Composition and Diversity of Salivary Microbiome Affected by Sample Collection Method

Purpose: The purpose of this study was to investigate whether various saliva collection methods affect the observed salivary microbiome and whether microbiomes of stimulated and unstimulated saliva and plaque differ in richness and diversity. Methods: Seven sampling methods for unstimulated saliva, stimulated saliva, and plaque samples were applied to six orally and systemically healthy participants. Bacterial 16S ribosomal RNA genes of 10 major oral bacterial species, namely, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, Streptococcus mitis, Streptococcus sobrinus, and Lactobacillus casei, were analyzed by real-time polymerase chain reaction. We comprehensively examined the dependence of the amount of bacterial ribosomal DNA (rDNA), bacterial-community composition, and relative abundance of each species on sample collection methods. Results: There were significant differences in the bacterial rDNA copy number depending on the collection method in three species: F. nucleatum, P. nigrescens, and S. mitis. The species with the highest richness was S. mitis, with the range from 89.31% to 100.00%, followed by F. nucleatum, P. nigrescens, T. denticola, T. forsythia, and P. intermedia, and the sum of the proportions of the remaining five species was less than 1%. The species with the lowest observed richness was P. gingivalis (<0.1%). The Shannon diversity index was the highest in unstimulated saliva collected with a funnel (4.449). The Shannon diversity index was higher in plaque samples (3.623) than in unstimulated (3.171) and stimulated (3.129) saliva and in mouthwash saliva samples (2.061). Conclusions: The oral microbial profile of saliva samples can be affected by sample collection methods, and saliva differs from plaque in the microbiome. An easy and rapid technique for saliva collection is desirable; however, observed microbial-community composition may more accurately reflect the actual microbiome when unstimulated saliva is assayed.