1 Professor Dennis Cvitkovitch Faculty of Dentistry Dental Research Institute University of Toronto The human microbiome We are a composite species: eukaryotic, bacterial, archeal Every human harbors over 100 trillion microbes These bacterial counts outnumber our own cells by more than ten-to-one; Most cells in the human body are bacteria 2 Bacteria that live in or on our bodies can affect the outcome of health vs. disease Learning objectives Understand: Oral environment is a complex, dynamic ecosystem affected by host and individual microorganisms, and by the relationships of microorganisms to each other and the host Oral infectious diseases have a complex etiology, caused by environmental changes resulting in the overgrowth of pathogenic species over time Structure & formation of the dental biofilm 3 1
Human dental plaque 4 Life in the mouth: oral biofilm Hundreds of species (less than half are cultured) Oral microbial infections Dental caries Gingivitis, Periodontal disease 5 Who is out there? 6 Classical genomics and microbiology largely rely on isolating individual microbial species in pure cultures - that is, cultures containing only microbes of a particular species Problem: Less than 50% of oral bacteria can be cultured 2
What is dental plaque? A complex microbial community/ microbiome (BIOFILM) Develops on tooth surface Embedded in a matrix of polymers of bacterial and salivary origin 7 Dental plaque http://dentistry.usc.edu What is so special about a biofilm mode of growth? Biofilm cells have: Increased resistance to antibiotics Increased resistance to mechanical forces Heterogeneous phenotypes/genotypes Numerous intra/interspecies interactions Biofilms are involved in 65% of infections 8 Biofilm formation Pellicle Pioneers Microcolonies Polysaccharide Anaerobes 9 3
What is a climax community? Microbial homeostasis Plaque composition is stable over time despite regular environmental challenges (dynamic balance established) Healthy plaque results from a community that has reached microbial homeostasis 10 Disruption/Dysfunction of microbial homeostasis in dental plaque = disease Jenkinson & Douglas; In Polymicrobial Diseases (Brogden & Guthmiller) 2002 11 12 4
Benefit for host Benefits of dental plaque Major benefit : Colonization resistance Prevent colonization and infection by pathogens (e.g. by producing acid) Suppress overgrowth of opportunistic pathogens (e.g. fungi) 13 Streptococcus mutans http://www.saishika.jp/biofilm/aa.html Benefits of dental plaque (2) Benefit for microbes 1. Synergistic catabolism of host macromolecules 2. Modulation of local environmental conditions (e.g. ph, oxygen tension) 3. Nutrient and energy cycling via cross-feeding and food webs 4. Co-aggregation (co-adhesion) 14 Microorganisms in the oral cavity Sub-topics 1. Diversity, abundance, and intrinsic biological properties of oral microbes 2. Interaction between microbes 3. Interaction between microbes and the host 15 5
1. Microorganisms of the oral cavity Who lives here? Bacteria, viruses, fungi http://www.scharfphoto.com/fine_art_prints/archives/000613.php 16 Thrush: Candida albicans Diversity 1. Microorganisms of the oral cavity (2) >700 species identified 100-200 species/person Streptococci, lactobacilli, staphylococci, bacteroides, spirochetes Abundance Each tooth surface: 1000-1 billion bacteria/tooth Most abundant? 17 Treponema denticola http://www.saishika.jp/biofilm/aa.html 2. Interactions between oral microorganisms Oral biofilm = Dental plaque The oral microbial community/microbiome 18 Dental plaque http://dentistry.usc.edu 6
2. Interactions between oral microorganisms (2) Microbial community interactions (intra- and inter-species) Competition Peptides, acid, H 2 O 2 : suppress growth of other species 19 Jenkinson & Douglas; In Polymicrobial Diseases (Brogden& Guthmiller); 2002 2. Interactions between oral microorganisms (3) Microbial community interactions (intra- and inter-species) Cooperation 1. Concerted action of 2 or more species to metabolize host molecules 2. Development of food chains 3. Co-aggregation Bacterial co-aggregation 20 3. Interactions between oral microorganisms and the host Physical habitat / environment in the host http://bacteriality.com Teeth are the only external body surface where shedding of surface epithelium does not occur Therefore teeth are a major site of long-term microbial colonization 21 7
3. Interactions between oral microorganisms and the host (2) Saliva Antibacterial lysozymes, peptides, agglutinins, secreted IgA Fluid shear forces (discourage bacterial adhesion) 22 Role of plaque bacteria in disease etiology Specific plaque hypothesis Only a few species from the diverse resident plaque microflora are actively involved in disease Non-specific plaque hypothesis Disease is outcome of the overall activity of the total plaque microflora Ecological plaque hypothesis Organisms associated with disease may also be present at non-diseased sites, but at levels too low to be clinically relevant Disease is a result of a shift in the balance of the resident microflora due to change in local environmental conditions 23 Ecological plaque model 24 Fig.6.1 Ecological shifts in the dental plaque microflora in health and disease 8
Dental caries: a multifactorial disease 25 Impact of dental caries 98% of population infected (50% of 5-9 year olds have at least one cavity) Annual dental spending $85 billion Most common chronic childhood disease 5X more common than asthma Pain and suffering often leads to problems eating, speaking and attending school 26 Progression of caries Douglas Brathall Mälmo University 27 9
Ecological plaque hypothesis Organisms associated with disease may also be present at sound sites, but at low levels Disease due to shift in balance of the resident microflora due to change in local environment conditions (e.g. repeated low ph) 28 Tooth surfaces 29 Bacteria associated with plaque 30 Fig. 5.9 Predominant groups of bacteria found at distinct sites on the tooth surface 10
31 Cariogenic bacteria Mutans Streptococci - initiators of caries S. mutans S. sobrinus S. cricetus, S. rattus Actinomyces - Acid producers associated with root caries: A. Odontolyticus A. Neslundii A. israelii Lactobacilli L. casei - associated with the progression of caries The first isolation of cariogenic bacteria Clark, 1924 ************* Isolation of cariogenic bacteria from caries lesions Discovery of Mutans Streptococci 32 Cariogenic factors of Streptococcus mutans Ability to adhere to tooth surface and colonize Water-insoluble glucans Ability to produce acid under [High sugar] Glycolytic pathway Efficient uptake of sugars and production of metabolic end products Lactic acid Ability to tolerate acid Capable of carrying out glycolysis and other cellular functions at low ph Proton extrusion (removal) from cell (via H + -ATPase) Inducible acid adaptation mechanism (ATR) 33 11
The Stephan curve After sugar intake the ph drops rapidly and may go below the critical ph of ph 5.5 Repeated hits of sugar will eventually lower the resting ph to levels sustained at or below the critical ph onset of caries 34 ph range for growth of oral bacteria Critical ph 35 Dental caries Carbohydrates (sucrose) Acid producing bacteria (e.g. S. mutans) Dental plaque Acid Demineralization 36 http://dent.umich.edu/media /VODI/html/01-dc/b-diseaseProcess /crowndecay/01.html Figure 17. Clinical illustration of arrested caries; The dentin is hard, darkly discolored dry looking and plaque free 12
Gingivitis Periodontal disease 37 Bacterial biofilms lead to chronic injury and delayed healing in the periodontium tooth Persistence of a biofilm here Causes inflammation and a destructive lesion here 38 gum Color Atlas of Periodontology Promotes chronicity and prolongs wound healing here Periodontal health: Bacteriology 39 Nonmotile cocci and bacilli [rods] Few motiles; Few if any spirochetes Mostly Gram-positive, facultative anaerobes 13
Chronic gingivitis: Clinical and histological features Swollen, poorly contoured, discolored margin Increased crevicular fluid flow; Bleeding Wide intercellular spaces in epithelium Vascular changes, edema Inflammatory cell infiltrate Collagen resorption limited to gingiva Intact alveolar crest, gingival attachment at CEJ 40 Color Atlas of Periodontology Chronic gingivitis: Bacteriology More complex flora than in health Prominent Gram-positive, facultative anaerobes Emergence of Gram-negative anaerobes, motile forms Emergence of spirochetes Growth of fastidious species in response to availability of nutrients in food web, GCF, blood, & reduced environment 41 Color Atlas of Periodontology Chronic periodontitis:, Clinical & histological features 42 14
Chronic periodontitis: Bacteriology 43 Greatly increased mass of bacteria Prominent motile rods Prominent spirochetes Increased Gram-negative anaerobes Prominent pigmented anaerobes Clusters of bacterial taxa in human subgingival pocket* 44 * Socransky and Haffajee, Periodontology 2000, 2002 Porphyromonas gingivalis and Treponema denticola cohabitate in subgingival biofilms 45 Kigure et al., 1995 15
Local aggressive (juvenile) periodontitis: Clinical & radiographic features 46 Local aggressive (juvenile) periodontitis: Bacteriology Aggregatibacter actinomycetemcomitans 47 Clinical state Pocket flora Changes in bacterial ecology Healthy / Hygienic Unhygienic/ Gingivitis Unhygienic/ Periodontitis Treated carrier Bacterial colonization of gingival crevice Increased bacterial mass Thicker, more extensive biofilm Proportional shift in microflora begins Metabolic (noxious) products increase Increased bacterial mass Major shift in proportions of species Overwhelming metabolic products Toxins, degradative enzymes Factors that suppress local immunity Removal/suppression of bulk of bacteria Proportional suppression of pathogens 48 Gram +ve Gram ve Facultative Strict anaerobe 16
Clinical state Pocket flora Periodontal tissue response Healthy / Hygienic Unhygienic/ Gingivitis Unhygienic/ Periodontitis Treated carrier Innate immunity locally protective Innate immunity chemotactic Cellular and vascular responses Increased crevicular fluid, bleeding Adaptive immune responses Pro-inflammatory cytokines intensified Suppression of protective immunity Pathogenic resorption of tissue Matrix (including collagen) degradation Alveolar bone resorption Resolution of inflammation, remodeling Sensitized immune system 49 Gram +ve Gram ve Facultative Strict anaerobe 50 17