Bacteriology and Oral Diseases; pptx 10

Dr. cugini slides

what are things to think about and keep in mind?

• current evidence is challenging the idea that we are born sterile

• when we emerge form the womb we become further inoculated

• microbes outnumber human cells at the level of 100:1 to 1:1(if ur including platelets)

• we live in harmony with out (commensal) flora-generally

• Disease, may times is a chance event

• A good pathogen does not kill its host

• bacteria are our friends 

where does our microbiome come from?

• acquisition form our mother and primary caregivers

  • window of infectivity

  • ~3/4 of our microbiome maternally-derived

  • Evidence of transmission along family and social networks 

• infant microbiome shaped by external factors beginning in utero

  • mode of delivery

  • maternal and pediatric diet composition and diversity

    • breast milk vs cow’s milk formula

  • antibiotic exposure

  • societal pressures can impact microbiome health

    • children born in industrialized nations present fewer species diversity in their gut flora

• The infant microbiome matures until ~2-3 years of age

  • gut microbiota takes on adult properties 

*look at the images and diagrams in pictures and read their descriptions

Consequences of microbiome alteration (Dysbiosis)

• Dysbiosis

  • a fundamental shift in the microbial flora generally from a population in homeostasis and health to one that is pathogenic 

• Many times low abundance organisms (pathobiont?) shift the microbiome by altering the metabolism thus changing the ecology

• causes of dysbiosis:

  • Host genetics (diabetes; immune dysfunction; polymorphisms)

  • Mechanical (anatomy; overhanging margins in then mouth)

  • antibiotic use (removal of commensal flora)

  • Pregnancy (generally reversable)\

  • Diet

  • Age

  • Chicken and egg conundrum

*still trying to understand what is it about this shift that goes from healthy to diseased microbiome (particularly for periodontal disease)

what does this picture table tell you?

It tells you the oral microbiome implications and children’s diseases (diseases of dysbiosis, shift in the flora present) the and it tables (from left to right)

• the health condition in the child

• the oral microorganisms with increased relative abundance

  • usually pathogenic bacteria

• oral microorganisms with decrease relive abundance 

• study type from which association identified

Dysbiosis result in:

• Diseases and syndromes associated with dysbiosis

  • Disruption or dysfunctional colonization during infancy linked to immune dysregulation and increased risk of disease into adulthood

  • Allergies

  • Diabetes

  • Leukocyte adhesion deficiency

  • Irritable bowel disease

  • Autism spectrum disorder

  • Heart disease

  • Rheumatoid arthritis

  • Periodontal disease....

Why does our microbiome fail us?

• This question is an active area of research.

• Researchers are actively trying to understand why the microbiome changes

• Can we then predict diseases of dysbiosis if we understand pre-dysbiotic events and microbial shifts?

  • Indicator species

  • Indicator host cytokines, other proteins, metabolites

  • Biomarker analysis

• Periodontitis as a model for understanding the shift

  • Inflammatory condition of infection of the gum (gingival mucosa) resulting in bone and tooth loss

Periodontitis and statistics about it

• Periodontal diseases are highly prevalent and costly

• Effects up to 90% of the worldwide population

• Effects half of the US population

• $108 billion dental services in the US

• $18 billion in Europe

• 5-10% of medical expenses

• ~50% of adults living in the USA have gum disease 

Most common infectious disease!!!

Eight classifications of Periodontal disease

• Gingivitis

  • Dental plaque-induced gingival diseases

  • Nonplaque-induced gingival lesions

• Chronic periodontitis - the focus for this lecture

  • Localized

  • Generalized

• Aggressive periodontitis

  • Localized

  • Generalized

• Periodontitis as a manifestation of systemic diseases

• Necrotizing periodontal diseases

  • Necrotizing ulcerative gingivitis

  • Necrotizing ulcerative periodontitis

• Abscesses of the periodontium

  • Gingival abscess

  • Periodontal abscess

  • Pericoronal abscess

• Periodontitis associated with endodontic lesions

• Developmental or acquired deformities and conditions

only really focusing on the first 3, chronic periodontitis for this lecture, aggressive periodontitis for next lecture. we are sticking with this classification structure for this class but make note of the new classification scheme 

New Classification Scheme for periodontal disease:

• Necrotizing periodontitis

• Periodontitis as a direct manifestation of systemic diseases

• Periodontitis (stage and grade designations)

very controversial, basically split opinion on this change, shift to more clinical classifications, presented in 2017

Chronic Periodontitis

1. Periodontal disease is a disease resulting from an inflammatory response to a microbial challenge

2. The challenge is initiated by bacteria that accumulate on the tooth surface above the gum forming plaque

3. There is a distinct progression to disease

• The bacteria is known to be inflammophilic

• The host responds it the dysbiotic biofilm through an inflammatory response, this leads to tissue and osseous support destruction

*look at picture to see the difference between normal health, gingivitis, periodontitis

subclinical gingivitis

• bacteria that colonize near the gingival margin

• PMNs need to migrate to the sulcus in order to fight the bacterial infection

usually okay as long as the body can maintain the bacteria or if you can remove the bacteria

early gingivitis

• If bacterial infection is not revolved early, the bacteria penetrate through the junctional epithelium into the underlying connective tissue

• the epithelium cells release inflammatory mediators to attract additional PMNs

• changes in the vasculature allow more PMNs to infiltrate and the loosening of the tissues allows more bacteria to come in

still reversable

Established Gingivitis

• subgingival plaque extends and disrupts the coronal-most portion of the junctional epithelium

• immune response can destroy healthy connective tissue; however, if the bacterial infection is controlled, the body is able to repair the tissue

  • the destroyed connective tissue also act as an additional food source for the bacteria

Periodontitis

• the host is no longer in control and tissue destruction occurs

• the plaque biofilm flows along the root surface

• the junctional epithelium extends into the connective tissue and migrates along the root

• inflammatory process leading to bone destruction

• Many of the same bacteria cause failures of implants, root canal therapy, etc

Periodontitis porgression

• localized gingivitis

• severe gingival inflammation overlying chronic periodontitis

• moderate periodontitis

• acute advanced periodontitis 

The balance between bacteria and host in periodontal disease

• Inflammation drives selection of obligate anaerobes

• Inflammation is an important source of nutrient:

  • Degraded collagen and heme containing products, used by proteolytic & asaccharolytic bacteria

  • GCF: rich serum exudate

• Inflammophilic bacteria or pathobionts have a survival advantage

• resistance factors

  • specifics in pic

• Risk factors

  • specifics in pic

*LOOK AT PICTURE

what is this picture basically representing

its representing a summery of periodontitis

chronic periodontitis

• A disease of dysbiosis

• Microbial imbalance on or inside the body

  • causes fundamental shift in the bacterial population

• outgrowth of pathobionts

gram positive, Aerobic, predominately streptococci ——> Gram negative, anaerobic

whare are changes that occur within the plaque

• Population increases

  • Number of bacteria

  • Eventual increase in species

• Change nutrient availability

  • Increase and decrease of different nutrients

  • Shift in the metabolism

• Plaque becomes more anaerobic

  • Drives shift in population and nutrients

• Host response to irritation

  • Also leads to nutrient changes for the bacteria

• Bacterial changes that allow for more growth, more virulence factor production, and better attachment

what are late colonizers can be referred to as

periodontopathogens or periodontopathogenic bacteria

Dr. cugini calls them pathobionts

colonized sites are not homogenous and each site have a different set of bacteria colonizing them, why?

• it is mainly because of the metabolic interactions present in the plaque

• the prevalence of virulence factors 

  • adherence factors on the bacteria

    • not all can bind to a specific surface or each other 

*when testing for identification of present bacteria, will get different signatures depending on site, see image for examples

what are the 3 zones of subgingival plaque biofilm?

• Unattached plaque

• epithelium attached plaque

• tooth attached plaque

this is a picture of bacteria that commonly associated with periodontal disease

Red - the main?

orange - can also be associated with disease

green - early colonizers 

what does this picture show u?

it shows the known interactions within the plaque biofilm and how they can metabolically help each other

What are the qualities for a good periodontal pathogen?

Most are pathobionts- Bacteria that are present in healthy people but increase in number in the plaque because of the environment or loss of host defense.

Their pathogenic qualities are attributed to the following:

• their ability to colonize subgingivally

• their invasive capacity

• their armamentarium of proteases and exotoxins

• their capacity to orchestrate destructive immune responses

Most do not grow well in oxygen-they are anaerobes etc.

what is promoting the shift of the population in chronic perio?

dysbiosis, the shift in the biofilm

If the biofilm is allowed to form on the tooth and it goes below the gingival crevice, what happens?

• As the bacteria migrate down in to the subgingival crevice, there is the creation of unique environment that allows for out growth of low-abundance Gram-, anaerobic bacteria

  • Red complex can thrive

  • These are highly proteolytic

  • The center of the biofilm is anaerobic, get a shift in the bacteria’s metabolism

  • GCF fluid is nutrient rich, good source of “food” for the bacteria

  • There is protection from the host immune defenses

since we have all these bacteria in our mouths anyway, why don’t we all have chronic periodontitis

• mechanical factors

  • how well u brush

• Host genetics

• non-permissive environment

• microbe-microbe interactions 

T/F only people that have poor OH can develop periodontitis

False, you can be healthy or have poor OH and can develop periodontitis; the reason for this is not clear yet

what are the facts about periodontitis

• it involves in a rise of the G- bacteria

• probably LPS initiation to trigger inflammatory responses

• from inflammatory response and tissue breakdown get the increase

  • more nutrients

    • allowing more to grow and an increase in bacteria that can grow deeper into the gingiva due to the presents of that nutrient

• we know what organism rise in perio:

  • T. denticola

  • T. forsythia

  • P. gingivalis

    • focusing on this for this lecture because it is the easiest to grow/study

Porphyromonas gingivalis

• gram-negative, nonspore forming, anaerobe

• distinct black pigment

  • is a protective mechanism 

    • takes 2 heme groups and combines them(forms bisheme) and then layers itself with bisheme which protects the bacteria from oxygen radicals 

• Asaccharoyltic

  • does not use sugars

  • prefers peptides, dipeptides, proteins

• associated with chronic periodontitis

• Natural member of the oral cavity...not only there with disease!

  • was confirmed by detection in children so we know it colonizes early 

• At low levels in health

• Increase detection in a disease state

• Not clonal in the oral cavity

• Pathobiont

what is the purpose of Socransky’s revision of Koch’s Postulates

koch’s postulates work for acute disease but don’t work for disease for dysbiosis or diseases in a microbiome so socransky revised them 

Socransky’s revision of Koch’s Postulates

• Isolate organism from disease

• Show that organism has virulent traits that are associated with pathogenesis of disease

• Show that the organism can produce disease in a healthy animal following Koch’s postulates

• Show that the host is responding to the organism

• Eliminate the organism and show that the disease resolves

• Show that if the organism returns the disease also returns

Does P. gingivalis fulfills Socransky’s revision of Koch’s Postulates?

yes

what does this chart show you?

it is a good example of the first postulate, also shows how porphyromonas gingivalis passes this postulate:

• found in high percentage of total plaque from patients shallow and deep pockets

• contrast with lower level in health 

what does this chart show you? (healthy vs diseased)

it is a good example of the second postulate, also shows how porphyromonas gingivalis passes this postulate:

• subgingival plaque samples from 7 healthy vs 6 periodontitis patients

• metatranscriptome (mRNA sequencing)

• Asked what virulence factors were found to be increased in the perio plaque 

what are P. gingivalis virulence factors

• Proteases- i.e. gingipains, collagenase, deiminase

  • Protein breakdown and nutrient acquisition

  • Immune system modulation and evasion

  • Biofilms, cell-cell attachment, adhesion

  • Surface attached, secreted, or in blebs/vesicles

• Capsule

  • Immune evasion

• Hemolysin

  • RBC lysis

  • Hemin acquisition

• Fimbria, hemagglutinin

  • Cell-cell aggregation

  • Biofilm formation

*has a very unstable genome; Virulence factors of P. gingivalis are focused on finding food and shelter!

p. gingivalis biofilm formation

very similar to other bacterial biofilm formation but can change its gene expression for capsule and fimbria depending on the surface it is in contact with and to or not to form a biofilm 

what promote biofilm formation? the gene expression of what bacterial strcutre?

the fimbriae, it facilitates aggregation, surface attachment and biofilm formation; this results in antimicrobial resistance, agent cannot get to the site!

what does these images show you?

it is a good example of the third postulate, also shows how porphyromonas gingivalis passes this postulate:

Shows a test showing how P. gingivalis can cause disease in mice and rabbits in a ligature-induced model(top) and how P. gingivalis can cause bone loss and a change in the microbial flora in mice (bottom half)

these tests basically show how the presents of P. gingivalis can cause bone loss and a shift in the CFUs for oral anaerobic bacteria 

*for the study done in mice SPF - specific pathogen free mice (means they were never exposed to PG until the study was performed), GF - germ free mice (means after the mice were born were put in a bacteria free environment; never encountered bacteria, immune system never adapted to encounter bacteria)

Does P. gingivalis make the host respond?

• The host produces antibodies against P. gingivalis virulence factors and other surface proteins including antibodies against:

  • Fimbria

  • Peptidyl arginine deiminase

  • Heat shock proteins

  • Gingipains

• The host produces cytokines when P. gingivalis is present (these cytokines are related to inflammation)

  • IL8

  • IL6

  • TNFα

• Neutrophils are recruited

*these show how porphyromonas gingivalis passes the fourth postulate

what do these pictures show you?

The pictures show how porphyromonas gingivalis passes the fifth postulate:

Upon antibiotic therapy and SRP treatment P. gingivalis is reduced(left) and how upon treatment symptoms resolve (right)

*for the left picture, the P. gingivalis was treated with Azithromycin metronisazole, SDD (sub-antibacterial dose doxycycline) and SRP (scaling and root planning)

what do these pictures show you?

The picture show how porphyromonas gingivalis passes the sixth postulate:

P. gingivalis increases over time in the absence of dental hygiene

• At the entry visit, after the initial monitoring and sampling, all periodontitis subjects received full mouth scaling and root planning (SRP) at a single visit, using manual curettes and ultrasonic devices, followed by polishing and flossing.

• Periodontally healthy subjects received a dental prophylaxis using rubber cup and paste, followed by dental flossing.

• After the initial prophylaxis or SRP, subjects refrained from oral hygiene procedures for 7 days

what are future plans researchers have for P. gingivalis

• Identify using techniques presented how the organism interacts with other member of the oral cavity

• Can we understand precisely what genes are changed in health to disease

• Can we use it to look for biomarkers at the chair side before we have disease-aim is for personalized treatments

How can the inform we gained on P. gingivalis be used in treatment plans?

• Generally microorganisms associated with pockets deeper than 4 mm are reduced to very low or even undetectable levels after periodontal treatment.

• Scaling and root planning (“deep cleaning”) or periodontal surgery, disrupt the microbiota of the periodontal pocket by mechanically scraping the biofilm off the root surface to a point that the microbial richness and biodiversity are significantly decreased.

• Antibiotic treatments alone or in combination with scaling and root planing disrupt the relative proportions of the taxa within the community.

• However, when the selective pressure is lifted, the community tends to return to equilibrium without persistence of antibiotic resistance in the various taxa