the short term, resulting in a less dysbiotic subgingival biofilm.

Background Treating oral squamous cell carcinoma (OSCC) introduces new ecological environments in the oral cavity. This is expected to cause changes in the oral microbiome. The purpose of this study was to gain new information on the salivary microbiome of OSCC patients in order to improve the aftercare of OSCC patients. The aims of this study were to investigate possible changes in the salivary microbiome profiles of OSCC patients before and after cancer treatment and to compare these changes with the profiles of healthy controls. Patients and methods Paraffin-stimulated whole saliva samples were collected, and the salivary flow rate was measured from 99 OSCC patients prior to surgical resection of the tumor and other adjuvant therapy. After treatment, 28 OSCC patients were re-examined with a mean follow-up time of 48 months. In addition, 101 healthy controls were examined and sampled. After DNA extraction and purification, the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using Illumina MiSeq. The merged read pairs were denoised using UNOISE3, mapped to zero-radius operational taxonomic units (zOTUs), and the representative zOTU sequences were assigned a taxonomy using HOMD. Descriptive statistics were used to study the differences in the microbial profiles of OSCC patients before and after treatment and in comparison to healthy controls. Results At baseline, the OSCC patients showed a higher relative abundance of zOTUs classified as Streptococcus anginosus, Abiotrophia defectiva, and Fusobacterium nucleatum. The microbial profiles differed significantly between OSCC patients and healthy controls (F = 5.9, p < 0.001). Alpha diversity of the salivary microbiome of OSCC patients was decreased at the follow-up, and the microbial profiles differed significantly from the pre-treatment (p < 0.001) and from that of healthy controls (p < 0.001). Conclusions OSCC patients’ salivary microbiome profile had a higher abundance of potentially pathogenic bacteria compared to healthy controls. Treatment of the OSCC caused a significant decrease in alpha diversity and increase in variability of the salivary microbiome, which was still evident after several years of follow-up. OSCC patients may benefit from preventive measures, such as the use of pre- or probiotics, salivary substitutes, or dietary counseling.

Dental caries lesions are a clinical manifestation of disease, preceded by microbial dysbiosis, which is poorly characterized and thought to be associated with saccharolytic taxa. Here, we assessed the associations between the oral microbiome of children and various caries risk factors such as demographics and behavioral and clinical data across early childhood and characterized over time the salivary and dental plaque microbiome of children before clinical diagnosis of caries lesions. Children ( N = 266) were examined clinically at ~1, 2.5, 4, and 6.5 y of age. The microbiome samples were collected at 1, 2.5, and 4 y. Caries groups consisted of children who remained caries free (International Caries Detection and Assessment System [ICDAS] = 0) at all time points (CFAT) ( n = 50); children diagnosed with caries (ICDAS ≥ 1) at 6.5 y (C6.5), 4 y (C4), or 2.5 y of age (C2.5); and children with early caries or advanced caries lesions at specific time points. Microbial community analyses were performed on zero-radius operational taxonomic units (zOTUs) obtained from V4 of 16S ribosomal RNA gene amplicon sequences. The oral microbiome of the children was affected by various factors, including antibiotic use, demographics, and dietary habits of the children and their caregivers. At all time points, various risk factors explained more of the variation in the dental plaque microbiome than in saliva. At 1 y, composition of saliva of the C4 group differed from that of the CFAT group, while at 2.5 y, this difference was observed only in plaque. At 4 y, multiple salivary and plaque zOTUs of genera Prevotella and Leptotrichia were significantly higher in samples of the C6.5 group than those of the CFAT group. In conclusion, up to 3 y prior to clinical caries detection, the oral microbial communities were already in a state of dysbiosis that was dominated by proteolytic taxa. Plaque discriminated dysbiotic oral ecosystems from healthy ones better than saliva.