Background
It is estimated that 2.4 billion people suffer from dental caries, and 621 million of them are children [
1]. Severe caries can affect their quality of life [
2]. Strategies to prevent caries are based on a comprehensive understanding of its etiology and effective control of the risk factors. It is recognized that causes of caries include microorganisms in the mouth and host factors. The oral cavity is one of the most diverse and complex microbial environments [
3]. Previous studies demonstrated that oral plaque film has high relevance in dental caries. The acid produced from bacteria break the balance of tooth mineralization and demineralization and the host have no rapid response to pH changes, which lead to organic degradation [
4]. Saliva is the main microenvironment of oral microorganisms, and to some extent, saliva microorganism determines the structure of plaque. Salivary protein has a crucial role in monitoring health status or monitoring disease [
5]. It was reported that the proteins in saliva could modulate the balance of oral health and homeostasis, maintain a stable ecosystem, and inhibit the growth of cariogenic bacteria [
6].
In the past few decades, several investigators have proposed several hypotheses regarding the etiology of caries [
7‐
9], the relationship between bacteria and dental caries, the complexity of the oral bacterial structure, and the difference of bacterial components. Previous studies also mentioned that some salivary proteomic molecules could regulate the oral cavity microbial flora and correlate to caries [
6,
10,
11]. Unfortunately, due to differences in samples, technologies, and analytical methods, the results remain controversial and the biomarker information unclear.
Thanks to recent advancements in molecular biology techniques, metagenomic and metaproteomic can be used to obtain a complete analysis of the oral bacteria and proteomic. Next-generation sequencing technologies have been successfully applied in oral microbial analysis [
12‐
14]. The isobaric tags for relative and absolute quantitation (iTRAQ) is a new technique which uses isotopes to label polypeptides for comparing proteomes quantitatively [
15,
16]. To the best of our knowledge, previous studies of caries-related microbiome and proteome were detached. Our present study uses metagenomic and metaproteomic analyses to explore the microbiological and proteinic biomarkers and investigate caries etiology in children.
In this study, we enrolled 6–8 years old children (isolated population) who come from Tujia and Miao minority autonomous county, Pengshui, Chongqing, China. These children have a simple and homogeneous diet; therefore, the impact of different diets and daily living habits is avoided. In the current study, the oral microecological diversity was studied using 16S rDNA pyrosequencing, and the salivary proteins were analyzed using the iTRAQ technique coupled with quantitative nano-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our study aimed to 1) detect the microbiological compositions and to investigate the core microbiome; 2) identify the salivary proteomic and characterize the functional classification in children with or without caries, and 3) attempt to identify microbiological or proteinic biomarkers helpful to prevent dental caries.
Discussion
The etiological concepts of oral infectious diseases, including caries and periodontal disease, has gradually changed from a single pathogen theory to a microecological imbalance theory [
4,
27,
28]. Therefore, a systems biology approach is required to explain the complex interactions between the microbiome and the host. As far as we know, an approximate of 1000 bacterial species have been found in the oral cavity [
29] due to the advent of molecular analysis methods. Recently, 16S rRNA sequence analysis was introduced in the study of uncultured oral microbial communities; this is an advantageous molecular analysis technology for investigating the oral bacteria diversity and microbial community composition in oral diseases. Meanwhile, salivary proteins play an essential role in the occurrence and development of caries. Proteomics has advanced significantly over the past decades, and it has been applied for the study of caries and other oral diseases [
28,
29]. In this study, we preliminarily explored microbiome and host factors in childhood caries using the high-throughput technique of 16S rDNA pyrosequencing and iTRAQ-coupled LC-MS/MS.
After 454 pyrosequencing the sequences were clustered into 14,076 OTUs and 18 phyla, 28 classes, 48 orders, 78 families,135 genera, and 410 species were detected. These results exceeded the data of the previous HOMIM analysis of our group [
30,
31]. The results of different sequencing technology methods could differ. To our knowledge, the HOMIM analysis has an emphasis on the predominant species of the bacterial community, while in the 16S rDNA pyrosequencing technology, the detection sensitivity of some species of bacteria is a little bit limited [
17]. In the present study, 16S rDNA pyrosequencing could be more favorable to investigate a complete profile of the oral microbiome and discover some rare and non-cultivated bacteria that could be related to caries.
According to the results of alpha diversity indices, the richness and diversity of the bacterial communities in caries groups were similar to the caries-free group, as previously found in other studies [
17,
19,
32]. However, Xiao et al., [
18] demonstrated a higher bacterial diversity of healthy dental plaques compared to dental caries. These controversial results could be influenced by the difference between individuals, the selection process of subjects, sequencing technology methods, and other factors. Moreover, we found the six most abundant phyla including
Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, Actinobacteria, and Candidate division TM7, which were in agreement with the results of previous studies [
13,
17,
18]. At the genus level, 135 genera were detected, including 13 prevalent genera, roughly similar to previous studies [
32‐
34]. These dominant bacterial communities at the phylum and genus level were similar in the caries-free and caries-active sample, and merely the relative abundance was different. This indicates that the activity of specific microorganisms does not cause dental caries, some cariogenic bacteria are also part of the normal oral flora, and their presence is a constant variable [
35].
Dialister, Selenomonas, Actinomyces, and
Mogibacterium were identified at significantly higher levels in the caries-active sample using the LEfSe analysis, which could be recognized as a potential bacterial biomarker in dental caries. We speculated that changing some metabolic pathways and these bacteria’ biological characteristics are relate to caries in children .
At the beginning of caries, several microorganisms gather on the tooth surface in an ordered way, and then the oral ecosystem is broken when caries occur. Acidogenic and acid-tolerating species shift toward community dominance [
36,
37]. In the current study, a higher abundances of Actinomyces were observed in the PH group compared to PN (
P < 0.05), with the detection rates of Actinomyces odontolyticus being the highest. Actinomyces viscosus, an acid-producing bacterium associated with biofilm formation, was significantly higher in both SH and PH than in the caries-free group with low abundance. This indicated that some low abundances also could play an important role in in the oral microenvironment [
25]. Actinomyces gerencseriae was higher in the caries-active group, which might be meaningful to investigate the correlation with caries in future studies. The Streptococcus genus had no significant difference between the caries-free and caries-active group. At the species level, Streptococcus sanguinis was higher in caries-free plaque than caries-active plaque. These outcomes could derive from different categories of severity. Streptococcus sanguinis settle on the tooth surface during early caries lesions and its population decrease with the development of caries [
38]. The detection rates of Streptococcus mutans were lower than 0.2%, and it was significantly higher in the SH and PH group compared with the caries-free group. It is widely recognized that Streptococcus mutans is an acidogenic and aciduric bacterial species interrelated with caries. However, previous studies proved that caries occurred without the presence of Streptococcus mutans [
8,
39]. The ecological plaque hypothesis emphasizes that the occurrence and development of dental caries result from an ecological imbalance between tooth mineral and microbial flora, and the upsurge in the acidogenic and aciduric component in the oral microenvironment would break the balance [
9]. The current study demonstrated that the diversity of the microbial community has little effect on caries and some rarely detected bacteria but at higher levels in the caries-active sample would play a critical role in caries development, supporting the “ecological plaque hypothesis.”
Oral health and disease are correlated with the interplay inside the oral microbial community. Saliva, as the main microenvironment of oral bacteria, is considered a significant influence on the colonization of microorganisms [
40]. The result of PCoA analysis revealed clear segregation between samples from dental plaque and saliva, meaning the distribution of microorganism structures in plaque were different from those of saliva. Ren et al., [
13] suggested that dental plaque had significant phylogenetic differences compared with saliva and tongue coating. The reasons for this situation are probably related to the physicochemical features at different sites, such as pH, oxygen concentration, and bacterial adherence [
41].
Human microbiological studies support the concept of a “core microbiome,” which is referred to the microbiome shared by most individuals in a specific environment of the body such as the skin, nasal cavity, intestinal tract, and oral cavity [
42‐
45]. In our study, the Venn diagram shows that 52.6% of all the genera were shared and 18 predominant genera uniform was identified in saliva and plaque subjects, indicating the existence of “oral core microbiome,” as suggested by a previous study [
18]. The core microbiome contributes to the functional stability and microecological balance of a healthy oral cavity.
For the result of salivary proteome analysis, we detected differentially expressed proteins and their functional classification between the SN and SH groups. Compared with the method of electrospray ionization ion-trap tandem mass spectrometry (ESI-MS/MS) used in our previous study, the number of proteins and peptides identified in our present study was higher [
46]. Two hundred and fifty-eight proteins were found to be differentially expressed, which might play a part in the process of childhood dental caries. Some important proteins were included in differentially expressed proteins, such as
lactoferrin, matrix metalloproteinase-9, cystatin-B, mucin-7, protein S100-A9, proline-rich protein and so on, which have demonstrated a potential relationship with caries in previous studies [
47‐
49]. Lactoferrin is an antibacterial protein with the iron-chelating property directly binding to bacteria and agglutinate
S. mutans. The combined bacteria are easy to be removed with the mechanical saliva action [
50,
51]. Also, it was reported that there was a high correlation between matrix metalloproteinase-9 and caries lesion depth [
49]. MMPs and cysteine cathepsins could affect the caries process in the early phases of demineralization [
52]. The result of the GO analysis shows that differentially expressed proteins were associated with metabolic process and regulation of the biological process, mainly in the protein binding. As common salivary proteins, mucin-7 binding to proline-rich protein could be adsorbed onto the tooth surface to form a pellicle that regulates the bacteria adhesion and modulate the demineralization/remineralization process [
53,
54]. Particularly,
azurocidin was identified in the differentially expressed proteins, which has been found to be associated with gingivitis and early inflammatory periodontal destruction, and is a potential biomarker for periodontitis. But its possible anti-caries effect is worth further exploration. In addition, there were other proteins that were detected in differentially expressed in SH group or SN group, and their potential to cause or prevent caries needs to be further confirmed. The molecular sequencing techniques make precise identification of proteins. However, because of the complexity of saliva and immature technologies, proteinic information in our current research is not complete, and some low abundance proteins from the microorganism and its metabolite were not explored. The specific mechanism and more detailed information about the proteins in the saliva need to be further investigated. There is still a long way to devise strategies that modulate interactions of microbiota and salivary proteins for the treatment of oral diseases.
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