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ORAL MICROBIAL FLORA PLAQUE

INTRODUCTION Over 500 different bacterial species have been isolated from the mouth At any one time there are 50-100 species present

"normal flora = species present..." normal flora = species present in a healthy mouth (usually also present in a diseased mouth) transient flora = species sometimes present but are usually only “passing through” (bacteria in food or drink or saliva from another person via a kiss)

"The normal flora is dominated..." The normal flora is dominated by anaerobic and facultative bacteria that grow optimally at 37OC

O2 Tensions in the Mouth Location O2 Tension Anterior Surface of Tongue 16.4% Posterior Surface of Tongue 12.4% Maxillary Buccal Fold     0.4% Mandibular Buccal Fold   0.3% Supragingival area 1.0% -20% Periodontal pockets     0.1-0.2%

ACQUIRING THE ORAL FLORA Prior to birth Birth to first few days Prior to tooth eruption Following tooth eruption Childhood and adolescence Adult and advanced age

Flora - Prior to Birth Prior to birth, the mouth is sterile

Flora - Birth to first few days During passage through the birth canal vaginal flora enters the mouth

Flora - Birth to first few days Later organisms from Mother Nurses Father Towels Feeding bottles A host of environmental sources

"Most do not to colonize" Most do not to colonize Those that do include streptococci (primarily S. mitis but also some S. salivarious) Neisseria Haemophilus Lactobacillus (aerobes or facultatives)

Flora - Prior to tooth eruption Little research done on anaerobes Prevotella melaninogenica = most frequently isolated anaerobe found in 70% of edentulous infants aged 1-7 months

"Other anaerobes commonly found" Other anaerobes commonly found Fusobacterium nucleatum, Veillonella spp., and other Prevotella species Streptococcus vestibularis - in predentate and dentate children, not adults

Flora in 30 edentulous infants Prevotella melaninogenica 70% Fusobacterium nucleatum 60% Veillonella spp. 57% nonpigmented Prevotella spp. 57% Bacteroides gracilis 23% Leptotrichia spp. 17% Capnocytophaga spp. 13% Prevotella loescheii 13% Prevotella intermedia   7% Wolinella spp.   3% (1 infant) Eikenella corrodens   3% A. actinomycetemcomitans   0% * * of 50 infants

Flora - Following tooth eruption The eruption of teeth changes things significantly Tooth surfaces are provided Gingival crevices develop

"Plaque is the name for..." Plaque is the name for the bacteria growing on the surface of a tooth

"Oral Surface Preferred for Colonization" Oral Surface Preferred for Colonization Organism Preferred surface S. mutans Hard surfaces S. sanguis Hard surfaces S. salivarius Soft tissues

"The gingival crevice provides both..." The gingival crevice provides both an anaerobic environment and nutrients essential to many anaerobic bacteria Spirochetes are found only when gingival crevices and some inflammation are present They require gingival inflammation to get alpha2 globulin

Flora - Childhood and Adolescence The flora is gradually acquired during childhood and adolescence By early adulthood almost all, if not all, the members of the permanent flora are present

Flora - Adult and Advanced age Black pigmented Gram-negative anaerobes are present in edentulous subjects with or without dentures Edentulous subjects do not harbor Porphyromonas gingivalis or     A. actinomycetemcomitans

"Prevotella melaninogenica," Prevotella melaninogenica, P. intermedia, and P. loescheii are common in the periodontally healthy mouth

"Comparing those over 70 to..." Comparing those over 70 to those under 40 increase in lactobacilli, staphylococci, and Candida in the saliva increase in A. viscosus in the plaque These changes were not related to denture wearing or disease

"Old age can result in..." Old age can result in cancers requiring irradiation or cytotoxic therapy These treatments reduce the salivary flow and thus affect the IgA levels and therefore may facilitate some bacterial attachment

NUTRIENT SOURCES IN THE ORAL CAVITY Diet Saliva Gingival Crevicular Fluid Microbial Products Host Products

Diet When we say diet, we think of it's chemical nature Chemical composition is important from a nutrition standpoint But, for oral health the physical consistency and frequency of ingestion are of equal or greater important

Diet-Physical consistency Ex. starches are very slowly broken down and normally would pass through the mouth unchanged But if they stick in the teeth, like potato chips, the starch will be partly degraded, resulting in acid production That acid will remain in contact with the tooth for a prolonged period

Diet-Frequency of ingestion Frequency of ingestion is also important Juices and other liquids with sugar -normally would be swallowed and cleared from the mouth But if sipped - teeth are continually bathed in the fluid If the fluid contains a fermentable sugar, acid will be produced that continually acts on the tooth and selects in favor of aciduric organisms

Saliva Saliva contains glycoproteins amino acids glucose other digestible chemicals materials that buffer the pH of the plaque (if there is not too much plaque)

Gingival Crevicular Fluid Gingival crevicular fluid contains tissue serum proteins amino acids vitamins glucose

"Normally the crevicular fluid is..." Normally the crevicular fluid is protective It flushes out the nonadherent bacteria and bacterial products Brings phagocytic cells and antibodies to the area But it can prove detrimental Provides a2-globulin which spirochetes require

Microbial Products Bacteria also add nutrients to the environment that are used by other bacteria Organism Requires Produced by Camp. sputorum H-formate Prev. oralis T. denticola Isobutyrate F. nucleatum T. denticola Spermine Actinomyces BPA Vit K Prev. oralis & C. sputorum

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Host Products The host sheds epithelial cells Phagocytic cells enter the oral cavity from the gingival crevice area These cells are lysed by the hypotonicity of the saliva That releases the contents, making them available to the bacteria

ECOLOGIC NICHES Supragingival Plaque Subgingival Plaque Tongue Other Mucosal Surfaces

Supragingival Plaque The 2 types of plaque are very different Supragingival Plaque starts with a salivary pellicle forming on the enamel

"They multiply with Gram +..." They multiply with Gram + rods attaching to the cocci Actinomyces spp. colonize at the gingival margin In the absence of periodontal disease, things stop here

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Eh of Developing Supragingival Plaque DAYS Eh (mv) 0 + 250 2   0 3 –   30 4 –  112 to – 140 7 –  140

"The Eh shift of developing..." The Eh shift of developing Supragingival Plaque from very aerobic to very anaerobic coincides with a shift of the flora from facultative or microaerophilic to very anaerobic

"However," However, even at an Eh of –140, Spirochetes can’t grow Spirochetes need an Eh of –180

"In the pits and fissures..." In the pits and fissures beneath the plaque, in addition to the other Gram-positive plaque-forming bacteria, there may be Gram-positive lactobacilli Lactobacilli cannot attach to the tooth or form plaque, but they can be mechanically retained in the pits and fissures

Subgingival Plaque Subgingival plaque is only present if there is some periodontal breakdown The Eh in subgingival plaque has been measured at –300 mv So spirochetes can grow in the subgingival plaque but perhaps not in supragingival plaque This plaque consists mainly of Gram-negative rods and spirochetes

Tongue Mainly S. salivarius and Veillonella Many others isolated in low numbers - not clear whether these are permanent or transient flora

Other Mucosal Surfaces The other mucosal surfaces such as Lips Cheeks Palate Gingiva Underside of the tongue Tonsils may harbor their own distinct floras but we haven't really studied them so we don't know

Saliva Note: saliva is not a niche Saliva merely bathes the outside of other bacterial mats or biofilms and thus passively contains those organisms that slough from those surfaces

Partial Composition Of Plaque, Saliva And Tongue Organism Plaque Saliva Dorsum of   Tongue S. mutans 1   0-50     1     1 S. sanguis 1 30-40 10-20   5-15 S. mitis 1 30-40 30-40 10-20 S. salivarius 1     1 40-60 40-60 Lactobacilli 2   0-0.04   0-1   0-2 Veillonella 2   1-3   5-10 10-15 1 Data expressed as % of streptococcal flora 2 Data expressed as % of total flora

FACTORS AFFECTING ACQUISITION AND RETENTION Attachment If cells aren’t attached they will wash away Retention Once attached, if not retained, they will also wash away

"3 Modes of Attachment" 3 Modes of Attachment Adherence Aggregation Mechanical

Attachment Modes Adherence - active attachment of a bacterium to a host surface S. sanguis attaches to hard surfaces S. salivarius attaches to epithelial surfaces, usually via fuzzy coat

"Proteoglycans or glycoproteins usually are..." Proteoglycans or glycoproteins usually are between the bacterium and the host surface These come from saliva and other bacteria

Attachment Aggregation - active attachment of one bacterium to another of the same or different kind Dextran facilitates this for S. mutans Salivary proteins do this for other microorganisms One unique aggregation is called a corncob - cocci are arranged around a central rod or filament

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Corncob formation between oral bacteria “Kernels” Core (Cob) F. nucleatum C. matruchotii S. sanguis + + S. mitis + - S. salivarius + - S. mutans + -

Attachment Mechanical - passive, nonspecific, entrapment of a bacterium In pits and fissures In carious lesions Around appliances (braces, others) Possibly in perio pockets The lactobacilli found in the mouth are a good example of this

Factors Affecting Retention 3 Factors Mechanical Nutritional Resist Inhibitors

Retention Factors Mechanical Attachment-cells must be attached to something that remains in the mouth Removal of braces Filling of caries

"Nutritional - organism must eat..." Nutritional - organism must eat or die Some nutrients come from other organisms Some come from the body

Some Nutritional Needs of Oral Bacteria Organism   Requires   Gets from T. denticola a-2 globulin Crevicular fluid BPA heme Blood BPA naphthoquinone Hormones

Resist Inhibitors Some from other bacteria pH - most bacteria grow best at pH 7.0 Most bacteria won’t grow below pH 6.0 Bacteria that lower the pH via acid production are called acidogenic Those that can survive or thrive at a low pH are called aciduric

"Eh - similar to the..." Eh - similar to the above but Strict anaerobes won't colonize until the facultatives have lowered the Eh sufficiently for them to grow That is why we can't effectively use germ-free animals to study periodontal disease - without a facultative flora, the Eh remains too high and the strict anaerobes won't colonize

"hydrogen peroxide - oxidizes some..." hydrogen peroxide - oxidizes some enzymes and bacterial membranes fatty acids - prevent the growth of some species bacteriocins - prevent the growth of some species unknown mechanisms - alpha strep inhibits Staph, beta strep, and others but we don’t know how

Inhibitors Some from the host Lysozyme - can kill many bacteria, Gm+ Antibodies - specific Lactoferrin - in saliva; binds iron

"Inhibitor neutralizers from bacteria" Inhibitor neutralizers from bacteria Mucin Coats some bacteria Protects them from reacting with antibodies and from being phagocytized Catalase Breaks down H2O2

CONTRIBUTIONS OF THE  ORAL FLORA Nutritional Protective

Contributions Nutritional GI tract organisms are our main source of niacin, thiamin, riboflavin, pyridoxine, cyanocobalamine, folic acid, pantothenic acid, biotin, and vitamin K

Contributions Protective via niche defense Organisms normally present in a niche prevent new organisms from coming in and taking over It normally takes one million Salmonella or Shigella to cause disease If bacteria like E. coli and other enterics are absent or significantly decreased, as few as ten Salmonella have been shown to initiate infection

See Table Some Bacterial Genera Commonly Present in the Mouth

Normal oral flora Gram-positive

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"Rods and Filaments" Rods and Filaments

"Spirals" Spirals

PLAQUE FORMATION There are many Factors Involved in Plaque Formation A. Ability of the bacterial cell to attach to the oral surface Hard vs Soft

"C." C. The retentiveness of the oral surfaces Tooth vs Epithelium Initial plaque bacteria require hard surfaces - permanent attachment Bacteria that attach to the primary plaque formers - don't know whether these need permanent attachment or not

"D." D. Saliva 1. Flow rate Stagnation leads to greater bacterial proliferation Heavy flow washes free floating cells away from surfaces and therefore prevents attachment - It also brings more SIgA which prevents attachment

"2." 2. Antibodies Can inhibit adherence to, at least, oral epithelial cells by blocking cell surface binding receptors can also foster aggregate formation which can bind new cells to already attached cells contribute to the pellicle (mostly salivary glycoproteins) - the pellicle coats the negatively charged tooth and bacterial surfaces

"3." 3. Mucinous- and glyco-proteins Contribute to pellicle & act as above 4. Lysozyme Kills some bacterial cells by breaking links in the cells wall 5. Lactoferrin The iron binding protein, competes with the bacteria for iron

"6." 6. Lactoperoxidase lactoperoxidase with salivary thiocyanate and H2O2, generates hypothiocyanate (OSCN), which is much less toxic to the bacterial cells than hydrogen peroxide but is a strong inhibitor of bacterial glycolytic enzymes, mainly streptococcal

"E." E. Gingival fluid May supply nutrients, antibodies, flushing action F. Diet Sucrose with S. mutans-formation of glucan G. Oxidation-Reduction potential (Eh)

"H." H. Host's bacterial flora Competition for binding sites-normal flora inhibits pathogens; i.e. C. diphtheria influence on pH-high carbohydrate intake-acid pH-get acidogenic and aciduric flora prior bacterial "attachment"-corn cobs provide nutrients and attachment sites Some organisms feed on the byproducts of other bacteria

"I." I. Availability of endogenous nutrients Some bacteria need blood components BPA need hemin (& hormones?) Spirochetes need I2-globulin

"J." J. Species of host Humans are similar to but not identical to others. S. salivarius can attach to teeth & cause caries in some animals but not humans Glucose formed on the pellicle by some bacteria serves as a binding site for adhesion for a rodent strain of A. viscosus but reduced the adhesion of human isolates

"K." K. Time of Day-in relation to oral habits Before or after eating Eating before sleeping

PLAQUE DEVELOPMENT Starts with A. Salivary Pellicle (glycoprotein + lysozyme) formation which involves the adsorption of salivary proteins to apatite surfaces

"Almost immediately after the beginning..." Almost immediately after the beginning of pellicle formation, B. Bacteria are "precipitated" or mechanically entrapped along with the pellicle material The first organisms form a cellular monolayer, either singly or in small groups

"These bacteria grow," These bacteria grow, eventually coalescing with neighboring patches of plaque During the first few hours, the attached bacteria proliferate and form small colonies of cocci With time, other types of microorganisms attach to the cocci and then other bacteria attach to them; eventually resulting in a characteristically complex mass

"C." C. In supragingival plaque Gm + Cocci è Gm + Rods. Actinomyces colonize at gingival margin Measurable amounts of supragingival plaque may form within 1 hour after the teeth have been cleaned, with maximum accumulation in about 30 days

"D." D. As gingivitis occurs spirochetes begin to form and subgingival plaque formation begins

"E." E. Subgingival plaque Contains many Gm - organisms that are "loosely adherent" to epithelium, which, in Periodontitis, overlies tooth attached Gm + and Gram - rods, cocci, and filaments In periodontosis there are the loosely attached Gm negatives, but no attached Gm +

"The subgingival plaque is formed..." The subgingival plaque is formed in the presence of gingival fluid & little or no saliva There is greater protection from mechanical cleansing a low redox potential a lack of direct dietary effect, and a different tooth surface-cementum

"F." F. Calculus Supragingival and subgingival plaque can become calcified from the calcium and phosphorus in the saliva This mineralized deposit is called calculus or tartar Calculus cannot be removed by brushing and provides a rough surface that presents an even greater surface area for bacterial colonization

PLAQUE INHIBITION Chlorhexidine Sanguinarine Cetylpyridinium Chloride Fluoride Essential oils Plax®

Chlorhexidine Attaches directly to the apatite and not the salivary pellicle Remains attached and is thus said to have substantivity Affects cell walls causing cell lysis and death Is poorly absorbed from the GI tract Didn't produce resistant organisms in 2 years

"Peridex®" Peridex® = Chlorhexidine gluconate 0.12%

Essential Oils Listerine® The active ingredients are methyl salicylate and the essential oils thymol, eucalyptol and menthol in 26.9% ethanol Acts on bacterial cell walls Has low substantivity Next to chlorhexidine, Listerine is better than the others listed here

Cetylpyridinium Chloride Cepacol® Scope® Viadent® A quaternary ammonium compound Acts by increasing cell wall permeability, which leads to cell lysis, reduction in cell metabolism and decreased bacterial ability to attach to tooth surfaces Most effective against Gram-positive bacteria but active against some Gram-negatives

"Show plaque reductions of 25..." Show plaque reductions of 25-35%

Cetylpyridinium Chloride Viadent(R) - Active Ingredients: Cetylpyridinium Chloride (0.05% w/w) Inactive Ingredients: Purified Water, Sorbitol, Propylene Glycol, SD Alcohol 38-B (6.1% w/w), Glycerin, PEG-40 Sorbitan Glycol, PEG-40 Sorbitan Diisostearate, Flavor, Sodium Benzoate, Hydroxyethylcelllulose, Sodium Saccharin, FD&C Yellow No. 6

Cetylpyridinium Chloride Scope® - Water, Alcohol (14.3 wt%), Glycerin, Flavor, Polysorbate 80, Sodium Saccharin, Sodium Benzoate, Cetylpyridinium Chloride, Domiphen Bromide, Benzoic Acid, Blue #1 Cepacol® - Cetylpyridinium Chloride, Alcohol, D&C Yellow #10, FD&C Green #3, Flavor, Glucono Delta Lactone, Glycerin, Poloxamer 407, Sodium Saccharin, Sodium Gluconate, Water

"Early reports indicated that this..." Early reports indicated that this was a good plaque preventive

Fluoride Stannous fluoride and sodium fluoride have been used Stannous fluoride is a more effective anti-plaque agent than sodium fluoride because the tin ion binds to the bacterial surface which affects their metabolic activity and ability to colonize Can inhibit plaque formation but not necessarily for a long time since all but one long-term study don’t show any plaque or gingivitis reduction

Plax® Water, Sorbitol Solution, Glycerin, Alcohol (8.6%), Tetrasodium Pyrophosphate, Benzoic Acid, Flavor, Poloxamer 407, Sodium Benzoate, Sodium Lauryl Sulfate, Sodium Saccharin, Xanthan Gum, FD&C; Red No. 40. No active ingredient

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