oral microbial ecology
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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 - PowerPoint PPT PresentationTRANSCRIPT
Oral Microbial Ecology
DENT 5301Introduction to Oral BiologyDr. 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 CareBiofilm Revolution
Scientific American, 2002The “natural habitat” of most oral bacteriaA structured multi-species communityBacteria embedded in matrix with water channelsAttachment - 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 single-species microcolonies
Growth requires “quorum sensing”
In vitro biofilm (and cat) by Streptococcus sanguinisSEM by Tracy Grossman
In vitro biofilm - in depth
http://www.eastman.ucl.ac.uk/research/MD/biofilms_ecology_confocal/index.html
QuickTime™ and aDV - PAL decompressor
are needed to see this picture.
Ecological succession
Kolenbrander et al. 2002, Microbiol Mol Biol Rev 66:486
3° colonizers (Gram-)Porphyromonas gingivalis
2° colonizers (Gram-)Bridge species - F. nucleatumBind other bacteria
1° colonizers (Gram+)Streptococci bind pellicle proteins from salivaDENT 5302
Inter-bacterial coaggregation
1μM 1μM
Streptococcus cristatus coaggregating with F. nucleatum - adhesins interacting with receptorsCoaggregation is important in ecological successionFusobacterium nucleatum is considered a bridge species because it is a promiscuous coaggregator
Edwards, Grossman, and Rudney, 2007, Oral Microbiol Immunol, in press
Interspecies collaboration - O2
In vitro three-species biofilm made by replicating an ecological succession
Grossman, Edwards, and Rudney 2006 AADR
Streptococcus cristatusFacultative speciesCan live w/ or w/o O2
Uses up O2 when available
Fusobacterium nucleatumRobust anaerobeBinding strep improves survival when O2 is present
Porphyromonas gingivalisSensitive anaerobeCoaggregation essential to survival when O2 is present
Inter-species competition
Picture courtesy of Dr. Jens Kreth
Many oral species produce substances that can kill closely related competitors
Overlay experiment:Streptococcus sobrinus lawnSpotted with wild-type Streptococcus mutans strain producing mutacins I and IVSingle knockout mutantsDouble knockout mutantsMeasure zones of growth inhibition
Inter-species communication
Egland, Paul G. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 16917-16922
Streptococci ferment CHOExcrete lactic acidVeillonella use lactate made by Strep for nutritionThey are biofilm buddies
Strep can make amylaseStarch-digesting enzymeEnhances lactate excretionVeillonella send a chemical signal to activate transcription of Strep amylase geneBacteria sense other species
Oral ecological zones
Mostly the same species present, but proportions differ High biomass sites
Non-shedding surfacesSupragingival tooth surfacesSubgingival tooth surfaces
Shedding surfaceThe tongue
Low biomass (reservoir) sites Shedding oral mucosal surfaces
Buccal, palate, external gingiva, floor of mouth Saliva as a transitional zone
Subgingival tooth surfaces
Emerging trends in Oral CareBiofilm Revolution
Scientific American, 2002
Narrow crevice between gingival epithelium and cementum
Low oxygen tensionFavorable for Gm- anaerobes
Major site for interaction between bacteria and host tissues
Species mix varies between each side and the center- distinct microenvironments
The tongue
A shedding surface-Cells slough offBUTStructure includes crypts and fissures
Favorable for Gm- anaerobes
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 cytoskeletonCan 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
Edwards, Grossman, and Rudney 2006, Infect Immun 74: 654
Tissue culture experiment
F. nucleatum invades epithelial cells
S. cristatus does not invade cells
After coaggregation, S. cristatus is carried inside by F. nucleatum
Salivary transportQuorum 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 surfaceExfoliated 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 functionInnate 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