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