In subjects with DS, early development of periodontal diseases has been reported, while the rate of dental caries is reportedly low [7, 8].
This study therefore compared the salivary microbiomes of children with and without DS aged 1 to 12 years, which is considered the crucial period for microbiome formation, with the aim to elucidate the relationship between the onset of dental caries and periodontal disease in DS.
Amano et al. and Sakellari et al. detected periodontopathic bacteria in 2- to 13-year-old and 8- to 28-year-old subjects with DS, respectively, using quantitative PCR and DNA–DNA hybridization, respectively. They reported that these bacteria were detected earlier in subjects with DS than in healthy subjects [9, 10]. With regards to cariogenic bacteria, Scalioni et al. tested for S. mutans in the saliva of 3- to 12-year-old children with in situ hybridization, and reported lower detection rates of the species in children with DS than in healthy children .
This study collected the saliva of 27 children with DS and age-matched controls to test salivary cariogenic and periodontopathic bacteria in these samples using quantitative PCR, and found low detection rates of these pathogens in children with PD (aged 1–4 years), and no differences in detection rates between DS and ND in the MD stage (aged 6–12 years) (Table 2).
Studies to date have measured periodontopathic bacteria in the subgingival plaque, whereas this study tested them in saliva; this difference may explain why we did not observe a difference because of a lower detection rate of the red complex, which are composed of strictly anaerobic bacteria. Moreover, rates of colonization of cariogenic bacteria in children in Japan have recently decreased because of the increase in awareness of the importance of oral hygiene ; this may have played a role in the difference between the findings of this study and those of the previous studies.
The comparison of bacterial microbiome by 16S rRNA high throughput sequencing did not reveal any difference between DS and ND in α diversity of the microbiomes in both PD and MD groups; however, a significant difference was observed in β diversity as shown on the PCoA PLot (Fig. 1, 2).
In both PD and MD groups, Corynebacterium and Cardiobacterium were dominant and TM7 was less abundant among the bacterial genera composing the microbiome in DS. Moreover, there were more genera with significant DS-ND differences in the MD stage than in the PD stage, suggesting that the DS-ND differences in salivary microbiomes may widen with age.
Willis JR et al. collected the oral rinse samples from individuals with DS aged 7–55 years for a comparison of the microbiome with healthy controls, and reported that the genera Kingella, Staphylococcus, Gemella, Cardiobacterium, Rothia, and
Actinobacillus were more abundant, and Alloprevotella, Atopobium, Candidatus, and Saccharimonas were less abundant in DS .
The results of this study also showed higher abundance of Cardiobacterium in children with DS in both the PD and MD groups, suggesting that the dominance of Cardiobacterium is a characteristic of the oral microbiome of DS from a very early stage.
At birth, the oral cavity is practically sterile, while bacterial flora develops under the influence of factors and events, such as mode of delivery, breast or bottle feeding, eruption of tooth, introduction of solids, and oral hygiene status [23, 24]. Among them, the oral hygiene status is one of the major factors of dental caries and onset of periodontal diseases as well as microbiome formation .
There are several limitations to this study related to insufficient data collection. First, we could not obtain clinical indices, such as the Oral Hygiene Index and the Gingival Index, and we were unable to study the relationships with the degree of dental plaque accumulation or gingival inflammation. However, the results of the questionnaire survey showed no major differences in the number of teeth or oral hygiene habits (Table 1), suggesting that the differences in the oral microbiome between children with and without DS are primarily due to differences in their oral environments, including salivary properties.
Many characteristics of the saliva of individuals with DS have been reported, including low secretion, increased oxidative stress, increased secretory IgA, and abnormality of inorganic salts [26–30], and all these factors are likely to affect the salivary microbiome [31, 32].
Effects of decreased salivary secretion on the oral microbiome have been discussed in the context of Sjögren's syndrome and side effects of radiotherapy and drug therapies [33–35]. Such studies have commonly reported higher detection rates of Lactobacilli and C. albicans; however, the present study showed a low detection rate of Lactobacilli and no significant difference between children with DS and ND group. In DS group, the observed high detection rate of C. albicans was consistent with the findings of the previous studies.
Overexpression of the SOD gene encoded on chromosome 21 has been reported to cause overproduction of hydrogen peroxide and associated hydroxy radicals in subjects with DS than in healthy individuals , and accordingly, oxidative stress marker levels in their saliva have been reported to be higher compared to those in healthy individuals [26, 27]. Furthermore, hydrogen peroxide produced by some bacterial species in the genus Streptococcus in the oral cavity are known to affect the surrounding microbiome, and hydrogen peroxide produced by S. sanguinis inhibits S. mutans colonization [37, 38]. Such increased oxidative stress in the saliva may indeed have an effect on the microbiome.
Among the bacterial species found at significantly higher abundance in children with DS in both PD and MD groups, Neisseria elongate, Rothia aeria, Rothia dentocariosa, and Corynebacterium durum are catalase-positive bacteria, except Gemella haemolysans . These bacteria are known to show resistance to high oxidative stress environments and may be a factor of dysbiosis in the salivary microbiome of the group of children with DS. Moreover, the aforementioned C. albicans also has a catalase gene, which may similarly explain their higher abundance in the DS group.
Dental plaque is formed through cell to cell interactions between bacteria from initial colonizers to the late colonizers in the oral cavity , and dental plaque adapted to the environment are known to develop in the supragingival and subgingival plaque [41, 42].
Khocht A et al. studied 40 bacterial species in the subgingival plaque of adults with DS using checkerboard DNA–DNA hybridization. They reported that Streptococcus sp. (i.e., S. oralis, S. mitis, and S. gordonii), which are initial colonizers, were more abundant in adults with DS than in healthy individuals . S. sanguinis, an initial colonizer, was also significantly more abundant in our participants with DS in the MD stage. These Streptococcus bacteria are known to be particularly important in the initial stage of dental plaque formation, and their differences in saliva are thus likely to influence the constituents in subsequent dental plaque formation.
C Xiao et al. analyzed supragingival dental plaque in adult patients with dental caries using 16S pyrosequencing and have reported the characteristic presence of Cardiobacterium and Corynebacterium bacteria in participants without dental caries . Waleed F Janem et al. studied the salivary microbiome of obese children with and without type 2 diabetes and have shown that Lautropia, Corynebacterium, and Cardiobacterium bacteria were detected in association with gingivitis .
In this study, the genera Corynebacterium and Cardiobacterium were dominant in the saliva of children with DS in the PD and MD stages, and the genus Lautropia was dominant in children with DS in the MD stage. The observed differences in salivary microbiomes, including the aforementioned differences in Streptococcus sp. affect the composition of plaque bacteria and may be associated with the onset of dental caries or periodontal diseases.
These findings suggest that the distinct characteristics of the salivary microbiome in subjects with DS from that in healthy individuals may be attributable to several factors; however, we were not able to measure the amount of salivary secretion or oxidative stress status in this study. Moreover, complications of DS or history of antibiotic administration within 1 week of specimen sampling can also affect the salivary microbiome , thus these factors should also be analyzed in future studies to get a better understanding of the dysbiosis of the salivary microbiome in DS. Furthermore, associations between the development of dental caries, periodontal diseases, and bacteria constituting dental plaque in the affected sites in subjects with DS should also be analyzed.
In conclusion, qPCR and high throughput sequencing provided basic data on the salivary microbiome in DS, and revealed dysbiosis in the salivary microbiome in children with DS when compared with that in healthy children. Moreover, the dominance of Corynebacterium, Cardiobacterium, and lower abundance of TM7 were identified as characteristic microbial markers of dysbiosis in children with DS in both the PD and MD stages.