Oral Biofilms Chapter 13

Contemporary Perspectives on Role of Bacteria

Recent advances in microbiology and immunology have altered beliefs, leaving us with two current hypotheses 1.Ecologic Plaque Hypothesis 2.Microbial Homeostasis--Host Response Hypothesis 3.Keystone Pathogen-Host Response Hypothesis

Socransky's Microbial Complexes

Level of pathogenicity organized by color (Orange and red as major etiologic agents) The theory was widely accepted until newer molecular-based approaches to microbe detection brought its validity into question.

Structure and Colonization of Dental Plaque Biofilms

1.Acquired pellicle 2.Initial colonizers attach 3.Additional bacteria coaggregate 4.Formation of extracellular slime layer 5.Major biofilm with bacterial microcolonies Pellicle: protein cover that protects and maintains an organism

Where bacteria live...lifestyles..

Aerobic bacteria: require O2 Anaerobic bacteria: cannot live in presence of O2 Facultative bacteria: can exist w/ or w/out O2 Free floating bacteria: planktonic bacteria (rare) Attached bacteria: Communities of bacteria that attach to each other. 99% of all bacteria live as attached bacteria. Attach to both living (root of tooth) and non-living surfaces (dental implant)

Bacterial Attachment Zones

Bacteria can attach subgingivally to: •Tooth surface •Epithelial lining Some bacteria may remain free-floating in the sulcus

Bacteria In the Oral Environment

Bacterium- simplest organisms; can only be seen with a microscope Innocuous-not harmful Pathogenic-capable of causing disease/virulent

Structure of the Bacterial Cell Membrane

GRAM + ~Single, thick cell membrane ~Most in a healthy mouth ~Retain purple color GRAM - ~Double cell membrane ~VIP in tissue destruction ~Show red stain RED=inflammation Gram-positive bacteria have a thick peptidoglycan cell wall that retains the purple color when stained with crystal violet dye. Gram-negative bacteria have a thin peptidoglycan layer which cannot retain the crystal violet dye. As a result, gram-negative bacteria appear read with the Gram staining method. Gram-negative bacteria are believed to play an important role in periodontal disease.

Biofilm Formation

Within minutes: free-floating microbes attach to surface 2-4 hours: microbes form strongly attached micro-colonies 6-12 hours: microbes produce protective matrix and become resistant to antiseptics and antibiotics 2-4 days: fully mature biofilm colonies can rapidly recover from mechanical disruption and reform within 24 hrs

Biofilms: Complex Bacterial Communities

~Bacteria are "social creatures" ~Biofilm: a living film of diverse species embedded with a slimy self-protective matrix of sugars and proteins ~Found nearly everywhere in nature ~Major impact on human health. It is estimated that 65% of all diseases are biofilm-induced: tuberculosis, cystic fibrosis, subacute bacterial endocarditis, and periodontal disease

Biofilm Protective Factors

~Blocking-protective matrix prevents large molecules and inflammatory cells from penetrating deeply into biofilm ~Mutual protection:antibiotic resistant bacteria secrete protective enzymes to protect neighboring bacteria ~Quiescence (hibernation): hibernating bacteria are unaffected by antibiotics Biofilms greatly enhance the ability of inhabiting microbes to withstand the host's immune system, antimicrobials, and environmental stresses. Microbes in a biofilm can resist factors that would easily kill these same microbes when in a free-floating state. The biofilm offers three inherent features (blocking/mutual protection/quiescence) that enable inhabiting microbes to tolerate various external stresses.

Keystone Pathogen Hypothesis

•A keystone species initiates a shift in microbes in the biofilm community. •This, in turn, triggers the host inflammatory response responsible for the tissue destruction seen in periodontitis.

Ecologic Plaque Hypothesis

•Accumulation of nonspecific bacteria triggers the host inflammatory response. •Host inflammatory response alters the local environment within the gingival sulcus. •As the local environment changes, it becomes more conducive to the growth of specific pathogenic bacteria which leads to growing specific periodontal pathogens

Stage 1: Initial Attachment of Microbes to Pellicle

•Acquired pellicle: variety of salivary glycoproteins and antibodies •Acquired Pellicle is to protect the enamel from acidic activity •Within minutes after cleaning, a film forms over the tooth surface •Double sided adhesive tape •Adheres to tooth surface on one side and provides a sticky surface on the other that facilitates attachment by bacteria to the tooth surface

Tissue-Associated Plaque Biofilms

•Adhere loosely to the epithelium of the pocket wall •Distinctly different bacteria colonize this area: Streptococcus oralis, Streptococccus intermedius, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Fusobacterium nucleatum •Bacteria from the tissue-attached plaque biofilms can invade the gingival connective tissue and be found within the periodontal connective tissues and on the surface of the alveolar bone ~Contains large numbers of spirochetes and flagellated bacteria and gram-negative cocci and rods Periodontal pockets also contains many free floating (planktonic) bacteria that are not part of the biofilm

Tooth-Associated Plaque Biofilms

•Attachment area extends from tooth surface at the gingival margin to junctional epithelium at the base of the pocket •Can invade dentinal tubules of cementum •Filamentous microorganisms, cocci, and rods, including S. mitis, S. sanguis, A. viscous

Detachment (Dispersion)

•Clumps break off and are carried away by fluid in the biofilm •Detached colonies can attach to other portions and form new colonies

Stage 3: Maturation Phase I/Self-Protective Matrix Formation

•Firmly attached bacteria secrete surrounding protective substance called extracellular protective matrix •Usually consists of proteins, glycolipids and bacterial DNA •Microbes become cocooned in the protective matrix •Protects against host-generated immune defenses •Chronic disease is established

Early Bacterial Colonizers: Important to Bacterial Succession

•First to colonize= nonpathogenic •Lays the foundation •Perio pathogens float until signals are received that conditions are favorable to join the biofilm •Streptococcus mitis and oralis species •Actinomyces viscosus and Streptococcus Sanguis

Stage 2: Permanent Attachment

•Microbes able to withstand hydrodynamic forces attain permanent attachment •Attached microbes produce substances to attract other microbes to the community •Co-aggregation is when genetically distinct bacteria become attached to one another

Stage 4: Maturation Phase II/Mushroom-Shaped Microcolonies

•Microbes cluster together and form mushroom-shaped microcolonies that attach to the tooth by a narrow base •Microbial blooms develop when specific species grow at an accelerated rate •Biofilm becomes thicker by stacking one microbial species on top of another forming polymicrobial colonies •Fluid channels penetrate the protective matrix •Quorum sensing - bacteria communicate with each other to share critical information to survive and thrive

Stage 5: Dispersion/Escape from the Matrix

•Microbes disperse from the colony to spread and colonize other tooth surfaces

Current Periodontal Etiology

•More than 600 bacterial strains have been identified in bacterial plaque •Only a small percentage are periodontal pathogens •A. actinomycetemcomitans •P. gingivalis •T. forsythia What do we believe? Plaque biofilm, itself, is necessary, but not sufficient to cause periodontal tissue destruction. It is more likely a host inflammatory and immune response to the biofilm that leads to this destruction. Seemingly, a shift from beneficial microbes to a pathogenic community triggers a potent host inflammatory response which contributes to tissue destruction.

•Intermediate and Late Colonizers

•Must join in the proper sequence •Fusobacterium nucleatum co-aggreates with the last colonizers •Many perio pathogens are late biofilm colonizers

Historical Perspective

•Nonspecific Plaque Hypothesis •Plaque at the gum line leads to gingival inflammation and subsequent periodontal inflammation •Failed to explain why most cases of gingivitis never progress to periodontitis •Specific Plaque Hypothesis •Specific pathogenic bacteria present in subgingival biofilms and their toxic products resulted in destruction of the periodontal tissues •As periodontitis develops, gram + aerobic community shifts to gram - anaerobes

5 Main Hypotheses on Role of Oral Biofilms in Periodontal Disease

•Nonspecific Plaque Hypothesis: Accumulation of bacterial biofilms lead to periodontal disease •Specific Plaque/Microbial Shift Hypothesis: Specific pathogenic bacteria and their products - rather than amount - lead to periodontal destruction •Ecological Plaque Hypothesis: Shifts in subgingival environment can favor an increase of pathogenic bacteria in biofilm •Microbial Homeostasis-Host Response Hypothesis: Host-related factors are major factors that contribute to initiation and progression of periodontal disease •Keystone Pathogen Hypothesis: Key species (even in small amounts) can exert a profound effect on the plaque biofilm

Bacterial Microcolonies

•Not evenly distributed, cluster together, tiny independent community •Different colonies contain different bacteria •Differences in oxygen concentration, pH, and temperature •That is how they can survive

Microbial Homeostasis--Host Response Hypothesis

•Plaque biofilms are the cause of initial inflammatory response leading to gingivitis •Pathogenic bacteria are not the direct cause of periodontal tissue destruction •Host-related factors are major factors that contribute to initiation and progression of periodontal disease •Page and Schroeder established that gingivitis does not progress to periodontitis unless another unknown factor tips the biofilm balance toward further tissue destruction

Growth

•Release substances that attract other free floaters to join the community •Secrete extracellular slime layer (protective matrix) •Multiply rapidly and form mushroom-shaped microcolonies of complex collections of different microbes •Movement of fluid results in extensions that stream from the main body of the biofilm