Human saliva plays an important role in protecting the teeth and mucosa of the oral cavity and in maintaining the human oral microbiome. Saliva contains a number of inorganic components including Ca2+ and F- ions as well as phosphates and carbonates. These ions are important for mineralising tooth enamel, and the bicarbonates present in saliva allow it to act as a buffer preventing acidification of the oral environment following the production of acid by bacteria when they metabolise dietary sugars (Larsen and Pearce, 2003; Dodds et
Saliva also contains organic components including proteins involved in food digestion. For example, α-amylase and lingual lipase are two such enzymes that respectively convert starch to oligosaccharides and triglycerides to glycerides and free fatty acids that may be used by oral microbes (Hamosh and Scow, 1973; Kaczmarek and Rosenmund, 1977). A number of antimicrobial proteins are also present in the saliva, including cationic peptides such as histatins, statherin and lysozyme, IgA and IgG antibodies and chelating agents such as lactoferrin. These salivary components act to prevent harmful exogenous pathogens from colonising the oral cavity as well as controlling commensal microorganism populations (Rudney and Smith, 1985; De Sousa-Pereira et al., 2013; Brandtzaeg, 2013).
Salivary components are the most important nutrient source for the majority of oral
microbes, particularly in early plaque (polymicrobial dental biofilm) development (Marsh et
al., 2016). Glycoproteins including mucins and IgA are a source of fermentable
carbohydrates while amino acids and DNA are also utilised by some oral bacteria (Kilian et
al., 1996; Wickstrom and Svensater, 2008; Palmer et al., 2012; Edlund et al., 2015). Saliva
also contains serum components from the gingival crevicular fluid (GCF) that flows from the gingival margin (AlRowis et al., 2014).
Analyses of the human saliva microbiome using 16S rRNA gene sequencing have been conducted to determine the diversity and stability of the human saliva microbiome. These studies have revealed that at the phylum level, Firmicutes typically dominate, followed by Bacteroidetes, Proteobacteria and Actinobacteria. Fusobacteria, TM7, Spirochaetes and the Synergistetes although prevalent are typically less abundant. At the genus level
Streptococcus, Neisseria, Veillonella and Prevotella are often identified as being the most
2012; Dassi et al., 2014). A number of studies have also shown that there appears to be temporal stability over periods of weeks to years in the human saliva and the oral cavity in general (Costello et al., 2009; Cameron et al., 2015).
The human saliva microbiome has also been shown to be stable following exposure to antibiotics. A recent study conducted by Zaura et al. (2015) showed the human saliva microbiome to exhibit stability following a single course of clindamycin, amoxicillin, minocycline or ciprofloxacin, as microbial shifts were identified 1 week after treatment compared with baseline results (no antibiotic treatment) but at no other time points over a 12-month period. This indicated that the human saliva microbiome was able to rapidly return to its baseline composition following cessation of antibiotic use (Zaura et al., 2015).
Although an earlier study conducted by Nasidze et al. (2009) showed no geographical clustering of saliva microbiomes, the survey used a 16S rRNA gene cloning and sequencing approach with relatively low sequencing depth (Nasidze et al., 2009). Later studies using next generation 16S rRNA gene sequencing with greater sequencing depth have provided evidence for the contrary. For example, Li et al. (2014) used 454 sequencing to demonstate clear geographic influence on the oral microbiota. They showed that the human saliva microbiota of Alaskan and German populations were more similar to each other (Firmicutes most dominant followed by Bacteroidetes and Proteobacteria) than African (Uganda, Sierra Leone and Democratic Republic of the Congo) populations (Proteobacteria most dominant followed by Firmicutes and Bacteroidetes) (Li et al., 2014). In another study by Takeshita et
al. (2014) the saliva microbiome of South Koreans was shown to be less diverse than that of
human saliva microbiome as people from both countries have been shown to have similar genotypes (Abdulla et al., 2009; Takeshita et al., 2014).
3.1.1.2.2 The Teeth
Teeth are the only exposed non-shedding surfaces in the human body. They are made of enamel and represent approximately 20 % of the surface area of the mouth (Collins and Dawes, 1987). They are coated with an acquired enamel pellicle (AEP) which is a layer of proteins and glycoproteins from saliva, plasma and the oral microbiota and mucosa. The AEP protects teeth from acid degradation and abrasion, as well as acting as a platform for early microbial colonisers to interact with during plaque formation (Hannig and Joiner, 2006).
Plaque can reside above the gum line (gingiva) on the occlusal surfaces (fissure plaque) and the surfaces on the side of the tooth (supragingival). Subgingival plaque is on or below the gingiva. Due to its location, fissure plaque mainly harbours bacteria that can survive in aerobic conditions with Streptococcus spp. dominating, although some anaerobes such as
Veillonella spp. are present due to the presence of anaerobic microenvironments (Wilson,
2005).
Compared with other environments in the oral cavity, greater diversity is seen in the supragingival and subgingival plaques. Primary colonisers of teeth such as Streptococcus
mitis, Streptococcus oralis, Neisseria mucosa, Veillonella parvum and Actinomyces spp. are
and Levin, 2002; Teixeira et al., 2006). Secondary colonisers interact with cells that are adhered directly to the AEP or with cells adhered to these primary colonisers. Later colonisers interact with the outer cell layers of the plaque and the extrapolymeric substances (EPS) that are produced by cells within the plaque, Figure 3.2 (Rosan and Lamont, 2000).
Figure 3.2
Figure 3.2 Dental Plaque Biofilm. A diagram showing the bacterial composition of dental plaque biofilms. The
acquired enamel pellicle is bound by the primary Streptococcus spp. colonisers to which secondary colonisers interact. As the biofilm develops microenvironments are created including low oxygen zones (as oxygen is used by bacteria in the plaque or does not diffuse through the biofilm) that promote the growth of anaerobic bacteria including Fusobacterium spp. and Prevotella spp. This figure was reproduced from Kolenbrander et al. (Kolenbrander et al., 2002).
The interaction of cells within the biofilm is primarily mediated by lectin-carbohydrate interactions and is called co-aggregation (Rosen and Sela, 2006; Schuler et al., 2012). Co- aggregation results from cells coming into close contact and the creation of metabolic communication networks. An example of such a metabolic communication network is P.
gingivalis producing free glycines that Treponema denticola metabolises to produce lactate
that is utilised by P. gingivalis (Lewis et al., 2009). Microenvironments also develop within the plaque where aerobes utilise oxygen creating zones of low oxygen and low redox potential allowing for the survival of anaerobes, Figure 3.2.
As the supragingival plaque matures, secondary colonisers including species of
Fusobacterium, Treponema, Prevotella and Corynebacterium are incorporated into the
biofilm, and the relative abundance of N. mucosa and Streptococcus spp. declines (Aas et al., 2005; Uzel et al., 2011; Segata et al., 2012; Teles et al., 2012). Compared with the saliva, Firmicutes are less dominant in the supragingival plaque although still abundant. As the subgingival plaque is in a more anaerobic environment than the supragingival plaque and receives its nutrients from the GCF, asaccharolytic anaerobic bacteria including species of
Fusobacterium, Porphorymonas, Prevotella, Tannerella, Parvimonas and Treponema are
more prevalent in this biofilm again at the expense of genera of Firmicutes (Aas et al., 2005; Segata et al., 2012; Ge et al., 2013; Y. Li et al., 2014).
Bacteria present in these oral biofilms have the potential to enter the saliva during biofilm dispersal. For example, dispersin B genes (dspB; encodes a protein involved in biofilm dispersion) are encoded by oral Aggregatibacter spp. and S. mutans produces a protein (surface-protein-releasing-enzyme; SPRE) that mediates its release from biofilm at low pH
(Kaplan, 2010). Thus, it is important to understand the bacterial composition of dental plaque when studying the saliva microbiota.