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SIBAR INSTITUTE OF DENTAL SCIENCES

DEPATMENT OF ORAL AND MAXILLOFACIAL

PATHOLOGY

ORAL FLORA AND ITS VIRULANCE

Dr. Sujatha .R ,

Post Graduate .

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Introduction:

In a healthy animal, the internal tissues, e.g. blood,

brain, muscle, etc., are normally free of

microorganisms.

surface tissues

oral mucous membrane skin

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The mixture of organisms regularly found at any

anatomical site is referred to as the normal flora.

Researchers - "indigenous microbiota".

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Normal flora:

• Normal flora refers to the populations of microorganisms

that inhabit the skin and the mucous membranes of normal

human body.

Resident flora

Indigenous flora

Supplemental flora

Transient flora

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Oral flora comprises a diverse array of organisms and

includes eubacteria, fungi, protozoa and possibly a viral

flora which persist from time to time.

Bacteria – 350 cultivable species

Oral bacteria classification

a. Gram-positive

b. Gram-negative

Oral flora

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Depending upon the effect of oxygen divided as:

Obligate aerobe

Micro aerophilic / Micro aerophilic strict anaerobe

Facultative anearobes

Strict obligate anaerobe

Capnophilic

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Bacteria commonly found on the surfaces of the human body

Bacterium Skin Nose Pharnyx MouthStaphylococcu

s epidermidis ++ ++ ++ ++

Staphylococcu

s aureus + + + +

Streptococcus 

salivarius  ++ ++

Streptococcus

mutans + ++

Enterococcus

faecalis +/- +

Streptococcus

pneumoniae +/- + +

Streptococcus

pyogenes +/- + +

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Bacterium Skin Pharnyx Mouth

Neisseria sp. ++ +

Neisseria

meningitidis ++

+Enterobacteriaceae

(Escherichia coli) +/- + ++

Pseudomonas

aeruginosa +

Haemophilus

influenzae +/- +

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Association between Humans and the Normal flora

Not much known

dynamic interactions

mutualistic

Some normal flora

parasitic

pathogenic

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Tissue specificity:

Most members of the normal bacterial flora prefer to

colonize certain tissues and not others.

This “tissue specificity” is usually due to properties of both

the host and the bacterium.

1. Tissue tropism

essential nutrients

growth factors, suitable oxygen, pH

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Lactobacillus acidophilus, informally known as "Doderlein's bacillus" colonizes the vagina because glycogen is produced which provides the bacteria with a source of sugar that they ferment to lactic acid

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2. Specific adherence

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Bacterium Bacterial adhesion Attachment siteStreptococcus pyogenes

Cell-bound protein (M-protein)

Pharyngeal epithelium

Streptococcus mutans

Cell- bound protein (Glycosyl transferase)

Pellicle of tooth

Streptococcus salivarius

Lipoteichoic acidBuccal epithelium of tongue

Streptococcus pneumoniae

Cell-bound protein (choline-binding protein)

Mucosal epithelium

Staphylococcus aureus

Cell-bound protein Mucosal epithelium

Neisseria gonorrhoeae

N-methylphenyl- alanine pili

Urethral/cervical epithelium

Enterotoxigenic E. coli

Type-1 fimbriae Intestinal epithelium

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3. Biofilm formation:

Some of the indigenous bacteria are able to construct

biofilms on a tissue surface, or they are able to colonize a

biofilm built by another bacterial species. 

Many biofilms are a mixture of microbes, although one

member is responsible for maintaining the biofilm and may

predominate.

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Cartoon depicting biofilm formation.

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The classic biofilm - oral cavity

dental plaque on the teeth.

Plaque is a naturally-constructed biofilm,

thickness of 300-500 cells - teeth.

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These accumulations subject the teeth and gingival

tissues to high concentrations of bacterial metabolites,

which result in dental diseases like caries.

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The Composition of Normal flora

The makeup of the normal flora influenced

genetics

age

sex

stress

nutrition

and diet of the individual.

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Three developmental changes in humans ,weaning,

the eruption of the teeth, and the onset and cessation of

the ovarian functions, invariably affect the composition

of the normal flora in the oral cavity.

The Bacterial Flora of Humans© 2007 Kenneth Todar University of Wisconsin-Madison

Department of Bacteriology 

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Table . Predominant bacteria at various anatomical locations in adults.

Anatomical Location Predominant bacteria

Skin staphylococci and corynebacteria

Conjunctiva sparse, Gram-positive cocci and Gram-negative rods

Oral cavity

            Teeth Streptococci, lactobacilli

            mucous membranes Streptococci and lactic acid bacteria

Upper respiratory tract

            nares (nasal membranes) staphylococci and corynebacteria

            pharynx (throat) streptococci, neisseria, Gram-negative rods and cocci

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Normal Flora of the oral Cavity:

                                                                               

Various streptococci in a biofilm in the oral cavity.

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Flora of the oral cavity:

GRAM POSITIVE COCCI:

Genus streptococcus:

Streptococcus mutans. Gram stain

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Main species Strep mutans, strep sorbinus, strep cricetus

Cultural characteristics Mitis Salivarius Agar (MSA)

Intra oral sites

and

infections

Teeth

Dental caries

The mutans group:

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Main species Strep salivarius,Strep vestibularis

Cultural characteristics Mitis Salivarius Agar (MSA)

Intra oral sites

and

infections

dorsum of the tongue saliva.

does not cause major oral pathogenesis

The salivarius group:

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Main species Strep constellatus, Strep intermedius, Strep anginosus

Cultural characteristics Mitis Salivarius Agar (MSA).

Intra oral sites

and

infections

Gingival crevice

Dentoalveolar and endodontic infections.

The anginosus group:

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Main species Strep mitis;Strep sanguisStrep gordonii Strep oralis

Cultural characteristics Mitis Salivarius Agar (MSA).

Intra oral sites

and

infections

dental plaque biofilms , tongue and cheek.

dental caries.

The mitis group

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Main species Micromonas micros Finegoldia magnus

Cultural characteristics

Intra oral sites

and

infections

teeth, especially the carious dentine.

periodontal and dentoalveolar abscesses

Anaerobic streptococci

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Main species Stomatococcus

Cultural characteristics facultative anaerobes

Intra oral sites

and

infections

tongue and the gingival crevice.

major opportunistic pathogen

Genus stomatococcus

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Genus stomatococcus

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Main species Staph aureus, staph epidermidis, Staph saprophyticus

Cultural characteristics blood agar

Intra oral sites

and

infections

Present on the buccal mucosa

Angular chelitis

Genus Staphylococcus

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Genus Staphylococcus

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GRAM POSITIVE RODS AND FILAMENTS:

These organisms are very commonly isolated

from the biofilms of dental plaque.

This group consists of the following organisms –

Actinomycetes

Lactobacilli

Eubacteria

Propionibacteria.

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Main species Actino israeliActino gerensceriaeActino odontolyticusActino naeslundii

Cultural characteristics facultative anaerobes.

Intra oral sites

and

infections

Genus actinomycetes

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Actinomycetes

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Main intraoral sites and infections:

They are seen on the gingiva, on the mucosal and also

on the teeth surfaces.

Actinomyces odontolyticus is related to the earliest

stages of enamel demineralization and the progression of

small caries lesions.

Actinomyces naeslundii has been related to root

surface caries and gingivitis.

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Actinomyces israeli is an opportunistic pathogen

causing cervicofacial and ileocecal actinomycosis.

Actinomyces gerensceriae and Actinomyces georgiae

are considered to be the minor components of healthy

gingival flora.

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Genus Lactobacillus -

Main species Lacto caseiLacto fermentumLacto acidophilusLacto salivariusLacto rhamnosus.

Cultural characteristics Rogosa agar.

Intra oral sites

and

infections

dental plaque biofilm, advancing front of dental caries

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Lactobacillus

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Main species Eubact brachyEubact nodatumEubact saphenumEubact yurii.

Cultural characteristics Obligate anaerobes

Intra oral sites

and

infections

Dental plaque biofilm, calculus

Periodontal disease

Eubacterium yurii is involved in the “corn-cob” formation in the dental plaque

Genus Eubacterium

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Rod shaped eubacterium

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Main species Propionibacterium acnes

Cultural characteristicsStrict anaerobes

Intra oral sites

and

infections

gingival pockets and plaque biofilms

root surface caries

Genus propionibacterium

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OTHER NOTABLE GRAM-POSITIVE ORGANISMS:

• Rothia dentocariosa - Gram-positive branching filament - strict aerobe

Found in plaque & isolated from patients with infective endocarditis.

• Bifidobacterium dentium - Gram-positive, strict anaerobe,

Regularly isolated from the biofilms of plaque, and its role in disease is unclear.

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GRAM-NEGATIVE COCCI

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Main species Neisseria subflavaNeisseria mucosaNeisseria sicca

Cultural characteristics facultative anaerobes

Intra oral sites

and

infections

tongue, saliva, oral mucosa and early plaque.

rarely associated with any oral disease.

Genus Nisseria

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Neisseria

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Main species Veillonella parvulaVeillonella disparVeillonella atypical

Cultural characteristics Rogosa vancomycin agar

Intra oral sites

and

infections

tongue, saliva, plaque biofilm. not associated with any oral disease.

Genus Veillonella

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GRAM-NEGATIVE RODS – FACULTATIVE ANAEROBIC AND CAPNOPHILIC GENERA

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Main species HaemophilusparainfluenzaeHaemophilus segnisHaemophilus aphrophilusHaemophilus haemolyticus

Cultural characteristics Facultative anaerobes

Intra oral sites

and

infections

tongue, saliva, plaque biofilm.

dentoalveolar infections, acute sialadenitis, and infective endocarditis

Genus Haemophilus

Gram-negative coccobacilli

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Main species Actinobacillus actinomycetemcomitans

Cultural characteristics

Intra oral sites

and

infections

Genus Actinobacillus

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Culture characteristics:

The freshly isolated strains contain fimbriae that are lost on subculture.

Actinobacillus produces many virulence factors like –

leukotoxin; epitheliotoxin; collagenase; protease that

cleaves immunoglobulin G (IgG).

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microscopic picture of Actinobacillus

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Main intraoral sites and infections: Mostly seen in the periodontal pockets.

They are implicated in aggressive forms of periodontal

disease (e.g. localized and generalized forms of aggressive

periodontitis)

They are often isolated as co-pathogens from the

cervicofacial Actinomyces infections.

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Main species Eikenella corrodens

Cultural characteristics blood agar

Intra oral sites

and

infections

plaque biofilms

chronic periodontitisdentoalveolar abscesses

Genus Eikenella

Gram negative coccobacilli

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Main species Capnocytophaga gingivalisC. SputigenaC. OchraceaC. GranuloseC. Haemolytica

Cultural characteristics Capnophilic organisms

Intra oral sites

and

infections

Plaque, mucosal surfaces, saliva.

Periodontal disease

Genus Capnocytophaga

Gram-negative fusiform rods

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GRAM-NEGATIVE RODS – OBLIGATE ANAEROBIC GENERA:

Form large portion of the plaque biofilms

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Main species Porphyromonas gingivalisP. EndodontalisP. Catoniae

Cultural characteristics Strict anaerobes

Intra oral sites

and

infections

Gingival crevice and the subgingival plaque

chronic periodontitis and dentoalveolar abscess.

Genus Porphyromonas

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Porphyromonas

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Main species Fusobacterium nucleatumF. AlocisF. SulciF. periodonticum.

Cultural characteristics Strict anaerobes

Intra oral sites

and

infections

Normal gingival crevice, tonsils

acute ulcerative gingivitis, halitosis

Genus Fusobacterium

Gram-negative rods

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SEM picture of Fusobacterium

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Main species Treponema denticolaTreponema macrodentiumTreponema skoliodontiumTreponema sokranskiiTreponema maltophilum

Cultural characteristics Strict anaerobes

Intra oral sites

and

infections

Gingival crevice

acute ulcerative gingivitis, destructive periodontal disease

Genus Treponema

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ORAL PROTOZOA:

Genus Entamoeba

Main species Entamoeba gingivalis

Cultural characteristics Strict anaerobes

Intra oral sites

and

infections

Periodontal disease

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Main species Trichomonas tenax

Cultural characteristics Strict anaerobes

Intra oral sites

and

infections

Gingival crevice

Unclear

Genus Trichomonas

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Beneficial effects of the normal flora:

1.Can synthesize and excrete vitamins

2.Prevent colonization by pathogens

3.May antagonize other flora

4.Stimulate the production of natural antibodies

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OBLIGATORY PARASITES WHICH CAN BE

COMMENSALS IN THE ORAL CAVITY

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Main species Mycoplasma pneumoniaeMycoplasma buccaleMycoplasma oralMycoplasma salivarium; Mycoplasma faucium

Cultural characteristics Special agar and broth media

Intra oral sites

and

infections

oro-pharyngeal region, saliva, dental plaque

Mucocutaneous lesions

MYCOPLASMAS

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FUNGI

Yeasts

Main species Cryptococcus Rhodotorula

Intra oral sites

and

infections

Mucosa

Mucosal ulcers in immunocompramised patients

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Main species Candida albicansCandida tropicalisCandida kruseiCandida glabrataCandida guillermondiiCandida parapsilosisCandida kefyr

Intra oral sites

and

infections

Mucosa

Mucosal ulcers in immunocompramised patients

Yeast-like fungi

genus Candida

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Virulence:

The degree of pathogenicity of a microorganism as indicated

by the severity of disease produced and the ability to invade

the tissue of the host ; by extension, the competence of any

infectious agent to produce pathologic effects.

( Dorland’s Medical Dictionary)

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The main etiological agent of periodontal disease is microflora

– dental plaque

Dental plaque:

It is defined as a structured, resilient, yellow-grayish substance

that adheres to the intraoral hard surfaces, including removable

and fixed restorations.

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Composition

Bacteria (matrix of salivary glycoproteins & extra

cellular poysaccharides)

1 gm = 10 11 bacteria

> 500 species

30% uncultivable species

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Dental plaque

Supra gingival plaque:

Demonstrates a multi-layered accumulation of

bacterial morphotypes

gram positive cocci + rods - tooth surface

gram negative rods, filaments - outer surface

Sub-gingival bacteria

anerobic bacteria

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Plaque formation of at the ultra-structural level

Saliva derived layer – acquired pellicle

- glycoproteins, proline-rich proteins

Streptococcus & Actinomycetes bind to specific

salivary proteins

Veillonellae, Capnocytophagae, Prevotella – bind to

Streptococci, Actinomycetes

Primary colonizers

Secondary colonizers

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Microorganisms associated with periodontal disease:

Bacteria associated –

gram +veStreptococcus( s.sanguis, s.mitis)Actinomycetes (A.viscosus, A.naeslendi)

gram –ve capnocytophageneisseria

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The currently recognized key Gram negative

periodontopathogens include:

Porphyromonas gingivalis (P.g),

Prevotella intermedia (P.i),

Bacteroides forsythus (B.f),

Aggregatibacter actinomycetemcomitans (A.a),

Fusobacteriumnucleatum (F.n),

Capnocytophaga species(C.sp),

Campylobacter rectus (C.r)

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Porphyromonas gingivalis

previously known as Bacteroides

strictly anaerobic, Gram negative

Virulence mechanism:

carbohydrate capsule – prevents opsonization

Virulence factors:

fimbriae - adhesion

proteases – degrade collagen fibers, cytotoxins

hemolysin

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Actinobacillus actinomycetemcomitans

Gram –ve

Virulence factors:

leukotoxin

forms pores – death – osmotic pressure

collagenase

destruction of connective tissue

lipopolysaccharides

macrophages – IL 1, IL2 – bone resorption

Microbial etiology of periodontal disease – a mini review

Medicine and Biology Vol.15, No 1, 2008 - Ljiljana Kesic

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Fusobacterium nucleatum

Virulence factors:

induce – apoptotic cell death – PMN

release – cytokines

As fusobacterium co-aggregates with most oral

microorganisms – binding organisms

Microbial etiology of periodontal disease – a mini review

Medicine and Biology Vol.15, No 1, 2008 - Ljiljana Kesic

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Capnocytophaga sp

Gram –ve

Vrulence:

lipopolysaccharides – activity on alveolar bone

proteolytic enzymes

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Spirochetes:

Motile, flagella

Treponema denticola

Treponema vincentii

• a lipopolysaccharide, and unusual metabolic endproducts,

like indole, hydrogen sulphide, ammonia, which are

potentially toxic to host cells.

• T.d – proteolytic enzyme – destroy (IgA, IgM, Ig G)

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Dental caries

Multifactorial

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Virulence factors:

Specific adherence to tooth surface using antigen I/II adhesin and GTF (glycosyl transferase )

Production of extracellular polysaccharides (dextran) allows the cariogenic bacteria to stick onto the teeth and form a biofilm.

acid-tolerance (aciduricity)

Able to maintain microbial growth and continue acid production at low pH values.

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Rapid metabolism of sugars to lactic and other

organic acids

Lower the pH to below 5.5, the critical pH. Drives

the dissolution of calcium phosphate (hydroxyapatite)

of the tooth enamel (acidogenicity)

Accumulation of intracellular polysaccharides

(carbon/energy reserve)

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Virulence properties

streptococus mutans

Adhesionsucrose – independent adhesion

sucrose – dependent adhesion

Acidogenicity

Acid-tolerance

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Adhesion

Sucrose – independent adhesion

• influenced by antigen I/II

• proteins of this family share structural similarity

• role of antigen I/II

adhesion of s. mutans to saliva – coated hydroxyapetite

Ohat et al : characterization of a cell – surface protein antigen of hydrophilic streptococcus mutans strain. J Gen Microbiology 135, 981-988

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Gram stain - streptococcus

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Interaction between antigen I/II & salivary components

alanine- rich & proline-rich domains

Proved - mutant lacking P1 ( antigen I/II)

- did not bind

Bowen et al : Role of a cell surface-associated protein in adherence and dental caries . Infect immunology 59, 4606-4609

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Sucrose-Dependent Adhesion

• The action of glucosyltransferases (GTFs) in the synthesis

of glucans is the major mechanism behind sucrose -

dependent adhesion.

• GTFs – sucrase activity

• sucrose glucose + fructose

added

growing polymer of glucan

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s.mutans - 3 GTFs -

encoded by gftb, gftc, gftd

So , GTFs – synthesize

Water- soluble glucan water- insoluble glucan

(dextran) (mutan)

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• The ability of glucan to facilitate adhesion of S.mutans

may be due to hydrogen bonding of the glucan polymers to

both the salivary pellicle and the bacteria.

• This glucan could be synthesized by extracellular GTFs

that bound the salivary pellicle, S. mutans that had

previously adhered via sucrose-independent means, or

perhaps by other oral streptococci.

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• It is not known why S. mutans requires multiple GTFs,

but there is evidence that the different GTFs have

differing affinities for the bacterial surface or salivary

pellicle , and that a particular ratio of each is necessary

for optimal sucrose-dependent adhesion.

Ooshima et al: contbutions of three glucosyltransferases

to sucrose-dependent adherence of streptococcus

mutans. J dental res :80 , 1672-1677

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• Another possibility - WapA ( antigen A)

its yet uncertain

• Carbohydrate Metabolism:

additional putative factorsGene product Hypothesized function

Ftf Catalize the synthesize of fructans

FruA Breakdown fructans for energy

DexA Glucan synthesis

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Acidogenicity:

S.mutans

lactate, formate, acetate, ethanol

( fermentation end-products)

Glucose

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•The velocity with which S. mutans produces acid when

tested at a pH in the range from 7.0 to 5.0 exceeds that of

other oral streptococci in most instances.

Soet, J.J., B. Nyvad, & M. Kilian: Strain-relatedacid production by oral streptococci. Caries Res 34, 486-490, (2000)

• The acidogenicity of s.mutans---- ecological changes

in the plaque flora that includes an increase in proportion of s.mutans

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Acid – tolerance

• Growth-inhibitory pH

• distinguish s.mutans

• this is largely mediated by

- F1F0 – ATPase ATR

- gene and protein expression

• acid – tolerance may be aided by the synthesis of

water-insoluble glucan and the formation of biofilm

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• s.mutans with in biofilm – outside

ATR , physical characteristics of the biofilm

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The role of lactobaccillli:

Absent – incipient lesions

Pioneer organisms in the advancing front of carious

process

Virulence :

synthesize sucrose

their ability to grow low- pH lactic acid

The exact role – not well defined

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The results of this study suggest that Lactobacilli colonizes sites in the oral cavity (including the tongue and saliva) other than the tooth surface in rats.

The effect of Lactobacilli in plaque toward reduction of S.mutans-induced dental caries in rats

Oral Ecology and Virulence of Lactobacillus casei and Streptococcus mutans in Gnotobiotic Rats : SUZANNE M. MICHALEK el alDepartment ofMicrobiology and Institute of Dental Research, The University ofAlabama in Birmingham, Birmingham, Alabama 3529

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Candidiasis:

The physiological state host – etiology

Virulence factors:

hyphal formation,

surface recognition molecules,

extracellular hydrolytic enzyme production

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Hydrolytic enzymes:

production – proteinases

CATALIZE

hydrolysis of peptide bonds in proteins

secreated aspartyl proteinase (SAP genes)

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Correlation between Sap Production In Vitro and Candida Virulence

Main focus points.

(i) The virulence of C. albicans species appears to correlate

with the level of Sap activity in vitro and may correlate with

the number of SAP genes.

(ii) Infected patients (oral or vaginal) harbor C. albicans

strains that are significantly more proteolytic than are

isolates from asymptomatic carriers.

.

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(iii) HIV infection appears to lead to the selection of C.

albicans strains with heightened virulence attributes such

as proteinase production

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Association of Sap Production with Other Virulence

Processes of C. albicans

Main focus points.

(i) Sap proteins facilitate C. albicans adherence to

many host tissues and cell types.

(ii) Hypha formation and SAP4 to SAP6 expression are

coordinately regulated, but the signaling pathways

remain to be elucidated.

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(iii) SAP1 appears to be regulated by phenotypic

switching, but the contribution of switching to C. albicans

virulence in vivo is not yet clear.

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How do Sap proteins contribute to adherence?

Not clear

Two hypothesis

i. C. albicans proteinases could act as ligands to

surface moieties on host cells, which does not necessarily

require activity of the enzymes.

ii. C. albicans utilizes Sap proteins as active

enzymes to modify target proteins or ligands on the fungal

surface or on host cells (i.e., epithelial cells), which may

alter surface hydrophobicity or lead to conformational

changes, thus allowing better adhesion of the fungus

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Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis Julian R. Naglik et al; Microbiology and Molecular biology

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