Temporal Stability of the Salivary Microbiota in Oral Health

OBJECTIVES:Saliva is a biological fluid suitable for biomarker analysis, and differences in the salivary microbiota in oral health and disease have been reported. For such comparative analyses, time of sampling is critical since the bacterial composition may vary throughout the day, i.e., diurnal variation. The purpose of this study is to compare the salivary microbiome over time to determine the optimal time for sampling. DESIGN:Stimulated saliva samples were collected from 5 orally healthy individuals in 4 h intervals for 24 h, and collection was repeated 7 days later (number of samples per person, n = 12, total number of samples, n = 60). Salivary microbiota was analyzed using the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using the Kruskal-Wallis test with Benjamini-Hochberg's correction for multiple comparisons, cluster analysis, principal component analysis and correspondence analysis. RESULTS:From a total of 60 saliva samples, 477 probe targets were collectively identified with a mean number of probes per sample of 207 (range: 153-307). Little or no variation in microbial profiles within subjects was observed over time. CONCLUSIONS:Although there was considerable variation between subjects, microbial profiles within subjects were stable throughout a 24 hour period and after 1 week. Since there is little or no evidence of diurnal variation of the salivary microbiome, time of sampling of saliva is not critical for perturbation or other microbial studies

Comparative analysis of bacterial profiles in unstimulated and stimulated saliva samples

Background and objective: The microbial profiles of stimulated saliva samples have been shown to differentiate between patients with periodontitis, patients with dental caries, and orally healthy individuals. Saliva was stimulated to allow for easy and rapid collection; however, microbial composition may not reflect the more natural, unstimulated state. The purpose of this study was to validate whether stimulated saliva is an adequate surrogate for unstimulated saliva in determining salivary microbiomes. Design: Unstimulated (n=20) and stimulated (n=20) saliva samples were collected from 20 orally and systemically healthy, non-smoking participants. Salivary bacterial profiles were analyzed by means of the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using Mann–Whitney test with Benjamini–Hochberg's correction for multiple comparison, cluster analysis, principal component analysis, and correspondence analysis. Results: From a total of 40 saliva samples, 496 probe targets were identified with a mean number of targets per sample of 203 (range: 146–303), and a mean number of probe targets of 206 and 200 in unstimulated and stimulated saliva samples, respectively (p=0.62). Based on all statistical methods used for this study, the microbial profiles of unstimulated and stimulated saliva samples collected from the same person were not statistically significantly different. Conclusions: Analysis of bacterial salivary profiles in unstimulated and stimulated saliva samples collected from the same individual showed comparable results. Thus, the results verify that stimulated saliva is an adequate surrogate of unstimulated saliva for microbiome-related studies

Salivary microbiota in individuals with different levels of caries experience

This study compared salivary bacterial profiles in two groups having a 10-fold difference in levels of caries experience, as it was hypothesized that the composition of the salivary microbiota might associate with the levels of caries experience. Bacterial profiles in stimulated saliva samples from 85 individuals with low levels of caries experience (healthy group) and 79 individuals with high levels of caries experience (caries group) were analyzed by means of the Human Oral Microbiome Identification Next Generation Sequencing (HOMINGS) technique. Subsequently, saliva samples from caries-free individuals in the healthy group (n = 57) and the caries group (n = 31) were compared. A significantly higher α-diversity (p < 0.0001) and a twofold higher relative abundance of Neisseria, Haemophilus, and Fusobacterium were recorded in saliva samples from the healthy group compared with the caries group. Differences observed were more pronounced when limiting the analyses to caries-free individuals in each group. Data from this cross-sectional analysis suggest that low levels of caries experience might associate with a characteristic salivary bacterial composition different from that in individuals with high caries experience. Consequently, longitudinal studies are required to determine if the composition of the salivary microbiota might be a predictive factor of caries risk at the individual level

Correspondence analysis.

<p>Correspondence analysis visualized 2-dimensionally with axes expressed as the 2 most crucial inertia values accounting for a cumulative inertia of 37.6%. Individual 1: blue square, individual 2: red square, individual 3: green triangle, individual 4: purple cross, individual 5: light blue cross.</p

Principal component analysis.

<p>Principal component analysis visualized 2-dimensionally with axes expressed as the second and third most crucial components accounting for 36.6% of the variation of the dataset. Individual 1: blue square, individual 2: red square, individual 3: green triangle, individual 4: purple cross, individual 5: light blue cross.</p

Analysis of oral bacterial communities: comparison of HOMINGS with a tree-based approach implemented in QIIME

Background: Molecular taxonomic assignments in oral microbial communities have been made using probe-matching approaches, but never compared to those obtained by more readily accepted tree-based approaches. Objective:  To compare community composition profiles obtained from a probe-matching approach (HOMINGS) to those from a closed-ended tree-based approach (QIIME using the eHOMD database). Design:  HOMINGS and QIIME were used for parallel analysis of ten mock community samples, and of 119 supragingival plaque samples from ecologically unique sites (sound tooth surfaces in healthy subjects, sound tooth surfaces in patients with primary Sjögren’s Syndrome, and carious lesions in Sjögren’s Syndrome patients). Linear discriminant analysis Effective Size (LEfSe) was used to identify discriminating taxa among the natural plaque samples. Results: Community composition profiles of all samples were congruent between the two analysis aproaches. Alpha and beta diversity of the natural plaque communities were likewise similar. Communities from pSS patients and those from individuals with normal salivary flow differed in alpha and beta diversity. Both classification approaches yielded differences in composition predicted for samples from these subject cohorts, and discriminating taxa were similar between approaches. Conclusions: A direct comparison demonstrates that HOMINGS is largely equivalent to the tree-based approach as implemented here. Trial registration: ClinicalTrials.gov identifier: NCT00001196. Trial registration: ClinicalTrials.gov identifier: NCT01425892. Trial registration: ClinicalTrials.gov identifier: NCT00001390

Cluster analysis.

<p>Cluster analysis based on Spearman Rank Correlation. Participant 1: green, Participant 2: blue, Participant 3: red, Participant 4: yellow, Participant 5: purple. Sample denotation: P1-P5 (Participant 1-Participant 5), D1-D2 (Day 1-Day 2), S1-S6 (Sample 1-Sample 6).</p