NORMAL ORAL MICROFLORA AND

PLAQUEDr. Amitha GOral Pathology And Microbiology

Contents :◦ Introduction◦ Terminologies◦ Oral ecosystem◦ Normal oral flora of the oral cavity development, factors

effecting it.◦ Plaque◦ Brief outline on oral microflora in diseases

INTRODUCTION The mouth is continually exposed to organisms from the external

environment, beginning with the passage through the birth canal. In time, ecological balance is reached that serves to establish a

resident microbial flora that remains fairly stable throughout life.

Introduction◦ It has been estimated that the human

body is made up of over 1014 cells of which only around 10% are mammalian. The remainder are the micro-organisms that comprise the resident micro flora of the host.

(Sanders & Sanders, 1984)

◦ Microorganisms in mouth were first described by Anton von Leeuwenhoek in 1683.

TerminologiesHabitat: Site where a microorganism grows

Ecology: Study of relationship between organisms and their environment.

Ecological niche: The functional position of an organism in its environment comprising the living space, periods of time during which it is active there and resources it obtains there.

TerminologiesAerobes: Organisms require oxygen for aerobic cellular respiration

to obtain energy.

Obligate aerobe: Organism that can survive and grow only in an oxygenated environment

Facultative anaerobes: Oraganism that use oxygen, but also have anaerobic methods of energy production.

Terminologies:Capnophiles: Organism which thrive in the presence of high

concentrations of carbon dioxide.

An Oligotroph: Organism that can live in an environment that offers very low levels of nutrients.

Microaerophiles: Organisms that may use oxygen, but only at low concentrations.

Terminologies:◦ Indigenous Flora(Resident):

It comprises those indigenous species that are almost always present in high numbers, greater than 1 % of the total viable count.

◦ Supplemental Flora: The supplemental flora are those bacterial

species that are nearly always present, but in low numbers, less than 1 % of total viable count

◦Transient Flora: Transient flora comprise organisms "just

passing through" a host. At any given time a particular species may or may not be represented in the flora.

Terminologies:◦ Pathogens

Micro organisms that have the potential to cause disease are termed pathogens.

◦Opportunistic Pathogens Micro-organisms that cause disease only under exceptional circumstances.

◦True Pathogens Micro-organisms that are consistently associated with a particular disease.

The Oral EcosystemsFour major ecosystems are present in the oral cavity

- Buccal epithelium

- Dorsum of the tongue

- Supragingival tooth surface

- Subgingival tooth and crevicular epithelial surfaces

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Tooth surface• mitis group• mutans group• anginosus group

Saliva• S. salivarius• S. mitis• S. oralis• mitis group• anginosus group

Tongue• S. salivarius• S. mitis

Pharynx• S. mitis• S. oralis• mitis group• anginosus group

Nasopharynx• S. pneumoniae

Vestibular mucosa• S. vestibularis

Buccal mucosa• S. mitis• mitis group• anginosus group

Tonsils• S. sanguis• S. mitis• S. mutans• mitis group• anginosus group

Micro Flora

◦The buccal epithelium has gram positive streptococci in contrast to tongue which has more of gram positive filaments.

◦The subgingival region is anaerobic as compared to the supragingival region.

◦Mutans streptococci (S. mutans, S. sorbinus, S. cricetus, S. rattus) and S. Sanguis are found in large number on teeth.

◦S. Salivarius is isolated mainly from the tongue.◦S. mutans and S. sanguis appear only after eruption of teeth.

◦Buccal mucosa The predominant bacterias is Streptococcus mitior with Streptococcus sanguis and salivarius.

◦Hard palate flora also resembles that of buccal mucosa,

With predominance of streptococci.

◦Soft palateHarbours respiratory tract bacteria Corynebacterium, Neisseria, Haemophilus.

◦Tongue Is an ideal site for retention of microorganisms to its keratinized dorsal surface.

◦Streptococcus salivarius Is the predominant flora accounting for upto 50% of the total.

◦Streptococcus mitior Is also common here.

Gingival Crevice:◦Gingival crevice has the most numerous bacterial population among all sites in mouth.

◦As many as 1010-1011 organisms are recovered per gram wet weight of gingival debris.

◦It is considered to be due to absence of dislodging forces and crevicular fluid acting as a rich nutrient medium.

Normal Flora of the Human oral Cavity :

The presence of nutrients, epithelial debris, and secretions makes the mouth a favorable habitat for a great variety of bacteria.

Oral bacteria include streptococci, lactobacilli, staphylococci and corynebacteria, with a great number of anaerobes, especially bacteroides.

The mouth presents a succession of different ecological situations with age, and this corresponds with changes in the composition of the normal flora

Development of oral flora◦ The process begins with the colonization of habitat by pioneer microbial populations.

◦ In oral cavity of newborns, streptococci are the pioneer organisms.

◦ They fill the niche of the new environment and modify the habitat and new population develops.

◦ When no additional niche is available for new population, a stable assemblage of bacterial population is achieved called as climax community.

At birth:

◦ The mouth of full term foetus is usually sterile, transient flora from the birth canal may be acquired.

◦ Mouth then rapidly acquires organisms from mother and from the environment.

◦ It consists of several streptococcal and staphylococcal species with Lactobacilli, Bacillus, Neisseria and Yeasts.

◦ Streptococcus salivarius is the most common and forms the pioneer community with Staphylococcus albus.

Infancy & Early Childhood:◦ The infant comes into contact with an ever-increasing range of

microorganisms and some become established as part of commensal flora.

◦ The eruption of deciduous teeth provides a new attachment surface and turns Streptococcus sanguis and mutans as regular inhabitants of oral cavity.

◦ Anaerobes are few in number due to absence of deep gingival crevice.

◦ Actinomyces , Lactobacilli are found regularly.

Adolescence:

◦ The greatest number of organisms in mouth occur when permanent teeth erupt.

◦ These teeth have deep fissures, larger inter proximal spaces and deeper gingival crevice, allowing a great increase in anaerobes.

Adulthood:

◦ Its chief characteristic is its complexity of oral flora.◦ There is an increase in Bacteroides and Spirochetes with maturity of

dental plaque.

◦ As the teeth are lost the available sites for microbial colonisation decreases and several species diminish disproportionately in numbers.

◦ Edentulous persons harbour few Spirochetes or Bacteroides but carriage of Yeast increases.

◦ S.sanguis & mutans disappear.

HUMAN ORAL FLORA

Gram-positive facultative cocci

Gram-negative facultative rods

Staphylococcus epidermidis Staph. aureusStreptococcus mutansStrep. sanguis Strep. MitisStrep. Salivarius Strep. FaecalisBeta-hemolytic streptococci

EnterobacteriaceaeHemophilus influenzaeEikenella corrodensActinobacillusActinomycetemcomitans

Gram-positive anaerobic cocci

Gram-positive anaerobic rods

Peptostreptococcus sp Actinomyces israelii A. odonotolyticusA. ViscosusLactobacillus

Gram-negative anaerobic cocci

Gram-negative aerobic or facultative cocci

DiphtheroidsCorynebacterium

Eubacterium Neisseria siccaN. Flavescens

Spirochetes Yeasts

Treponema denticolaT. Microdentium

Candida albicansGeotrichum sp.

Protozoa Mycoplasma

Entamoeba gingivalisTirchomonas tenax

Mycoplasma oraleM. pneumoniae

Factors determining bacterial distribution

1. Physicochemical factors

a. - Temperature

b. - Oxygen tension

c. - Hydrogen ion concentration

2. Host factors

3. Nutrient sources

a. Temperature:◦Average temperature in the oral cavity is approx 37' C, temperatures can vary considerably, especially on the mucosal surfaces and on the clinical crowns of teeth.

Periodontal pockets with active disease have a higher temperature – up to 39 C compared with healthy sites (mean value 36.80 C).

Such changes in temperature affect gene expression in periodontal pathogens, such as Porphyromonas gingivalis.

b. Oxygen tension :

◦The oxygen concentrations at different locations in the oral cavity varies.

◦Dorsum of the tongue, buccal and palatal mucosa are aerobic.

◦In periodontal pocket: Oxygen tension inside is very low, with the species having a tendency to become reduced rather than oxidised, explaining the survival of obligate anaerobe.

◦Therefore obligate aerobic organisms (which require oxygen) cannot survive, whereas obligate anaerobic organisms (which cannot tolerate the presence of oxygen) are able to thrive.

c. Hydrogen ion concentration:

The term pH refers to the negative logarithm of hydrogen ion concentration Lower the pH value, the higher is the hydrogen ion concentration.

The buffering capacity of plaque and saliva maintain the pH in oral cavity at about 7.

pH can vary due to: 1) Exogenous materials placed in the mouth2) Production of hydrogen ion by bacteria as a result of

carbohydrate fermentation3) Buffering system in saliva (bicarbonates)

The second reason is responsible for dramatic drop in pH leading to Dental Caries.

Most oral bacteria grow best at a pH of about 7 (essentially neutral).

Most of the time the pH in the oral cavity is maintained at about 7 by the buffering systems in plaque and saliva.

Host factors :

1) Saliva 2) Crevicular fluid

1. Saliva :

◦When Salivary components interact with oral flora it either enhance or inhibit ability of these organisms to survive.

◦IgG is seen in gingival inflammation contributed by GCF.

◦These make it more difficult for bacteria to bind to oral epithelium or hard tissue surfaces.

Non-specific factors:These are lysozyme, lactoferrin and lactoperoxidase.◦Lysozyme degrades bacterial peptidoglycan i.e the cell wall, rendering bacteria susceptible to osmotic disruption and death.

◦Lactoferrin binds to iron which is a growth limiting substance in bacteria. Making iron unavailable to bacteria lactoferrin limits bacterial growth.

◦Lactoperoxidase catalyses the formation of hypothiocyanate ion ,inactivating bacterial enzymes and finally death.

2. Crevicular fluid :◦It is an inflammatory exudate derived from plasma with large amount of antibody and complement proteins.

◦The predominant immunoglobulin is IgG, derived from plasma cells located in periodontal tissues as well as from circulating plasma.

◦These antibodies keep the subgingival flora in check by inhibiting colonization or activating complement system.

Nutrient sources in the oral cavity :

MICROBIOLOGY OF DENTAL PLAQUE According to WHO :Plaque is a highly specific and selective but structurally variable clinical entity characterized by sequential colonization of microorganisms on the surface of teeth, restoration and other parts of the oral cavity. It is made up of ◦Mucins◦Desquamative epithelial cells◦Microorganisms embedded in an extracellular matrix.

MICROFLORA IN DISEASE

Interrelationship that leads to dental disease

DEFENITION

Dental plaque can be defined as the soft deposits that form the biofilm adhering to the tooth surfaces or other hard surfaces in the oral cavity, including removable and fixed prosthesis.

The term Biofilm is used to describe communities of micro-organisms attached to a surface.

Plaque:

CLASSIFICATION OF PLAQUE

Dental Plaque Sub gingival

Tooth associated

Tissue associated

Supra gingival

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• Gram +ve cocci and short rods predominant at tooth surfaces.

• Gram –ve rods, filaments, spirochetes, at outer surfaces.Supra gingival

• Filamentus microorganisms dominate.• Cocci and rods also present .• Gram +ve rods and cocci:• St. mitis, St. sangius• A. Viscosus , naeslundii• Eubacterium.

Sub gingival Tooth associated

Subgingival Tissue associated

St. oralis, St. intermedius Peptostreptcoccus micros P. gingivalis, P. intermedia T. Forsythis, F. Nucleatum

Development of dental plaque:

◦Pellicle formation◦Attachment of single bacterial cell (0-4h)◦Growth of attached bacteria leading to formation of distinct micro colonies.

(4-24h)◦Microbial succession and co-aggregation .(1-14 days)

◦Climax community plaque.(2 weeks or older)

Plaque Formation:

Pellicle formation:◦ Microrganisms don’t colonize on the mineralised tooth surface.

◦ The teeth are always covered by an acellular proteinaceous film ,the pellicle that forms on the naked tooth surface within mins to hours.

◦ The bacteria colonize the tooth surface only when pellicle is in place for hours.

Function of pellicle◦ Protect enamel.◦ Reduces friction.◦ Provide matrix for re-mineralization.

Pellicle contains-lysozyme,albumin,IgA,IgG.

Early colonisation :◦ Plaque builds up first in small defects or pits on the

enamel surface and then spreads over the tooth surface.

Early succession evolves adhesion between pellicle and pioneer organism.S.sanguis, A.viscosus, A.naeslundii and pepto streptoccous attaches within 1 hr.

◦The first organism to attach include Streptococcus sanguis, Streptococci and gram-negative cocci (Neisseria and Branhamella).

◦After 24 hours,

plaque consists largely of Streptococci and Veillonella, Corynebacterium, Actinomyces, Lactobacillus and Rothia.

Veillonella is first anaerobe to appear followed by facultative Actinomycetes and anaerobic Actinomyces israelli.

◦As plaque ages,

number of anaerobes increases and after 7 days Fusobacteria and Bacterioides can be detected.

Late stage of plaque succession

◦Responsible of causing specific disease.◦Early stage lack pathogenicity-aerobic, lack sufficient production of damaging metabolites.

◦Change of aerobic to anaerobic environment.◦Secondary colonizers are the microorganisms that do not initially colonize clean tooth surfaces, including P. intermedia, P. loescheii, Capnocytophaga spp., F.nucleatum, and Porphyromonas gingivalis.

◦These microorganisms adhere to cells of bacteria already in the plaque mass.

◦Extensive laboratory studies have documented the ability of different species and genera of plaque microorganisms to adhere to one another, a process known as coaggregation.

◦Coaggregation is based on the specific interaction of a proteinaceous adhesion procedure by one bacterium and a respective carbohydrate or protein receptor found on the surface of another bacterium.

◦ F. nucleatum with S. sanguis,

◦ P. loescheii with A. viscosus,

Tooth Habitats For Pathogenic Plaque

Tooth surface is stable and covered with pellicle , so it is an ideal surface for the attachment of many oral streptococci .Tooth habitats favorable for harboring pathogenic plaque includes :Pits and fissures- Community is dominated by S.sanguis and other streptococci.

Smooth surface- Proximal areas of very young patient are less favorable habitat for MS.

Root surface Gingival recession favors the formation of plaque in this area.

Sub gingival areas◦Initial occupants of sulcus are merely extension of immediate tooth surface

◦Plaque community changes from masses of cocci to a community dominated by filamentous bacteria and spirochetes in sub gingival area .

◦B. melaninogenicous can explore this habitat, because protein and heme is available.

Differences between early and mature supra gingival plaque

Characteristic Early Mature

Gram reaction + +/-

Morphotypes Cocci, branching rods cocci, rod,

spirochetes Energy metabolism Facultative F- anaerobic

Tolerated by host Well Can cause caries and gingivitis

CHRONIC GINGIVITISGram positive:Actinomyces viscosusActinomyces naeslundiiStreptococcus sanguisStreptococcus mitisPeptosreptococcus microsGram negative:Fusobactecterium nucleatumP. intermediaVeillonella parvulaWolinellaHaemophilus species

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Acute necrotising ulcerative gingivitis

Fusobacterium (fusobacterium nucleatum)

Oral spirochaetes (treponema species)

Fusospirochaetal complex

Others – Provetella intermediaVeillonella spirochaetes 52

CHRONIC PERIODONTITIS:

Caused by

P. gingivalis

P. intermedia

A. actinomycetemcomitans

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P. gingivalis◦ Gram negative ,non motile pleomorphic short rods

◦ Gram negative obligate anaerobe.

◦ P. gingivalis is a member of "black-pigmented Bacteroides" group

◦ Size – 0.5 * 1 microns

◦ Doesn’t ferment carbohydrate.

◦Major site of colonization – gingival sulcus of human oral cavity

◦ Aggressive periodontal pathogen.

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Localized aggressive periodontitis

◦Microbiota is predominantly composed of gram –ve anaerobic, capnophilic rods.

◦Microbiological studies indicate- All disease sites harbor A.comitans which may compose as much as 90 % of total cultivable microbiota.

◦Other organisms found include P. GingivalisC. Rectus F. Nucleatum. 55

A. actinomycetemcomitans This is a small, non-motile, Gram-negative saccharolytic, capnophilic, round-

ended rod that forms small, convex colonies with a "star-shaped" center when grown on blood agar plates.

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Actinomycetemcomitans

◦ May be isolated on non selective blood agar incubated anaerobically.

◦ Its growth is stimulated by addition of carbon dioxide

◦ Ferments glucose and fructose

◦ Site – subgingival sites in oral cavity

◦ Virulence factors – LPS, leukotoxin,

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PRE-PUBERTAL PERIODONTITIS

P. Intermedia

A. Actinomycetemcomitans

Fusobacterium species

P. Gingivalis

JUVENILE PERIODONTITIS

A. actinomycetemcomitans

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REFRACTORY PERIODONTITIS A. actinomycetecomitans

P. gingivalis

P.intermedia

B.forsythus

PAPILLON-LEFEVRE SYNDROME

A. actinomycetemcomitans

P. intermedia

P.gingivalis

F.nucleatum

E.corredens

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PERIODONTAL ABSCESS

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• The micro flora contains mainly gram –ve anaerobic rods

• There is high prevalence of putative pathogens F.nucleatum , P. intermedia , P. gingivalis & T. forsythia.

OSTEOMYELITISCaused by Staphylococcal aureus or enterobacteria

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MICROBIOLOGY OF DENTAL CARIES

◦Dental caries is a specific and treatable bacterial infection due to mutans streptococcus (MS) and in the later stages to  lactobacillus.” (Oral Sci Rev. 1976).

◦“Caries is a dynamic process of demineralization of  the dental hard tissues by the products of bacterial metabolism alternating with periods of remineralization.”Larsen and Bruun (Clinical Cariology, 1994)

◦In 1976 Loesch (Oral Sci Rev, 1976) postulated that dental caries is a specific and treatable bacterial infection due primarily to MS and in the later stages to lactobacillus

◦There is evidence that some bacteria – S. mutans, Lactobacilli and actinomyces- are more important than others.

S. Mutans: ◦The feature that supports its role as cariogenic organisms are its:

◦Rapid generation time ◦Acidogenic nature ◦Production of extra cellular polysaccharides from sucrose which aids on adherence and acts as a nutrient

◦Isolated in high numbers in caries active mouth in incipient lesions

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S. Mutans: ◦ Capacity to attain critical pH for enamel demineralization more rapidly than

other plaque bacteria.

◦ S. mutans can produce glucan by using glucosyl transferase enzyme.It is this glucans which help in the attachment of the bacteria to the tooth.

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Mutans Streptococci

◦Gram positive cocci forming chains◦Non motile, facultative anaerobes.◦Size – 0.5-0.75 microns in diameter◦Usually alpha hemolytic◦Selectively cultured in Mitis salivarius (20% sucrose + 0.2% unit/ml of bacitracin)Culture◦blood agar– grey to white◦sucrose-containing media –produce extracellular polysachharides

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Collection of seven different species

1. S. mutans2. S. sobrinus3. S. cricetus4. S. ferus5. S. rattus6. S. macacae7. S. downei

Primary habitat – human oral cavity to colonize smooth surface of teeth. Doesn’t appear prior to eruption of teeth and disappear after eruption

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Lactobacilli◦Gram negative ,non spore forming rods◦Grow under micro-aerophillic conditions ◦They are both acidogenic and aciduric◦They are secondary invaders◦Lactobacilli for many years was believed to be the causative agent of dental caries as

◦High numbers were obtained in most enamel caries

◦Able to synthesis extra cellular and intra cellular polysaccharides form glucose.

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◦They produces lactic acid at pH < 5 ◦Although these properties seemed valuable to a cariogenic organism, it was also seen that their affinity for the tooth surface and their number prior to development of caries was low.

◦They were, in fact, absent from incipient lesions while present is significant numbers in developed caries.

◦Thus, they were categorized as secondary invaders which caused progression of caries due to their acidogenic and aciduric properties.

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Actinomyces

◦Gram positive non acid fast non motile non spore.

◦Strict anaerobic or facultative anaerobes.

Main species –◦[A. naesulundi + A. viscosus] : facultative anaerobes

◦ [A.israelli +A.odontolyticus] : strict anaerobes

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Actinomysis:

◦Found in increase numbers in plaque overlying root surface and sound root surface.

Actinomyces species: A.viscous, some other Gram +ve bacilli are involved in the initiation of lesions on root surface.

◦Role played by a large number of other bacteria isolated from caries such as Arachnia, Eubacterium, Rothia etc are not yet clearly known.

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Fluoride and Cariogenic bacteria

◦ Antibacterial effects of fluoride are attributable to the weak acid nature of hydrofluoric acid allowing its permeation at low pH into bacterial cells.

◦ The unionised hydrofluoric acid here dissociates due to alkaline intracellular pH, which positions fluoride to affect a variety of vital enzymatic cell functions.

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Fluoride and Cariogenic bacteria◦ The significant ones are inhibition of enolase, potassium and inorganic

phosphate transport.

◦ The mutans streptococci are considerably more sensitive to fluoride inhibition than A. viscosus or Lactobacilli.

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Fungi In The Oral Cavity◦Most commonly found:- candida species (C.albicans, C. tropicalis, C. stellatoidea, C. parapsilosis, C. guilliermondi)

◦Other rhodotorula & torulopsis ( denture wearer)◦Conditions – thrush ,erythematous candidiasis,hyperplastic candiasis,angular chelitis

◦Oral sampling – imprint culture◦Medium used – sabouraand’s agar(peptone-glucose)◦Indentification – psuedohyphae, septate hyphae and germ tubes

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Parasites In The Oral Caity

Entamoeba gingivalis Trichomonas tenax

◦E. gingivalis – ◦found in soft calculus,periodontal pockets and

infection of tonsils◦Can become opportunistic ◦pathogen◦ T.tenax – only parasitic flagellate in◦ oral cavity◦ --number increases in periodontitis

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Role of oral flora in systemic disease◦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 includes:Cardiovascular diseases:

◦Infective endocarditis• Coronary heart disease (atherosclerosis, MI)• stroke

Bacterial pneumoniaDiabetic mellitusAIDS

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Diabetes Mellitus:◦capnocytophaga species◦P. intermedia◦A. actinomycetemcomitans◦P. gingivalisAIDS◦ fusobacterium species◦ A. actinomycetemcomitas◦ P. micros◦ P. intermedia

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Brief Outline Of Oralmicroflora In Disease

INFECTIONS OF THE MOUTH

Infection OrganismDental caries Streptococcus mutansPeriodontal diseases Bacteroides, ActinomycesSurgical infectiona) Dry socketb) Dental abscessc) Osteomyelitisd) Ludwig’s anginae) Pericoronitis

ActinomycesOral streptococciStaphylococcus aureusβ -haemolytic streptococciBacteroides

INFECTIONS OF THE MOUTHInfection Organism

Soft tissue infectionsa) Diphtheriab) ANUGc) Cancrum orisd) Tuberculosise) Leprosy

C. DiphtheriaeFuso-spirochaetesFuso-spirochaetesM. TuberculosisM. Leprae

Viral infectionsa) Herpetic stomatitisb) Herpes Zosterc) Mumpsd) Measles

Herpes simplexVaricella-zosterMumps virusMeasles virus

INFECTIONS OF THE MOUTHInfection Organism

Fungal infectionsa) Candidosisb) Histoplasmosis

Candida albicansH. Capsulatum

Miscellaneousa) Erythema multiformeb) StevensJohnson

syndrome

Conclusion 

There are wide variety of organisms present, each with a distinctive property. This determines the ways in which they

will react with their hosts therefore contribute to the characteristics of the disease they cause.The normal flora

play a very important role in protection against these established pathogenic microbes.

References 

1. Textbook of Microbiology- Anantnarayan2. Carranza’s clinical periodontology. 9th edition3. Textbook of cariology – Ernest Newburn