Periodontal microbiology

Periodontal Microbiology

Human fetus is sterile but after passing through

birth canal it acquires vaginal and fecal

microorganisms

By 2nd day Anaerobic flora can be detected in

infant edentulous mouth

Dr Saif Khan 204/07/23

Within 2nd week a nearly mature microbiota

is established in the gut of newborn

After weaning(>2yrs), entire human microbial

flora formed by complex collection of more

than 400 types of bacteria.

04/07/23 Dr Saif Khan 3

Oral cavity is an “Open growth system”


Oral cavity divided into 5 major niches


Most species colonize on all above described

niches with the exception of spirochetes

Adherence of bacteria to oral epithelial cells

is directly related to its virulence


Teeth and Implants are unique from a microbiological point of view for two reason– Provide hard, non-shedding surface that allows for

the development of extensive structured baterial deposit

– Form a unique ectodermal interruptionA special seal of epithelium and connective

tissue exists between external enviroment and internal part of body


Teeth are primary habitat for periopathogens

Thus teeth can be considered as “Port of entry for Periopathogens

Cariogenic species like S mutans remain restricted to solid surfaces and are called “obligate periphyte”


Dental Plaque

Dental plaque is a specific, amorphous, granular

deposit which accumulates on the surface of

teeth, dental restoration and dental calculus. - -Glickmann


WHO defnition

“Dental plaque is a specific but highly variable

entity resulting from growth and colonization of

micro-organisms on surfaces of teeth,restoration

and soft- tissue consisting of various species

microorganisms entangled in extracellular matrix.”


Dental plaque• Defined clinically as structured, reselient,

yellowish-grayish substance that adhere tenaciously to the intraoral hard surface,including removable and fixed restoration.

• Plaque is primarily composed of bacteria in matrix of salivary glycoproteins and extracellular polysaccharides

• 1gm of dental plaque(wet weight) contains 1011

bacteria



Thin supragingival dental plaque of a 32-year-old man who had not brushed his teeth for 7 days. A, Unstained plaque is not readily apparent. B, Extent of the plaque becomes apparent when stained with a disclosing solution (i.e.,erythrosine dye)

No of bacteria inSupragingival plaque on single tooth – 109

Gingival Crevice- 103

Deep periodontal pocket- 108

More than 500 distinct microbial species are found in dental plaque

Non bacterial microorganism found in plaque include Mycoplasma species, yeasts, Protozoa and Viruses


Dental plaque is broadly Classified into


Supragingival plaque visualized by Disclosing solution



Plaque-bacteria interaction with tooth surface and Periodontal tissuesDr Saif Khan 1704/07/23



The enviromental paramaters of subgingival region is different from supragingival region

– Gingival crevice is bathed by GCF which may contains many substances that bacteria may use as nutrients

– Low oxygen tension in the subgingival area– Host inflammatory cells and mediators have

considerable influence on establishment and growth of bacteria


The apical border of the plaque mass is

separated from junctional epitheliun by a layer

of host leukocytes and the bacteria of this

apical-tooth associated region show an

increased concentration of Gm-ve rods


Dental Plaque as a Biofilm

• Biofilm is a highly organized structure• Consists of microcolonies of bacterial cells

randomly distributed in a shaped matrix or glycocalyx.

• Lower plaque layer are dense in which microbes are bound together in polysaccharide matrix with other organic and imorganic materials


• On top of lower layer, loose layer can be seen which can extend into surrounding medium (for teeth and saliva)

• The fluid layer bordering the biofilm has a stationary sublayer and a fluid layer in motion

• Nutrient components penetrate this fluid medium by molecular diffusion


• The dental plaque biofilm has open fluid filled channels running across the plaque mass

• Act as primitive “circulating system”


Intercellular Matrix

• Organic Constituent- Consists of– Polysacchrides, Proteins, Glycoprotein & Lipid– Albumin derived from GCF– Lipid material consists of debris from the membranes

of disrupted bacterial & Host cells and possibly Food debris

– Glycoprotein from saliva important component of pellicle

– Polysacchrides produced by bacteria of which Dextran is most predominant form & play major role in maintaining integrity of Biofilm


Inorganic component• Predominantly Ca and P• Trace minerals Na, K and F• Source of inorganic constituent of supragingival

plaque is primarily saliva• As mineral content increases plaque calcifies to

Calculus• Inorganic portion of subgingival fluid are derived

from crevicular fluid• F content of plaque is basically derived from

external sources such as floridinated toothpaste, rinses, water etc


Plaque formation at Ultrastructural level1. Formation of Pellicle

– Thin, saliva derived layer– Consists of Glycoprotein (mucins), Proline rich protein,

Phosphoproteins (Statherin), Histidine rich proteins,enzymes

– Forms within seconds of prophylaxis– Forms by selective adsorption of enviromental

macromolecules– Mechanism involved in pellicle formation include

Vanderwall forces & Hydrophobic forces




2. Initial Adhesion and attachment of bacteriaPhase I : Transport to the surface•Initial contact of bacterium to tooth surface•Random contacts through Brownian movement (40μm/hr)•Through Active bacterial movement

Phase II: Initial Adhesion•Reversible adhesion•Interaction b/w bacteria and surface at certain distance (50nm) through long range and short range forces•Including Vanderwall attractive and electrostatic repulsive forces•Gibbs total energy GTOT = GA + GE

•GTOT is a function of separation distance between negatively charged particle and negatively charged surface in a medium ionic suspension (saliva)•For most bacteria reversible binding takes place 5-20 nm from surface


Schematic representation of interactions involved in

bacterial adhesion to solid substrata


Phase III: Attachment•After intial adhesion firm adhesion is established by specific interactions (covalent,ionic or hydrogen•Rough surfaces are more conducive for attachment as bacteria are better protected against sheer force leading change from reversible to irreversible bonding•The bonding between bacteria and pellicle is mediated by by specific extracellular protein components

eg; Streptococci (S sangius) early colonizer binds to acidic proline-rich proteins, also α-amylase and sialic acid


3. Colonization and plaque maturation

Primary Colonizers: Streptococci and Actinomycetes

Secondary Colonizers: P intermedia, P loescheii, Capnocytophaga, F nucleatum, P gingivalis


All oral bacteria possess surface molecule or receptor which foster cell to cell interaction

Highly specific stereo chemical interaction of proteins and carbohydrate molecules located on bacterial cell surfaces leading to Coaggregation

Most Co aggregation are among strains of different genera are mediated by Lectin like adhesins and inhibited by Lactose and other galactosides


Examples of Co-aggregation

Fusobacterium nucleatum with steptocooci sangius,Prevotella loescheii with A viscosus Capnocytophaga ochraceus with A viscosus

Intrageneric co-aggregation in streptocooci


Dr Saif Khan 36

Fusobacterium nucleatum

S sangius

P loeschiiA viscosus

04/07/23

Coaggregation



Growth dynamics of Dental Plaque

• First 2-8 hrs, adherent pioneering streptococci saturate the salivary pellicular binding sites and cover 3-30% of enamel surface

• After 1 day the term Biofilm is fully deserved because organization takes place in it

• After 3 days plaque growth increases at rapid rate and then slows down


• There is shift towards more anaerobic and gram-negative flora, including an influx of Fusobacteria, filaments, Spiral forms and spirochetes

• In ecological shift within of the biofilm, there is a transition from the early aerobic environment characterized by Gm+ve facultative species to a highly oxygen- deprived environment in which Gm-ve anaerobic microorganism predominate


Topography of Supragingival Plaque

• Early plaque formation follow typical topograhic pattern with initial growth along gingival margin and interdental space

• Later further there is extension in coronal direction

• This pattern changes when tooth contains surface irregularities such as grooves,cracks, perikymata, or pits

• Surface irregularities can give rise to “individualized plaque growth pattern


Surface Microroughness

• Rough intraoral surface (crown,implant abutments, denture bases)accumulate and retain more plaque and calculus in terms of thickness area and colony forming unit

• [Ra=0.2µ] is threshold for surface roughness above which bacterial adhesion is facilitated


Individual variables influencing plaque formation

Heavy(fast) plaque formers Rapid plaque formers demonstrate higher proportion

of Gm-ve rods (35% vs 17%) in 14-day old plaque

Light (slow) plaque formers


Intersubject variation in plaque formation can be explained by factors such as

• Diet• Food• Smoking• Presence of copper amalgam• Tongue& palate brushing• Colloid stability of bacteria in the saliva• Antimicrobial factors present in saliva• Chemical composition of the Pellicle• Retention depth of dentogingival area


Variation within the Dentition

Early plaque formation occurs faster in •lower jaw compared to upper•Molar areas•Buccal tooth surfaces as compared to oral sites (esp in upper jaw)•Interdental region compared to buccal or oral surfaces


Impact of Gingival inflammation

• Early invivo plaque formation is more rapid on tooth surfaces facing inflamed gingival margins than those adjacent to healthy gingival margins

• Increase in crevicular fluid production enhances plaque formation


Impact of Age

Recent studies show that subject’s age does not

influence de novo plaque formation


Spontaneous tooth cleaning

• Firm attachment between bacteria and surface this is unlikeky

• Even occlusal part of molars, plaque remains after chewing fibrous food

• Only negligible differences in plaque extension could be observed


De novo subgingival plaque formation

Recent studies suggest that complex subgingival microbiota, including most periopathogens, is established within 1 week after abutment insertion

Smooth abutments[Ra<0.2µ] were found to harbour less bacteria than less ones, with a slightly higher density of coccoids (i.e nonpathogenic) cell


Physiological properties of Dental Plaque

The transition from Gm+ve to Gm-ve microorganism observed in structural development of plaque is paralled by physiologic transition in the developing plaque

Early colonizers use oxygen and lower redox potential of the environment which then favors growth of anaerobic species


Early colonizers use sugar as energy source and saliva as carbon source

Bacteria which predominate in mature plaque or late colonizers are Asaccharolytic and use amino acids and small peptides as energy source

Lactate and formate are by products of metabolism of streptococci and actinomycetes may be used by other microorganism


Hemin a breakdown product from host hemoglobin is important in metabolism of P gingivalis

Increase in steroid hormone is associated with increase in proportions of Prevotella intermedia in subgingival plaque


Metabolic interaction among different bacteria species found in plaque and also between host and plaque bacteria


Ecological plaque Hypothesis Given by Marsh & Co- Workers in 1990

Total amount of dental Plaque and the specific microbial composition of plaque contribute to transition from health to disease

Health –associated dental plaque microflora is considered to be relatively stable overtime and in state of dynamic equilibrium or “ microbial

homeostasis ” Dr Saif Khan 5404/07/23

Ecological plaque Hypothesis

Change in nutrient status of a periodontal pocket or

Chemical and physical changes to habitat can lead over growth of pathogens

Eg: Increase in GCF flow can lead to enrichment of proteolytic species(periopathogens) by providing essential nutrients such as heme containing molecules


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Schematic representation of the ecologicalplaque hypothesis in relation to periodontal disease

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Dr Saif Khan 57

Relationship between the microbial composition of dental plaque in health and disease.

04/07/23

Non- specific plaque hypothesis Periodontal disease results from elaboration of

noxious products from entire plaque flora

Large amount of plaque produces large amount of noxious product that would overwhelm host’s defense


Dr Saif Khan 59

Control of periodontal disease depend on control of amount of plaque deposit

The current standard treatment of periodontitis still focuses on the removal of plaque and its product founded on non-specific plaque hypothesis

04/07/23

Non-specific plaque hypothesis has been discarded because

Some individuals with considerable amount of plaque and calculus as well as gingivitis never developed destructive periodontitis

Individuals with periodontitis demonstrated considerable site specificity in pattern of disease

Individuals with very less plaque developed destructive periodontal disease as in Aggressive periodontitis


Specific plaque Hypothesis

States that only certain plaque is pathogenic

And its pathogenecity depends on presence of or

increase in specific microorganism



Plaque harboring specific bacterial

pathogen results in periodontal disease

because these organism produce

substance that mediate the destruction

of host tissue

Eg: A actinomycetemcomitans as pathogen in

localized aggressive periodontitis

Specific Bacterial behaviour in Biofilm:Antibiotic resistance

Microorganisms in biofilm are 1000 to 1500 times more resistant to antibiotics than in their planktonic stage

The mechanism of this increased resistance differs from species to species, from antibiotic to antibiotic, and for biofilm growing in different habitats


Resistance of bacteria to antibiotics is affected by their

• Nutritional status• Growth rate• Temparature• pH• Prior exposure to subeffective concentration

of anti microbial agents



Also slower growth of bacterial species in biofilm is

another important mechanism of antibiotic resistance

Biofilm matrix although not significant barrier in itself to

diffusion of antibiotics but have certain properties to

resist diffusion

Biofilm act as ion-exchange resin removing antibiotics

from solution


Also extracellular enzymes such as β lactamases,

formaldehyde lyase and formaldehyde

dehydrogenase may become trapped and

concentrated in the extracellular matrix thus

inactivating some antibiotics(especially positive

charged hydrophilic antibiotics)


Some antibiotics such as Macrolide which

are positive charged but hydrophobic

are unaffected by this process


“Super-resistant” bacteria have been identified

within a biofilm and these cells have multidrug-

resistant pump that can extrude antimicrobials

from the cell

Quorum SensingBacteria in biofilm communicate with each

other

This involves the regulation of expression of specific genes through accumulation of signalling compounds that mediate intercellular communication

When these signalling compounds reach a threshold level(quorum cell density) gene expression is activated


• Quorum sensing seems to play a role in

expressing genes for antibiotic

resistance and encouraging growth of

beneficial species to the biofilm and

discouraging the growth of competitors


Schematic representation of the types of interaction that occur in a microbial community, such as dental

plaque


Dr Saif Khan 72

High density of bacterial cells in biofilm facilitates the exchange of genetic information among cell of the same

species and genera through;

1. Conjugation (sex pilus)2. Transformation (movement of small pieces of DNA

from enviroment into bacterial chromosome)3. Plasmid tranfer4. Transposon transfer (DNA sequence which can

change sequence within the genome)

04/07/23


Translocation and Mechanical Debridement

• To reduce chance of intraoral transmission, one stage, Full mouth disinfection has been introduced by Leuven group in the 1990s

• This strategy attempts to eradicate, or atleast suppress, periopathogens in short time not only from the periodontal pockets, but also from all their intraoral habitats(mucous membrane, tongue, and saliva)


One stage, Full mouth disinfection

Full mouth scaling and root planning within 24 hrs to reduce number of subgingival pathogenic organisms

Subgingival irrigation of all pockets with 1% chlorhexidine gel to kill remaining bacteria

Tongue brushing with an antiseptic to suppress the bacteria in the niche

Mouth rinsing with antiseptic to reduce the bacteria in the saliva and on the tonsil


Benefits of the One stage, Full mouth disinfection

• Pocket depth reduction

• Gain in clinical attachment level

• Microbiologic shift


The current concept on etiology of Periodontitis considers three factors that determine whether active periodontitis will occur

1. Susceptible Host2. Presence of a Pathogenic species3. Absence or small numbers of beneficial

species



Role of Beneficial species

Passively occupying

niches otherwise

occupied by pathogenic

bacteria

Actively limiting

pathogens ability to adhere to

appropriate tooth

surface

Adversely affecting

the vitality or growth

of pathogens

Affecting the ability

of pathogenic species to produce virulence

factor

Degrading Virulence

factor produced

by pathogen


Dr Saif Khan 80

Beneficial species such as S sangius,

Veillonella parvula and C ochreus are

typically found in higher number at

periodontal sites with no attachment loss

where as lower in number where there is

active periodontal destruction

04/07/23

Criteria for identification of Periodontal pathogen

Kochs PostulatesGiven by Robert Koch as classic criteria by which microorganims are judged causativeMust be routinely isolated from diseased individualMust be grown in pure culture in laboratoryMust produce similar disease when inoculated in susceptible laboratory animalMust be recovered from lesions in diseased laboratory animalStreptococcus mutans has been shown to follow Koch’s postulate as an etiologic agent of dental caries


koch’s criteria are difficult to apply in periodontal disease because of 3 reasons

1. Inability to culture all the microorganism that have been associated with disease (eg: spirochetes)

2. The difficulties inherent in defining and culturing sites of active disease

3. Lack of good animal model for study of Periodontitis


Socransky criteria • Proposed criteria by which periodontal microorganism

may be judged to be potential pathogens1. Must be associated with disease, as evident by increase in

the number of organisms at diseased sites2. Must be eliminated or decreased in sites that demonstrate

clinical resolution of disease with treatment3. Must demonstrate a host response, in the form of an

alteration in the host cellular or humoral immune response4. Must be capable of causing disease in experimental animal

model.5. Must demonstrate virulence factors responsible for

enabling the microorganism to cause destruction of periodontal tissue


Microorganism associated with specific periodontal disease

Fewer coccal cells and more motile rods and spirochetes are found in diseased state than healthy sites by means of phase-contrast or dark-field microscopy

All most all periodontal pathogens except Campylobacter rectus are immobile

Bacteria from healthy periodontal sites consists of gram +ve facultative rods and cocci


Periodontal Health

Gm+ve facultative species of genera Streptococcus and Actinomycetes(S sangius, S mitis, A viscosus, A naeslundi)

Small proportions of Gm-ve speciesa are also found (P intermedia, F nucleatum,

Capnocytophaga, C ochareus)


GingivitisMicro biota of dental –plaque induced gingivitis(chronic gingivitis) consists of equal proportions of Gm+ve(56%) & Gm-ve(44%) species

Facultative(59%) & anaerobic(51%) microorganisms

Predominant Gm+ve microorganisms are S sangius, S mitis, S intermedius, S oralis, A viscosus, A naeslundii and P micros


Dr Saif Khan 87

The predominant Gm-ve microorganisms are F

nucleatum, P intermedia, V parvula as well as

Haemophilus, Campylobacter and Capnocytophaga

Pregnancy associated gingivitis is acute inflammation of

gingivae associatedin pregnancy. There is increase in steroid

hormones in crevicular fluid and dramatic increase in

Prevotella intermedia which uses steroid as growth factor

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Chronic periodontitis

Microscopic examination of plaque from sites with chronic gingivitis consistently revealed elevated proportions of spirochetes

Cultivation of plaque microorganisms from sites chronic periodontitis have reveal high percentages of anaerobic(90%) and gram negative (75%) bacterial species


Dr Saif Khan 89

Bacteria most often cultivated include P. gingivalis

, T. forsythus,,C rectus, E corrodens, F.

nucleatum, A actinomycetemcomitans, P

micros, Treponema and Eubacterium

C rectus, P gingivalis, P intermedia, F nucleatum & T

forsythia are elevated in active sites

04/07/23

Dr Saif Khan 90

Detectable levels of P gingivalis, P intermedia,

T forsythia, C rectus and A

actinomycetemcomitans are associated with

disease progression and their elimination by

therapy is associated with improve clinical

outcome

04/07/23

Dr Saif Khan 91

Also recent studies have documented

association between chronic periodontitis

and viral microrganisms of Herpes

group, most notably Epstein barr virus-1

(EBV-1) and Human cytomegalovirus

(HCMV) are associated with putative

pathogens P gingivalis, T forsythia, P

intermedia and T denticola04/07/23

Microbial Shift during disease


Localized Aggressive Periodontitis

A actinomycetemcomitans compose of 90% of total cultivable microbiota

P gingivalis, E corrodens, C rectus, F nucleatum, B

capillus, Eubacterium brachy, Capnocytophaga sp

and Spirochetes are also found in significant levels

Herpes viruses, including EBV-1 and HCMV have been associated with LAP


Necrotizing Periodontal disease

Microbiologic studies indicate high levels of Prevotella intermedia and especially Spirochetes in NUG lesions

Spirochetes penetrate deep into necrotic tissue and unaffected connective tissue


Microbial specificity in Periodontitis• There is no “black-or-white” situation; most

pathogens might be present, but do not necessarily have to be present for specific form of periodontitis

• Microbial composition can not be used to differentiate different forms of periodontal disease


Dr Saif Khan 96

Most pathogens can also be detected

in healthy subjects with frequencies

ranging from 10% to 85%. This

automatically reduces the specificity

of microbiologic testing in

periodontology

04/07/23

Periimplantitis

Inflammatory process affecting the tissue around an

already osseointegrated implant resulting in loss of

supporting bone Healthy periimplant pockets are characterised by

high proportions of coccoid cells, low anaerobic/aerobic ratio, low number of gram anaerobic species and low detection frequency for

periodontal pathogens



Key characteristics of specific Periopathogens



