1. Oral Microbial Ecology
DENT 5301
Introduction to Oral Biology
Dr. Joel Rudney

2. Oral microbial diversity
The “we know what we can grow” bias
Emphasis on species recovered from culture
Revolutionized by molecular methods for species ID
Species signatures in 16S ribosomal RNA sequences
Does not require microbial culture
Perhaps 700+ distinct oral species
Most have never been cultured
Gram+, Gram-, cocci, rods, filaments, spirochetes, etc.
Include some very exotic taxonomic groups (Archea)
Termite guts and other extreme environments
Fungi and viruses are all in the mix

3. Biofilm on tooth surfaces
Emerging trends in Oral Care
Biofilm Revolution
Scientific American, 2002
The “natural habitat” of most oral bacteria
A structured multi-species community
Bacteria embedded in matrix with water channels
Attachment - growth - ecological succession - maturation

4. Adherence and microcolonies
Biofilms are initiated by bacterial adherence to a surface
Isolated cells bind receptors on surface
Replication (growth) is required to form single-species microcolonies
Growth requires “quorum sensing”
In vitro biofilm (and cat) by Streptococcus sanguinis
SEM by Tracy Grossman

5. In vitro biofilm - in depth

6. Ecological succession
3° colonizers (Gram-)
Porphyromonas gingivalis
2° colonizers (Gram-)
Bridge species - F. nucleatum
Bind other bacteria
1° colonizers (Gram+)
Streptococci bind pellicle proteins from saliva
DENT 5302
Kolenbrander et al. 2002, Microbiol Mol Biol Rev 66:486

7. Inter-bacterial coaggregation
1μM
Edwards, Grossman, and Rudney, 2007, Oral Microbiol Immunol, in press
Streptococcus cristatus coaggregating with F. nucleatum
- adhesins interacting with receptors
Coaggregation is important in ecological succession
Fusobacterium nucleatum is considered a bridge species because it is a promiscuous coaggregator

8. Interspecies collaboration - O2
Streptococcus cristatus
Facultative species
Can live w/ or w/o O2
Uses up O2 when available
Fusobacterium nucleatum
Robust anaerobe
Binding strep improves survival when O2 is present
Porphyromonas gingivalis
Sensitive anaerobe
Coaggregation essential to survival when O2 is present
In vitro three-species biofilm made by replicating an ecological succession
Grossman, Edwards, and Rudney 2006 AADR

9. Inter-species competition
Many oral species produce substances that can kill closely related competitors
Overlay experiment:
Streptococcus sobrinus lawn
Spotted with wild-type Streptococcus Mutans strain producing mutacins I and IV
Single knockout mutants
Double knockout mutants
Measure zones of growth inhibition
Picture courtesy of Dr. Jens Kreth

10. Inter-species communication
Streptococci ferment CHO
Excrete lactic acid
Veillonella use lactate made by Strep for nutrition
They are biofilm buddies
Strep can make amylase
Starch-digesting enzyme
Enhances lactate excretion
Veillonella send a chemical signal to activate transcription of Strep amylase gene
Bacteria sense other species
Egland, Paul G. et al. (2004) Proc. Natl. Acad. Sci. USA 101,

11. Oral ecological zones
Mostly the same species present, but proportions differ
High biomass sites
Non-shedding surfaces
Supragingival tooth surfaces
Subgingival tooth surfaces
Shedding surface
The tongue
Low biomass (reservoir) sites
Shedding oral mucosal surfaces
Buccal, palate, external gingiva, floor of mouth
Saliva as a transitional zone

12. Subgingival tooth surfaces
Narrow crevice between gingival epithelium and cementum
Low oxygen tension
Favorable for Gm- anaerobes
Major site for interaction between bacteria and host tissues
Species mix varies between each side and the center
- distinct microenvironments
Emerging trends in Oral Care
Biofilm Revolution
Scientific American, 2002

13. The tongue A shedding surface Cells slough off BUT
Structure includes crypts and fissures
Favorable for Gm- anaerobes

14. Mucosal reservoir sites
Smooth exfoliating surfaces
How do bacteria keep from being swept away?
Some oral species can invade epithelial cells
Requires communication between bacteria and cells
Bacteria “subvert” the cell to take them in
Take control of the cytoskeleton
Can live and grow inside
Can direct the cell to export them to other cells
Multi-species intracellular flora resembles mixed biofilm

15. Invaded buccal cells
Rudney, Chen, and Zhang 2005 J Dent Res 84:1165

16. Collaborative invasion
Tissue culture experiment
F. nucleatum invades epithelial cells
S. cristatus does not invade cells
After coaggregation, S. cristatus is carried inside by F. nucleatum
Edwards, Grossman, and Rudney 2006, Infect Immun 74: 654

17. Salivary transport
Quorum sensing tells bacteria when to grow, and when it’s time to go
Bacteria at the outer surface of mature biofilms are signaled to detach and become planktonic
The goal is to find a new home
Different genes are active in planktonic and biofilm states
Saliva is the transport medium for planktonic oral bacteria
-They don’t grow unless they encounter another surface
Exfoliated epithelial cells in saliva can also transport bacteria
-A protected environment

18. Bacteria during the life cycle
Oral colonization begins in the birth canal
Reservoir populations on the tongue and mucosa
Established during infancy - include anaerobes
Tooth eruption provides non-shedding surfaces
The “window of infectivity” concept
Colonization from reservoir sites and caregiver saliva
Hormonal shifts - puberty and pregnancy
Can alter proportions of Gm- anerobes
Complete loss of teeth shifts flora towards infant state
Dentures restore supragingival non-shedding sites
Implants restore supra- and subgingival sites

19. Relationships with the host
Host defenses in the mouth
Epithelial cells
Barrier function
Innate immunity - sensors (Toll-like receptors)
Inflammatory mediators, antimicrobial peptides
Salivary antimicrobial factors - DENT 5302
Mucosal antibodies (secretory IgA)
Cell-mediated immunity (T-cells)
In most cases, host defenses tolerate oral bacteria
The predominant relationships are commensal

20. Are there true oral pathogens?
Classic concept of a pathogen
Not normally present
Produces “virulence factors”
Damage host directly (e.g. toxins)
Induce host to damage itself (immune responses)
Presumed oral pathogens don’t quite fit that model
Normally present throughout life
Damage requires presence in large numbers
Ecological concept of oral microbial diseases
Ecological shifts lead to changes in proportions
Balance shifts in favor of “pathogens”/disease