Oral Microbial Ecology Ecology Ecology (from Greek) is the scientific study of interactions among organisms and their environment, such as the interactions organisms have with each other and with their abiotic environment; The ecosystem is composed of microbial communities living on specific sites surrounded by a different physical and chemical elements. Ecosystems Ecosystems are composed of dynamically interacting parts including organisms, the communities they make up, and the non-living components of their environment; Ecosystem processes, such as primary production, nutrient cycling, and various niche construction activities, regulate the flux of energy and matter through an environment. Econiche Definitions of Hutchinson: “The set of biotic and abiotic conditions in which a species is able to persist and maintain stable population sizes." Niche In ecology, a niche is a term describing the way of life of a species; Each species have a separate, unique niche; The ecological niche describes how an organism responds to the distribution of resources and competitors (e.g., by growing when resources are abundant, and when predators, parasites and pathogens are scarce) and how it in turn alters those same factors (e.g., limiting access to resources by other organisms, acting as a food source for predators and a consumer of prey). Termite Termite mounds with varied heights of chimneys regulate gas exchange, temperature and other environmental parameters that are needed to sustain the internal physiology of the entire colony. Microflora Living microorganisms that are so small that they can be seen only with a microscope and that maintain a more or less constant presence in a particular area; Includes bacteria, viruses, protozoa, fungi. Oral microflora Oral microflora refers to the community of microorganisms coexisting in the oral cavity as its primary habitat; These strains of bacteria colonize the various different surfaces present in the oral cavity, and communicate between each other through complex cell signaling processes; The body’s own defenses also play a role in maintaining a balance and ensuring a healthy oral environment. Normal - resident flora In a healthy body, the internal tissues - blood, brain, muscle, etc., are normally free of microorganisms; However, the surface tissues - skin and mucous membranes, are constantly in contact with environmental organisms and become readily colonized by various microbial species; The mixture of organisms regularly found at any anatomical site is referred to as the normal flora or resident flora, some researchers prefer the term "indigenous microbiota". Resident microflora Typical microflora of a econiche; Microorganisms are separated and grouped according to the different conditions of life; Resident microflora has an important function in host: Digestive and nutritional; Competition with pathogenic microflora. Transient Microbiota Transient microbes are just passing through; Although they may attempt to colonize the same areas of the body as do resident microbiota, transients are unable to remain in the body for extended periods of time due to: Competition from resident microbes; Elimination by the body’s immune system; Physical or chemical changes within the body that discourage the growth of transient microbes. Opportunistic microbes Under normal conditions, resident and transient microbes cause the host no harm; However, if the opportunity arises, some of these microbes are able to cause disease and become opportunistic pathogens. This can happen due to a number of different conditions: When the immune system isn’t working properly, normal flora can overpopulate or move into areas of the body where they do not normally occur; When the balance of normal microbes is disrupted, for example when a person takes broad spectrum antibiotics, microbes that are normally crowded out by resident microbes have an opportunity to take over; Disease can result when normal flora are traumatically introduced to an area of the body that they do not normally occur in. Endogenous microflora That is microflora already present in the body, but has previously been inapparent or dormant. Bacterial flora is endogenous bacteria, which is defined as bacteria that naturally reside in a closed system. Exogenous microflora Exogenous bacteria are microorganisms introduced to closed biological systems from the external world; They exist in aquatic and terrestrial environments, as well as the atmosphere; Microorganisms in the external environment have existed on Earth for 3.5 billion years; Exogenous bacteria can be either benign or pathogenic. The origin of Oral Microflora The microflora present in our mouths is acquired from birth, as newborn babies have no bacteria in their mouths; Over time, the oral cavity becomes colonized by the strains of bacteria that eventually form the resident microflora, which establish a permanent population in the mouth; Over time as the individual is further exposed to external sources of bacteria, the biodiversity of the oral cavity increases, to a point where stability is reached. This is termed the climax community. The source of microorganisms The source of these microorganisms mainly comes from saliva, although it also includes the food and water consumed by the individual; This process takes place within the first few hours of life; Majority of children obtain their resident microflora from their mothers, as they often possess identical strains of bacteria; This is known as vertical transmission; Horizontal transmission also takes place as children interact with their peers, and later in life between spouses and partners. Formation of the ecosystem The development of microbial community comprises an alternation of populations; The process starts with the colonization of the environment of the first microbial population; It fills the new space, modify it and makes it convenient for resettlement of new microbial population. This process continues Growing microbial community was enriched with different species living in complex; The process of alternation of populations and enrichment of the community stops only when no available niches for colonization of new microbial species. Climax community The settlement of all econiches occurs relative stability of the system by the microorganisms – called climax community; The stability of the community is based on the homeostasis, including compensatory mechanisms against the effects of various factors. Bacteria during the life cycle Oral colonization begins in the birth canal: Populations on the tongue and mucosa; Established during infancy - include anaerobes; Tooth eruption provides non-shedding surfaces: The “window of infectivity” concept; Colonization from source 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. Microflora at birth At birth, the oral cavity is composed only of the soft tissues of the lips, cheeks, tongue and palate, which are kept moist by the secretions of the salivary glands. At birth the oral cavity is sterile but rapidly becomes colonized from the environment, particularly from the mother in the first feeding. Streptococcus salivarius is dominant and may make up 98% of the total oral flora until the appearance of the teeth (6 - 9 months in humans). Periods of formation of the oral ecosystem The neonatal period: Mostly aerobes; Candida albicans - 54%; Obtained from the mother and the surrounding area; Period before eruption: Facultative anaerobes and anaerobes; Streptococcus - 90%; Actinomyces; Candida albicans. After eruption The eruption of the teeth during the first year leads to colonization by S. mutans and S. sanguis; These bacteria require a nondesquamating (nonepithelial) surface in order to colonize; They will persist as long as teeth remain; Other strains of streptococci adhere strongly to the gums and cheeks but not to the teeth. Period after eruption Anaerobes: Actinomyces; Lactobacillus; Veillonella; Fusiformis; Spirochetes; Candida albicans. Oral surfaces The oral cavity contains various different forms of tissue which have different properties; The microflora in the mouth is distributed according to the different types of surfaces present, which provide each particular species with its optimal growing environment; Certain breeds of bacteria are dependent on specific surfaces to adhere to. Diverse ecological niches in the oral cavity The heterogeneity of tissue types in the oral cavity, such as teeth, tongue and mucosa, means that a variety of sites are available for colonization by oral microorganisms; Each site has unique characteristics and allows those microorganisms best suited to the environment to inhabit the site; The role of microorganisms in a habitat is referred as an ecological niche and a number of ecological niches exist in the oral cavity, including supragingival plaque, subgingival plaque and tongue coating. These ecological niches can be characterized by the environmental factors and the metabolic characteristics of the microbial flora occupying these sites. Mucosal Surfaces: These include the palate, cheek and tongue, which have cells which are constantly replaced due to the normal wear-andtear of the mouth; The different mucosal surfaces also have different properties which contribute to the presence of different types of bacteria: Areas such as the palate contain keratinized squamous epithelium and the softer non-keratinized areas such as the cheeks; The tongue is a unique surface with papillary structures, which provides ample crevices for a wide variety of different strains of bacteria to reside; The presence of these crypts allows for bacteria to be protected from the normal shedding and removal by saliva flow, and hence species not found elsewhere, such as obligate anaerobes, can be found on the dorsum of the tongue. 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. Oral econiches Hard structures – teeth, providing various locations for colonization: Subgingival; Supragingival; Soft structures – mucosa: Cheeks, lips, tongue, gingiva, palate; Keratinized and nonkeratinized mucosa; Epithelium of the gingival sulcus. Mucosa of the gingiva, palate, cheek, floor of the mouth Streptococcus: S. oralis, S. sanguis; Neisseria; Haemophilus; Veillonella. Surface of the tongue Streptococcus salivarius, S. mitis; Veillonella spp.; Peptostreptococcus spp. Gram + rods - Actinomyces spp.; Gram - rods - Bacteroides spp.; Obligate anaerobes black - rods and spirochetes associated with periodontal diseases. Dental econiches Supragingival plaque: On approximal surfaces; On occlusal surfaces - fissures, pits and grooves; Cervical; Gram positive MO; Facultative anaerobes: Streptococcus; Actinomyces; Gram negative: Veillonella, Haemophilus, Bacteroides Subgingival plaque From healthy gingival sulcus isolate: Gram negative rods: Porphyromonas gingivalis; Porphyromonas endodontalis; Prevotella malaninogenica; Prevotella intermedia; Prevotella loesheii; Prevotella denticola. 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. Microorganisms in saliva Depend on the microorganisms of all ecological niches; Microbial composition of saliva is the most similar to the tongue. Oral fluids Oral surfaces washes of two liquids: Saliva; Gingival fluid; They are the basis for maintaining oral ecosystem: Provide water; Nutrients; Adhesion; Antimicrobial factors. Pathogenicity of microflora Microflora usually is non-pathogenic and form an integral part of the host; The presence of friendly resident microorganisms on oral surfaces contributes to the body’s defense against foreign pathogens, which are generally transient and can give rise to harmful infections. This is known as colonization resistance; Function of Microflora Colonization resistance is firstly achieved through the saturation of oral surfaces with preexisting resident bacteria, which reduces the available sites left for the attachment of exogenous organisms; Essential nutrients derived from the saliva and various proteins in the oral cavity are also more effectively utilized by the resident microflora, which inhibits infections via competition for resources; Resident microflora modified the conditions of the mouth It maintains unfavorable conditions for invading microbes; Producing substances such as hydrogen peroxide that actively inhibits pathogen growth; As a result, the harmony between the oral microflora and the body’s natural defenses acts as an effective deterrent to infections, and thus maintains the health of the host. Variations in Microflora Everyone has microflora, but it varies between people depending on the condition of their oral cavity; Not all individuals would have the same microflora profile; Not all bacteria are ubiquitous in all individuals. Gingival crevicular fluid: At the gingival margin, serum flows from the inside the gingiva into the mouth; The flow of this fluid removes foreign microbes which do not adhere to these surfaces; For the resident population, the flow of crevicular fluid provides these organisms with a source of nutrition; They have specialised enzymes which can break down these proteins to provide them with the nutrients necessary for growth. Teeth and artificial hard surfaces: Microbes can accumulate on teeth; This generally forms a dense layer of bioflim known as dental plaque; Fissures and deep grooves in the teeth provide ideal conditions for the formation and build-up of dental plaque; Most of the bacteria found in dental plaque is absent in individuals without teeth, such as babies. The role of saliva in the maintenance of a healthy oral environment By acting as a buffer, saliva maintains the pH of the mouth, ensuring the optimal growth of the resident colonies; Most of the microbes present in the mouth utilize the glycoproteins and proteins in the saliva as their main source of nutrition; Proteins and glycoproteins of saliva are responsible for the formation of the pellicle, a film on the surface of the tooth enamel where the microorganisms of the microflora can adhere to. Salivary flow It is responsible for the removal of non-endogenous bacteria which is unable to adhere to specific sites in the mouth; Saliva acts a collector of these cells and facilitates their removal; Areas which receive markedly less saliva flow, such as deep gingival crevices, proximal spaces and occlusal fissures, tend to have significantly higher levels of bacterial buildup; Saliva is carrying bacteria - the main source of microbial transmission between individuals; Saliva also circulates bacteria within the oral cavity, resulting in re-colonization of oral surfaces where the microflora might be removed via mechanical forces such as cleaning. The normal bacterial flora of the oral cavity Benefit from the host who provides nutrients and habitat; Occupy available colonization sites which makes it more difficult for other microorganisms to become established; Contribute to host nutrition through the synthesis of vitamins, and they contribute to immunity by inducing low levels of circulating and secretory antibodies that may cross react with pathogens; Exert microbial antagonism against exogenous species by production of inhibitory substances such as fatty acids, peroxides and bacteriocins. The oral flora are the usual cause of various oral diseases Abscesses, dental caries, gingivitis, and periodontal disease; If oral bacteria can gain entrance into deeper tissues, they may cause abscesses of alveolar bone, lung, brain, or the extremities; Such infections usually contain mixtures of bacteria with Bacteroides melaninogenicus often playing a dominant role; If oral streptococci are introduced into wounds created by dental manipulation or treatment, they may adhere to heart valves and initiate subacute bacterial endocarditis. DEFINITIONS: • Commensal: An organism living in/on an organism of another species without injuring the host; Parasite: An organism living in/on and at the expense of another organism (the host). Equivalent to a pathogen; Opportunistic Pathogen: A commensal organism that can cause disease in certain circumstances ; Symbiosis: The mutually beneficial association between two organisms ; Aerobic Organism: requiring oxygen for growth and replication; Anaerobe: Organism that grows and replicates in the absence of oxygen; not necessarily killed by oxygen; Strict anaerobe: Anaerobe killed by oxygen; Facultative anaerobe: Organism capable of growth and replication in the presence or absence of oxygen; Various streptococci in a biofilm in the oral cavity. Members of Human Resident oral microflora Gram- positive bacteria Aerobes, facultative anaerobes, microaerophils Anaerobes Streptococcus Peptostreptococcus S. anginosus P. anaerobius S. cricetus P. micros S. gordonii S. mitis S. mutans S. oralis S. rattus S.salivarius S. sanguinus S. sorbinus S. vestibularis Stomatococcus – S. mucilaginosus Important Oral Bacteria 1. Gram Positive organisms: • Rods (bacilli), cocci or irregular shape (pleomorphic); • Oxygen tolerance varies from aerobes to strict anaerobes; • Most are fermentative; • Cell wall has thick peptidoglycan layer (penicillin has effect by interfering production of this layer). Streptococci: Isolated from all sights of the mouth; Large proportion of resident microflora; Majority α-haemolytic. Strep mutans: Associated with caries; Associated with bacterial endocarditis. Streptococcus mutans. Streptococcus mutans is the primary bacterium involved in plaque formation and initiation of dental caries; Viewed as an opportunistic infection, dental disease is one of the most prevalent and costly infectious diseases Strep salivarius: Colonise mucosal surfaces especially the tongue. Strep angiosus: Isolated dental plaque & mucosal surfaces; Seen in maxillofacial infections, brain, liver etc. Strep mitis: Opportunistic pathogens e.g. endocarditis. Dental plaque Scanning electron micrograph illustrating the diversity of microbes in plaque Three important genera: Actinomyces, facultative anaerobe; • Lactobacillus, produce lactic acid, facultative anaerobe, role in dentine caries rather than enamel caries; • Streptococcus facultative anaerobic cocci, produce lactic acid some implicated in caries. Lactobacillus species Lactobacilli in the oral cavity probably contribute to acid formation that leads to dental caries. Important of Streptococci in the oral and their properties Important Oral Species Growth on Prohard duction of surfaces Insol. Extracellular Polysaccharide Production of Acid Cariogenic Endocarditis isolates S mutans + + + +++ + S sanguis + + + ++ ++ S mitior + _ + _ + + _ +++ S milleri + _ + + + S salivarius _ _ + _ _ Distribution of Streptococci in the oral cavity Species Cheek Tongue Saliva Tooth S.mutans _ _ +/- ++ S. mitior +++ +++ +++ +++ S. salivarius _ ++ ++ _ Biofilm formation Biofilms usually occur when one bacterial species attaches specifically or non specifically to a surface, and then secretes carbohydrate slime (exopolymer) that imbeds the bacteria and attracts other microbes to the biofilm for protection or nutritional advantages. Biofilm The classic biofilm that involves components of the normal flora of the oral cavity is the formation of dental plaque on the teeth; Plaque is a naturally-constructed biofilm, in which the consortia of bacteria may reach a thickness of 300-500 cells on the surfaces of the teeth; These accumulations subject the teeth and gingival tissues to high concentrations of bacterial metabolites, which result in dental disease. Gram-negative bacteria Aerobes, facultative anaerobes, microaerophils Cocci Neisseria N. flavescens N. mucosa N. sicca N. subflava Branhamella B. catarrhalis Anaerobes Veillonella V. atypica V. dispar V. parvula 2. Gram Negative organisms Many Gram-negative bacteria found in the mouth, especially in established/subgingival plaque; Cocci, rods, filamantous rods, spindle shaped or spiral shaped; Range of oxygen tolerance but most important strict or facultative anaerobes; Some fermentative, produce acids which other organisms use acids as an energy source, others produce enzymes which break down tissue; Most important Gram negative bacteria: Porphyromonas: P. gingivalis major periodontal pathogen; Prevotella: P. intermedia a periodontal pathogen; Fusobacterium: F. nucleatum periodontal pathogen; Actinobacillus/Aggregatibacter: A.actinomycetemcomitans associated with aggressive periodontitis; Treponema: group important in acute periodontal conditions i.e ANUG; Neisseria; Veillonella. Rods Actinomyces A. naeslundii A. odontolyticus A. viscosus A. israelii A. meyeri Actinomyces and various proteolytic bacteria are commonly found in human carious dentin and cementum, which suggests that they are secondary invaders that contribute to the progression of the lesions Actinomyces: Short pleomorphic rods with branching; Major proportion of plaque; Increase in gingivitis; Associated with root caries. Microbial habitats Habitat Comment Lips, cheek, palate •Biomass limited by desquamation; •Some surfaces have specialised host cell types; Tongue •Highly papillated surface ; •Acts as a reservoir for obligate anaerobes . Teeth and Gingival crevice Habitat Teeth Gingival crevice /Pocket Comment Non-shedding surface enabling large masses of microbes to accumulate (dental plaque biofilm); Teeth have distinct surfaces for microbial colonisation (Smooth surfaces, pits & fissures etc) will support distinct micro flora due to their biological properties; Health - aerobic, disease - anaerobic. Cheeks, lips, palate -descquamation; -Saliva is major influence; -Microflora has small deversity; -Sterptococcus spp. Predominate; -Some periodontal pathogens persist by invading buccal cells Teeth - Non-shredding surfaces; -Stagnant sites – food impaction possible; -Diverse microflora –site variation; -Many obligate anerobes; -Influenced by saliva and GCF; -Streptococcus, Actinomyces, Veillonella, Fusobacterium, Prevotella, Treponema, unculturable organisms. Tongue High papillated surface; Some anaerobic sites; Desquamation; Diverse microflora; Facultative and obligate anerobes; Streptococcus, Actinomyces, Rothia, Nisseria, some Gr- anaerobes. The tongue A shedding surface; Structure includes crypts and fissures; Favorable for Gm- anaerobes. Smooth surface plaque Approximal plaque Fissure plaque Sub-gingival plaque Gingival crevice plaque Supra-gingival plaque Factors Affecting Growth of Microorganisms in the oral cavity 1. Temperature; 2. REDOX Potential / Anaerobiosis; 3. pH; 4. Nutrients (endogenous & exogenous (diet); 5. Host Defences (Innate & Acquired immunity); 6. Host genetics (changes in immune response etc); 7. Antimicrobial agents & inhibitors. Temperature Relatively constant - 34˚-36 ˚; Life allows a large number of microorganisms; The temperature is variable on the teeth and mucosa; When eating microorganisms colonized there are exposed to extreme temperatures; No evidence of metabolic disorders in microorganisms to these short periods. pH, or hydrogen ion concentration Affects their metabolism; In the mouth pH varies between 6.7 and 7.3; It is maintained by saliva through: salivary flow; buffer systems; In an acid medium in the eco niches are developed: Lactobacillus; Str. mutans; In the gingival sulcus pH is alkaline -7.5 - 8.5; In the gingival fluid pH is 7,5 - 7,9 . It develops periodonto-pathogenic microorganisms. Redox potential and anaerobiosis Under the action of the enzymes some of the components are subjected to oxidation, and others to reduction; These processes depend on the oxygen and are redox potential; In a predominance of reduction processes have a negative redox potential and develop anaerobic microorganisms; With positive redox potential are buccal and palatal mucosa and the back part of the tongue; With negative redox potential are approximal surfaces, fissures and gingival sulcus. Nutrients Desquamated epithelial cells; Gingival fluid; Saliva; Residues from host`s food; Products of metabolism of other microbial species. Host factors Host defense mechanisms; Hormonal changes; Stress; Genetic factors. 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. Host defense mechanisms Removing the microorganisms through stimulation of salivary flow; Specific protection: SIg A; Nonspecific protection: Mucin; Antimicrobial factors: Lysozyme; Lactoferrin; Salivary peroxidase; Histatine-rich peptides; Cistatin; Leukocytes; Complement. Removing of the microorganisms The majority of microorganisms in the mouth is removed by washing action of saliva; Salivary flow is stimulated by muscle activity of lips and tongue. Specific protection - Secretory IgA system SIgA-antibodies reduce microbial adhesion to enamel epithelium and through: Neutralizing enzymes microorganisms; Neutralize toxins and viruses; Synergy with other antibacterial agents such as lysozyme, lactoferrin, peroxidase and mucin; Protects the mucosa of penetration of antigens; Helps complement activation. Mucin Provides a protective coating of enamel and mucosa; Catches microorganisms and antigens like in a trap; Limits microorganisms penetration into tissues; Eliminates microorganisms with continuous updating of mucin layer combined with washing action of saliva flow; As part of peliculla protects teeth from demineralization. IgG, (IgA, IgM) complement polymorphs. -----------------Gingival crevicular fluid Acquired pellicle sIgA saliva Flow rate; Mucin/agglutinins; Lysozyme-proteaseanion system; Lactoferrin; Salivary peroxidase system; Histatin-rich polypeptides. Flora of normal, healthy dentate mouth % (approx) 85% Remainder Bacteria Streptococci Veillonella Gram positive Diptheroids Gram negative anaerobic rods 5-7% Neissaeria 2% Lactobacilli 1% Staphylococci & Micrococci Other bacteria, fungi, protozoa & viruses Difference between mature supra & sub-gingival plaque Characteristic Supra gingival Sub gingival Grams stain Gram + or -ve Mainly Gram –ve Morphotypes Cocci, branching rods, filaments & spirochaetes Mainly rods & spirochaetes Energy Metabolism Facultative, some anaerobic Mainly anaerobic Energy source Mainly ferment carbohydrate Many proteolytic forms Motility Few Many Pathology Caries & gingivitis Gingivitis & periodontitis Oral flora changes with age Time during a lifetime MAJOR COMPONENTS & CHANGES IN ORAL FLORA Newborn Oral cavity sterile. Soon colonised by facultative and aerobic organisms; esp S. salivarius 6 months Flora becomes more complex & includes anaerobic orgs eg. Veillonella sp. & Fusobacteria Tooth eruption Increase in complexity. S sanguis, S mutans and A viscosus appear. New habitats include hard surfaces and gingival crevice. Child to adult Various anaerobes frequently found inc. Members of the Bacteroidaceae. Spirochaetes isolated more frequently Loss of teeth Disappearance of S mutan, S sanguis, spirochaetes and many anaerobes Dentures etc Reappearance of bacteria able to grow on hard surfaces 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. Ecological succession 1° colonizers (Gram+) Streptococci bind pellicle proteins from saliva DENT 5302; 2° colonizers (Gram-) Bridge species - F. nucleatum Bind other bacteria 3° colonizers (Gram-) Porphyromonas gingivalis Biofilm on tooth surfaces - 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 singlespecies microcolonies; Growth requires “quorum sensing”. In vitro biofilm by Streptococcus sanguinis SEM by Tracy Grossman 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. 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. 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 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. 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. Criteria for colonization Adhesion Major milestone allowing resistance of the microorganisms against washing action of the saliva; Provides from adhesins of the microbial surface and receptors on oral surfaces. microbial adhesins Constructed of: Polysaccharides; Lipoteichoic acid; Glycosyltransferase; Carbohydrate-binding proteins; They are located in the cell wall in the form of: Fimbriae; Fibrils; Capsules. Receptors on the oral structures salivary components bacterial components Mucin; Glycosyltransferase; Glycoproteins; Glucans. Amylase; Lysozyme; IgA, IgG; Proline-rich proteins. MECHANISMS OF ADHESION BY ORAL BACTERIA Adherence mechanisms of oral bacteria are essential to bacterial colonization of the oral cavity; In their absence, bacteria become part of the salivary ﬂow and are swallowed; Consequently, oral bacteria have evolved several mechanisms to fulﬁll this role. The ﬁrst organisms to attach are the primary (early) colonizers and primary colonization is mediated through speciﬁc or non-speciﬁc physio-chemical interactions with components of an adsorbed, organic conditioning ﬁlm; Mechanism of adhesion Nonspecific chemical and physical interactions: Example: Lipoteichoic acid at the microbial surface interacts with the negative charged components of the host by Ca, H ions or hydrophobic coatings; Then act strong stereo-chemically interactions; Microorganisms can adhere to the surface of the already colonized microorganisms – coaggregation. Inter-bacterial coaggregation 1μM 1μM 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. Mechanism of the coaggregation Coaggregation interactions are believed to contribute to the development of bioﬁlms by two routes: The ﬁrst route is by single cells in suspension speciﬁcally recognizing and adhering to genetically distinct cells in the developing bioﬁlm; The second route is by the prior coaggregation in suspension of secondary colonizers followed by the subsequent adhesion of this coaggregate to the developing bioﬁlm; In both cases, bacterial cells in suspension (planktonic cells) speciﬁcally adhere to cells in the bioﬁlm in a process known as coadhesion. Ecological signiﬁcance of bacterial coaggregation Speciﬁc coaggregation processes are likely to have an important ecological role as an integral process in the development and maintenance of mixed-species bioﬁlm communities; Allows adhesion of the microorganisms, who are not able to direct adhesion; Strengthens bacterial attachment; Increases the stability of the plaque matrix. Bacterial attachment to a surface can be divided into several distinct phases Primary and reversible adhesion; Secondary and irreversible adhesion; Biofilm formation. Adhesion is dependent on: A contact: Start of interaction; A dose: There is a certain amount of micro-organisms; Frequency of exposure: Partial colonization; Adsorption: By electrostatic forces to the surface of pellicle; With specific receptors on the cell surfaces; With fibrillere coupling end. Retention Adaptation; Growth; Reproduction; Accumulation. Ecological relationships Independence Life free from influences, management or control of other organisms; Favorable relationships – commensal; One type of benefit, but others did not suffer; Pathological. Microbial relationships There are a complex of relationships between different microbial species: Neutral; Antagonistic microbial interactions; Synergistic. Neutral Relationship between two species living together without affecting each other - neither favorable nor unfavorable. Synergy Relationships between the two microbial species living together, wherein both are mutually favored, leading to enhancement of the effects of each of these. Example of synergy Various microorganisms may cooperate to use substances which are not able to metabolise themselves; P. gingivalis and F. Nucleatum are unite to hydrolyze casein. Antagonism Relationship of two microbial species living together, yielding inhibition of their function, and less effect on the independent operation of each one of them. Mechanisms of microbial antagonism Competition for adhesion receptors; Competition for nutrients; Production of inhibitory substances: Organic fatty acids; Hydrogen peroxide: Dairy complex; Antibiotics; Enzymes; Bacteriocins. An example of microbial antagonism S. mutans and Lactobacillus produce lactic acid; It is caused an acidic environment; It inhibits the growth of S.sanguis and S. oralis, and gram negative microorganisms; Separation bacteriocins inhibit gram positive microorganisms. Host – microorganisms relationships Symbiosis; Comensal; Mutualism; Parasitism. Symbiosis Symbiotic relationships include those associations in which one organism lives on another, or where one partner lives inside the other . Commensalism Commensalism describes a relationship between two living organisms where one benefits and the other is not significantly harmed or helped; Commensal relationships may involve one organism using another for transportation or for housing , or it may also involve one organism using something another created, after its death. Mutualism Mutualism is any relationship between individuals of different species where both individuals benefit; During mutualistic symbioses, the host cell lacks some of the nutrients, which are provided by the endosymbiont; As a result, the host favors endosymbiont's growth processes within itself by producing some specialized cells; These cells affect the genetic composition of the host in order to regulate the increasing population of the endosymbionts and ensuring that these genetic changes are passed onto the offspring via vertical transmission. Mutualistic relationship Coaggregating cells can possess a combined metabolic advantage over single cells; Mutualistic relationship that allowed each to grow where neither grew alone; Example: coaggregating partnership of S. oralis and Actinomyces naeslundii formed a nutritionally beneﬁcial, mutualistic relationship that allowed each to grow where neither grew alone. Parasitism A parasitic relationship is one in which one member of the association benefits while the other is harmed; This is also known as antagonistic or antipathetic symbiosis. Influence of external factors Diets; Oral hygiene and antimicrobial agents; Drugs and diseases. Diets Food high in carbohydrates increases the development of Str. mutans and Lactobacillus; A diet high in protein inhibits the development of Lactobacillus. oral Hygiene Good oral hygiene: Facultative aerobes; Acidogenious microorganisms; Neglected oral hygiene: Anaerobes; Proteolytic microorganisms. Antimicrobial agents Fluorides have depressing effect on microbial p- ment; Reducing sugar transport; Reduce their glycolytic activity; Suppress the acid tolerance of gram + microorganisms, Drugs and diseases Patients with reduced salivation have reduced capacity to remove sugars, reduced buffering capacity and reduced protection; Antibiotics suppress the resident microflora, leading to over-development of antibiotic resistant species Candida and allow colonization of exogenous pathogens such as Enterobacteriaceae. Beneficial effect of the microflora on the body Prevention of exogenous and endogenous infections; Stimulating an immune response; Renewal and healing of the epithelium; Delivery of certain biological factors to the host. Adverse effects Source of endogenous infection; Change the local environment to physiological conditions facilitating exogenous microbial overdeveloped; Create hypersensitivity in macroorganism to microbial agents. Protective mechanisms in the mouth Protective mechanisms in the mouth 1. Protection of enamel; 2. Protection of the oral mucosa; 3. Protection of the oral physiological fluid; 4. Protective mechanisms of microbial life. Enamel Protection Protects underlying tooth structure and tissue; Provides the physiological mineralization; Opportunity for remineralization. Protection of the oral mucosa Protection of the epithelium by keranitization cell exfoliation Renewal and regeneration intercellular connections Relationship between cell layers Langerhans cells receptors Protection of other layers of the mucousa Basement membrane: Lamina Propria: Connective tissue cells; Blood element;s Collagen fibers; Matrix; Receptors; Innervation. Protection of the physiological content - saliva Mechanical washing; Salivary anti-caries activity; Buffer systems; Immunoglobulins; Enzymes. Protective mechanisms of gingival fluid Microphages; Macrophages; Immunoglobulins; Urea; ALP; Lysozyme; Lactoferrin; Epithelial timoactive factor. Protective mechanisms of microorganisms Prevention of exogenous and endogenous infections; Renewal and healing of the epithelium; Stimulating an immune response; Development of oral tolerance; Delivery of certain biological factors to the host.