**1. Introduction**

The human oral cavity is the second largest microbial habitat comprising fungi, bacteria, and viruses, after the intestines, and has a special niche consisting of soft tissue of the gingiva and oral mucosa and the hard tissue of the teeth [1]. Although the oral cavity and intestines are a distance apart, they are connected and show a marvelous diversity of microorganisms. The oral microbiome refers to the collective genetic materials of oral microorganisms. The microorganisms in the oral cavity are referred to as oral microbiota, oral microflora, and oral microbiome [2]. Microorganisms coexist in our bodies and live through symbiosis, dysbiosis, and pathogenic relations. The microbiome not only refers to the microorganisms involved, but also includes areas of activity that result in the formation of specific ecological niches. Significant changes in the local environment can disrupt the microbe–microbe interaction, which can alter the host–microbe equilibrium, increasing the risk of disease [3]. The oral cavity is the most accessible habitat for studying the relationship between the host and microorganisms. Different oral structures such as the teeth, the gingiva, the palate, the cheek, and the lips are colonized by distinct microbial communities, and the surfaces of the oral cavity are covered with bacterial biofilm [4]. The special environment of the oral cavity, with its stable pH of 6.5–7.0 of saliva, moisture, and its temperature of an average of 37 ◦C, creates the favorable conditions necessary for the growth of microorganisms [5]. For this reason, various kinds of microorganisms are well distributed in the mouth. The oral cavity is one of the best-studied microbiomes to date, with research focusing on its role as a part of human microbiome development and how it influences systemic health and disease. About 700 species of prokaryotes have been identified [6], and 392 taxa with at least one reference genome exist [7]. In the near future, the advances in our understanding of oral microbiomes may change our way of life through

**Citation:** Cho, Y.-D.; Kim, K.-H.; Lee, Y.-M.; Ku, Y.; Seol, Y.-J. Oral Microbiome and Host Health: Review on Current Advances in Genome-Wide Analysis. *Appl. Sci.* **2021**, *11*, 4050. https://doi.org/ 10.3390/app11094050

Academic Editor: Yoshiaki Nomura

Received: 30 March 2021 Accepted: 27 April 2021 Published: 29 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in-depth principles of oral biology and novel therapeutics. In this review, we provide a general understanding of the oral microbiome, establish the link between oral microbiomes and general health based on a genome-wide analysis through the latest technique, and discuss the perspective and future directions for both oral and systemic health.

## **2. Paradigm Shift: Microorganisms to Microbiomes**

The term "microorganism" simply refers to very small living things such as archaea, bacteria, protists, fungi, and viruses, while "microbiome" is a collective and comprehensive term for microorganisms. The community of microbial residents is referred to as the microbiome, and recent research on microbiomes has demonstrated an important role of the communities of microorganisms in human homeostasis (Figure 1A) [8]. The term "microbiome" was coined by Joshua Lederberg, a Nobel Prize laureate, "to signify the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease" [9]. As a collection of information, the microbiome includes the microorganisms' genomic data, structural elements, metabolites, and environmental conditions (Figure 1A) [10]. With the emergence of new technology, including next-generation sequencing (NGS), and in-depth information, such as sequence profiles of microbial communities, numerous insights on the relation between the human microbiome and disease have been obtained [11].

**Figure 1.** From microorganism to microbiome; a paradigm shift with advanced technology. (**A**) The term, microbiome, refers not only to the microorganisms but also to their theatre of activity. (**B**) Timeline of microbiome research.

In the 1670s, the study of human microbiology was initiated with the invention of the microscope, and the consequent observation of bacteria in pond water and dental plaque, by Antoni Philips van Leeuwenhoek [12,13] (Figure 1B). He was the first to observe microorganisms and determine their sizes. After that revolutionary invention, a culturebased method that is dependent on the growth of viable and culturable microbes was introduced to identify microbes with biochemical subtyping in the 1800s [14]. After the 1980s, the emergence of new technology, such as DNA sequencing, which enables acquisition of more than 1 trillion sequences of genetic information, provided a powerful unculturable method for understanding human health and disease [15]. Based on this advance in sequencing technology, the Human Genome Project (HGP) was started in 1990 and completed in 2003. The first sequence of the human genome was published in 2001 and constituted about 90% of the genome [16]. With the HGP, the elucidation of the human

genome initiated a new era in "precision medicine", which is the ideal concept of health care [17]. The basis of precision medicine includes the genomes of humans and of the microorganisms, such as fungi, viruses, and bacteria, that reside in the human body. As the extension of the HGP, the Human Microbiome Project (HMP) was launched in 2007 with the goal of establishing 3000 microbial genomes from the representative six regions of the body (the mouth, the esophagus, the stomach, the colon, the vagina, and the skin) as reference genomes [18].

#### **3. Methodology of the Microbiome Research**

Completion of the HGP was accompanied by parallel and ongoing development of commercially available high-throughput DNA sequencing tools, which eventually facilitated the acquisition of genomic information from human samples. High-throughput techniques such as NGS have replaced the culture-based traditional methods for identifying and characterizing microbes [15]. Past studies without NGS and relying on the laboratory culture system were limited to small parts, such as the biochemical or physiological properties of microbiomes, called opportunistic pathogens [19]. In the beginning, microbiology research had focused on identifying pathogens within the commensal microbiota and deciphering their virulence in relation to health and disease. In the 1980s, a sequencing technique was introduced that enabled nucleotide sequences to be determined. This remarkable advance provided new and diverse information about microbial taxonomic profiling regardless of cultivability [19]. In taxonomic profiling, two methods, amplicon sequencing (16S rRNA sequencing) and direct shotgun sequencing (metagenomics), are used [19]. In the oral microbiome studies, samples were taken from dental plaque and crevicular fluid from the periodontal pocket. This technology has allowed the characterization of microbial diversity in the human microbiome with unprecedented depth and coverage [20]. These oral microbiome studies originated from HMP's 16S rRNA sequencing data, published in the Human Oral Microbiome Database (HOMD), which identified oral bacteria from specific locations in the mouth such as the teeth, the gingival sulcus, the tongue, the cheek, the tonsils, and the soft and hard palates [2]. These early sequencing studies focused on the 16S rRNA sequence, and this conventional sequencing method was performed using a cultivated clonal culture; however, there were many microorganisms that were unculturable and thus could not be identified with this conventional method (Figure 2) [21]. This cultivation-based method could find < 1% of microbial species; therefore, metagenomics was introduced to identify non-isolated organisms via DNA sequencing without the need for cultivation and isolation [21]. In addition to metagenomics, DNA barcoding or metabarcoding is also used for the identification of species, using a short section of DNA from a reference library database [22]. The term "metabarcoding" is used when DNA barcoding is used to identify organisms from samples containing DNA from more than one organism [23]. Metagenomics also enables the metabolic and functional diversity of microbial communities to be accessed via metatranscriptomics, metaproteomics, and metabolomics [24]. Metaproteomics has emerged as a complementary approach to identify the functions of microbial communities [25]. Mass spectrometry-based proteomics has been widely used for studying the composition of proteins in oral microbial communities and has been applied to characterize oral biofilms [26]. Many metaproteomic studies have been performed in periodontal disease and endodontic infections associated with apical periodontitis using saliva and crevicular fluids [27]. Metabolomics is the study of metabolites within a biologic system to discover what happens in cells. Several studies have been conducted on the salivary metabolome; however, these have offered less specific explanations due to difficulties regarding disentangling host reactions and microbial contributions [27]. As such, metaproteomics and metabolomics complement the foundations established by metagenomics and metatranscriptomics in the comprehensive understanding of microbiomes.

**Figure 2.** Methodology of the microbiome research.

### **4. Core, Pathologic, and Healthy Microbiomes**

Microbiomes are largely divided into two types: core and variable microbiomes. The core microbiome is common to all individuals, while the variable microbiome is unique to individuals according to their lifestyle [28]. One of the goals of the HMP was to identify "core microbiomes" defined as microbial taxa or genes shared by most people [29]. The core microbiomes are described via five types: common core, temporal core, ecological core, functional core, and host-adapted core [28]. In order to diagnose and treat disease at an early stage, it is necessary to explain the commensal microbiome associated with health [30]. The commensal human microbiome is estimated to be 10 times greater in number than the quantity of human cells. These microbial communities are normal residents of the oral cavity, the skin, and the intestinal mucosa, and have a wide range of functions that are essential to the survival of the host. When the symbiotic balance between the host and the microbes is disrupted and disease is obvious, the presence of pathologic microbes stands out [31]. On the other hand, if the healthy microbiome is dominated by a "core microbiome", the homeostasis of health would be maintained [32]. Zaura et al. defined the healthy core microbiome of oral microbial communities from several intraoral niches using 454 pyrosequencing, showing a major proportion of oral bacterial sequences of healthy individuals as identical, which supports the concept that there are key microbiomes in health [30]. In addition, Bao et al., identified the microbiome and proteomic profiles in the gingival tissue of healthy individuals and individuals with periodontitis by a pressure cycling technology-assisted workflow, which is an emerging platform for tissue homogenization and sequence retrieval coverage [33]. The results showed that 69 proteins were differentially expressed in periodontitis, and Treponema sp. HMT253 and Fusobacterium naviforme were strongly associated with disease sites, indicating the existence of a tissue-specific microbiome signature.

#### **5. Human Microbiome Project**

The omics era, with its innovative sequencing techniques, accelerated all aspects of biological research, and its effects were particularly evident in studies on microbial communities and the human microbiome. Genomic sequencing has revealed the diversity of microbial communities in the body, and more information has been obtained as the National Institute of Health Human Microbiome Project was launched in 2007 [18] and completed its work in 2018. The HMP was conducted in two phases over a decade to provide resources, methods, and discoveries linking human-to-microbiome interactions with health- and disease-related outcomes. The first phase of HMP (HMP1) focused on the characterization of the microbial community of numerous targeted body parts, including the oral cavity, the nose, the gut, the skin, and the vagina, from healthy adult subjects, and demonstration projects focusing on specific diseases [34,35]. The HMP1 produced abundant community resources including nucleotide sequences of microorganisms [36,37], protocols for reproducible microbiome sampling and data curation [38,39], bioinformatics methodology, and epidemiology [40,41]. One of the important findings of HMP1 was that the taxonomic composition of microbiomes alone was not strongly associated with the phenotype of the host. This tended to be better predicted by a wide range of microbial molecular functions or personalized strain-specific compositions. Based on this, the Integrative HMP (iHMP), the second phase of HMP (HMP2), was promoted to understand host–microbiome communications, including molecular mechanisms and immunity [42,43]. Unlike HMP1, which focused on the microbiome, HMP2 expanded the scope to include both the microbiome and the host in three longitudinal cohort studies on preterm birth based on vaginal microbiomes, inflammatory bowel diseases based on gut microbiomes, and prediabetes based on gut and nasal microbiomes [43]. The studies of HMP2 included both microbial and host-specific multi-omics data such as genome, epigenome, transcriptome, metabolome, and proteome. In addition to HMP, Metagenomics of the Human Intestinal Tract (MetaHIT) has produced the resources and specialty needed to understand human microbiomes.
