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1
SIBAR INSTITUTE OF DENTAL SCIENCES
DEPATMENT OF ORAL AND MAXILLOFACIAL
PATHOLOGY
ORAL FLORA AND ITS VIRULANCE
Dr. Sujatha .R ,
Post Graduate .
2
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
3
The mixture of organisms regularly found at any
anatomical site is referred to as the normal flora.
Researchers - "indigenous microbiota".
4
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
5
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
6
Depending upon the effect of oxygen divided as:
Obligate aerobe
Micro aerophilic / Micro aerophilic strict anaerobe
Facultative anearobes
Strict obligate anaerobe
Capnophilic
7
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 +/- + +
8
Bacterium Skin Pharnyx Mouth
Neisseria sp. ++ +
Neisseria
meningitidis ++
+Enterobacteriaceae
(Escherichia coli) +/- + ++
Pseudomonas
aeruginosa +
Haemophilus
influenzae +/- +
9
Association between Humans and the Normal flora
Not much known
dynamic interactions
mutualistic
Some normal flora
parasitic
pathogenic
10
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
11
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
12
2. Specific adherence
13
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
14
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.
15
Cartoon depicting biofilm formation.
16
The classic biofilm - oral cavity
dental plaque on the teeth.
Plaque is a naturally-constructed biofilm,
thickness of 300-500 cells - teeth.
17
These accumulations subject the teeth and gingival
tissues to high concentrations of bacterial metabolites,
which result in dental diseases like caries.
18
The Composition of Normal flora
The makeup of the normal flora influenced
genetics
age
sex
stress
nutrition
and diet of the individual.
19
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
20
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
21
Normal Flora of the oral Cavity:
Various streptococci in a biofilm in the oral cavity.
22
Flora of the oral cavity:
GRAM POSITIVE COCCI:
Genus streptococcus:
Streptococcus mutans. Gram stain
23
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:
24
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:
25
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:
26
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
27
Main species Micromonas micros Finegoldia magnus
Cultural characteristics
Intra oral sites
and
infections
teeth, especially the carious dentine.
periodontal and dentoalveolar abscesses
Anaerobic streptococci
28
Main species Stomatococcus
Cultural characteristics facultative anaerobes
Intra oral sites
and
infections
tongue and the gingival crevice.
major opportunistic pathogen
Genus stomatococcus
29
Genus stomatococcus
30
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
31
Genus Staphylococcus
32
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.
33
Main species Actino israeliActino gerensceriaeActino odontolyticusActino naeslundii
Cultural characteristics facultative anaerobes.
Intra oral sites
and
infections
Genus actinomycetes
34
Actinomycetes
35
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.
36
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.
37
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
38
Lactobacillus
39
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
40
Rod shaped eubacterium
41
Main species Propionibacterium acnes
Cultural characteristicsStrict anaerobes
Intra oral sites
and
infections
gingival pockets and plaque biofilms
root surface caries
Genus propionibacterium
42
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.
43
GRAM-NEGATIVE COCCI
44
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
45
Neisseria
46
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
47
GRAM-NEGATIVE RODS – FACULTATIVE ANAEROBIC AND CAPNOPHILIC GENERA
48
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
49
Main species Actinobacillus actinomycetemcomitans
Cultural characteristics
Intra oral sites
and
infections
Genus Actinobacillus
50
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).
51
microscopic picture of Actinobacillus
52
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.
53
Main species Eikenella corrodens
Cultural characteristics blood agar
Intra oral sites
and
infections
plaque biofilms
chronic periodontitisdentoalveolar abscesses
Genus Eikenella
Gram negative coccobacilli
54
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
55
GRAM-NEGATIVE RODS – OBLIGATE ANAEROBIC GENERA:
Form large portion of the plaque biofilms
56
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
57
Porphyromonas
58
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
59
SEM picture of Fusobacterium
60
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
61
ORAL PROTOZOA:
Genus Entamoeba
Main species Entamoeba gingivalis
Cultural characteristics Strict anaerobes
Intra oral sites
and
infections
Periodontal disease
62
Main species Trichomonas tenax
Cultural characteristics Strict anaerobes
Intra oral sites
and
infections
Gingival crevice
Unclear
Genus Trichomonas
63
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
64
OBLIGATORY PARASITES WHICH CAN BE
COMMENSALS IN THE ORAL CAVITY
65
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
66
FUNGI
Yeasts
Main species Cryptococcus Rhodotorula
Intra oral sites
and
infections
Mucosa
Mucosal ulcers in immunocompramised patients
67
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
68
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)
69
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.
70
Composition
Bacteria (matrix of salivary glycoproteins & extra
cellular poysaccharides)
1 gm = 10 11 bacteria
> 500 species
30% uncultivable species
71
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
72
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
73
Microorganisms associated with periodontal disease:
Bacteria associated –
gram +veStreptococcus( s.sanguis, s.mitis)Actinomycetes (A.viscosus, A.naeslendi)
gram –ve capnocytophageneisseria
74
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)
75
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
76
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
77
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
78
Capnocytophaga sp
Gram –ve
Vrulence:
lipopolysaccharides – activity on alveolar bone
proteolytic enzymes
79
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)
80
Dental caries
Multifactorial
81
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.
82
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)
83
Virulence properties
streptococus mutans
Adhesionsucrose – independent adhesion
sucrose – dependent adhesion
Acidogenicity
Acid-tolerance
84
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
85
Gram stain - streptococcus
86
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
87
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
88
s.mutans - 3 GTFs -
encoded by gftb, gftc, gftd
So , GTFs – synthesize
Water- soluble glucan water- insoluble glucan
(dextran) (mutan)
89
• 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.
90
• 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
91
• 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
92
Acidogenicity:
S.mutans
lactate, formate, acetate, ethanol
( fermentation end-products)
Glucose
93
•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
94
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
95
• s.mutans with in biofilm – outside
ATR , physical characteristics of the biofilm
96
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
97
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
98
Candidiasis:
The physiological state host – etiology
Virulence factors:
hyphal formation,
surface recognition molecules,
extracellular hydrolytic enzyme production
99
Hydrolytic enzymes:
production – proteinases
CATALIZE
hydrolysis of peptide bonds in proteins
secreated aspartyl proteinase (SAP genes)
100
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.
.
101
(iii) HIV infection appears to lead to the selection of C.
albicans strains with heightened virulence attributes such
as proteinase production
102
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.
103
(iii) SAP1 appears to be regulated by phenotypic
switching, but the contribution of switching to C. albicans
virulence in vivo is not yet clear.
104
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
105
Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis Julian R. Naglik et al; Microbiology and Molecular biology
106
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