*3.4. Core Microbiome Analysis*

Figure 2 shows the prevalence of taxa in terms of abundance. Abundant species belonged to *Firmicutes*, *Proteobacteria*, and *Bacteroides*. In this study, 29 species were detected from all subjects in denture plaque and the tongue. Table 1 shows the species detected from all subjects, and Table S3 shows the 15 species in plaque and 7 species in the tongue. The most abundant bacteria were Firmicutes at the phylum level and the *Streptococcus salivarius* group at the species level in both plaque and the tongue. The *Fusobacterium nucleatum* group was detected from all subjects. A heatmap of the candidates for the core microbiome is shown in Figure S3.


**Figure 2.** Prevalence of taxa in terms of abundance for 14 different phyla.

## *3.5. Analysis of Co-prevalent Species*

Then, the levels of species co-prevalence were analyzed by t-distributed stochastic neighbor embedding (tSNE). As shown in Figure 3, species were constructed in clusters that were not dependent on the phylum. The list of classified species and detection in 3 of 4 subjects (75%) is shown in Table S2.

**Figure 3.** t-distributed stochastic neighbor embedding (tSNE) plot of species: (**A**) denture plaque, and (**B**) the tongue.

#### **4. Discussion**

This study describes microbial profiles of the oral cavities of four female centenarians by high throughput sequencing. Highly prevalent and abundant species are described. In addition, differences in sampling sites and co-prevalence of species are presented.

A total of 406 species were detected. Studies indicate that the diversity of bacteria is reduced with the frailty of older adults [3,46]. All participants in this study were female centenarians. Bacterial diversity was not reduced when compared to another study [16].

*Firmicutes* was the most abundant phylum for both the denture plaque and tongue samples. Several studies have shown that *Firmicutes* is the most abundant phylum in the saliva of infants [16] and young children aged 6–8 years [47], swabs of the oral cavities of youths and adults [48], and dental plaque from older adults [34]. *Firmicutes*, *Streptococcus*, and *Veillonella* were detected in all subjects in both the denture plaque and the tongue. The result that *Firmicutes* was the most abundant, followed by *Veillonella*, is consistent with the proportions of oral microorganisms in the Human Oral Microbiome Database [49]. The abundant genera after *Veillonella* vary between studies. *Streptococcus*, *Veillonella*, and *Neisseria* were the predominant bacterial genera present in infants. Abundant genera that coat the tooth surface first are *Streptococcus* and *Veillonella* [50]. *Streptococcus*, *Veillonella*, and *Neisseria* are the predominant bacterial genera present in infants [16]. *Streptococcus* and *Veillonella* are known as early colonizers. *Neisseria* settles down at the age of 1–2 years [18].

In this study, *Neisseria* was not detected in all subjects. Differences at the species level in previous reports may be due to the sample difference. All subjects surveyed in this study wore complete dentures, so the plaque sample was denture plaque, not dental plaque attached to the tooth surface. Additionally, the small sample size may have affected the difference.

In this study, *Streptococcus salivarius* was the most abundant species in both the denture plaque and the tongue. This result is consistent with another study that investigated the tongues of Japanese older adults [16]. The results of the study show that more than 5% of abundant species were *Streptococcus salivarius*, *Prevotella melaninogenica, Rothia mucilaginosa, Veillonella atypica,* and *Neisseria flavescens*. Except for *Neisseria flavescens*, the other four species were common to our results. Another study showed that *Streptococcus oralis* was by far the most prevalent species [51]. As described above, Firmicutes and *Veillonella* are abundant phyla. Each study has different bacterial profiles at the genus or species level.

The bacterial profile of the denture plaque was similar to that of oral mucosa. Actinobacteria was abundant in the denture plaque and Bacilli were abundant in the dental plaque [52]. In this study, the proportion of these bacteria varies between samples: 5.4%, 15.5%, 10.8%, and 6.5% for Actinobacteria and 82.45%, 44.4%, 21.5%, and 48.1% for Bacilli. The small sample size may have affected the results.

A previous study had shown that oral microbiome profiles were related to the risk of death by pneumonia for older persons residing in nursing homes [53]. *Neisseria flavescens*, the *Fusobacterium periodonticum* group, and the *Haemophilus parainfluenzae* group were abundant in the risk group. In this study, one subject (Sample ID 4) had higher levels of these bacteria than the mean values of the risk group.

As shown in Figure 3 the bacterial community formed clusters with a low prevalence or abundance of bacteria. The clustering was not phylum dependent.

The clusters were composed of bacteria with a wide range of prevalence and abundance. This indicates that some bacteria were the core of the cluster. There may be an interaction or symbiotic mechanism between core bacteria and low-prevalence or low-abundance bacteria. Therefore, there may be healthy microbiome candidates within the core bacteria of each cluster.

A limitation of this study was the small sample size of four people. The design was a cross-sectional study. The data presented in this study were from Japanese older persons. Even though dietary habits have little effect on the oral microbiome [54], regional or cultural differences could have an effect on the oral microbiome [44]. The health status of the subjects who participated in this study was limited. Subjects with specific diseases were not included. For comparison, healthy and pathogenic conditions should be considered. Longevity is not simply explained by microflora. Further study is needed to evaluate the host factors such as nutraceutical agent [29], serum malondialdehyde [30], and progenitor cell levels [31]. In addition, the subjects investigated in this study were all female and edentulous.
