1. Introduction to oral microbiology

2. Oral ecology
oral cavity is complex and diverse ecosystem (consist of different microenvironment)
microorganisms do not exist as single species; rather they are almost always present in communities.
The teeth, gums, tongue, throat and cheeks all provide different surfaces for microbial colonization (shedding &non shedding surface)
The constant production of saliva and intermittent provision of sugars and amino acids from ingested food provides nutrients for microbial growth (a lot of metabolic activity) (saliva &Gingival Crevicular Fluid)

3. The human oral cavity is home to about 700 identified species of and may reach to 1000 in the future, at least 30 species of fungi (mainly of the genus Candida), several species of protozoa , and various intracellular viruses. in a single subject it is usual to find between 20–50 species of bacteria at healthy oral sites. While at diseased sites different species to be present, perhaps 200 or more.
Wide range of PH& nutrient

4. oral flora are a mixture of commensals and pathogenic m.o
Microorganism
oral flora are a mixture of commensals and pathogenic m.o
commensal m.o one that lives on or within a host with out cause any apparent disease. But as many commensal under certain conditions, may causes disease. in immunocompromised, So commensal referred to as opportunistic pathogens.
Many of the cultivated bacteria present in the mouth contribute to oral diseases to a greater or lesser extent, because these diseases are almost always associated with polymicrobial infections

5. Gram-positive cocci Genus Streptococcus mutans group
• : Streptococcus mutans serotypes c,e, f,·
S. sobrinus serotypes d, g;
salivarius group
anginosus group
mitis group

9. Genera Staphylococcus and Micrococcus

11. Eubacterium

15. Oral protozoa Genus Entamoeba
Large, motile amoebae about 12 11m in diameter.
• Main species: Entamoeba gingivalis.
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.

16. Genus Trichomonas
Flagellated protozoa, about m in diameter.
• Main species: Trichomonas tenax.
strict anaerobe; complex medium; difficult to grow in pure culture.
• Main intraoral sites and infections: gingival crevice; its role in disease is unclear.
**** mycoplasma and fungi in the oral cavity,

17. The oral ecosystem
Ecology is the study of the relationships between living organisms and their environment.
The oral environment
The human mouth is lined by str. Squ. Epith. 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.

18. Oral habitats The major oral habitats are: @@ buccal mucosa
dorsum of tongue
tooth surfaces (both supragingival and subgingival)
crevicular epithelium
prosthodontic and orthodontic appliances, if present

19. *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 Gram-negative anaerobes implicated in periodontal disease.
**The surfaces of the teeth are the only nonshedding area of the body that harbours a microbial population.
Large masses of bacteria and their products accumulate on tooth surfaces to produce dental plaque, present in both health and disease.

20. dental plaque
Plaque is a classic example of a natural biofilm and is the major agent initiating caries and periodontal disease.

21. The nature of the bacterial community varies depending on
(1) the tooth concerned and
(2) the degree of exposure to the environment:
smooth surfaces are colonized by a smaller a number of species than pits and fissures; subgingival surfaces are more anaerobic than supragingival surfaces.

22. @@Crevicular epithelium and gingival crevice.
Althaough this habitat is only a minor region of the oral environment, bacteria that colonize the crevicular area playa critical role in the initiation and development of gingival and periodontal disease. A vast literature on this subject is available.
and orthodontic appliances.
If present and not kept 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.

23. Factors modulating microbial growth
1-Anatomical factors
Bacterial stagnation areas are created as a result of:
A- the shape of the teeth
B- the topography of the teeth (e.g. occlusal fissures)
C- malalignment of teeth
D- poor quality of restorations (e.g. fillings and bridges)
E- non-keratinized sulcular epithelium.
These areas are difficult to clean, either by the natural flushing action of saliva or by tooth-brushing.

24. Role of saliva
@ 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 formation •
@ growth inhibition of exogenous organisms by non-specific defence factors (e.g. lysozyme, lactoferrin and histatins which are bactericidal and fungicidal and specific defence factors (e.g. Igs, IgA and salivary I leukocyte protease inhibitor (SLPI) which destroys human immunodeficiency virus) •
@ maintenance of pH with its excellent buffering capacity .

25. There is a continuous but slow flow of gingival crevicular
Gingival crevicular fluid
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 non-specific defence factors of serum.

26. 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, a. a. and CHO 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 neutrophil

27. Microbial factors
Microbes can interact with each other both in promoting and suppressing the neighbouring bacteria.
Mechanisms that accomplish this include:
@competition for receptors for adhesion
@ prevention of attachment of 'late-comers'
@ production of toxins, such as bacteriocins, that kill cells of the same or other bacterial species; e.g. St. salivarius inhibits S. pyogene s
@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 St. mutans)
@ coaggregation with the same species (homotypic) or different species (heterotypic) of bacteria, e.g. corn-cob formation .

28. Miscellaneous factors
@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 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) while others are eliminated by competitive inhibition.
@Oxidation-reduction potential. The oxidation-reduction potential of the environment (Eh) varies in different locations of the mouth. For instance, redox potential falls during plaque 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.

29. @Antimicrobial therapy
@Antimicrobial therapy. 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.
@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 facilitates adherence of organisms to surfaces, while the intracellular polysaccharides serve as a food resource.

30. latrogenic factors.
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.

31. Nutrition of oral bacteria
Oral bac.obtain their food from a no. of sources.These include
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)
resources:
extracellular microbial products of the neighbouring bacteria, especially in dense communities such as plaque
intracellular food storage (glycogen) granules.

32. Acquisition of the normal oral flora
1. The infant mouth is sterile at birth.
2. A few hrs later m.o.s from mother's (or nurse's) mouth and environment are established in the mouth.
3. These pioneer species are streptococci, which bind to mucosal epithelium (e.g. St. salivarius).

33. 4. The metabolic activity of the pioneer community then alters the oral environment to facilitate colonization by other bacterial genera and species.
S. salivarius produces extracellular polymers from sucrose, to which other bacteria such as Actinomyces spp. can attach
5. When the composition of this complex ecosystem reaches equilibrium, a climax community is said to exist.

34. 6. Oral flora on the child's first birthday usually consist of strept
6. Oral flora on the child's first birthday usually consist of strept., staphy., neisseriae and lactobacilli, together with anaerobes ( Veillonella and fusobacteria). Less frequently isolated are Lactobacillus, Actinomyces, Prevotella and Fusobacterium species.

35. The next evolutionary change in this community occurs during and after tooth eruption,
two niches are provided for bac. colonization:
@ surface of enamel and
@ gingival crevice.
St.mutans, S. sanguis and Actinomyces spp., then selectively colonize enamel surfaces, and Prevotella spp., Porphyromonas spp. and spirochaetes, colonize the crevicular tissues.

36. 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 years or over. The denture plaque is somewhat similar to enamel plaque; it may also harbour significant quantities of yeast

37. 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.

41. composition
The microbial composition of dental plaque can vary widely between individuals; some people are rapid plaque formers, others slow. 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.

42. 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

43. 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 del' Waals forces
and electrostatic repulsion produces a 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 irre-versible phase in which polymer bridging between m.o. and the surface helps to anchor the m.o., after which the m.o. multiply on the virgin surface. Doubling times of plaque 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.

44. 5. Coaggregation or coadhesion:
6. Biofilm formation
Calculus formation

45. The role of oral flora in systemic infection
• cardiovascular disease:
• infective endocarditis
• coronary heart disease: atherosclerosis and myocardial
infection
• stroke
• bacterial pneumonia
• diabetes mellitus
• low-birth-weight babies.

51. Bacteria

52. Lecture 49 Lecture 49Department of Biochemistry and Molecular Biology
New Jersey Medical School
Dr. S. Kumar
Lecture 49

53. Oral diseases
Almost every member of the human population is afflicted at some stage of their lives with an oral disease
The incidence of dental caries has declined generally in the developed world, due to fluoride in the water supply, in toothpaste, or taken in tablet form .However there are many groups within societies that are still seriously afflicted with caries.
Polymicrobial infections of the gingiva and sub-gingival area (periodontitis, implantitis and pulpitis) are major conditions requiring clinical intervention.
Halitosis is often caused by bacteria on the tongue processing proteins into volatile sulfur compounds.
Pharyngitis and tonsillitis are common diseases in children and are caused by bacteria or by viruses
Osteonecrosis of the jaw is associated with the use of bisphosphonates particularly in cancer patients with multiple myeloma.
Fungal infections, most frequently by the yeast Candida albicans, are associated with reduced salivary flow, ill-fitting dentures, hormonal changes, or compromised immune function.
Viral infections of the oral mucosa include HPV, EBV and HSV.

54. General properties of saliva

55. Around 0.5 to 1.5 liters of saliva are secreted into the mouth each day.
an adequate flow of saliva is essential for the maintenance of both hard and soft tissue integrity
Saliva is hypotonic, with an average pH of around 6.7
Saliva contains both organic compounds (2–3 g/l): protein, enzyme amylase) and inorganic compounds :bicarbonate, chloride, potassium and sodium.
Salivary flow rate and composition can be affected by some infectious diseases, clinical conditions, e.g. wearing of dentures, radiation therapies for oral cancer, or pharmaceutical drugs. The symptoms of dry mouth (xerostomia), due to deficiencies in salivary flow, are frequently accompanied by increased susceptibility to oral microbial diseases

56. Function of saliva
1 million and 100 million bacteria present in 1 ml of saliva, depending upon oral hygiene, frequency of food consumption, and salivary flow rate.
Saliva flush out bacteria from the mouth. Saliva contains agglutinins that aggregate bacteria, thus preventing adherence to surfaces, and the bacterial clumps are removed by swallowing or expectoration
possesses a number of additional antimicrobial components

57. Salivary pellicle
Saliva form a coating protective film on all the surfaces in the mouth, a thick mucus coating forms on the soft tissues. consist of proteins, glycoproteins, glycolipids and lipids
The salivary films formed on hard surfaces, such as teeth, or dentures, are very thin (less than 1 micron) and are known as acquired pellicle . &it is composition differs according to the surface on which it is formed. Enamel is ionic so binds to charged molecules. Acrylic materials hydrophobic, so bind to uncharged molecules
some components from bacteria and host cells can be incorporated into pellicle. Transglutaminase from epithelial cells. GTF and FTF enzymatically active, increasing the levels of glucan and fructan polymers, amylase in pellicle remains active and may continue to hydrolyze starch to glucose that can be used by oral streptococci for fermentation with resultant acid production.
The acquired pellicle provides receptors for bacterial adhesion. ( agglutinins) the agglutinins that aggregate bacteria & also promote adhesion when deposited in the enamel pellicle, eg. PRPs, will only bind bacteria when deposited on surfaces, as a conformational change in the protein occurs which exposes previously hidden binding sites (cryptitopes).Proteolytic activity by some organisms such as P. gingivalis can also expose cryptitopes.

58. Saliva as a nutrient Saliva can provide growth nutrients for bacteria:
bacteria produce proteases that degrade salivary proteins into peptides and amino acids, which can be used by the bacteria when exogenous nutrients are limiting.
Bacteria can also produce glycan hydrolases that cleave sugar residues from the salivary glycoproteins, so that the sugars can be used for bacterial growth.

59. Anti-microbial properties of saliva
(1) Lysozyme is a basic protein found in most secretions,including saliva( high concentrations).
Salivary lysozyme originates from both the salivary gland secretions and from gingival crevicular fluid (GCF).
Lysozyme digests the cell walls of Gram-positive bacteria by breaking the β(1-4) bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan
Lysozyme can also bind and aggregate bacteria and facilitate clearance by swallowing or expectoration. In
lysozyme contains small amphipathic sequences in the C terminal
region that are capable of killing bacteria

61. (2) Salivary peroxidase is derived from the salivary glands and PMNs, and catalyzes the oxidation of thiocyanate (SCN−) to hypothiocyanite (OSCN−) by hydrogen peroxide, which is produced by the aerobic metabolism of oral bacteria.
At acid pH, OSCN−becomes protonated (and uncharged) and readily passes through bacterial membranes. Hypothiocyanite oxidizes SH groups in bacterialenzymes and inhibits bacterial metabolism.
Reduction of hydrogen peroxide to water by peroxidase also prevents oxidative damage to the host soft tissues.
(3) Lactoferrin Lactoferrin is an iron binding glycoprotein produced
from glandular acinar cells, epithelial cells and phagocytic cells Lactoferrin inhibits bacterial growth by binding and sequestering Fe2+ ions, and in the apo (iron free) state can be toxic to bacteria and interfere with bacterial adhesion.

62. (4) Histatins are cationic histidine rich proteins that kill Candida albicans and some bacteria
At least 12 histatins are present in saliva, ( proteolysis of the genetically distinct histatins 1 and 3. Histatin 5 (the N-terminal 24 amino acids of histatin 3) is a major salivary histatin and is very effective in killing yeast. Histatins bind to a Candida membrane receptor, then the peptide is taken up by the cells. This results in arrest of the cell cycle and the cells lose ATP by efflux.
Histatins can also regulate hydroxyapatite crystal growth,
inhibit bacterial cysteine proteinases
prevent bacterial coaggregation.
(5) Cystatins are cysteine rich peptides that inhibit bacterial cysteine proteases. Cystatins also regulate inflammation by inhibiting host proteases and up-regulating cytokines.