Transcript Oral Microbial Ecology - University of Minnesota

Oral Microbial Ecology
DENT 5301
Introduction to Oral Biology
Dr. Joel Rudney
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
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
Adherence and microcolonies
Biofilms are initiated by
bacterial adherence to
a surface
Isolated cells bind
receptors on surface
Replication (growth) is
required to form singlespecies microcolonies
Growth requires
“quorum sensing”
In vitro biofilm (and cat) by
Streptococcus sanguinis
SEM by Tracy Grossman
In vitro biofilm - in depth
QuickTime™ and a
DV - PAL decompress or
are needed to see this picture.
http://www.eastman.ucl.ac.uk/research/MD/biofilms_ecology_confocal/index.html
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
Inter-bacterial coaggregation
1μM
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
Interspecies collaboration - O2
Streptococcus cristatus
Facultative species
Can live w/ or w/o O2
Uses up O2 when available
In vitro three-species biofilm
made by replicating an
ecological succession
Fusobacterium nucleatum
Robust anaerobe
Binding strep improves
survival when O2 is present
Porphyromonas gingivalis
Sensitive anaerobe
Coaggregation essential to
survival when O2 is present
Grossman, Edwards, and Rudney 2006 AADR
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
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, 16917-16922
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
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
The tongue
A shedding surface
-Cells slough off
BUT
Structure includes
crypts and fissures
Favorable for Gmanaerobes
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
Invaded buccal cells
Rudney, Chen, and Zhang 2005 J Dent Res 84:1165
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
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
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
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
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