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 single-species microcolonies Growth requires “quorum sensing” In vitro biofilm (and cat) by Streptococcus sanguinis SEM by Tracy Grossman

In vitro biofilm - in depth 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 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 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

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 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 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