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  • br Discussion br Some studies have


    4. Discussion
    Some studies have assessed the characteristics of the microbiota in OSCC patients and healthy controls by comparing those groups [20e23]. However, due to the small number of research subjects and lack of research in Japanese people, we have conducted the present study comparing between OSCC patients and noncancer controls using salivary samples that reflect the entire oral micro-biota and performing metagenome analysis by 16S rRNA sequencing.
    4.1. a-diversity and b-diversity
    We analyzed the microbiota Y-27632 dihydrochloride and compared the con-stituent bacteria. a-diversity compares the differences of in-dividuals and b-diversity compares the differences among groups. a-diversity is calculated from several diversity indexes. The
    Fig. 1. Comparison of a-diversity and b-diversity in OSCC patients and controls. In Observed and Chao 1 index (A) (B), we recognized diversity abundance, but there was no significant difference between the Simpson index and Shannon index (C) (D). PCoA based on weighted UniFrac distance (E). PCoA based on unweighted UniFrac distance (F). PCoA revealed that b-diversity in OSCC was significantly different in the controls.
    Fig. 2. Composition of bacterial communities across the samples at genus level (A) Relative abundance between individuals (B) Relative abundance between OSCC and the controls (C) Rearrangement of bacteria from the controls in descending order.
    Table 1
    Bacteria that showed a significant difference in OSCC patients and the controls.
    D:1::Bacteroidetes_D:2::Flavobacteriia_D:3::Flavobacteriales 0.0221
    D:1::Bacteroidetes_D:2::Flavobacteriia_D:3::Flavobacteriales_D:4::Flavobacteriaceae 0.021857
    D:1::Bacteroidetes_D:2::Flavobacteriia_D:3::Flavobacteriales_D:4::Flavobacteriaceae_D:5::Capnocytophaga 0.004448
    D:1::Fusobacteria_D:2::Fusobacteriia_D:3::Fusobacteriales_D:4::Fusobacteriaceae_D:5::Fusobacterium 0.027351
    D:1::Proteobacteria_D:2::Gammaproteobacteria_D:3::Pasteurellales_D:4::Pasteurellaceae 0.017721
    D:1::Proteobacteria_D:2::Gammaproteobacteria_D:3::Pasteurellales_D:4::Pasteurellaceae_D:5::Haemophilus 0.011794
    Fig. 3. Comparison of relative abundance of the genera between OSCC and the controls. Y-27632 dihydrochloride Peptostreptococcus, Fusobacterium, Alloprevotella, and Capnocytophaga were frequently observed in OSCC patients (A)e(D), whereas, Rothia and Haemophilus were less (E) (F). Each black dot shows the individual OTUs.
    observed index counts the number of OTUs, comparing the di-versity with the relative number of bacteria. Chao 1 index compares diversity using the abundance data of bacterial types; Shannon index and Simpson index compare the diversity using the unifor-mity degree of existent bacterial numbers. Regarding b-diversity, a phylogenetic tree was created from OTUs, and similarity was compared using phylogenetic distance.
    Weighted UniFrac distance does not consider the number of reads whereas unweighted UniFrac distance does. The former re-flects the presence or absence of bacterial species, and the latter reflects the composition ratio of bacteria.
    In this study, a-diversity between OSCC patients and controls was observed with a significant difference in the observed and Chao 1 indexes. A significant difference could not be found for the Shannon or Simpson indexes (Fig. 1 A e D). The results indicated 
    that more bacterial species were observed in the OSCC patients than in the controls, also including small counts of rare bacteria.
    b-diversity results showed significant differences in Unifrac unweighted distance and Unifrac weighted distance (Fig. 1 E, F), confirming that the variation of diversity is different between the OSCC patients and controls. From these results, we concluded that the oral environment of OSCC patients is more complex with various kinds of bacteria.
    Two previous studies compared healthy tissue samples and cancer tissue samples in the same patient and reported a significant difference in the Shannon index in one report [24], but not in the other report [11].
    Regarding b-diversity, both reports state that Unifrac un-weighted distance demonstrated an increase in the diversity of the microbiota in cancer tissue, which was similar to our results in that
    Table 2
    Relationship between bacteria and other factors that showed significant difference between OSCC patients and the controls(p-value).
    sex smoke drink dentures
    Fig. 4. Relative abundance of genera associated with impact factor and cancer incidence. Peptostreptococcus was frequent in females, and Haemophilus was frequent in males and drinkers (A)e(C). Each black dot shows the individual OTUs.
    Table 3
    Determination of risk factors involved in cancer incidence using logistic regression analysis.
    the complexity of the microbiota was increased. The results of this study also revealed the characteristics of the microbiota (Figs. 2e4 Tables 1 and 2).