10. Microbiology of Endodontic Infections Flashcards
Endodontics - branch of dentistry that includes the study of the biology of the normal ____ as well as the etiology, diagnosis, prevention and treatment of diseases located within the coronal ____ and extending into the ____ and ____ tissues.
dental pulp
pulp chamber
root canal
periradicular
Overview of Tissue Inflammation and Destruction
Apical (Periapical/Periradicular) Periodontitis - an ____ or ____ inflammatory disease of ____ etiology primarily caused by infection of the pulp and root canal system that can extend to the development of a lesion around the apex of the tooth root.
• Enlarged pulpal area ○ \_\_\_\_ at tips of roots ○ PA abscess § Tip of root ○ Infection spreads into surrounding tissue § Inflammation • Untreated carious lesion > pulp > infection
acute
chronic
microbial
radiolucency
Timeline of Characterization of Endodontic Lesions
• \_\_\_\_ ○ First observed bacteria in root canal • \_\_\_\_ ○ Treated pulp to preserve teeth • \_\_\_\_r ○ Various bacterial morphotypes in root canals ○ Diversity § Spread as a gradient, difference in location (coronal vs. root, etc.) • \_\_\_\_ ○ Restorations caused problems § Oral sepsis/inflammation and disease • \_\_\_\_ ○ Pulp necrosis > microorganisms • \_\_\_\_ ○ Necrotic pulps were a result of anaerobic bacteria § Deeper in tooth, oxygen is lacking • \_\_\_\_ ○ Pathological changes in periradicular occurs when the pulp is devitalized and infected (sick, blood flow is degraded) • \_\_\_\_ ○ Bacterial organizations = biofilms • \_\_\_\_ ○ Mixed species biofilms are key in endo infections
van Leewonhoek Fauchard Miller Hunter Kakehashi/Fitzgerald Sundqvist Muller Nair Ricucci
Inflammation and Tissue Destruction is Due to Infectious Microorganisms
• Kakahashi • Germ-free rats ○ Nonexistant \_\_\_\_ ○ Exposed pulp § ~100 days: remained vital, tissue repair § Dentin bridge separates enamel from pulp § Invading pulp: macro's, PMN's • Conventional rats ○ Exposed pulp § ~100 days: pulp \_\_\_\_ § Apical perio lesions □ \_\_\_\_ stained area □ Stained bacterial cells • Shows that microorganisms are essential for tissue destruction
flora
pulp necrosis
darkly
Inflammation and Tissue Destruction is Due to Infectious Microorganisms
Inflammation and Tissue Destruction is Due to Infectious Microorganisms
• Muller • Infected devitalized pulps with micro's ○ Tissue \_\_\_\_ apparent in pulp infected with micro's • No micro's with non-infected pulp ○ \_\_\_\_ tissue is normal ○ Periradicular = outside the tooth, only the soft tissue
pathology
periradicular
Entry of Microorganism
• Common entry: caries ○ Result of \_\_\_\_ activity • Iatrogenic restorative ○ Result of \_\_\_\_ treatment ○ Not necessarily due to bad \_\_\_\_ ○ PA abscess at tip of root • Traumatic occlusion ○ \_\_\_\_, or breakdown in \_\_\_\_
bacterial dental practice cracking elderly
Consequences of Microbial Entry
Necrotic Pulp Conditions That Favor Microbial Colonization
- Necrotic pulp provides bacteria with moist, warm, nutritious and ____ environment for colonization.
- Environment protected from host defenses because of lack of active ____ in the necrotic pulp tissue.
- Root canal walls are non-shedding surfaces conducive to persistent colonization and formation of ____.
- Major ecologic factors include ____ tension, type and amount of available ____ and bacterial interactions. (temperature, ____, and receptors for ____ may also be important)
- About ____ bacterial species, out of about 600 to 700 in the oral cavity, are consistently selected out for growth and survival in necrotic pulp tissue.
anaerobic blood circulation complex communities (biofilms) oxygen nutrients pH adhesins 10-20
Consequences of Microbial Entry
• ____ major type of microorganism implicated in the pathogenesis of
apical periodontitis.
• Root canal microbiota dominated by bacterial morphotypes that include ____, rods, ____ and spirochetes.
• Bacteria usually part of the normal oral microbiota (“____ pathogens”).
• Opportunistic pathogens ○ Not normally pathogenic, but under right conditions they can cause disease § Because of change in environment or in organism itself • Left image: ○ Cocci ○ Large objects = cells, yeast • Right image: ○ Primarily cocci, but it is mixed
bacteria
cocci
filaments
opportunistic
Complex Microbial Communities (Biofilms)
• Walls of canal have \_\_\_\_ staining ○ Bacteria: rods, but it is \_\_\_\_ ○ Tightly \_\_\_\_ to the walls of the canal
dark
mixed
adhered
Complex Microbial Communities (Biofilms)
- ____ biofilms generally observed in apical segment of approximately 80% of the root canals of teeth with primary or post-treatment apical periodontitis.
- Morphology of biofilms ____ consistently from individual to individual by thickness, morphotypes and bacterial cells/extracellular matrix ratio
____ underneath biofilms were often invaded by bacterial cells
from the bottom of the biofilm community.
• In most cases of AP perio ○ Bottom portion of roots: biofilms • Dentin invaded by bacterial cells from bottom of biofilm community ○ Deepest part of biofilm: area just above the dentin ○ Invade the dentin and can invade the pulp
intraradicular
differed
dentinal tubules
Complex Microbial Communities (Biofilms) Criteria Used to Establish Causal Link Between Biofilms
and an Infectious Disease
- Infecting bacteria ____ to or associated with a surface.
- Direct examination of infected tissue shows bacteria forming ____ or ____ encased in an extracellular matrix.
- Infection is generally confined to a particular site. Although dissemination may occur, it is a ____ event.
- Infection is difficult or impossible to eradicate with ____ in spite of the responsible microorganisms being susceptible to killing in the planktonic state.
- Ineffective host ____ is evident as suggested by the location of bacterial colonies in areas of host tissue associated with host inflammatory cells (polymorphonuclear neutrophils (PMN) and macrophages).
- Elimination or significant disruption of biofilm structure and ecology leads to remission of the disease process.• Importance: establishing a causal link
○ Most information implicates microorganism in disease by association, not ____
§ Koch’s postulates proves causality
1. Definition of biofilm
2. Definition of biofilm
3. Dissemination occurs by breaking off of clumps of cells from biofilm
4. Biofilm is protective region, which doesn’t allow the infiltration of antibiotics (the deeper the more difficult)
5. NA
6. Koch’s postulate - remove microorganism, the inflam/disease is resolved, or introduce into a healthy animal you reestablish
a. Endodontic biofilms: in studies 5/6 are met, but ____ is not met
b. Not fully proven that biofilms are the ____ agent of disease bc haven’t satisfied last tenant
adhered clusters microcolonies secondary antibiotics clearance
causality
last
causative
Microbial Identification Methods
• Currently: \_\_\_\_ generation to identify endodontic infections • 1st generation ○ \_\_\_\_ § "cultivable species" • 2nd generation ○ \_\_\_\_ methods § PCR/checkerboard □ Sakransky ○ \_\_\_\_-specific analysis ○ Confirmed data from 1st generation, and added more species • 3rd generation ○ \_\_\_\_-sequencing, \_\_\_\_ (terminal-RFLP, gives you patterns of molecular content of micro's) ○ \_\_\_\_ range § Wide scope of samples, identify a large number ○ Increased diversity ○ Uncultivated and uncharacterized § Did not need to culture the bacteria (like 1st gen) • 4th generation ○ \_\_\_\_, reverse-capture checkerboard ○ \_\_\_\_-specific (like 2nd) ○ Cultivable and uncultivated species in larger scale studies • 5th generation ○ Current stage ○ \_\_\_\_ ○ Rapid, \_\_\_\_ range, easy to complete ○ Comprehensive analysis § Identifying microorganisms bc they cannot be cultured in lab bc we do not know what nutrients they need
5th culturing moelcular species cloning T-RFLP broad DNA microarrays species pyrosequencing broad
Microbial Identification Methods
Culture methods
• Sample taken via \_\_\_\_ point • Placed into transport medium ○ Not a growth medium, but a \_\_\_\_ medium § Viscous material, contains \_\_\_\_ to support anaerobes § \_\_\_\_ enviornent § Aerobes won't be killed off, but \_\_\_\_ through ○ Keep microorganisms alive • Dispersed via mild \_\_\_\_ ○ Bc bacteria are sticking together • Grown under \_\_\_\_ and \_\_\_\_ conditions • Plated ○ Single colonies isolated on various growth medium § Here: blood agar • Phenotypic analyses ○ \_\_\_\_ ○ \_\_\_\_ § Battery of enzymatic tests/fermentative tests that can be automated in plastic strips ○ \_\_\_\_ analysis § Fluorescence, or FISH ***• Must be able to be grown in \_\_\_\_***
paper supportive glycerol anaerobic carried sonication anaerobic aerobic gram-stain API-20 serotypic laboratory
Microbial Identification Methods Fluorescence in situ Hybridization (FISH)
• Four bacteria found in endodontic infections • Detected with ab's that are conjugated to fluorscent dyes ○ Streptococcus gordonii = green ○ actinomyces naeslundii = blue ○ lactobacillus salivarius = red ○ enterococcus faecalis = purple
The antibodies that
detect EF and LS, they’re
____, aka they
detect each other, so the
antibodies used for EF will detect LS. In order avoid it, two different dyes are coupled to the same antibody and the EF then comes out ____ instead of red
Therefore, even though there is cross-reactivity, you can detect all 4 genera.
You get the different spatial organization of different bacteria and their different morphologies.
cross-reactive
purple
Advantages of culture methods
____-range nature, identification of unexpected species
Allow ____ of all major viable
cultivable microorganisms in samples
Allow determination of antimicrobial ____ of isolates
____ studies are possible
____ studies are possible
Widely available
broad quantification susceptibilities physiologic pathogenicity
Limitations of culture methods
Impossibility of culturing a ____ number of extant bacterial species
Not all viable bacteria can be ____
Once isolated, bacteria require identification using a number of ____
____ of strains with ambiguous or aberrant phenotypic behavior
____ sensitivity
Strict dependence on the mode of ____
Samples require immediate ____
Costly, time consuming and laborious (especially ____)
Specificity is dependent on ____ of microbiologist
Extensive expertise and specialized equipment needed to isolate anaerobes
Takes several ____ to identify most anaerobes
large recovered techniques misidentification low sample transport processing anaerobes experience days to weeks
Microbial Identification Methods
Broad range molecular analysis
• Two major groups: ○ \_\_\_\_ range: looking for large number of species ○ \_\_\_\_-specific • \_\_\_\_ point collection • Placed into solution, and DNA is extracted from total sample, no separation • \_\_\_\_ to amplify • Denaturing gradient • Gel electrophoresis ○ Separtated by \_\_\_\_ • T-RFLP ○ Pattern for number of \_\_\_\_ • Pyrosequencing • \_\_\_\_ analysis by identifying genera
broad species paper PCR size RFLP broad
Microbial Identification Methods
Species-specific molecular analysis
• The top half is the same • Different primers used to idenitfy species • Checkerboard DNA-DNA hybridization ○ Probes can be analzyed on one \_\_\_\_ ○ \_\_\_\_ • Multiplex \_\_\_\_ ○ Similar to CB • RT-PCR ○ More \_\_\_\_ • DNA microarray ○ Processing of large number of samples • Manipulating primers in PCR, and number of primers, you can identify down to the species level
chromatogram
Socransky
PCR
quantitative
Microbial Identification Methods
Advantages of molecular methods
Detect both cultivable and ____ species or strains
High specificity and accurate identification of strains with ambiguous or aberrant phenotypic behavior
Detect species directly in clinical samples
____ sensitivity
____; most assays take no more than a few hours to identify a microbial species
Do not require carefully controlled ____ conditions during sampling and transportation
Can be used during ____ treatment
Anaerobic handling and expertise not required
Samples can be stored ____ for later analysis
DNA can be transported ____ between laboratories
uncultivated high rapid anaerobic antimicrobial frozen easily
Microbial Identification Methods
Limitations of molecular methods
Most assays are ____ or
____ (exceptions: ____)
Most assays only detect one species or a few different species at a time (exceptions: broad-range PCR, checkerboard, microarray)
Most assays detect only the ____ species and fail to detect unexpected species (exception: ____)
Some assays can be laborious (broad-range PCR)
____ in broad-range PCR introduced by homogenization procedures, preferential DNA amplification and differential DNA extraction
Hybridization assays using whole genome probes detect only ____ species
Can be very ____
qualitative
semiquantitative
RT-PCR
target
broad-range PCR
biases
cultivable
expensive
Diversity of the Endodontic Microbiota
Bacterial Phyla in Endodontic Samples Based on Detection Method
• Identification of the major taxa and/or phyla that are found in endo infections ○ Lists bacteria: major large groups (phyla) • Compilation of analyses via culturing alone, molecular methods alone, or a combination of both techniques • In a compilation of samples: [LOOK UP TEXTBOOK] ○ AB down to PB the numbers are \_\_\_\_ (45% of it detectable by molecular, 23% of combo, and 32% of culturing) ○ Spirochetes to SR1 § Not \_\_\_\_ § \_\_\_\_ methods are necessary in order to detect these species § Synergistetes of small proportion used \_\_\_\_ • Molecular methods are more effective in detecting a \_\_\_\_ flora ○ Detecting closer to 100% of the time ○ Depends on what you're looking for…
same cultivable molecular both diverse
Diversity of the Endodontic Microbiota
Distribution of Bacterial Phyla in Endodontic Samples
• Any of the >600 bacterial species/phylotypes in the human oral cavity has the ____ to be an endodontic pathogen or at least play a role in the ecology of the endodontic microbial community.
• PB ○ \_\_\_\_ • TM7/SR1 ○ Identified primarily based on the sequence of \_\_\_\_ • Not responsible for all the genera and species ○ Know the major taxa • Firmicutes/bacteriodetes ○ Account for \_\_\_\_ of species identified in abscesses • Relationships among the taxa
potential
gram-negative
DNA
70%
Diversity of the Endodontic Microbiota
Bacterial Phyla in the Domain Bacteria of the Phylogenetic Tree
• PB ○ Divided into multiple groups, based on \_\_\_\_ alphabet
greek
Types of Endodontic Infections
• Endodontic infections into two types: ○ Intraradicular: \_\_\_\_/\_\_\_\_ § Primary □ Infections of \_\_\_\_ colonization ® Pulpal \_\_\_\_ in root □ Untreated carious lesions, iatrogenic lesion, or traumatic occlusion § Secondary/persistent □ Resolve first infection, but it \_\_\_\_ □ Arises after \_\_\_\_, but difficult to treat infection (persistent) □ May or may not be difficult to treat ○ Extraradicular: \_\_\_\_ surrounding teeth § Dependent □ Depend on \_\_\_\_ § Independent □ Do not depend on \_\_\_\_
pulp root canal initial tissue revisits treatment soft tissues intraradicular intraradicular
