Presented by – Dr. Priyanka Singh MDS-IInd year, Department of Oral Pathology, SPIDMS.
CONTENTS
Introduction Prokaryotic cell & eukaryotic cell Bacteria Staining of bacteria Anatomy of bacteria Growth and multiplication of bacteria Growth regulation Flora of the oral cavity Dental plaque bio film Calculus formation Role of oral flora in systemic infection Conclusion
Introduction
Human beings like other animals, harbour a wide array of microorganisms either on or in their bodies. A knowledge of normal flora of body is essential to an understanding of the interaction between human beings and their pathogen laden environment.
The oral cavity is accessible to the introduction of many different types of microorganisms and consist of large number of microorganisms including mycoplasma, fungi, protozoa nad viruses.
Microorganisms – A heterogeneous group of several distinct classes of living beings – Originally classified under plant and animal kingdoms – 3rd kingdom protista was formed for them – Kingdom protista – Prokaryotes – bacteria and blue-green algae Eukaryotes – fungi, algae, slime moulds and protozoa
Prokaryotic cell & eukaryotic cell Characteristic Nucleus
Prokaryote Absent
Diameter of cell Cytoskeleton
Present About 1µm
Absent
Cytoplasmic organelle DNA content
Eukaryote
10-100µm Present
Absent Present
Unorganized
Organized
Chromosomes Single circular Multiple linear DNA molecule DNA molecule
BACTERIA
Prokaryotic microorganisms
Do not contain chlorophyll
Unicellular
Do not show true branching except in Actinomycetes
Staining of bacteria
Live bacteria do not show much structural detail due to lack of contrast
Stained to produce colour contrast
Depending upon the effect of stains on the viability of microorganisms – Supravital staining Toxic Kill the cells Intravital staining Nontoxic Cells remain viable
Routine methods Simple stain – Methylene blue & basic fuchsin – Do not stain bacterial capsules – Imparts same colour to all bacteria
Negative stain – India ink or nigrosin – Uniformly colors background – Unstained bacteria in contrast – Useful for demonstration of bacterial capsules – Spirochetes which are very slender can be stained
Impregnation – Thin cellular structures can be seen – Thickened by impregnation of silver on the surface – Spirochetes and bacterial flagella
Differential stains – Imparts different colour to different bacteria or bacterial structures – 2 most widely used differential stains are – Gram stain Acid fast stains
GRAM’S STAIN Devised by – Christian Gram (1884)
Procedure – – Primary staining with pararosaniline dye e.g. – crystal violet, methyl violet or gentian violet – Dilute solution of iodine – Decolorisation with an organic solvent e.g.- ethanol, acetone or aniline – Counterstaining with a dye of contrasting colour e.g. – carbol fuchsin, Safranin or neutral red
Purpose of gram stain Identification of bacteria Therapy of bacterial infections - gram +ve bacteria are more susceptible to penicillins
Principle GRAM +ve
Relates to the permeability of the bacterial cell wall and cytoplasmic membrane to the dye Gram –ve cells permits the outflow of the dye during decolorisation Gram positive cells resist decolorisation and also has got a more acidic protoplasm and so retain the primary stain, appearing violet Gram – ve bacteria are decolorised by organic solvents and so take the counter stain, appearing red GRAM -ve
ACID FAST STAIN – Discovered by Ehrlich – after staining with aniline dyes tubercle bacilli resist decolorisation with acids – Modified by Ziehl and Neelson – Smear stained with carbol fuchsin with application of heat Decolorised with 20 % sulphuric acid Counterstained with a contrasting dye i.e. methylene blue Acid fast bacteria retain fuchsin (red) colour while others take the counter stain
SIZE OF BACTERIA
0.2 – 1.5 m in diameter 3 – 5 m in length
SHAPE OF BACTERIA -
Cocci (kokkos – berry) – spherical or oval Diplococci (in pairs) Streptococci (in chains) Grape – like clusters (staphylococci)
Bacilli (bacillus – rod) – rod shaped Streptobacilli (in chains) Corynebacteria (arranged at angles to each other, presenting a cuneiform or Chinese letter pattern)
Vibrios (comma shaped, curved rods) – characteristic vibratory motility Spilrilla – rigid spiral forms Spirochetes (speira=coil ; chaite=hair) – flexuous spiral forms
Actinomycetes (actis = ray, mykes = fungus) – branching filamentous bacteria – radiating rays of the sun like appearance - due to presence of rigid cell wall
Mycoplasma – Do not posses a stable morphology – protoplasts, spheroplasts or L-forms – Deficient cell wall due to drugs like penicillin – Round or oval bodies
Some are pleomorphic (pleo : many, morphic shaped)
ANATOMY OF BACTERIA
Consists of – Outer layer or cell envelop Cell wall Plasma membrane
Inner cytoplasm and cytoplasmic inclusions Ribosomes and Mesosomes Granules Vacuoles Nuclear body
Some bacterial cell – – Enclosed by a viscid slimy layer or organized as a capsule – Filamentous appendages protruding from the cell surface – flagella – organs of locomotion – Fimbriae – organs of locotion
STRUCTURES EXTERNAL TO CELL WALL Flagella
Whip like long filaments Used for locomotion Guide the bacteria towards nutritional and other sources Composed of subunits of a single protein flagellin 3 parts – Filament Hook Basal body 3-20 m long 0.01-0.013 m in diameter
Flagella of different bacteria have same chemical composition but are antigenically different
Flagellar antigens induce specific antibody in high titers
Flagellar antibodies are useful in serodiagnosis
Flagella – – Peritrichos – Polar – Monotrichos – Lophotrichos – Amphitrichos
Presence is usually detected by motility of bacteria
Motility – spreading type of growth on a semisolid agar medium
Present in all motile bacteria except in spirochetes (move with axial filament – a flagellum like structure)
Fimbriae & Pili – – Fine hair like surface appendages – Shorter than flagella – 0.5 m long and 10 nm thick – Present mainly on gram –ve bacteria
– Consists of self aggregating monomers of pilin – Adhesion of bacteria to receptors on human cell surface – first step in initiation of infection – Cause hemagglutination – a method of detection – Antigenic in nature
Sex pili – – A specialized type of fimbriae – Forms attachment between male (donor) and the female (recipient) through conjugation tube – Genetic material is transferred from donor to recipient cell
Glycocalyx (slime layer)
Loose undemarcated
Polysaccharide or polypeptide covering
Adhesion of bacteria to structures like oral mucosa, teeth, etc.
E.g. - leuconostoc
Capsule
An amorphous gelatinous layer
Surrounds entire bacteria
Composed of mainly polysaccharides
Antigenic
Demonstrable by serological methods by antigenic differences of sugar in polysaccharide capsule
Mediates adhesion of bacteria to human tissues
Hinders or inhibits phagocytosis
Quelling reaction – – Described by Neufeld (1902) – Presence of antiserum against capsular polysaccharide, swelling of the capsule – Protects the bacteria from deleterious agents in nature. – Helps in virulence of pathogenic bacteria – Inhibits phagocytosis – E.g. – streptococcus mutans
Spores
E.g. – bacillus & clostridium
Highly resistant resting stages
One bacteria forms one spore – on germination forms one vegetative cell
Not a method of reproduction
Called endospores as are formed inside the parent cell
Cause – in response to adverse conditions
Contains bacterial DNA, cytoplasm, cell membrane, peptidoglycan, water and a thick, keratin like coat – Consists of calcium dipicolinate Resistant to heat, dehydration, chemicals
Metabolically inert
Remain dormant for many years
Types of spores – Central, budging – Central, not budging – Sub terminal, budging – Sub terminal, not budging – Terminal, budging – Terminal, not budging
When spores are transferred to favorable conditions
Spore loses its refractility
Swells
Spore wall is shed
Germ cell appears
Elongates to form vegetative bacterium
Can be destroyed by autoclaving at 120 for 15 mins
Bacillus stearothermophilus – ensures sterilization efficacy of autoclaves
Appears unattained in gram stain
Can be stained by Ziehl-Neelson stain due to its acid fast nature
Cell wall
Confers rigidity upon bacterial cell
Multilayered structure outside protoplasmic membrane
Porous and semi permeable
Important in virulence and immunity
Demonstrable by plasmolysis – Hypertonic solution Osmosis Cytoplasm loses water Shrinkage of cell But cell wall retains its original shape
Inner layer – Peptidoglycan – Scaffoldings formed by Nacetyl glucosamine and Nacetyl muramic acid – Present in chains which are cross-linked by peptide chains
Outer layer – Depends upon gram staining property of bacteria
Bacteria with deficient or defective cell walls
Mycoplasma – Do not posses cell wall – May cause human disease e.g. – pneumonia
L - forms – – – –
E.g. – streptobacillus moniliformis Swollen cells Named after Lister Institute, London Develops – Spontaneously Presence of penicillin Other agents that interfere with cell wall synthesis Cause urinary and suppurative infections
Spheroplasts (gram –ve bacteria) & protoplasts (gram +ve bacteria) Lack cell walls Cannot replicate on laboratory media Unstable Osmotically fragile Require hypertonic conditions for maintenance Produced in laboratory by action of enzymes or antibiotics Spheroplast
Protoplast
Conversion of bacteria into protoplast and spheroplast
Cytoplasmic membrane
Thin inner layer lining the inner surface of the cell wall
5 – 10 nm thick
Separates cell wall from cytoplasm
Semi permeable membrane
age through the membrane depends upon– Molecular size of the particles – Presence of specific enzymes (permeases)
Consists of – – Lipoprotein with small amounts of carbohydrate – Sterols are absent except in mycoplasma
Cytoplasm
Colloidal system of organic and inorganic solutes in a viscous watery solution
Differs from eukaryotic cytoplasm – – Absence of protoplasmic streaming – Absence of endoplasmic reticulum and mitochondria – Stains uniformly with basic dyes – Contains ribosome, mesosome, inclusions (volutin granules or Babes Ernst granules) and vacuoles, metabolites and various ions
Ribosomes
Centers of protein synthesis
Smaller than in eukaryotes
Sedimentation rate – 70 s
Integrated in linear strands of mRNA to form polysomes
Mesosomes (chondroids)
Convoluted or multilaminated structures
Invaginations of plasma membrane into the cytoplasm
More prominent in gram + ve bacteria
Principal site of respiration
Analogous top mitochondria of eukaryotes
Nucleus
Demonstrated by acid or ribonuclease hydrolysis and subsequent staining
Oval or elongated
No nuclear membrane or nucleolus
Genome consists of a single molecule of double stranded DNA arranged in a form of a circle
Chromosome is haploid and replicates by simple binary fission
Plasmids or episomes – Extra nuclear genetic material – Carried by some bacteria – Consists of DNA – Cytoplasmic carriers of genetic information
GROWTH AND MULTIPLICATION OF BACTERIA
Divide by binary fission
On reaching certain size Nucleus divides Cell divides to form 2 daughter cells
Generation time or population doubling time – time interval between two cell divisions –generally vary from 20 mins in E. coli to 24 hrs in M. tuberculosis
Can be arrested by deficiency of nutrients Accumulation of toxins Antibiotics like penicillin and acriflavin
Bacterial growth curve
Growth regulation
Regulated by nutritional environment – Intracellular Extracellular
Intracellular End-product inhibition – first enzyme in metabolic pathway is inhibited by end product of that pathway Catabolic repression – enzyme synthesis is inhibited by catabolites
Extracellular factors
Temperature – – Can grow in a wide range of temperature – Mesophiles – 25 – 40 C – majority of bacteria Thermophiles – 55 – 80 C Psychrophiles – below 20 C
pH – Optimal growth at physiological pH – 7.2 – 7.4 Vibrio cholera – high degrees of alkalinity
Oxygen requirement – Obligate aerobes – require oxygen – ATP generating system is oxygen dependent – e.g.-M. tuberculosis Facultative anaerobes – can survive in the absence of oxygen - e.g.- Strep. Mutans & E. coli Obligate anaerobes – do not require oxygen e.g. – Porphyromonas gingivalis Microaerophiles – grow best at low oxygen concentration e.g. – Campylobacter fetus
Light Grows well in absence of light Sensitive to ultraviolet and other radiations
Osmotic effect – More tolerant to osmotic variation Due to mechanical strength of the cell walls Sudden exposure to hypertonic solutions – plasmolysis – mostly in gram –ve bacteria Plasmoptysis – sudden transfer from conc. Solution to distilled water – swelling and rupture of cell
Moisture and drying – Moisture is essential Drying – lethal Highly sensitive for drying – Treponema palladium
Antimicrobial therapy – Broad-spectrum antibiotics Wipe out most of the endogenous flora Favor fungal infection
FLORA OF THE ORAL CAVITY
Gram positive cocci Streptococcus Gram positive Non motile, flagella usually absent Occur in chains Generally non capsulated Facultative anaerobes Causes α haemolysis Selective medium – mitis salivarius agar Various types are – – Mutans group
High, convex, opaque colonies Produce extra cellular polysaccharide Site – tooth surfaces Causes – Dental caries
– Salivarious group
Appear in mouth of infants as early as 18 hrs to 1 month after birth. Large mucoid colonies Produce extra cellular fructans (polymer of fructose with a levan structure) Site – dorsum of tongue and saliva (especially in infants) Not a major oral pathogen – Mitis group
Small, rubbery or non adherent colonies Site – tongue and cheeks Causes – Dental plaque bio films, Dental caries, Infective endocarditis
– Anaerobic streptococci
Strict anaerobes Slow growing Usually non hemolytic Site – teeth (carious dentin) Causes – Periodontal or Dentoalveolar abscess
Gram positive rods and cocci – Actinomyces
Strict and facultative anaerobes Branched – sun ray appearance Ferments glucose – characteristic patterns of short chain carboxylic acids Causes – Dental caries (especially root caries) & Gingivitis Actinomycosis
– Lactobacillus Micro-aerophiic Aciduric (optimal ph=5.5-5.8) Selective medium – Rogosa agar Catalase negative Site – common oral inhabitats Causes – Dental plaque bio film Advancing front of dental caries – Eubacterium Pleomorphic Obligatory anaerobes Site – plaque bio films and calculus Causes – Role in dental caries
– Propionibacterium
Strict anaerobe Produces propionic acid from glucose Causes – Root surface caries and Dento-alveolar infections
Gram negative cocci – Neisseria Facultative anaerobe Site – tongue, oral mucosa, saliva and early plaque Causes - not a major oral pathogen – Veillonela Strict anaerobe Site – tongue, saliva and plaque bio film
Gram negative rods (Facultative anaerobe an capnophilic genera) – Hemophilus
Growth on heated blood agar Requires X (haemin) and V (NAD) for growth Site - oral mucosa, saliva and plaque bio film Causes – Dentoalveolar infections, Acute sialadenitis Infective endocarditis
– Actinobacillus Produces virulent factors – leucotoxin, epitheiotoxin, cardotoxin Site – periodontal pockets Causes - Periodontal disease
Gram negative rods (obligate anaerobic genera) Produce brown black pigment on blood agar Black pigmented anaerobes – Prevotella
Pleomorphic rods Non-motile Strict anaerobe Require Vit. K and haemin for growth Site – gingival crevice and sub gingival plaque Causes – Chronic periodontitis and Dentoalveolar abscess
– Fusobacterium Sender, cigar shaped Gm –ve rods with rounded ends Site – gingival crevice and tonsils Causes – periodontal infections acute ulcerative gingivitis dentoalveolar abscess
– Treponema
Motile Helical cells (large, medium, small) Strict anaerobes Require enriched media with serum to grow Site – gingival crevice Causes - acute ulcerative gingivitis destructive periodontal disease
Other bacterial genus present are –
Angiogenous group Stomatococcus group Propionibacterium group Actinobacillus group Eikenella group Capnocytophaga group Leptotrichia group Wolinella
Fungi and protozoa present in the oral cavity are – – Fungi – Candida albicans Candida tropicalis Candida pseudo tropicalis Cryptococcus – Protozoa Entameoba gingivalis
Factors in the oral cavity affecting growth of micro flora
Anatomical factors Shape of teeth Malalignment of teeth Poor quality of restorations Non-keratinized sulcular epithelium – Difficult to clean – Favors accumulation of bacteria
Saliva – – Consist of proteins, glycoproteins and various other ions & metabolites – Modulate bacterial growth by – Adsorption on the tooth surfaces – pellicle – facilitates bacterial adhesion Acts as a primary source of nutrition Helps in aggregation of bacteria – plaque formation Inhibits growth – Due to presence of defense factors – – Lysozyme, lactoferrin, IgA, etc – Bactericidal and fungicidal
Gingival crevicular fluid – Continuous but slow flow Flushes microbes out of the crevice Maintains pH level Provide defense factors – IgG ( also IgM & IgA) Causes phagocytosis due to presence of neutrophils
Microbial factors – – Interaction of bacteria with each other may promote or suppress the neighboring bacteria by – Competition for receptors for adhesion Production of toxins by one may destroy same or other bacterial species Production of metabolic end products – lowering of pH Co-aggregation to form corn-cob formation with – Same (homotypic) or Different (heterotypic) species
Distribution of oral flora
Oral micro flora does not represent a homogenous collection of organisms through out the oral cavity but it is localized in 3 different foci – – Gingival crevices – Dorsum of tongue – Areas that are not cleaned by mastication – Other areas that may be involved are – Lips Palate Saliva Cheeks or buccal mucosa
Gingival crevices – – The gingival crevice is not sterile, but harbors a microbial flora in both health and disease – In healthy sulci, gram positive cocci are major macrophyte and compose almost 2/3rd of total flora. Filamentous forms and all spirochetes fusiforms are found. – In diseased conditions, gram positive as well a sgram negative rods such as capnocytophaga, bacteroids, compylobacter, fusobacterium can be isolated.
Tongue – – The tongue with its complex surface structures (crypts and papilla) forms large surface areas that growth of a wide variety of bacteria and serves as a reservior of microorganisms in the mouths. – Predominant cultivative bacteria isolated from the tongue falls in the following list of microorganisms which are -
– – – – – – – – – – –
Facultative streptococci Veillonelle Facultative diptheroids Anaerobic diptheroids Bacteroides Peptostreptococcus Neisseria Vibrio Fusobacterium Provetella intermedia Provetella melanogenicus
Lips – – The lips form the border between the skin flora and consists predominantly of staphylococcus. – Facultative anaerobic micro flora comprises a large part of flora of the lips. – Veillonella and neisseria have been found – If commissures are moistened by saliva, then Candida albicans, Staph. aureus and Strept. Pyogenes are common.
Cheeks/buccal mucosa – – Predominant bacteria are – Strept. sanguis and Strept. salivarious. – Yeasts may also be isolated from carriers. Other organisms may be hemophilus and neisseria sp.
Palate – – Hard palate s a streptococcal flora resembling the cheek flora – Haemophillus and lactobacilli are also found regularly – Yeasts and lactobacilli are increased dramatically, in some denture wearers and flora may alter substantially when the palate is protected from the action of tongue and saliva by denture base. – Soft palate – also harbor respiratory tract bacteria like hemophilus, corynebacterium, neisseria.
Saliva – – Adult human saliva contains approx. 6 billion micro flora and include – Streptococci Peptostreptococci Veillonella Corynebacteria Neisseria Nocardia Fusobacteria Lactobacilli Actinomycosis Spirochetes Fungi like Candida and Cryptococci
Teeth – – On eruption, teeth are covered by organic structure of embryonic origin. – After eruption, organic deposits form on the surfaces of teeth which contain organic acids, bacterial and cytotoxic agents. – Williams demonstrated microscopically, the presence of thick mass of microorganisms covering the surface of teeth – G.V. Black introduced the term gelatinous microbial plaque to describe microbial colonies on the surface of teeth. – Microorganisms do not attach themselves directly to mineralized tooth surface as teeth are covered by teeth by an acellular proteinaceous film.
DENTAL PLAQUE BIOFILM
“Dental plaque is a tenacious microbial community which develops on soft and hard surfaces of mouth, comprising of living, dead and drying bacteria and their extra cellular products, together with host compounds mainly derived from saliva.”
Composition – – Organisms surrounded by organic matrix – 30% – Matrix acts as a food reserve – for adhesion of further bacteria – Composition varies – At different sites on same tooth At same site on different teeth At different time on the same tooth site
Distribution – – Found on dental surfaces and appliances – Mainly in absence of oral hygiene – May be Supra gingival – mainly aerobic bacteria Sub gingival – mainly anaerobic bacteria
Plaque has been found to contain the following microorganisms in percentages – – Supragingival plaque Facultative streptococcus – 87% Facultative diphtheroids – 23% Anaerobic diphtheroids – 18% Peptostreptococcus – 13% – Subgingival plaque Veillonella – 6% Bacteroids – 4% Fusobacteria – 4% Neisseria – 3% Vibrio – 2%
Stages of Plaque Bio film formation Pellicle formation – Thin layer of salivary glycoproteins Deposited on the surface Forms within minutes of exposure to the oral environment Oral bacteria attach to pellicle for the initiation of plaque formation
Transport – Bacteria are transported to pellicle site by – Natural salivary flow – Brownian motion – Chemo taxis
Long range interactions – Between microbial cell surface and pellicle coated tooth Called pioneer group-gram positive cocci and rods Through Vander waals forces Reversible
Short range interactions – Polymer bridging between organisms and the surface After which organisms come and accumulate – to increase the microorganism mass Forms corn-cob arrangements Secondary invaders - gram negative cocci and rods and later by fusobacteria, spirals, and spirochetes Irreversible
Co-aggregation or co-adhesion – – Fresh Bacteria now attach to the primary invaders – May be of same genus or different genera – Process of plaque bio film mass reaches a critical size at which – Deposition = Loss of bacterial accumulation – Climax community – The bacteria in climax community- detached – Plank tonic phase – suspended in saliva – transported to new colonization site
Bio film formation – – – –
Due to resultant confluent growth Matures in complexity as time progresses Occurs at inert surfaces e.g.- tooth enamel, dentures, etc. Organisms may be arranged in columns or mushroom shaped structures – Water channels that carry metabolites and nutrition are interspersed within the organic matrix
“A bio film is defined as a complex functional community of one or more species of microbes encased in and exopolysachharide matrix and attached to one another or to solid surface”
Major component of bio film is extra-cellular matrix – Microbial polysaccharides Salivary glycoproteins
Calculus formation
Due to deposition of calcium and phosphate ions from saliva within dental plaque
Bacteria in plaque as seeding agents of mineralization
Accelerated by bacterial phosphatases and proteases – degrade calcification inhibitors (Statherin and proline rich proteins) in saliva
– Formation of insoluble calcium phosphate crystals – Coalesce to form a calcified mass of plaque – calculus – Mature calculus – Mineralized material – 80% Organic compounds – 20%
Structure of calculus – Predominant flora – cocci, bacilli, and filaments – outer layers – Supra gingival calculus – gram positive bacteria – Sub gingival – gram negative bacteria – Outer surfaces of calculus – cocci attach grow on surface of filamentous microorganism corn cob arrangements
– Filaments orient themselves at right angles to enamel surface – Palisade effect (like books on a shelf) – Cytoplasm of bacteria is Reduced Contains glycogens like food storage granules Ready source of nutrition during periods of adversity – Has enough surface – Porous – Ideal reservoir for bacterial toxins – harmful to periodontium
Role of oral flora in systemic infection
Plaque related oral diseases, especially periodontitis may alter – – Cardiovascular disease Infective endocarditis Coronary heart disease – Bacterial pneumonia – Diabetes mellitus – Low birth weight babies
According to ‘focal infection theory’ – 3 mechanisms lining oral infections to secondary systemic are –
1.
Metastatic infection
2.
Gain entry into vascular system through breach in oral vascular barrier E.g. - bacteraemias during tooth extractions
Metastatic injury
3.
disease
Products of bacteria (catalytic enzymes, endotoxins, exotoxins) gain access to cardiovascular system
Metastatic inflammation
Caused by immunological injury due to antigens produced by oral organisms – enter blood stream from oral route - react with circulating antibodies - immune mediated disease
Factors for periodontitis predispose risk for systemic disease
Sub gingival bio films – reservoir for gram –ve bacteria – continuous source of lipopolysachharides (endotoxin) causes – Induction of major vascular responses Up regulates endothelial cell adhesion molecules Secretion of interleukin-1 and tumour necrosis factor-α Gain entry into vascular system
Although, there are various controversies regarding exact roe of oral flora in dental health, it can definitely be stated that good oral health is important not only to prevent oral disease but also to maintain good general health.