Oral Microbiology

Normal Oral MicrofloraMicroflora of Dental Caries

Microflora of the Root Canal ( endo )Microflora of the Periodontal Pocket (perio)

The Importance of Studying Bacterial Pathogenesis• A human body has 1 X 1013 eukaryoticcells and 1 X 1014 bacterial cells• Microbial infections are the most epidemic diseases and the leading cause of death– Diarrhea and enteric bacteria– Tuberculosis and Mycobacterium– Ulcer and Helicobacter infection– Urinary tract infection– STDHow microbiology is related to dentistry?

The first microbes observed: Anton Van Leeuwenhoek (1632-1723)

Developed the microscope and was the first to discover oral bacterial flora: “I didn’t clean my teeth for three days and then took the material that had lodged in small amounts on the gums above my front teeth…. I found a few living animalcules..”

What causes dental caries?• Pre-microbiology era– Dental caries is the death (decay) of a tissue• Microbiology period era– Dental caries is a microbe related disease

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Plaque is a complex microbial community

How to identify bacteria within dental plaque?

• Culture methodstake saliva or plaque, dilute and plate on

appropriate plates, grow to single colonies,identify by microscopic and biochemical methods

• 16S DNA/RNA based detectionuse 2 oligo-nucleotide primers universal to ALL

bacteria 16S rDNA, PCR amplification of the total saliva or plaque DNA pool, clone the PCR product and sequence, phylogenetic analysis using computer database

Supragingival PlaquePredominant cultivable microflora obtained from occlusal fissures

Bacteria Median percentage Range % isolation

Streptococcus 45 8-86 100

Staphylococcus 9 0-23 80

Acinomyces 18 0-46 80

Propionibacterium 1 0-8 50

Eubacterium 0 0-27 10

Lactobacillus 0 0-29 20

Veilonella 3 0-44 60

Supragingival PlaquePredominant cultivable microflora obtained from occlusal fissures

Specie Median percentage Range % isolation

S.mutans 25 0-86 70

S. Sanguis 1 0-15 50

S. Oralis 0 0-13 30

S. Anginosus 0 0-3 10

A.Naeslundi 3 0-44 70

L. Casei 0 0-10 10

L. plantarum 0 0-29 10

Subgingival plaque

Bacteria Mean percentage Frequency %

Streptococcus 23 100

Actinomyces 42 100

Prevotella 8 93

Veilonella 14 93

S. Sanguis 6 86

A. naeslandii 19 97

Current knowledge about bacteria in plaque• Both culture and DNA/RNA-based techniquesare used for identification and quantification oforal microorganisms• Overall, there are ~700 species exist in the oralcavity• ~20% of these 700 species have been cultivated• Both Gram-positive and Gram-negative exist• Some archaea are found• Most anaerobic or facultative anaerobicWho are the bad guys?

The first isolation of cariogenic bacteria by Clark, 1924

Isolation of cariogenic bacteria from caries lesions

Discovery of Mutans streptococci

Keyes and Fitzgerald, 1962’sRe-isolation of “Mutans streptococci”:

• Streptococcus mutans (human) (samespecies Clark isolated in England in 1924)

• Streptococcus sobrinus (human)• Streptococcus rattus (rats)• Streptococcus cricetus• Streptococcus ferus• Streptococcus macacae• Streptococcus downeii

• The “cariogenic bacteria” – bacteria associated with dental caries

• Actinomyces – early colonizers and root caries• A. odontolyticus• A. naeslundii genospecies 2• A. isrealii• A. gerensceriae

• Lactobacilli (L. casei) – caries progression• Mutans streptococci (S. mutans) – caries initiation

The virulence factors ofcariogenic bacteria

1. Acid production (acidogenicity)• Lower the pH to below 5.5, the critical pH, drives the dissolution of calcium phosphate(hydroxyapatite) of the tooth enamel• Inhibit the growth of beneficial bacteria, promote the growth of aciduric bacteria.• Further lower the pH, promote progression of the carious lesion

2. Acid tolerance (aciduricity)

– Allows the cariogenic bacteria to thrive under acidic conditions while other beneficial bacteria are inhibited. This results in dominance of the plaque by cariogenic bacteria

• Glucan formation– Allows the cariogenic bacteria to stick onto the

teeth and form a biofilm– Glucan mediated biofilms are more resistant

tomechanical removal– Bacteria in these biofilms are more resistant to

antimicrobial treatments

Dental Caries

Carbohydrate (Sucrose)

Cariogenic bacteria such as strep. mutans

Glucans/levansPlaque

formation

AcidsDemineralizatio

n

Dental caries is a bacterial infectious disease

Transmission• Mother – Child (vertical transmission) -true for most oral bacteria• Persons in close contact to the baby• Horizontal transfer (between spouses) is rare, only observed in some periodontal pathogens (i.e. P.gingivalis)

The most common vehicle is saliva

New problem: everybody has S. mutans!

• So…….Why not every body who has S. mutans develop dental caries?

S. mutans is not present in high portionsAcid produced is neutralized by urea or ammonia

produced by other bacteria in the plaqueS. mutans is away from the tooth surface so acid

produced is diffused

The ecologic plaque hypothesis

Both pathogenic and commensal (nonharmful) bacteria exist in a natural plaque. At sound site, the pathogenic bacteria may exist in low numbers to cause any clinical effect, or they may exist in higher numbers, but the acid produced is neutralized by the action of other bacteria.

Disease is a result of a shift in the balance of the residence microflora driven by a change in the local environment (frequent sugar intake etc).

Human genome project

Oral bacteria genome projects

Metagenomics of the oral cavity

Complex genetic makeup of man

Human genome only has 200,000

genes

Each oral bacterium 2000 to 6000 genes. • With over 1000000

bactrial genes in the oral cavity

The dental plaque is a complexmultispecies biofilm

Transmission Attachment &

Colonization

Growth ofPioneer species Environment

alModification

pH, EhExpose new

receptorsGenerate

new nutrients

Microbialsuccession

G+, G-(periodontal pathogens

Mother to Child

Pioneer colonizers:

S. oralisS. mitis

S. salivariusS. sanguis

S. anginosusS. gordonii

Increased species

diversity

Climaxcommunity

That’s all folks! ! ! Have fun ! !