This chapter describes in detail the oral flora and its living habitat. Particular emphasis is placed on the oral ecosystem, and how it modulates the life and times of the microbial populations living within our oral cavity.
The human body is composed of approximately hundred trillion cells, of which 90% comprise the resident microflora of the host and only 10% are mammalian. Bacteria are by far the predominant group of organisms in the oral cavity, and there are probably some 700 common oral species or phylotypes of which only 50%-60% are cultivable in the laboratory. Of these, approximately 54% are officially named, 14% unnamed (but cultivated) and 32% are known only as uncultivated phylotypes or uncultivable flora. The latter are currently identified using molecular techniques, especially those based on 16S ribosomal RNA (rRNA) sequencing, pyrosequencing, and next-generation sequencing (NGS) technology. These new genomic technologies together with bioinformatics tools provide a powerful means of understanding the role of the oral microbes in health and disease.
The humans are not colonized at random and the microbial residents we harbour and provide shelter have coevolved with us over millions of years. This has led to the realization that the host and its residents together contribute to health and disease as a holobiont.
A perplexing array of organisms can be found in the oral cavity, which is one of the most heavily colonized parts of the human body. This is due to its unique anatomical structures found nowhere else in the body, such as teeth and gingivae. Under normal circumstances this vast array of organisms including bacteria, archaea, fungi, mycoplasmas, protozoa and a viral flora usually live in harmony. However, diseases such as caries and periodontal disease ensues when there is an ecological imbalance in the oral cavity either due to intrinsic or extrinsic causes.
The totality of the oral microbes, their genetic information, and the oral environment in which they interact are called the oral microbiome, whereas all living microbes constituting the oral microbiome is termed the oral microbiota. The oral microbiome in turn could be divided into three major subcompartments as the (Fig. 31.1):
■ oral bacteriome (bacterial component)
■ oral mycobiome (fungal component; Greek: mykes fungus)
■ oral virome/virobiome (viral component)
Apart from the aforementioned three distinct compartments of the oral microbiome, recent reports indicate the existence of a multitude of yet-to-be cultured (or uncultivable) ultra-small bacteria that may fall within the bacteriome group, and these have been classified into a subsector called the 'candidate phyla radiation (CPR)' group. Organisms in the CPR group are ultra-small (nanometre range, compared with micron-scale bacteria) and highly abundant (>15% of bacteriome). They are characterized by reduced genomes and unusual ribosomes, and appear to be obligate symbionts, living attached to either host bacteria or fungi.
In recent studies of the structure, function and the diversity of human oral microbiome, evaluated using NGS technology, it has been clearly shown that the oral microbiome is unique to each individual. Even healthy individuals differ remarkably in the composition of the resident oral microbiota. Although much of this diversity remains unexplained, diet, environment, host genetics and early microbial exposure have all been implicated in the constituent flora of the climax community.
The oral microbiota exists either suspended in saliva as planktonic phase organisms or attached to oral surfaces, in the biofilm phase (also called the sessile phase, i.e., the plaque biofilm). In general, despite the high diversity in the salivary microbiome within and between individuals, little geographic variations can be noticed. Individuals from different parts of the world harbour similar salivary microbiota, indicating that host species is the primary determinant of the oral microbiome.
Some oral microbes are more closely associated with disease than others (e.g., Porphyromonas gingivalis, a periodontal pathogen) although they commonly lurk within the normal oral flora without harming the oral health. This symbiosis between beneficial and pathogenic organisms is the key factor that contributes to the maintenance of oral health. In other words, when this homeostasis and the symbiotic equilibrium break down, for example, on taking broad-spectrum antibiotics, a state called dysbiosis sets in, leading to disease such as caries, periodontal disease and candidal infections. Essentially, in a dysbiotic microbiome, the diversity and relative proportions of species or taxa within the microbiota are disturbed.
Fig. 31.1 Major compartments of the oral microbiome. Oral bacteriome is by far the biggest component and oral virobiome (syn: oral virome) is the smallest. Note the difference between mycobiome and microbiome (Greek: Mykes fungus).
On occasions, specific microbes (mainly lactobacilli) could be administered to help restore a natural healthy microbiome in a given habitat (i.e., to convert a dysbiotic state to a symbiotic state). Such organisms are known as probiotics (see Chapter 26).
Oral bacteria can be classified primarily as Gram-positive and Gram-negative organisms, and secondarily as either anaerobic or facultatively anaerobic according to their oxygen requirements. Some oral microbes are more closely associated with disease than others, although a vast proportion of these appear to be uncultivable. The following is a synopsis of the major bacterial genera isolated from the oral cavity. Students should refer to the appropriate chapters in Part 3 for detailed information on these organisms.
A note on the nomenclature of oral flora
Because of continuing advances in molecular technology, especially those based on 16S rRNA sequences and NGS technology, microbial taxonomy is always in a state of flux. This poses a challenge to both the student and the scientist alike. Despite these changes, some prefer using the traditional nomenclature, whereas others use the new terminology, leading to further confusion. Hence in the following text, both the old and the recent taxonomic changes of oral bacteria are highlighted.
Finally, those who wish to pursue the subject of the oral microbiota and the oral microbiome in further detail need to visit the Human Oral Microbiome Database (HOMD; http:// www.homd.org). The HOMD presents a provisional naming scheme for the currently unnamed species so that strain, clone and probe data from any laboratory can be directly linked to a stably named reference scheme.
Gram-positive cocci in chains, non-motile, usually possessing surface fibrils, occasionally capsulate; facultative anaerobes;
variable haemolysis but a-haemolysis most common; selective medium: mitis salivarius agar (MSA).
■ Main species: Streptococcus mutans serotypes c, e, f, k; Streptococcus sobrinus serotypes d, g; Streptococcus criceti (previous Streptococcus cricetus) serotype a; Streptococcus ratti (previous Streptococcus rattus) serotype b. Oral isolates from monkeys: Streptococcus ferus; Streptococcus macacae; Streptococcus downei serotype h.
■ Cultural characteristics: high, convex, opaque colonies; produce profuse extracellular polysaccharide in sucrose-containing media (Fig. 11.3); selective medium: MSA + bacitracin agar.
■ Main intraoral sites and infections: tooth surface, dental caries.
■ Main species: Streptococcus salivarius; Streptococcus vestibularis.
■ Cultural characteristics: large, mucoid colonies on MSA due to the production of extracellular fructans (polymer of fructose with a levan structure). Streptococcus vestibularis do not produce extracellular polysaccharide from sucrose; they produce urease and hydrogen peroxide, which lowers the pH and contributes to the salivary peroxidase system, respectively.
■ Main intraoral sites and infections: dorsum of the tongue and saliva; Streptococcus vestibularis mainly reside in the vestibular mucosa (hence the name); not a major oral pathogen.
■ Main species: Streptococcus constellatus; Streptococcus intermedius; Streptococcus anginosus.
■ Cultural characteristics: carbon dioxide dependent; form small, non-adherent colonies on MSA.
■ Main intraoral sites and infections: gingival crevice; dentoalveolar and endodontic infections.
■ Main species: Streptococcus mitis, Streptococcus sanguinis (previously Streptococcus sanguinis); Streptococcus gordonii, Streptococcus oralis, Streptococcus cristatus (previously Streptococcus crista), Streptococcus parasanguinis, Streptococcus oligofermentans, Streptococcus sinensis, Streptococcus australis, Streptococcus peroris, Streptococcus infantis.
■ Cultural characteristics: small, rubbery (Streptococcus sanguinis) or non-adherent (Streptococcus oralis and Streptococcus mitis) colonies on MSA.
■ Main intraoral sites and infections: mainly dental plaque biofilms, tongue and cheek, possibly dental caries, infective endocarditis (except Streptococcus mitis).
■ Main species: Peptostreptococcus anaerobius, Micromonas micros (previously Peptostreptococcus micros), Finegoldia magna (previously Peptostreptococcus magnus) and Peptoniphilus asaccharolyticus (previously Peptostreptococcus asaccharolyticus); group acronym GPAC (Gram-positive anaerobic cocci).
■ Cultural characteristics: strict anaerobes, slow-growing, usually non-haemolytic.
■ Main intraoral sites and infections: teeth, especially carious dentine, periodontal and dentoalveolar abscesses in mixed culture.
■ Main species: Stomatococcus (formerly Micrococcus) mucilaginous.
■ Cultural characteristics: coagulase-negative; forms large colonies adherent to blood agar surface, facultative anaerobes.
■ Main intraoral sites and infections: tongue mainly, gingival crevice; not a major opportunistic pathogen.
■ Genus Granulicatella (previously termed 'nutritionally variant streptococci')
■ Main species: Granulicatella adiacens, Granulicatella elegans, Granulicatella balaenopterae.
■ Cultural characteristics: Gram-positive cocci, non-motile, catalase- and oxidase-negative, non-spore-forming, facultatively anaerobic requiring pyridoxal or L-cysteine for growth (Fig. 31.2). When grown on media without these components, their cell morphology and Gram stainability changes.
■ Main intraoral sites and infections: A component of normal oral flora and inhabits plaque biofilms, periodontal pockets, and root canals. Increased prevalence in caries, periodontitis and endodontic infections has been noted. Importantly, they cause serious infections such as infective endocarditis.
Genera Staphylococcus and Micrococcus
See Chapter 11.
Gram-positive rods and filaments
These organisms are common isolates from dental plaque biofilms and include actinomycetes, lactobacilli, eubacteria and propionibacteria.
Fig. 31.2 Scanning electron micrograph of Granulicatella species showing nonspore-forming coccal forms (Picture courtesy Dr M. Karched).
Short, Gram-positive pleomorphic rods:
■ Main species: Actinomyces israelii, Actinomyces gerencseriae, Actinomyces odontolyticus, Actinomyces naeslundii (genospecies 1 and 2), Actinomyces meyeri, Actinomyces georgiae. The most important human pathogen is Actinomyces israelii.
■ Cultural characteristics: ferments glucose to give characteristic patterns of short-chain carboxylic acids useful for speciating; strict or facultative anaerobes.
■ Main intraoral sites and infections: Actinomyces odontolyticus, earliest stages of enamel demineralization, and the progression of small caries lesions appear related; Actinomyces naeslundii implicated in root surface caries and gingivitis; Actinomyces israelii is an opportunistic pathogen causing cervicofacial and ileocaecal actinomycosis (Chapter 13). Actinomyces gerencseriae and Actinomyces georgiae are minor components of healthy gingival flora.
■ Main species: Lactobacillus casei, Lactobacillus fermentum, Lactobacillus acidophilus (others include Lactobacillus salivarius, Lactobacillus rhamnosus).
■ Cultural characteristics: catalase-negative, microaerophilic; complex nutritional requirements; aciduric, optimal pH 5.5-5.8. Selective medium: Rogosa agar.
■ Main intraoral sites and infections: common oral inhabitants, but comprise less than 1% of the oral flora. Dental plaque biofilms, usually in small numbers; advancing front of dental caries. As levels of salivary lactobacilli correlate well with intake of dietary carbohydrates, they are used to detect the cariogenic potential of the diet.
Pleomorphic, Gram-variable rods or filaments:
■ Main species: Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum. (Note: Eubacterium timidum and Eubacterium lenta, previously in this group, have now been reclassified as Mogibacterium timidum and Eggerthella lenta, respectively).
■ Cultural characteristics: obligatory anaerobes, characterization ill-defined.
■ Main intraoral sites and infections: plaque biofilms and calculus; implicated in caries and periodontal disease but role unclear; comprise over 50% of the anaerobes of periodontal pockets; Eubacterium yurii is involved in 'corn-cob' formation in dental plaque (see Fig. 31.3).
■ Main species: Propionibacterium acnes (includes Propionibacterium propionicus, formerly Arachnia propionica).
Fig. 31.3 Scanning electron micrograph of a supragingival plaque biofilm showing clearly corn-cob arrangements of cocci aggregated around axial filamentous organisms (arrows), together with other filamentous and coccal forms; Inset: a high-power picture of corn-cob arrangement (magnification x2000; bar 10 μm).
■ Cultural characteristics: strict anaerobe; morphologically indistinguishable from Actinomyces israelii but produces propionic acid from glucose, unlike Actinomyces israelii.
■ Main intraoral sites and infections: root surface caries, plaque biofilms. Possible involvement in dentoalveolar infections.
Other notable Gram-positive organisms
Rothia dentocariosa, a Gram-positive branching filament, is a strict aerobe, found in plaque and occasionally isolated from infective endocarditis.
Bifidobacterium dentium is a Gram-positive strict anaerobe regularly isolated from plaque biofilms; its role in disease is unclear.
■ Main species: Neisseria subflava, Neisseria mucosa, Neisseria sicca.
■ Cultural characteristics: asaccharolytic and non-polysaccharide-producing, facultative anaerobes.
■ Main intraoral sites and infections: isolated in low numbers from the tongue, saliva, oral mucosa and early plaque biofilm; may consume oxygen in early stages of plaque formation and provide conditions conducive for the growth of anaerobes; rarely associated with disease.
Small, Gram-negative cocci:
■ Main species: Veillonella parvula, Veillonella dispar, Veillonella atypica.
■ Cultural characteristics: strict anaerobes; selective medium: Rogosa vancomycin agar. Lack glucokinase and fructokinase and hence unable to metabolize carbohydrates; they therefore use lactate produced by other bacteria and raise the pH of the plaque biofilm, and are thus considered to be beneficial in relation to dental caries.
■ Main intraoral sites and infections: isolated from most surfaces, including the tongue, saliva and plaque biofilms. No association with disease.
Gram-negative rods: facultative anaerobic and capnophilic genera
■ Main species: Haemophilus parainfluenzae, Haemophilus segnis, Haemophilus aphrophilus, Haemophilus haemolyticus, Haemophilus parahaemolyticus.
■ Cultural characteristics: all isolates are facultative anaerobes; growth is enhanced on heated blood agar (chocolate), requires haemin (X factor) and nicotinamide adenine dinucleotide (V factor) for growth.
■ Main intraoral sites and infections: plaque biofilms, saliva and mucosae; dentoalveolar infections, acute sialadenitis, infective endocarditis.
Gram-negative coccobacilli, microaerophilic or capnophilic (carbon dioxide dependent).
■ Main species: Aggregatibacter actinomycetemcomitans (serotypes a-e).
■ Cultural characteristics: freshly isolated strains contain fimbriae that are lost on subculture. Produces many virulence factors: leukotoxin, epitheliotoxin, collagenase, protease that cleaves immunoglobulin G (IgG).
■ Main intraoral sites and infections: periodontal pockets; implicated in aggressive forms of periodontal disease (e.g., localized and generalized aggressive periodontitis). Often isolated from cervicofacial Actinomyces infections as co-pathogens.
■ Main species: Eikenella corrodens.
■ Cultural characteristics: factor X dependent and microaerophilic; produces corroding colonies on blood agar.
■ Main intraoral sites and infections: plaque biofilms; dentoalveolar abscesses, infective endocarditis; possibly implicated in some forms of chronic periodontitis.
Carbon dioxide dependent, Gram-negative fusiform rods with 'gliding' motility:
■ Main species: Capnocytophaga gingivalis, Capnocytophaga sputigena, Capnocytophaga ochracea, Capnocytophaga granulose, Capnocytophaga haemolytica.
■ Cultural characteristics: capnophilic, medium-sized colonies with an irregular spreading edge.
■ Main intraoral sites and infections: plaque, mucosal surfaces, saliva; infections in immunocompromised, possibly destructive periodontal disease. Some strains produce IgA1 protease.
Gram-negative rods: obligate anaerobic genera
These comprise a large proportion of the plaque biofilms. The classification of this group of organisms is fraught with difficulties, but the advent of new tests such as lipid analysis and molecular approaches have eased the problem to some extent. Most of the oral anaerobes were previously classified under the genus Bacteroides. However, advances in taxonomic methods have shown that they belong to two major genera, now termed Porphyromonas and Prevotella, which differ in their ability to metabolize sugar. Some of these organisms produce characteristic brown-black pigments on blood agar and are referred to collectively as 'black-pigmented anaerobes' (see Fig. 17.1).
Gram-negative pleomorphic rods, non-motile; six serotypes
based on capsular polysaccharides (K antigen); asaccharolytic:
■ Main species: Porphyromonas gingivalis, Porphyromonas endodontalis, Porphyromonas catoniae.
■ Cultural characteristics: strict anaerobes, require vitamin K and haemin for growth.
■ Main intraoral sites and infections: gingival crevice and subgingival plaque biofilm in small numbers. Associated with chronic periodontitis and dentoalveolar abscess; Porphyromonas gingivalis is highly virulent in experimental infections, producing proteases, a haemolysin, collagen-degrading enzymes and cytotoxic metabolites; its capsule is an important virulent attribute; fimbriae helps adhesion. Porphyromonas endodontalis is mainly recovered from infected root canals.
Gram-negative pleomorphic rods, non-motile; moderately asac-charolytic, producing acetic, succinic and other acids from glucose:
■ Main species: pigmented species include Prevotella intermedia, Prevotella nigrescens, Prevotella loescheii,
Prevotella corporis, Prevotella melaninogenica; non-pigmented species include Prevotella buccae, Prevotella oralis, Prevotella oris, Prevotella oulora, Prevotella veroralis, Prevotella dentalis (Bacteroides forsythus, another non-pigmented species considered an important periodontal pathogen, has now been reclassified as Tannerella forsythensis).
■ Cultural characteristics: strict anaerobes, usually require vitamin K and haemin for growth.
■ Main intraoral sites and infections: periodontal pockets, dental plaque biofilm; chronic periodontitis and dentoalveolar abscess.
Slender, cigar-shaped Gram-negative rods with rounded ends (see Fig. 18.1):
■ Main species: Fusobacterium nucleatum, Fusobacterium alocis, Fusobacterium sulci, Fusobacterium periodonticum.
■ Cultural characteristics: require rich media for growth and are often asaccharolytic, strict anaerobes, usually non-haemolytic; Fusobacterium nucleatum can produce ammonia and hydrogen sulphide from cysteine and methionine and is implicated as an odorigenic organism in halitosis.
■ Main intraoral sites and infections: most common isolate is Fusobacterium nucleatum; normal gingival crevice, tonsils (Fusobacterium alocis and Fusobacterium sulci) or periodontal infections (Fusobacterium periodonticum); acute ulcerative gingivitis, dentoalveolar abscess.
Gram-negative filaments with at least one pointed end:
■ Main species: Leptotrichia buccalis.
■ Cultural characteristics: strict anaerobes, with colonies resembling fusobacteria.
■ Main intraoral sites and infections: dental plaque biofilm. No known disease association.
Gram-negative curved bacilli, motile by polar flagella:
■ Main species: Wolinella succinogenes (Wolinella recta and Wolinella curva are now assigned to the Campylobacter genus).
■ Cultural characteristics: strict anaerobe.
■ Main intraoral sites and infections: gingival crevice. Possible involvement in destructive periodontal disease.
Gram-negative curved cells with tufts of flagella:
■ Main species: Selenomonas sputigena, Selenomonas noxia, Selenomonas flueggei, Selenomonas infelix, Selenomonas diane.
■ Cultural characteristics: strict anaerobe.
■ Main intraoral sites and infections: gingival crevice. No known disease association.
Motile Gram-negative helical cells, in three main sizes (large, medium and small):
■ Main species: Treponema denticola, Treponema macrodentium, Treponema skoliodontium, Treponema socranskii, Treponema maltophilum, Treponema amylovarum, Treponema vincentii.
■ Cultural characteristics: all treponemes are strict anaerobes and difficult to culture. Require enriched media with serum. Characterization poor; Treponema denticola is asaccharolytic; Treponema socranskii ferments carbohydrates to acetic, lactic and succinic acids.
■ Main intraoral sites and infections: Treponema denticola is more proteolytic than others and possesses proline aminopeptidase and arginine-specific protease; it also degrades collagen and gelatin. Found in the gingival crevice; closely associated with acute ulcerative gingivitis, a destructive periodontal disease.
A note on uncultivable bacteria
As stated earlier, it is now estimated that only about 50% of the oral bacteria that can be visualized by microscopy can be cultivated through traditional laboratory culture techniques. The identity and the role of these so-called uncultivable bacteria are mostly an enigma. There are two major reasons why these bacteria cannot be cultured. First, their nutritional requirements are unknown, and second, they coexist in a supportive ecosystem in tandem with neighbouring organisms that sustain them nutritionally as well as physically (through an intricate architectural hierarchy; Figs 31.3 and 31.4). Some examples of novel species and clones of bacteria detected from subgingival plaque biofilm using 16S rRNA and other techniques such as pyrosequencing are given in Table 31.1.
For many generations, it was generally thought that fungi belonging to the Candida species were the only organisms that reign supreme within the oral microbiome (Chapter 22), and if other fungi are isolated from oral samples they were merely transient oral colonizers. Indeed, numerous studies using conventional culture techniques have clearly shown that approximately one half of the population, irrespective of their origin, carries commensal Candida species in the oral cavity. However, in the 'very young, the very old and the very sick', these fungi may proliferate, leading to various forms of opportunistic oral candidal infections (see Chapter 35).
Table 31.1 Examples of novel species and clones of bacteria detected in subgingival plaque using 16S ribosomal RNA (rRNA) and other techniques such as pyrosequencing
TM7 (clone 1025)
Note: The significance of these isolates and their role in oral disease is still
The notion that Candida is the supreme inhabitant of the oral microbiome has been over turned by recent studies using NGS technology. It is now clear that apart from Candida there is a so- called basal oral fungal microbiome, hence called 'mycobiome' (Greek: Mykes, fungus), comprising many fungi, in both health and disease. Some studies have identified up to 80 genera, including cultivable and non-cultivable species! They range from common, rather innocuous species such as Aspergillus and Penicillium to Cryptococcus that cause invasive infections, especially in debilitated and compromised populations.
The pathogenic role of these fungi, apart from Candida species is yet to be defined. It is, however, important to note that in terms of the population size Candida species are yet the predominant fungal residents of the oral microbiome.
It is now thought that viruses and bacteriophages may be indigenous to the oral microbiome, constituting the oral virobiome or oral virome. The viruses may be dormant, and silently reside within the lining epithelium or arrive via the nerve trunks of the trigeminal nerve from time to time to cause disease. The oral virome is highly variable between individuals but remarkably stable over time within each individual. Herpesviridae and Papillomaviridae are the most common virus families present in healthy oral cavities.
Herpes group of viruses are the predominate constituents of the oral virome as described in Chapter 21. Herpes simplex, for example, causes gingivostomatitis or a subclinical infection and the virus can subsequently enter a dormant state in the trigeminal ganglion. It may be reactivated in response to external stimuli such as stress and cold weather, and recrudescent infection may manifest as herpes labialis (cold sores). The human papilloma virus, by contrast, resides in some 10% of the human population and may cause papillomas, condylomas and focal epithelial hyperplasia for reasons that are yet unclear. The role of human papilloma and related viruses in oral cancer is currently a hot topic of research.
Fig. 31.4 A schematic picture illustrating the various interactions of oral microbial species that lead to plaque biofilm formation. See text for the generic name of the species listed. (Reproduced, with permission, from Kolenbrander, P E., Andersen, R. N., Blehert, D. S., et al. (2002). Communication among oral bacteria. Microbiology and Molecular Biology Reviews, 66, 486-505.)
Viruses with a short, transient oral presence include organisms associated with systemic disease, and these include the mumps and rabies viruses that infect the salivary glands and are secreted in the saliva of affected individuals. Further, viruses causing upper respiratory tract infections are commonly present in the mouth during the acute phase of these diseases Similarly, bloodborne viruses such as the hepatitis viruses (Hepatitis B, C, D and F) and human immunodeficiency virus (HIV) can enter the mouth via gingival crevicular fluid in affected individuals. Hence it is of critical importance to implement standard infection control measures in clinical dentistry (see Chapter 37).
Oral bacteriophages: There is a rapidly expanding literature on oral bacteriophages. The most common phage families identified are Siphoviridae, Myoviridae and Podoviridae.
Phages are thought to play a major role in modulating the oral microbiome through mechanisms such as lysogeny (destroying the host or incorporating its nucleic acid into the host genome) and spreading antibiotic-resistance genes from one organism to the other.
A note on Archaea: They constitute a minor component of the oral microbiome with a very small number of species/ phylotypes, all of which are methanogens. Although detectable in healthy mouths, their numbers in subjects with periodontitis vary. All oral archaea are methanogens and examples include Methanobrevibacter oralis and Methanosarcina mazei.
Oral protozoans were first noted in very early microscopic studies of plaque samples of patients with very poor oral hygiene and periodontal disease. Hence they were thought to be agents of periodontal disease, a notion that has not been proven. They are currently considered as harmless saprophytes and mere passengers lurking within unhealthy mouths with plenty of nutritional resources such as food debris and bacteria.
Two main protozoon species of the normal microbiome are Entamoeba gingivalis and Trichomonas tenax.
Large, motile amoebae about 12 μm in diameter:
■ Main species: Entamoeba gingivalis.
■ Cultural characteristics: strict anaerobe; complex medium; cannot be easily cultured.
■ Main intraoral sites and infections: periodontal tissues, especially in patients who have received radiotherapy and are on metronidazole. Its role, if any, in periodontal disease is unclear.
Flagellated protozoa, about 7.5 μm in diameter:
■ Main species: Trichomonas tenax.
■ Cultural characteristics: strict anaerobe; complex medium; difficult to grow in pure culture.
■ Main intraoral sites and infections: gingival crevice of unhygienic mouths; its role in disease is unclear.
The oral ecosystem
Ecology is the study of the relationships between living organisms and their environment. An understanding of oral ecology is essential to comprehend the pathogenesis of diseases, such as caries and periodontal disease, caused by oral bacteria.
The oral environment
The human mouth is lined by stratified squamous epithelium.
This is modified in areas according to function (e.g., the tongue) and interrupted by other structures such as teeth and salivary ducts. The gingival tissues form a cuff around each tooth, and there is a continuous exudate of crevicular fluid from the gingival crevice. A thin layer of saliva bathes the surface of the oral mucosa.
The mouth, being an extension of an external body site, has a natural microflora. This commensal (or indigenous, or resident) flora exists in harmony with the host, but disease conditions supervene when this relationship is broken. The predominant dental diseases in humans (caries and periodontal disease) are caused in this manner. In addition to the commensal flora, there are others (such as coliforms) that survive in the mouth only for short periods (transient flora). These transient species of flora cannot get a foothold in the oral environment due to the ecological pressure, that is, the colonization resistance exerted by the resident flora. Indeed, the latter are considered critical in defending the key portal of entry into the digestive system, by offending pathogens.
The oral ecosystem comprises the oral flora, the different sites of the oral cavity where they grow (i.e., habitats) and the associated surroundings.
The major oral habitats are:
■ buccal mucosa
■ dorsum of the tongue
■ tooth surfaces (both supragingival and subgingival)
■ crevicular epithelium
■ prosthodontic and orthodontic appliances and dental fillings, if present.
Buccal mucosa and dorsum of the tongue
Special features and niches of the oral mucosa contribute to the diversity of the flora; for instance, the cheek mucosa is relatively sparsely colonized, whereas the papillary surface of the tongue is highly colonized because of the safe refuge provided by the papillae. The papillary surface of the tongue has a low redox potential (Eh), promoting the growth of anaerobic flora, and thus may serve as a reservoir for some of the Gramnegative anaerobes implicated in periodontal disease. Further, the keratinized and non-keratinized mucosae may offer refuge to variants of oral flora.
The surfaces of the teeth are the only non-shedding area of the body that harbours a microbial population. Masses of bacteria and their products constantly accumulate on tooth surfaces to produce plaque biofilms, in both healthy and disease states. Plaque is a classic example of a natural biofilm and is the major agent initiating caries and periodontal disease. In the latter situations, there is a shift in the composition of the plaque flora from a symbiotic equilibrium that predominates in the healthy state to a dysbiotic disease state (see Chapters 32 and 33).
A range of habitats are associated with the tooth surface (Fig. 31.5). The nature of the bacterial community varies depending on the tooth concerned and the degree of exposure to the environment: smooth surfaces are colonized by a smaller number of species than pits and fissures; subgingival surfaces are more anaerobic than supragingival surfaces.
Crevicular epithelium and gingival crevice
Although this habitat is only a minor region of the oral environment, bacteria that colonize the crevicular area play a critical role in the initiation and development of gingival and periodontal disease. A vast literature on this subject is available.
Prosthodontic and orthodontic appliances, and dental fillings
If present and not kept scrupulously clean, dental appliances may act as inanimate reservoirs of bacteria and yeasts. Yeasts on the fitting surface of full dentures can initiate Candida-associated denture stomatitis due to poor denture hygiene.
Fig. 31.5 Habitats associated with tooth surfaces and the nomenclature of plaque biofilm derived from these habitats.
If not properly cleaned, plaque biofilms may grow to varying extents on amalgam or composite dental fillings depending on their size, shape, location and the quality. These may cause secondary caries at the margins of fillings, or gingivitis if located near gingivae.
Factors modulating microbial growth
Different microenvironments in the mouth support their own microflora, which differ both qualitatively and quantitatively. The reasons for such variations are complex and include anatomical, salivary, crevicular fluid and microbial factors, among others.
Bacterial stagnation areas are created as a result of:
■ the shape of the teeth
■ the topography of the teeth (e.g., occlusal fissures)
■ malalignment of teeth
■ poor quality of restorations (e.g., fillings and bridges)
■ non-keratinized sulcular epithelium.
These areas are difficult to clean, either by the natural flushing action of saliva or by tooth-brushing.
Whole (mixed) saliva bathing oral surfaces is derived from the major (parotid, submandibular and sublingual) and minor (labial, lingual, buccal and palatal) salivary glands. It is a complex mixture of inorganic ions, including sodium, potassium, calcium, chloride, bicarbonate and phosphate; the concentrations of these ions vary diurnally and in stimulated and resting saliva. The major organic constituents of saliva are proteins and glycoproteins (such as mucin), which modulate bacterial growth (Table 31.2) in the following ways:
■ adsorption on the tooth surfaces forms a salivary pellicle, a conditioning film that facilitates bacterial adhesion
■ acting as a readily available, primary source of food (carbohydrates and proteins)
■ aggregation of bacteria, thereby facilitating their clearance from the mouth, or deposition on surfaces, contributing to plaque biofilm formation
Table 31.2 Specific and non-specific host defence factors of the mouth
Physical removal of microbes
Physical removal of microbes
Physical removal of microbes
Cell lysis (bactericidal, fungicidal)
Iron sequestration (bactericidal, fungicidal)
Iron sequestration (bactericidal, fungicidal)
Hypothiocyanite production (neutral pH), hypocyanous acid production (low pH)
Antibacterial and antifungal activity
Secretory leukocyte protease inhibitor (SLPI)
Blocks cell surface receptors needed for entry of human immunodeficiency virus (HIV)
Chitinase and chromogranin
Intraepithelial lymphocytes and Langerhans cells
Cellular barrier to penetrating bacteria and/or antigens
Secretory immunoglobulin A (IgA)
Prevents microbia l adhesion and metabolism
IgG, IgA, IgM
Prevent microbial adhesion, opsonins, complement activators
Note: See also Tables 8.1,8.2 and 8.3.
■ growth inhibition of exogenous organisms by non-specific defence factors, for example, lysozyme, lactoferrin and histatins, which are bactericidal and fungicidal and specific defence factors (e.g., Igs, mainly IgA and salivary leukocyte protease inhibitor (SLPI), which destroys HIV)
■ maintenance of pH with its excellent buffering capacity (acidic saliva promotes growth of cariogenic bacteria).
Gingival crevicular fluid
There is a continuous but slow flow of gingival crevicular fluid in healthy state, and this increases during inflammation (e.g., gingivitis). The composition of crevicular fluid is similar to that of serum, and thus, the crevice is protected by these 'surrogate'-specific and non-specific defence factors of serum. Crevicular fluid can influence the ecology of the crevice by:
■ flushing microbes out of the crevice
■ acting as a primary source of nutrients: proteolytic and saccharolytic bacteria in the crevice can utilize the crevicular fluid to provide peptides, amino acids and carbohydrates for growth; essential cofactors (e.g., haemin) can be obtained by degrading haem-containing molecules such as haemoglobin
■ maintaining pH conditions
■ providing specific and non-specific defence factors: IgG predominates (IgM and IgA are both present to a lesser extent)
■ phagocytosis: 95% of leukocytes in the crevicular fluid are neutrophils.
Microbes in the oral environment can interact with each other both in promoting and suppressing the neighbouring bacteria. Mechanisms that accomplish this include:
■ competition for receptors for adhesion by prior occupation of colonizing sites and prevention of attachment of 'late-comers'
■ production of toxins, such as bacteriocins, that kill cells of the same or other bacterial species; for example, Streptococcus salivarius produces an inhibitor (enocin) that inhibits Streptococcus pyogenes
■ production of metabolic end products such as short-chain carboxylic acids, which lower the pH and also act as noxious, antagonistic agents
■ use of metabolic end products of other bacteria for nutritional purposes (e.g., Veillonella spp. use acids produced by Streptococcus mutans)
■ coaggregation with the same species (homotypic) or different species (heterotypic) of bacteria, for example, corn-cob formation (Fig. 31.3)
■ production of specific messenger chemicals called quorum-sensing molecules (such as homoserine lactone) that helps the resident bacteria to communicate (i.e., crosstalk) with each other within a biofilm community, and maintain homeostasis of the biofilm.
These mechanisms, which enable the commensal oral flora to suppress or inhibit the growth of exogenous, non-oral organisms and thereby exclude them from their communal neighbourhood habitats, are called colonization resistance.
Local environmental pH
Many microbes require a neutral pH for growth. The acidity of most oral surfaces is regulated by saliva (mean pH 6.7). Depending on the frequency of intake of dietary carbohydrates, the pH of plaque biofilm can fall to as low as 5.0 as a result of bacterial metabolism. Under these conditions, acidophilic bacteria can grow well (e.g., lactobacilli), whereas others are eliminated by competitive inhibition.
The oxidation-reduction potential of the environment (Eh) varies in different locations of the mouth. For instance, redox potential falls during plaque biofilm development from an initial Eh of over +200 mV (highly oxidized) to -141 mV (highly reduced) after 7 days. Such fluctuations favour the growth of different groups of bacteria.
Systemic or topical antibiotics and antiseptics affect the oral flora; for instance, broad-spectrum antibiotics such as tetracycline can wipe out most of the endogenous flora and favour the emergence of yeast species.
Fermentable carbohydrates are the main class of compounds that alter the oral ecology. They act as a major source of nutrients, promoting the growth of acidogenic flora. The production of extracellular polysaccharides facilitates adherence of organisms to surfaces, whereas the intracellular polysaccharides serve as a food resource.
Procedures such as dental scaling can radically alter the composition of the periodontal pocket flora of diseased sites and shift the balance in favour of colonization of such sites by flora that are associated with health.
Nutrition of oral bacteria
Oral bacteria obtain their food from a number of sources. These include host resources:
■ remnants of the host diet always present in the oral cavity (e.g., sucrose, starch)
■ salivary constituents (e.g., glycoproteins, minerals, vitamins)
■ crevicular exudate (e.g., proteins)
■ gaseous environment (although most require only a very low level of oxygen) and microbial resources:
■ extracellular microbial products of the neighbouring bacteria, especially in dense communities such as the plaque biofilm
■ intracellular food storage (glycogen) granules.
Acquisition of the normal oral flora
1. The infant mouth is sterile at birth, except perhaps for a few organisms acquired from the mother's birth canal.
2. A few hours later, the organisms from the mother's (or the nurse's) mouth and possibly a few from the environment are established in the mouth.
3. These pioneer species are usually streptococci, which bind to mucosal epithelium (e.g., Streptococcus salivarius).
4. The metabolic activity of the pioneer community then alters the oral environment to facilitate colonization by other bacterial genera and species. For instance, Streptococcus salivarius produces extracellular polymers from sucrose, to which other bacteria such as Actinomyces spp. can attach (Figs 31.3 and 31.4).
5. When the composition of this complex ecosystem (comprising several genera and species in varying numbers) reaches equilibrium, a climax community is said to exist. (Note: this is a highly dynamic system.)
6. Oral flora on the child's first birthday usually consists of streptococci, staphylococci, neisseriae and lactobacilli, together with some anaerobes such as Veillonella and fusobacteria. Less frequently isolated are Lactobacillus, Actinomyces, Prevotella and Fusobacterium species.
7. The next evolutionary change in this community occurs during and after tooth eruption when two further niches are provided for bacterial colonization: the hard-tissue surface of enamel and the gingival crevice. Organisms that prefer hard-tissue colonization, such as Streptococcus mutans, Streptococcus sanguinis and Actinomyces spp., then selectively colonize enamel surfaces, and those preferring anaerobic environments, such as Prevotella spp., Porphyromonas spp. and spirochaetes, colonize the crevicular tissues. However, the anaerobes do not appear in significant numbers until adolescence. For instance, only 18%-40% of 5-year olds have spirochaetes and black-pigmented anaerobes compared with 90% of 13- to 16-year olds.
8. A second childhood (in terms of oral bacterial colonization) is reached if all teeth are surgically extracted. Bacteria that colonize the mouth at this stage are very similar to those in a child prior to tooth eruption.
9. Introduction of a prosthetic appliance at any stage changes the microbial composition once again. Growth of Candida species is particularly increased after the introduction of acrylic dentures, while it is now recognized that the prevalence of Staphylococcus aureus and lactobacilli is high in those aged 70 or over. The denture plaque biofilm is somewhat similar to plaque biofilm on enamel surface; it may also harbour significant quantities of yeast.
The plaque biofilm
The plaque biofilm is a tenacious microbial community embedded in an extracellular polysaccharide matrix, and attached to either the soft- or hard-tissue surfaces of the mouth, comprising living and dead bacteria and their extracellular products, together with host compounds, mainly derived from the saliva.
The organisms in plaque biofilm are embedded in an organic matrix, which comprises about 30% of the total volume. The matrix is derived from the products of both the host and biofilm constituents. In the gingival area, proteins from the crevicular exudate become incorporated into the plaque biofilm. This matrix acts as a food reserve and as a cement, binding organisms both to each other and to various surfaces.
The microbial composition of dental plaque biofilm can vary widely between individuals; some people are so-called rapid plaque formers and others slow plaque formers. Further, there are large variations in plaque composition within an individual, for example:
■ at different sites on the same tooth
■ at the same site on different teeth
■ at different times on the same tooth site.
Plaque biofilm is found on dental surfaces and appliances especially in the absence of oral hygiene. In general, it is
found in anatomical areas protected from the host defences, for example, occlusal fissures, interproximally or around the gingival crevice. Plaque samples are described in relation to their site of origin and are categorized as supragingival:
■ fissure plaque: mainly in molar fissures
■ approximal plaque: at contact points of teeth
■ smooth surface: for example, buccal and palatal surfaces subgingival, or appliance associated:
■ full and partial dentures (denture plaque)
■ orthodontic appliance-related plaque.
Microbial adherence and plaque biofilm formation
Adherence of a microbe to an oral surface is an essential prerequisite for colonization and biofilm formation. It is also the initial step in the path leading to subsequent infection and invasion of tissues. There are a number of intrinsic host factors that prevent microbial colonization on oral surfaces and these include (Fig. 31.6):
■ the mucosal barrier with constant desquamation of the epithelium that dislodges the attached organisms from soft-tissue surfaces.
■ the dynamic salivary flow patterns in different oral niches
■ the muscular movements of the tongue and cheeks that physically dislodge the biofilms
■ the non-specific and specific defines factors (such as IgA) in saliva
■ the resident community of microbiota that offers 'colonization resistance' to invading extraneous organisms.
Fig. 31.6 Factors affecting microbial colonization of the oral mucosa.
Plaque biofilm formation
Plaque biofilm formation is a complex process comprising a number of different stages:
1. Pellicle formation. Adsorption of host and bacterial molecules to the tooth surface forms the acquired salivary pellicle. A thin layer of salivary glycoproteins is deposited on the surface of a tooth within minutes of exposure to the oral environment. Oral bacteria initially attach to the pellicle and not directly to enamel (i.e., hydroxyapatite).
2. Transport. Bacteria approach the vicinity of the tooth surface prior to attachment, by means of natural salivary flow, Brownian motion or chemotaxis.
3. Long-range interactions involve physicochemical interactions between the microbial cell surface and the pellicle-coated tooth. Interplay of van der Waals forces and electrostatic repulsion produces a primary reversible phase of net adhesion.
4. Short-range interactions consist of stereochemical reactions between adhesins on the microbial cell surface and receptors on the acquired pellicle. This is an irreversible phase in which polymer bridging between organisms and the surface helps to anchor the organism, after which they multiply on the virgin surface. Doubling times of plaque biofilm bacteria can vary considerably (from minutes to hours), both between different bacterial species and between members of the same species, depending on the environmental conditions.
5. Coaggregation or coadhesion. Fresh bacteria now attach on to the already attached first generation of cells (also called pioneer or initial colonizers) these may be bacteria of the same genus or different but compatible genera (Fig. 31.4).
6. Biofilm formation. The attached organisms now grow horizontally on the surface and form micro-colonies at first whilst the aforementioned process continues with a resultant confluent growth and the formation of a biofilm, which matures in complexity as time progresses. Simply defined, biofilm is a complex functional community of one or more species of microbes encased in an extracellular polysaccharide matrix and attached to one another or to a solid surface. The latter could be an inert surface such as tooth enamel, denture acrylic or a plastic catheter or alternatively an organic/living surface such as a heart valve. Architecturally, the biofilm is not a flat compact structure resembling an inert piece of concrete. The aggregates of organisms are arranged in columns or mushroom-shaped structures interspersed with water channels that carry metabolites and bring in nutrients (Figs 5.2 and 5.3).
Thus, biofilm formation is a complex, competitive, sequential and dynamic colonization process, and in plaque biofilms, this complexity is further compounded due to the participation of different categories of oral bacteria. Specifically, the pioneer group of organisms that selectively colonize the salivary pellicle during plaque biofilm formation are Gram-positive cocci and rods. These are followed by Gram-negative cocci and rods, and finally by filaments, fusobacteria, spirils and spirochaetes. Such an example of a natural succession of plaque flora has been elegantly demonstrated in 'experimental gingivitis' studies, where groups of individuals, initially subjected to meticulous oral hygiene, were then followed up during a phase of no oral hygiene, and the freshly developing plaque flora was monitored closely. Results of such a study are shown in Fig. 31.7.
One major component of a biofilm is the extracellular matrix. This comprises microbial polysaccharides and additional layers of salivary glycoprotein (or crevicular fluid components, depending on the site). The metabolic products of the early plaque biofilm colonizers can radically alter the immediate environment (e.g., create a low redox potential suitable for anaerobes), leading to new colonizers inhabiting the biofilm, with a resultant gradual increase in microbial complexity, biomass and thickness. As a result of this dynamic process, the plaque biofilm mass reaches a critical size at which a balance between the deposition and loss of plaque bacteria is established; this community is termed the climax community (Fig. 31.8).
Fig. 31.7 Results from an experimental study showing the predominant groups of organisms comprising the pioneer and the climax community of plaque. Note the relationship between the plaque index and the gingival index.
The molecular biology of biofilm formation is complex. Biofilm bacteria appear to maintain their complex structure through continuous secretion of low levels of molecules called quorum-sensing molecules (e.g., homoserine lactone, autoinducer-2) that coordinate gene expression. As the number of organisms in the biofilm increases, there is a simultaneous, proportionate increase in the quorum-sensing signals. These activate genes that may be related to additional extracellular polysaccharide production, or reduction of metabolism (for bacteria at the bottom of the matrix) or production of virulent factors, including drug-destroying genes.
Fig. 31.8 Micrographs of (A) smooth-surface plaque showing the many relationships between different bacterial forms, including palisading and corn-cob formation and (B) mature plaque with compact bacteria and calcification at the base (approximately x5000). (C) Mature subgingival plaque biofilms stained by fluorescent in situ hybridization (FISH) technique showing non-specific bacteria (green), group 1 treponemes (orange) and Fusobacterium species (magenta) colonizing distinct parts of the biofilm. Some gingival host cell nuclei are stained blue with a nucleic acid stain. (Image courtesy Dr Annette Motte.)
The bacteria that colonize this climax community may detach and enter the planktonic phase (i.e., suspended in saliva) and be transported to new colonization sites, thus restarting the whole cycle.
Further notes on biofilms
The realization of the fact that up to 65% of human infections are caused by organisms encased in biofilms (i.e., sessile organisms) as opposed to planktonic or free-living forms has resulted in much research and a vast literature on the behaviour of these two rather divergent lifestyles of microbes. There is also a preponderance of biofilms in nature, for instance, as slimy coats that grow in stagnant water or water pipes (see Chapter 38 for biofilms in dental unit water lines). In clinical terms, it is recognized that biofilm organisms are more resistant to antibiotics and chemotherapeutic agents than their planktonic counterparts (see Chapter 5). The problem of drug resistance, however, is not a major concern in dental plaque biofilms due to their ready accessibility to mechanical cleansing measures. However, drug resistance due to biofilms in other diseased states (e.g., airways infection by Pseudomonas aeruginosa in cystic fibrosis) is a major therapeutic problem.
Calcium and phosphate ions derived from saliva may become deposited within deeper layers of dental plaque biofilm (as saliva is supersaturated with respect to these ions). If the plaque biofilm is allowed to grow undisturbed, then the degenerating bacteria in a climax community may act as seeding agents of mineralization. The process is accelerated by bacterial phosphatases and proteases that degrade some of the calcification inhibitors in saliva (statherin and proline-rich proteins). These processes lead to the formation of insoluble calcium phosphate crystals that coalesce to form a calcified mass of plaque, termed calculus.
Many toothpastes now contain pyrophosphate compounds that adsorb excess calcium ions, thus reducing intraplaque mineral deposition. In general, mature calculus is composed of 80% (dry weight) mineralized material, mostly hydroxyapatite and the remainder (20%) organic compounds.
The structure of calculus is shown in Fig. 31.8. Predominant flora are cocci, bacilli and filaments (especially in the outer layers), and occasionally spiral organisms. The bacteria near the enamel surface tend to have a reduced cytoplasm-to-cell wall ratio, suggesting that they are metabolically inactive. Supragingival calculus contains more Gram-positive organisms, whereas subgingival calculus tends to contain more Gram-negative species.
In some areas (especially the outer surface), cocci attach and grow on the surface of filamentous microorganisms, giving a 'corn-cob' arrangement (Fig. 31.3). The filamentous bacteria tend to orient themselves at right angles to the enamel surface, producing a palisade effect (like books on a shelf). The cytoplasm of some bacteria (mainly cocci) may contain glycogen-like food storage granules, available as a ready source of nutrition during periods of adversity.
Calculus has a rough surface and is porous, thus serving as an ideal reservoir for bacterial toxins that are harmful to the periodontium (e.g., lipopolysaccharides (LPSs)). Hence removal of calculus is essential to maintain good periodontal health.
The role of dental plaque biofilm in caries and periodontal disease is discussed in Chapters 32 and 33, respectively.
The role of oral flora in systemic infection: the oral-systemic axis
Over the last decade or so, it has been recognized that plaque biofilm-related oral diseases, especially periodontitis, may alter the course and pathogenesis of a number of systemic diseases. This is a resurgence of a common belief called 'focal infection theory' popular in the late 19th and early 20th century, where clinicians believed that oral foci of infection may lead to systemic diseases including heart disease and diabetes. However, it is important to note that definitive evidence for such outcomes are not available as yet, for most of these postulated oral-systemic links. They seem to be mere associations, and associations do not necessarily prove causality.
Numerous research groups have now studied the oral- systemic connectivity and up to some one hundred diseases ranging from dementia and Alzheimer's disease to erectile dysfunction have been explored for possible linking pathways! There is a convincing body of data to indicate that the following diseases have a stronger association than others with a dysbiotic oral microbiome, particularly periodontal inflammation. These include:
■ cardiovascular disease:
• infective endocarditis
• coronary heart disease: atherosclerosis and myocardial infection
■ hospital-acquired (i.e., nosocomial) bacterial pneumonia
■ diabetes mellitus
■ adverse pregnancy outcomes (e.g., low-birth-weight babies).
Several mechanisms linking oral infections to secondary systemic disease have been postulated (Fig. 31.9):
1. Metastatic infection and infestation:
• Microbes gaining entry into the circulatory system through breaches in the oral vascular barrier, as in the case of bacteraemias produced during tooth extractions (see Chapter 24), or indeed in the case of periodontitis, through the sites of gingival ulcerations.
• Periodontal bacteria, such as Fusobacterium nucleatum, can translocate across the foeto-placental barrier, causing adverse pregnancy outcomes.
• Oral microbes that are constantly swallowed via the saliva into the gut may cause alterations to the gut microbiota, thereby leading to increased gut epithelial permeability and endotoxaemia, which in turn causes systemic inflammation.
Fig. 31.9 A schematic diagram depicting postulated linking pathways between periodontal inflammation and systemic disease. In particular, the links between periodontitis and atherosclerotic heart disease and adverse pregnancy outcomes are shown (please see text for details). (Adapted, with permission from Macmillan Publishers Ltd., from HajishengaJlis, G. (2015). Periodontitis: From microbial immune subversion to systemic inflammation. Nature Reviews Immunology, 15, 30-44.). CRP1 C-reactive protein; IL, interleukin; TNF, tumour necrosis factor.
2. Metastatic injury: products of bacteria, such as cytolytic enzymes, exotoxins and endotoxins (i.e., LPSs), gaining access to the cardiovascular system in individuals suffering from periodontitis.
3. Metastatic inflammation: caused by immunological injury due to oral organisms.
• In periodontitis, in particular, locally produced pro-inflammatory cytokines (tumour necrosis factor (TNF), interleukin-1β (IL-1β), IL-6) can enter the systemic circulation and induce an acute-phase response in the liver (characterized by increased levels of C-reactive protein (CRP fibrinogen and serum amyloid A) and in turn contribute to atherosclerosis or intra-uterine inflammation.
• Soluble antigens may enter the blood stream from the oral route, react with circulating specific antibodies and form macromolecular complexes, leading to immune-mediated disease such as Behçet's syndrome.
Of these, the mechanisms linking systemic infection and periodontal disease have been studied the most and the following are now known:
1. Factors that place individuals at high risk for periodontitis may also place them at high risk for systemic disease such as cardiovascular disease. These include tobacco smoking, stress, ageing, race or ethnicity, and male gender.
2. Subgingival biofilms are vast reservoirs of especially Gram-negative bacteria, and they are a continuous source of LPSs (i.e., endotoxins), which induce major vascular responses (so-called atherogenic response). Further, LPSs upregulate endothelial cell adhesion molecules, and secretion of IL-1 and TNF-a.
3. Periodontium is a reservoir of cytokines: the proinflammatory cytokines TNF-a and IL-1β, y-interferon and prostaglandin E2 reach high concentrations in periodontitis. Spillover of these mediators into the circulation may induce or aggravate systemic effects, as explained earlier.
Apart from the well-established link between endocarditis and dental bacteraemias, there is no firm evidence to indicate that the other postulated diseases mentioned earlier are either initiated or perpetuated by oral flora and their by-products. The evidence available is circumstantial at best, with a multitude of confounding factors. Therefore, further research is necessary to confirm or refute these observations. Nonetheless, it is beyond doubt that good oral health is important not only to prevent oral disease but also to maintain good systemic health.
• The oral microbiota comprises a diverse group of organisms and includes bacteria, fungi, mycoplasmas, protozoa and possibly viruses.
• The three major sub-divisions of the oral microbiome are, the bacteriome, mycobiome and the virome (virobiome).
• There are probably some 350 different cultivable species and a vast proportion of unculturable flora, currently identified using molecular techniques.
• Streptococci are the predominant supragingival bacteria; they belong to four main species groups: mutans, salivarius, anginosus and mitis.
• The predominant cultivable species in subgingival plaque biofilm are Actinomyces, Prevotella, Porphyromonas, Fusobacterium and Veillonella spp.
• The oral ecosystem comprises the oral flora, the different sites of the oral cavity where they grow (i.e., habitats) and the associated surroundings.
• The major oral habitats are the keratinized and unkeratinized buccal mucosa, including the dorsum of the tongue, tooth surfaces, crevicular epithelium and prosthodontic and orthodontic appliances, if present.
• Adherence of a microbe to an oral surface is a prerequisite for colonization and is the initial step in the path leading to subsequent infection or invasion of tissues.
• Saliva modulates bacterial growth by (1) providing a pellicle for bacterial adhesion, (2) acting as a nutrient source, (3) coaggregating bacteria, (4) providing non-specific (e.g., lysozyme, lactoferrin and histatins) and specific (e.g., mainly immunoglobulin A (IgA)) defence factors and (5) maintaining pH.
• Microbes interact with each other by competing for receptors for adhesion, production of bacteriocins plus antagonistic metabolic end products and by coaggregation.
• Large masses of bacteria and their products accumulate on tooth surfaces to produce plaque biofilms, present in both healthy and disease states; plaque is an example of a natural biofilm.
• Stages in the plaque biofilm formation are transport and adhesion/coadhesion of bacteria leading to irreversible attachment with concomitant extracellular polysaccharide matrix formation.
• Dental plaque biofilm can be defined as a tenacious, complex microbial community, found on tooth surfaces, comprising living, dead and dying bacteria and their products, embedded in a matrix of polymers mainly derived from the saliva.
• Sessile organisms in biofilms are generally more resistant to antimicrobials than their planktonic counterparts due to properties conferred by the thick biofilm matrix and the differentials in the genetic and phenotypic make-up of the sessile forms.
• Recently, it has been recognized that plaque biofilm related to oral diseases, especially periodontitis, may alter the course and pathogenesis of a number of systemic diseases. These include cardiovascular disease, infective endocarditis, bacterial pneumonia, diabetes mellitus and low-birth-weight babies. This is known as the ‘focal infection theory’.
• However, apart from the well-established link between endocarditis and dental bacteraemias, there is no firm evidence to indicate that the other postulated diseases mentioned earlier are either initiated or perpetuated by oral flora and their by-products.
Review questions (answers on p. 367)
Please indicate which answers are true, and which are false.
31.1 Streptococci comprise a considerable proportion of the normal oral flora. The predominant streptococci found in supragingival sites include:
A. Streptococcus pneumoniae
B. Streptococcus mutans
C. Streptococcus salivarius
D. Streptococcus pyogenes
E. Streptococcus mitis
31.2 Which of the following statements on saliva are true?
A. a salivary pellicle is always found on the surfaces of the healthy oral cavity
B. saliva provides nutrition for bacteria
C. salivary lactoferrin is an antimicrobial agent
D. coaggregation of bacteria is facilitated by saliva
E. salivary leukocyte protease inhibitor (SLPI) is antibacterial in nature
31.3 Which of the following are true of plaque biofilms?
A. organic matrix comprises more than 70% of the mass
B. the matrix facilitates development of antimicrobial resistance
C. biofilms on the molar fissures are called supragingival plaque
D. more than 80% of the mature calculus consists of mineralized material
E. natural salivary flow is the only mechanism used by organisms to access tooth surfaces
31.4 Which of the following are true with respect to intraoral plaque biofilms?
A. the initial colonizers are often Gram-negative rods
B. plaque Eh fluctuations are critical for caries development
C. early plaque biofilm colonizers reduce the redox potential so that the growth of anaerobes is promoted
D. climax community refers to the planktonic cells
E. the degenerating plaque biofilm bacteria may act as nuclei for calculus formation