1. Oral Microbiology II: Oral Micro flora and Plaque
Oral flora
Ecologically diverse
350 different cultivable species.
100 million organisms per millilitre of saliva
Unique habitats: teeth, mucosal surfaces and gingival crevices
Resident flora including anerobes
Any disturbance in oral environment leads to instability of the flora/changes in the flora leading to disease.

2. Study of the oral micro flora
Sample collection
Aspirates, Paper point samples, Oral rinses
Samples can easily be contaminated
Transportation to the lab
Different transport media used
Most bacteria will not survive long outside normal habitat-must be as fast as possible.
Visualisation of sample
Gram staining (general classification), dark field microscopy (spirochetes), fluorescence microscopy.
Help in identification of organisms
Sample Processing
Disaggregation and dilution.
Semi-selective growth media-Sabouraud's agar (Candida), mitis salivaris bacitracin MSB (Strep. Mutans)
Anaerobic media.
Requirement for special growth media and growth factors and atmospheric conditions.
Identification of organism
Enzyme profiles, biochemical profiling, serological profiling, DNA probes and PCR
Time consuming and expensive especially if wide a variety

3. Composition of the Oral Flora: Gram Positive
Streptococcus spp.
Cocci.
Facultative anerobes/obligate anaerobes
50% of cultivable flora from saliva and tongue. 30% of cultivable flora from gingival crevices
Anaerobic spp associated with purulent infections.
Staphylococcus spp
Cocci
Facultative anerobes
Coagulase negative part of normal flora
E.g. Staphylococcus epidermidis
Actinomyces spp
Bacilli
Facultative anaerobe
Mainly in plaque especially approximal sites. Numbers increase in gingivitis and root surface caries
E.g. A. neuslundii,

4. Composition of the Oral Flora: Gram Positive
Lactobacillus spp.
Bacilli
Facultative anaerobe
Small percentage of the flora. Numbers increase in carious lesions
E.g. L. acidophilus
Eubacterium spp
Obligate anerobes
Found n periodontitis and dental abscesses
E.g. E. brachy

5. Composition of the Oral Flora: Gram Negative
Nesseria spp
Cocci, Anaerobic
Early colonisers of teeth. Isolated in low numbers from most sits in oral cavity. N.sublava
Veillonella spp
Cocci, Obligate anaerobe. High numbers on tongue and plaque. E.g. V. parvula
Haemophillus spp
Bacilli, Facultative anaerobe. In saliva, plaque and mucosal surfaces. E.g. H. aphrophilus
Eikenella spp
Bacilli. Facultative anerobe. Mainly in sub gingival plaque. Increase in gingivitis. E.g. E. corrodens

6. Composition of the Oral Flora: Gram Negative
Capnocytophaga spp.
Bacilli, capnophilic(strictly anaerobic=can’t survive even with very few oxygen). Isolated in periodontal disease. E.g. C. gingivalis
Actinobacilus spp.
Bacilli. Capnophillic. In periodontal pockets and implicated in juvenile periodontitis. E.g. A. actinomycetecomitans
Porphyromonas spp.
Bacilli. Obligate anaerobe. In sub gingival plaque. Implicated in adult periodontitis. E.g. P. gingivalis
Prevotella spp.
Bacilli. Obligate anerobe. In sub gingival plaque. Implicated in adulthood periodontitis. E.g. P. intermedia
Fusobacterium spp
Bacilli. Obligate anerobe. In sub gingival plaque. Implicated in adult periodontitis. E.g. F. nucleatum
Spirochaetes spp
Spiral. Obligate anerobe. In periodontal Pockets. Difficult to stain and culture. E.g. Treponema denticola

7. Composition of the Oral Flora: Classification of oral streptococci.
Mutans
Alpha-haemolytic
S. mutans
S. sabrinus
S. cricetus
Fissure caries
Rare
Salivarus
S. salivarius
S. vestibularis
Keratinised surfaces
Vestibular mucosa
Oralis
S. oralis
S.sanguis
S. mitis
S. Gordonii
S. Parasanguis
Produce IgA protease and glucans. Found in dental plaque. Opportunistic pathogens in infective endocarditis.
Aginosus
Beta-haemolytic
S. anginosus
S. Intermedius
S. constellatus
dental plaque. Common and important cause purulent disease e.g. dental and brain abscesses.

8. Dental Plaque formation
Adherence to tooth surface is essential for plaque formation.
Pellicle layer
Complex of salivary glycoprotein's, minerals and immunoglobulin.
is essential for attachment to enamel surface.
Stages:
I: Transport to site
II: Initial adhesion: weak electrostatic forces
III: Attachment:
Covalent, ionic or electrostatic bonds to specific receptors on the host surface (ligands) and bacterial adhesins.
iv. Colonisation and biofilm formation.

9. Factors affecting growth of micro flora
Diet: Chemical composition, physical consistency and frequency
Saliva: flow rate, pH and antimicrobial factors e.g. lysozyme
Gingival crevicular fluid: antimicrobial. IgG
Microbial interactions.
Gaseous environment
Host factors: systemic disease, antibiotic us and oral hygiene practices.

10. Defence mechanisms of the mouth.
Integrity of the oral mucosa
Oral Lymphoid tissues
Extra and intra oral.
Saliva
Mechanical cleansing
Chemical: Lysozyme, peroxdase, lactoferrins
Leucocytes
Secretory IgA
Gingival crevicular fluid.
Immunoglobulin, complement, enzymes, electrolytes, wbcs

11. Oral Microbiology III.

12. Diagnostic Oral Microbiology-General Principles
Microbiological specimens
Accuracy of report is only as good as the quality of the specimen
Specimen types
For Culture:
Fluid (Blood, pus, tissue)
Collected in sterile container
Need rapid transportation to lab.
Detection of Microbial products:
E.g. cell wall antigens, toxins, genetic sequences
Viability of organisms not essential
Rapid
Antibody detection (Serology):
Serum, CSF, saliva
Used for non cultivable organisms or hazardous organisms
Detect increase in IgG or IgM antibody titre during course of disease.
IgM response diagnostic for most diseases.

13. Diagnostic Oral Microbiology-General Principles
Sampling and normal flora
Specimens collect from sterile and non sterile sites.
Normal flora may contaminate specimens
Surface isolation, cleaning or disinfection may be required before sample collection.

14. Diagnostic Oral Microbiology-General Principles
Transportation of Microbiological specimens
All specimens transported as rapidly as possible to ensure viability of organisms on arrival in lab.
Avoid growth of bacteria/fungi that may mask fastidious causative agent
Ensure the survival of delicate organisms that may not survive outside oral environment
Fluids and tissue specimens: Sterile container without fixative
Swabs:
Use media that preserves viability of the of organisms but do not allow growth
Stuart or Amies transport media which contain charcoal to adsorb toxic agents.
Swabs for viral culture transported in antibiotic media to reduce bacterial contamination.

15. Diagnostic Oral Microbiology-General Principles
Laboratory processing of specimens
Non culture methods:
Microscopy:
Bright field microscopy: Wet preparation or stained specimens
X1000 magnification using oil immersion
Dark ground microscopy: Rapid diagnosis of Spirochaetes. Organisms illuminated against a dark background.
Fluorescence microscopy: Fluorescent antibodies tagged with a fluorescent dies are used.

16. Diagnostic Oral Microbiology-General Principles
Detection of Microbial antigens
More rapid than cultural tests
Use of monoclonal or polyclonal antibodies raised against a particular antigen
Antibodies coated onto RBCs, latex particles (agglutination tests)
Antibodies labelled with radioisotopes, enzyme or fluorescent marker.
Assays for toxins using antigenic or bioassays

17. Diagnostic Oral Microbiology-General Principles
Culture Methods:
Bacterial and fungi: Solid and liquid media
Viruses: Tissue culture cells.

18. Diagnostic Oral Microbiology-Diagnosis of purulent infections
Aspirates preferred to swabs.
Gram stain performed on arrival of aspirate at lab.
Inoculation of specimen in sterile broth and blood agar plates for aerobic and anaerobic incubation.
Antibiotic sensitivity testing.

19. Diagnostic Oral Microbiology-Diagnosis of Oral mucosal infections
Fungal Infections
Swab or oral rinse
Sabouraud’s agar used for the general isolation of yeasts
CHROMagar used to isolate separate species
Germ tube tests to identify C. albicans and C. dubliniensis
Antifungal sensitivity testing

20. Diagnostic Oral Microbiology-Diagnosis of Oral mucosal infections
Viral infections
HSV commonest infection
Tissue culture cells
Serological tests of limited value:
Too slow for diagnosis of primary infection
No serological markers of reactivation of disease. due to Swab or oral rinse

21. Diagnostic Oral Microbiology-Diagnosis of Caries and periodontal disease.
Saliva Lactobacillus and S.mutans counts
Rogosa and MSB agar
Poor predictors of future caries activity if taken n isolation
Lactobacillus counts are a useful marker patient compliance with low carbohydrate diet
Periodontal disease.
Laboratory diagnosis more complex because of multiple organisms involved
Smears for spirochetes in cases of ANUG useful
Screening of plaque for putative organisms
e.g. Porphorymonas gingival is, Prevotella, Bacteroides forsythus, Actinbacillus actinomycetemconitans.
Anaerobic culture, antigen detection test and gene probing.

22. Diagnostic Oral Microbiology-Diagnosis of viral infections.
Tissue culture cells
Serological tests of limited value:
Too slow for diagnosis of primary infection
No serological markers of reactivation of disease. due to Swab or oral rinse

23. Oral microbiology IV

24. Infection control in dentistry Bacterial infections of the oral Mucosa
Group Assignments: To be handed in on 23/04/2010 at 1200 noon. Late assignments will not be accepted or marked.
In groups of three write prepare a 500 word essay on the microbiology of:
Hepatitis virus
Retroviruses
Infection control in dentistry
Bacterial infections of the oral Mucosa
Viral infections of the oral mucosa
Salivary gland infections
Oral fungal infections.
Infections of the pulp, dento alveolar abscesses and osteomyelitis.
Cellulitis and Ludwig’s angina

25. Microbiology of Dental caries
Plaque associated bacteria.
Results from ecological shift of bacterial composition of plaque:
Increase in lactobacillus species, streptococcus mutans and Actinomyces spp
Lower plaque pH due associated with increased intake of fermentable sugars and normal to reduced salivary flow and factors
Tooth surface demineralisation

26. Microbiology of Dental caries
Mutans streptococci:
S. mutans, S. sobrinus, S. cricetus, S.ferus, S. rattus, S. macae, S.downei
8 serotypes (a-h). S. mutans (c/e/f) and S. sobrinus (d/g) commonest isolates found in humans.
Factors related to carcinogenicity of mutans streptococci:
Correlation between counts in saliva and plaque with caries prevalence, incidence and progression
Isolated from carious lesions
Production of water soluble and insoluble polysaccharide from sucrose which promote colonisation of tooth surfaces.
Out compete other bacteria in sugar uptake at lower pH
Growth in low pH.
Acidogenic, aciduric, and an sustain low pH needed to cause enamel demineralisation
Immunisation in animals with S. mutans decreases incidence of caries.

27. Microbiology of Dental caries
Lactobacillus species:
Two groups
Homofermentative lactic acid producing species (L. acidophilus, L. casei)
Heterofermentative species which produce both lactic and acetic acid (L. fermentum)
Factors related to carcinogenicity of lactobacilli.
Present increased numbers in most carious lesions of enamel and root surfaces.
Associated with extension of caries into dentine rather than initiation.
Correlation between counts in saliva and with caries prevalence, incidence and progression
Production caries in gnotobiotic rats
Aciduric and acidogenic
Some strains synthesise extracelluar polysaccharide.

28. Microbiology of Dental caries
Actinomyces species:
Form micro flora of approximal sits and gingival crevice
Associated with the formation root surface and enamel caries.
Factors related to carcinogenicity of Actinomyces spp.
Present increased numbers in most carious lesions of root surfaces.
Production root caries in gnotobiotic rats
Appear later in the progression of demineralisation.

29. Role of micro organisms in periodontal disease:
Associated with progressively diverse plaque micro flora.
Associated with an increase in plaque mass
Associated with a shift from streptococcal domination to one in which Actinomyces spp, capnophilic organisms and obligate anaerobic gram –ve organisms.
Organisms implicated included
Actinobacillus actinomycetemcomitans, Porphyromonas gingival is, Prevotella intermedia, Bacteroides forsythus, Fusobacterium nucleatum and Capnocytophaga spp.
Periodontitis may be a result of:
Specific bacteria
Non specific inflammatory response to plaque bacteria
Establishment of conditions that favour expression bacterial virulent factors.