Normal Oral Microbial Flora

- Dr. Neeraj R Mehta. Dept. of Orthodontics and Dentofacial Orthopedics.

The oral cavity is comprised of many surfaces, each coated with a plethora of bacteria, the proverbial bacterial biolm. Some of these bacteria have been implicated in oral diseases such as caries and periodontitis, which are among the most common bacterial infections in humans. Natural microflora in the oral cavity become established during childhood and then change throughout the stages of life under the influence of various environmental and behavioural factors. If the profile of microbial communities is altered, pathogenic activity may be exhibited, resulting in dental caries or periodontal disease. Furthermore, recent studies have demonstrated that microorganisms in dental plaques can play an etiological role in systemic diseases such as diabetes and atherosclerosis. Dental plaque is now recognized as a complex microbial biofilm consisting of over 600 different bacterial species in the human oral cavity.

Normal flora: Microorganisms that are frequently found on or within the body of healthy persons; some of these are found only in association with the body of humans or animals, while others live freely in the environment. Oral flora is unique and selective in that microorganisms normally found in the neighbouring ecosystems such as skin are not found in the mouth. Types of flora: i) ii) iii) Indigenous: These are microbes regularly found at any anatomical site. Exogenous: Microbes that are not the residents of a given site, and cause infection. Transient: Microbes that are not residents of given site, but may be found there as commensals, for a short period, from time to time.

Beneficial effects of normal flora: Commensals do provide us benefits, such as A. Preventing invasion by pathogenic bacteria: Normal bacterial flora is able to prevent entry of pathogenic microorganisms by two mechanisms: i) By sheer physical advantage of having been resident at the site, esp. on the epithelial surface. ii) By production of substances (e.g. antibiotics, lethal proteins) that inhibit pathogenic microorganisms that enter the region.

B. Immune stimulation: Microorganisms play an important role in development and expansion of lymphoid tissues and maintenance and regulation immunity. The antigens stimulate the immune system, resulting in formation of antibodies against bacteria, fungi that inhabit the body. These antibodies, although low in number, serve as a defence mechanism preventing commensals from colonizing deeper tissues. C. Possible Role in Human Nutrition and Metabolism: Several intestinal bacteria, for e.g. E. coli and bacteroides species are known to synthesize Vitamin K and vitamins belonging to B complex group. Several bacteria are also known to make glucoronidases and sulphatases that can deconjugate sex steroid hormones and bile salts that are excreted through the bile in conjugated form.

Harmful Effects of normal flora: A. Source of infection: Normal flora can act as a source of many opportunistic infections. Microbes that are commensals to one part of body gain access to region where they do not usually reside, may cause a disease. For e.g. Streptococci which are commensal in mouth, if gain access to blood stream can colonise the heart valve and cause infective endocarditis. B. Initiation of pathological mucosal inflammation in a susceptible host: Certain microbes (commensals to the gut) may cause inflammation in susceptible host. This may lead to inflammatory bowel disease. For e.g. Cohns Disease. C. Possible Source of Carcinogens: Normal intestinal flora may produce carcinogens from compounds that an individual ingests. For e.g. Cyclamate (Cyclohexamine sulphate) an artificial sweetener is converted into active carcinogen cyclohexamine by bacterial sulphatases.

Normal Oral Flora:

It consists of following microorganisms:

Bacteria Gram Positive : Cocci : Streptococcus Staphylococcus Gemella species Peptostreptococcus Enterococcus Micrococci

Rods and filaments : Actinomycetes Eubacteria Lactobacillus

Other Genera Propionibacterium Cornybacterium

Gram Negative Cocci Neiserria Velionella Haemophilus Aggregatibacter Eikenella Capnocytophaga Kingella Simonsiella

Obligatory Anaerobic Genera Prevotella Porphyromonas Fusobacterium Leptotrichia Campylobacter Spirochaetes

Mycoplasma M. salivarium M. pneumoniae M. hominis M. buccale M. orale

Viruses Herpes simplex type 1 Cytomegalovirus Bacteriophage

Fungi Candida spp. Geotrichum

Protozoa Plasmodium Entamoeba histolytica Entamoeba gingivalis Cryptosporidium

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). 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. 6. Oral flora on the child's first birthday usually consist 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. 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 13-16-year-olds. 8. A second childhood (in terms of oral bacterial colonization) is reached if all teeth are lost as a result of senility. Bacteria that colonize the mouth at this stage are very similar to those in a child before tooth eruption. 9. Introduction of a prosthetic or orthodontic appliance at this stage changes the microbial composition once again.

The oral ecosystem:

The mouth as a habitat for microbial growth:Not all of the micro-organisms that enter the mouth are able to colonize. The properties of the mouth make it ecologically distinct from all other surfaces of the body, and dictate the types of microbe able to persist. Ecological conditions include mucosal surfaces such as the lips, cheek, palate and tongue and teeth. Ecological conditions within the mouth will also vary during the change from the primary to the permanent dentition

The major oral habitats are: Teeth Buccal mucosa Dorsum of the tongue Saliva Gingival Crevicular Fluid (GCF). Appliances orthodontic and prosthodontic

Teeth Are the only normally accessible site in the body that has hard nonshedding surface for microbial colonization. These unique tissues allow the accumulation of large masses of microorganisms (predominantly bacteria) and their extra cellular products, termed dental plaque. Plaque is an example of a biofilm, and, while it is found naturally in health, it is also associated with dental caries and periodontal disease. In disease, there is a shift in the composition of the plaque microflora away from the species that predominate in health. The ecological complexity of the mouth is increased still further by the range of habitats associated with the tooth surface. Teeth do not provide a uniform habitat but possess several distinct surfaces, each of which is optimal for colonization and growth by different populations of micro-organisms. This is due to the physical nature of the particular surface and the resulting biological properties of the area. The areas between adjacent teeth and in the gingival crevice afford protection from adverse conditions in the mouth. Both sites are also anaerobic and, in addition, the gingival crevice region is bathed in the nutritionally rich GCF, particularly during inflammation, and so these areas support a more diverse community. Smooth surfaces are more exposed to the environment and can be colonized by only a limited number of bacterial species adapted to such extreme conditions The properties of a smooth surface will differ according to whether it faces the cheek (buccal surface) or the inside (lingual surface) of the mouth.

Pits and fissures of the biting (occlusal) surfaces of the teeth also offer protection from the environment. Such protected areas are associated with the largest microbial communities and, in general, the most disease.

Mucosal surfaces and dorsum of tongue Although the mouth is similar to other ecosystems in the digestive tract in having mucosal surfaces for microbial colonization, the oral cavity does have specialized surfaces which contribute to the diversity of the microflora at certain sites. The cheek mucosa is sparsely colonized whereas the papillary structure of the dorsum of the tongue provides refuge for many microorganisms which would otherwise be removed by mastication and the flow of saliva. Such sites on the tongue can also have a low redox potential, promoting the growth of anaerobic flora. Thus the tongue may serve as a reservoir for some of the Gramnegative anaerobes that are implicated in the etiology of periodontal disease The mouth also contains keratinized (e.g. the palate) as well as nonkeratinized, stratified squamous epithelium which may affect the intra-oral distribution of micro-organisms.

Saliva It is a complex mixture of inorganic ions, including sodium, potassium, calcium, chloride, bicarbonate and phosphate, the concentrations of these ions varies diurnally and, in stimulated and resting saliva. The major organic constituents of saliva are proteins and glycoproteins (such as mucin), which modulate bacterial growth in the following ways: 1) Adsorption on the tooth surfaces forms a salivary pellicle, a conditioning film that facilitates bacterial adhesion 2) It acts as a readily available primary source of food for microorganisms (carbohydrates and proteins) and cause aggregation of bacteria, thereby facilitating their clearance from the mouth or deposition on surfaces, contributing to plaque formation

3) Growth inhibition of exogenous organisms by non-specific defense factors and specific defense factors 4) Maintenance of pH with its excellent buffering capacity (acidic saliva promotes growth of cariogenic bacteria)

Gingival Crevicular Fluid (GCF) There is a continuous but slow flow of gingival crevicular fluid in health, 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 nonspecific defence factors of serum. Crevicular fluid can influence the ecology by: 1. Flushing microbes out of the crevice 2. Acting as a primary source of nutrients 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 haem containing molecules such as haemoglobin

3. Maintaining pH conditions 4. Providing nonspecific and specific defense factors 5. Phagocytosis: 95% of leukocytes in the crevicular fluid are neutrophils.

Distribution of the resident oral microflora:

Factors affecting the growth of micro-organisms in the oral cavity: Temperature The human mouth is kept at a relatively constant temperature (35-36 C), which provides conditions suitable for the growth and metabolism of a wide range of micro-organisms. Temperature can also affect key parameters associated with the habitat, such as pH, ion activity, aggregation of macro-molecules and gas solubility. Periodontal pockets with active disease have a higher temperature (up to 390 C) compared with healthy sites (mean value 36.80 C). Such changes in temperature affect gene expression in periodontal pathogens, such as Porphyromonas gingivalis.

A large rise in temperature down regulates expression of fimbriae (which mediate attachment of the bacterium to host cells) and the major proteases of this micro-organism, and up regulates synthesis of superoxide dismutase, which neutralizes toxic oxygen metabolites. Temperature has been shown to vary between different sub gingival sites, even within the same individual, and may influence the proportions of certain bacterial species, such as the putative periodontal pathogens P. gingivalis, Bacteroides forsythus and Campylobacter rectus.

pH Many micro-organisms require a pH around neutrality for growth, and are sensitive to extremes of acid or alkali. The pH of most surfaces of the mouth is regulated by saliva so that, in general, optimum pH values for microbial growth are provided at sites bathed by this fluid. Bacterial population shifts within the plaque microflora can occur following fluctuations in environmental pH. After sugar consumption, the pH in plaque can fall rapidly to below pH 5.0 by the production of acids (predominantly lactic acid) by bacterial metabolism, the pH then recovers slowly to base-line values. Depending on the frequency of sugar intake, the bacteria in plaque will be exposed to varying challenges of low pH. Many of the predominant plaque bacteria from healthy sites can tolerate only brief conditions of low pH, and are inhibited or killed by more frequent or prolonged exposures to acidic conditions. This can result in the enhanced growth of, or colonization by, acidtolerant species, especially mutans streptococci and Lactobacillus species, which are normally absent or only minor components in dental plaque at healthy sites. Such a change in the bacterial composition of plaque predisposes a surface to dental caries. In contrast, the pH of the gingival crevice becomes alkaline during the host inflammatory response in periodontal disease, e.g. following deamination of amino acids and ammonia production. The mean pH may rise to between pH 7.2 and 7.4 during disease, with a few patients having pockets with a mean pH of around 7.8. Microbial factors

Anatomical factors 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.

Microbial factors Microbes in the oral environment can interact with each other both in promoting and suppressing the neighbouring bacteria.

Antimicrobial therapy Systemic or topical antibiotic 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.

Diet 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 facilitate adherence of organisms to surfaces, while the intracellular polysaccharides serve as a food resource.

Dental plaque biofilm

Dental plaque is a tenacious microbial community which develops on soft and hard-tissue surfaces of the mouth, comprising living, dead and dying bacteria and their extracellular products, together with host compounds mainly derived from saliva. The organisms in dental plaque are surrounded by an organic matrix which comprises about 30% of the total plaque volume. The matrix is derived from the products of both the host and plaque constituents. In the gingival area, proteins from the crevicular exudate become incorporated into the plaque. This matrix acts as a food reserve and as a cement, binding organisms both to each other and to various surfaces. 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, e.g. 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 - e.g. buccal and palatal surfaces

Subgingival, or Appliance-associated: full and partial dentures (denture plaque)

orthodontic appliance-related plaque

Microbial adherence, plaque biofilm formation Adherence of a microbe to an oral surface is a prerequisite for colonization and it is the initial step in the path leading to subsequent infection or invasion of tissues. Plaque biofilm formation is a complex process comprising a number of different stages. 1) Pellicle formation. a) Adsorption of host and bacterial molecules to the tooth surface forms the acquired salivary pellicle.

b) A thin layer of salivary glycoproteins is deposited on the surface of a tooth within minutes of exposure to the oral environment. c) Oral bacteria initially attach to the pellicle and not directly to enamel (i.e. hydroxyapatite). 2) Transport a) Bacteria approach the vicinity of the tooth surface prior to attachment, by means of natural salivary flow. b) III. Long-range interactions c) involve physicochemical interactions between the microbial cell surface and the pellicle-coated tooth. d) Interplay of van der Waals forces and electrostatic repulsion produces a reversible phase of net adhesion. 3) Short-range interactions a) consist of stereo chemical reactions between adhesins on the microbial cell surface and receptors on the acquired pellicle

4) Coaggregation or coadhesion a) fresh bacteria now attach on to the already attached first generation of cells 5) Biofilm formation The above process continues with a resultant confluent growth and the formation of a biofilm, which matures in complexity as time progresses. 6) Detachment. 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.

Calculus formation Calcium and phosphate ions derived from saliva may become deposited within deeper layers of dental plaque (as saliva is supersaturated with respect to these ions). If the plaque 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 which coalesce to form a calcified mass of plaque, termed calculus.

Predominant microflora of the dental plaque

The role of oral flora in systemic infection Recently it has been recognized that plaque-related oral diseases, especially periodontitis, may alter the course and pathogenesis of a number of systemic diseases. These include: 1. Cardiovascular disease: a) Infective endocarditis b) coronary heart disease: atherosclerosis and myocardial infection c) stroke 2. Bacterial pneumonia 3. Diabetes mellitus 4. Low-birth-weight babies.

Three mechanisms linking oral infections to secondary systemic disease have been proposed:

1.Metastatic infection: microbes gaining entry into the circulatory system through breaches in the oral vascular barrier, as in the case of bacteraemia produced during tooth extractions and resultant disease, such as infective endocarditis . 2. Metastatic injury: Products of bacteria, such as cytolytic enzymes, exotoxins and endotoxins gaining access to the cardiovascular system in individuals suffering from periodontitis. 3. Metastatic inflammation: It is caused by immunological injury due to oral organisms. Thus 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. Of these, the mechanisms linking oral 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: these enormous reservoirs of especially Gramnegative bacteria comprise a continuous source of lipopolysaccharide (LPS, i.e. endotoxins) which induces major vascular responses. Further, LPS upregulates endothelial cell adhesion molecules, and secretion of interleukin-l and tumour necrosis factor-a (TNF-a). 3. Periodontium is a reservoir of cytokines: the proinflammatory cytokines TNF-a and interleukin-l, gamma interferon and prostaglandin E2 reach high concentrations in periodontitis. Spill-over of these mediators into the circulation may induce or aggravate systemic effects

A study of the effects of orthodontic appliances on the oral microbial flora Rodney H. Bloom, Lee R. Brown Jr. (Science Direct, Volume 17, Issue 5, May 1964)

1. Prior to orthodontic therapy the total anaerobic population was numerically the highest bacterial category studied. In decreasing numerical order, this population was followed by the total aerobic, the streptococci, veillonella, staphylococci, lactobacilli, and yeast populations. 2. Although all categories showed a numerical increase after the placement of orthodontic appliances, only the lactobacilli increased by a statistically significant amount. 3. The subjects whose treatment involved the greatest number of orthodontic bands and auxiliaries manifested the greatest quantitative increase in the microbial populations studied.

References: 1. Essential Microbiology for Dentistry; Lakshman Samarnayake; Third Edn. 2. Textbook of Microbiology; Ananthnarayan and Paniker ; Eight Edn 3. Oral Microbiology; Martin, Marsh; Fifth Edn. 4. Science Direct 5. Journal of Clinical Microbiology, Nov. 2005