1 2018 Endodontic Board Review and Scientific Update University of Texas Health, San Antonio Endodontic microbiology Christine Sedgley MDS, MDSc(Endo), FRACDS, MRACDS(ENDO), PhD February 2018 Overview Part 1 Basic microbiology update Part 2 Methods used for evaluating endodontic microflora Part 3 Microorganisms in primary endodontic infections Part 4 Microorganisms and unsuccessful endodontic treatment Part 5 Microorganisms in periapical lesions Part 6 Microorganisms and regenerative endodontics Part 7 Antimicrobial efficacy of endodontic treatment Overview 1 Part 1 (Very) basic microbiology update Bugs Biofilms Virulence Basic microbiology Prokaryotes bacteria No nucleus Eukaryotes fungi, animals Chromosomes in nucleus Gram-positive Gram-negative Classification - cell wall Gram positive and Gram negative What are biofilms? ØAggregates of microbial cells enclosed in a self-produced matrix adherent to a surface Biofilm Composition Bacterial cells ± 20% by volume EPS/glycocalyx, biofilm matrix ± 80% by volume
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2018 Endodontic Board Review and Scientific Update �University of Texas Health, San Antonio�
Part 2 Methods used for evaluating endodontic microflora
Part 3 Microorganisms in primary endodontic infections
Part 4 Microorganisms and unsuccessful endodontic treatmentPart 5 Microorganisms in periapical lesionsPart 6 Microorganisms and regenerative endodontics Part 7 Antimicrobial efficacy of endodontic treatment
Overview 1
Part 1 (Very) basic microbiology update
Bugs Biofilms Virulence
Basic microbiology
ProkaryotesbacteriaNo nucleus
Eukaryotes fungi, animalsChromosomes
in nucleus
Gram-positive
Gram-negative
Classification - cell wall �Gram positive and Gram negative
What are biofilms?
Ø Aggregates of microbial cells enclosed in a self-produced matrix adherent to a surface
Biofilm CompositionBacterial cells ± 20% by volume
EPS/glycocalyx, biofilm matrix
± 80% by volume
2
EPS - Extracellular Polymeric Substance� What’s happening in the EPS?
Flemming 2016
• Means of regulating gene expression and diverse physiological activities within community
How is activity in the EPS regulated?
Quorum sensing = how bacteria talk
• Regulation is dynamic:§ Stressors
§ Population density
• Spatial wisdom of crowds:§ Texts, tweets etc
Ø Microbes in biofilms can easily interact with each other and exchange genes encoding:Ø Virulence factorsØ Antibiotic resistance
Ø Biofilm microorganisms are more resistant to antimicrobial agents than planktonic bacteria
Biofilms and antimicrobial resistance Intracanal biofilms
Ø First observed by Nair using microscopy in 1987
Ø Described as clusters of “self-aggregating” colonies of one distinct type or “coaggregating” communities of several types
Ø Described as “biofilms” by Svensater & Bergenholtz (2005)
LM, TEM: Nair 1990
Nair et al. 2005
Root canal biofilms are complex, polymicrobial and heterogenous
Biofilm morphology can vary between and within root canal systems
“Entombed biofilms” in the root canal system
Ø Proposed that “apical periodontitis” be included in the set of “biofilm-induced diseases”
Ricucci and Siqueira 2010
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What is virulence?
Ø The degree of pathogenicity or disease-producing ability of a microorganism
Ø The pathogenicity of an organism is determined by its virulence factors
Potential virulence factors associated with a bacterial cell
Sedgley 2016
Microbial virulence �Specific virulence factors and endodontics
Endotoxin (LPS)
Others?Hemolysin (Sedgley et al. 2005)Lipoteichoic acid (Baik et al. 2008)Peptidoglycan (Hahn and Liewehr 2007)Gelatinase (Sedgley 2007, Sato et al. 2009)Short chain fatty acids (Ho and Chang 2007, Provenzano et al. 2015)Fimbriae (Figdor and Davies 1997, Rôças and Siqueira 2010)Proteases (Ogawa et al. 2006, Nandakumar et al. 2009)
Endotoxin is an established pathogenic factor in endodontic infections
Microbial virulence �Endotoxin and endodontics
Endotoxin is positively associated with:
ü Pulpal pain and periapical inflammationSchein and Schilder 1975, Horiba et al. 1991,
Yamasaki et al. 1992, Khabbaz et al. 2001, and others
ü Bone destruction Dahlén et al. 1981, Dwyer and Torabinejad 1981,
Pitts et al. 1982, Mattison et al. 1987and others
Ø Periapical pathology is multifactorial
Ø The host determines the response to microorganisms ………….
Microbial virulence �What about host factors?
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Periapical pathology is multifactorial
Pulpal infection
Immune response
Systemic factors
Bone resorption
Cytokines
Adapted from Stashenko
Overview 2
Part 1 Basic microbiology updatePart 2 Methods used for evaluating endodontic microflora
Methods used for evaluating endodontic microflora
1. Histology
2. Culturing and biochemical tests
3. Microbial bioinformatics
Evaluating endodontic infections �1. Histology
Limited to observing morphology, and more recently, viability
• Light microscopy (LM) (Ricucci and Bergenholtz 2003, Ricucci et al. 2015)• Transmission electron microscopy (TEM) (Nair 2005)• Scanning electron microscopy (SEM) (Leonardo et al. 2002)• Environmental SEM (Bergmans et al. 2005)• Confocal laser SEM (Parmar et al. 2011)• Combination LM, TEM, SEM (Richardson et al. 2009)
LIVEDEAD
à Standardized sampling methodà Use of clinical sterility controls
à Pre-reduced transport mediaà Anaerobic culturing
à Pure culture and identification
sampletransfer
analyses
Evaluating endodontic infections �2. Culturing
anaerobic culturingidentification
Evaluating endodontic infections – now and the future �3. Microbial bioinformatics
Anderson et al. 2012Tzanetakis et al. 2015Siqueira et al. 2016
and others…
Overview 5
Part 1 Basic microbiology update
Part 2 Methods used for evaluating endodontic microfloraPart 3 Microorganisms in primary endodontic infections Part 4 Microorganisms and unsuccessful endodontic treatment
Part 5 Microorganisms in periapical lesions
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Ricucci et al. 2015
Periapical lesions�Histology study - humans
Ø Not detected in granulomas
Ø Bacteria only detected in abscesses or cysts
Ricucci et al. 2006
Microorganisms in asymptomatic periapical lesions�Tronstad et al. 1987
“Anaerobic bacteria are able to survive and maintain an infectious disease
process in periapical tissues”
8 PA lesionsCulture study
Microorganisms in periapical lesions�Sampling
Microbiological sampling of periapical tissue presents difficulties
J Submarginal incision better than marginal incision to avoid contamination of surgery site
Sunde et al. 2000, Gatti et al. 2000
However…..L Disinfection procedures do not “destroy” the
DNA
Ø Evaluated 39 PA lesions (root-filled teeth with asymptomatic apical periodontitis)
Ø Used CLSM for 3D observations, and FISH to detect specific microorganisms
ü Observed microcolonies throughout
ü Detected P. gingivalis, P. intermedia, T. forsythia, Streptococcus spp. and unidentified morphotypes
Sunde et al. 2003 Subramanian and Mickel 2009
Ø Examined 34 apicoectomy samples for bacterial DNA in root end and soft tissue (including one “control” sample which was neg for DNA)Ø Persistent PA lesions – 1/3 symptomaticØ All except one root end and six periradicular tissue samples showed bacterial DNA
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ü Sampling challenges
ü Polymicrobial
ü Predominantly Gram positive and Gram negative anaerobes
Part 2 Methods used for evaluating endodontic microfloraPart 3 Microorganisms in primary endodontic infections Part 4 Microorganisms and unsuccessful endodontic treatmentPart 5 Microorganisms and periapical lesions
Part 6 Microorganisms and regenerative endodontics
Verma et al. 2017
“Residual bacteria have a critical negative effect on the outcome of regenerative
endodontic procedures.”
Verma et al. 2017
Tissue engineering
approach
Traditional revascularization
approach
Source of bacteria and
bacterial antigens
Why were these cases unsuccessful?Ø Residual biofilm promoted osteoblastic
(BSP) versus dentinogenic (DSPP) gene expression of SCAP
Ø But, E. faecalis antigens inhibited SCAP osteogenic differentiation
Vishwanat et al. 2017
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Diogenes and Hargreaves 2017
Microbes take control!
Overview 7
Part 1 Basic microbiology update
Part 2 Methods used for evaluating endodontic microfloraPart 3 Microorganisms in primary endodontic infections Part 4 Microorganisms and unsuccessful endodontic treatmentPart 5 Microorganisms and periapical lesionsPart 6 Microorganisms and regenerative endodontics
Part 7 Antimicrobial efficacy of endodontic treatment
?
Clinical management of infected root canals
Aiming to remove:Ø Microorganisms
Ø in biofilm and planktonic state Ø dead and alive
Ø whole cells and parts of cells
Ø Debris from instrumentation, smear layer Ø Pulpal remnants - cellular, fibrousØ Metabolic by-productsØ Previous root filling material Siqueira and Rôças 2011
Pathways of the Pulp
Biofilms are involved in all stages of root canal infection
Biofilms grow on root canals walls and in dentinal tubules
Siqueira and Rôças 2011Pathways of the Pulp
Microbe tactics and strategies �Why it’s hard to get rid of biofilms
Howard Hughes Medical Institute
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Microbe tactics and strategies
WinnersVirulence factors
Biofilm interactionsResistance
LosersAccessible?
DisruptibleAntimicrobials
Antimicrobials
Mechanical
removal
Mechanicaldisruption
Clinical management of infected root canals
Antimicrobials
Mechanical
removal
Mechanicaldisruption
Clinical management of infected root canals
Ø Conventional irrigation
Ø Activation of irrigant
Ø Different file systems
Clinician tactics and strategies �Mechanical disruption
Clinician tactics and strategies �Conventional irrigation of root canals
Do irrigants reach apical part of the canal?
L There is little fluid exchange and displacement of particles beyond the tip of the needle �
Chow 1983
Needle tip positioned 3mm short of WL
Boutsioukis et al. 2010
�Apical pressure varies according to needle type�
�
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�What influences irrigant flow?�
Ø Increased canal taper and apical size improves
irrigant replacement and wall shear stress and reduces irrigant extrusion
Albrecht et al. 2004, Falk and Sedgley 2005, Boutsioukis et al. 2010Ø Needle depth placement influences irrigation
efficacySedgley et al. 2005, Boutsioukis et al. 2010
Ø Irrigation significantly less effective in curved compared to straight canals
Nguy and Sedgley 2005
Ø Conventional irrigation
Ø Activation of irrigant
Ø Different file systems
Clinician tactics and strategies �Mechanical disruption
Mechanical disruption
Sonic activation
Ø Lower frequency than ultrasonic EndoActivatorTM Sonicare CanalBrushTM VibringeTM GentleWaveTM☺ All remove debris/smear layer in vitro ☹ Need independent data on antimicrobial activity
and biofilm removal in clinical situations Salman et al. 2010, Kanter et al. 2011, Rodig et al. 2011, Molina et al. 2015
Photon-Initiated Photoacoustic Streaming (PIPSTM)
Uses pulsed erbium:YAG laser to create photon-initiated photoacoustic streamingIn vitro study – culture of samples infected with oral flora
Ø Extracted premolars prepared to apical #20.07Ø PIPSTM generated more negative bacterial
samples than conventional and ultrasonic activation
Ø Limited clinical information available on the use of PDT in root canal disinfection
Ø If supported by future clinical research, PDT may have efficacy for additional root canal disinfection, especially in the presence of multi–drug-resistant bacteria
Chrepa et al. 2014
Ø Conventional irrigation
Ø Activation of irrigant
Ø Different file systems
Clinician tactics and strategies �Mechanical disruption
In vitro studies comparing different file systems to reduce microbial load
No difference between:u SAF, Twisted File and Reciproc
Siqueira et al. 2013
u WaveOne and One ShapeNabeshima et al. 2014
u Hand (K-file) and ProTaperNakamura et al. 2013
u Single Reciproc file and BioRaCe seriesAlves et al. 2012
u WaveOne and ProTaperPinheiro et al. 2016
Clinical studies comparing different file systems to reduce microbial load
No difference between:u Hand NiTi using alternated rotation motion
or BioRaceRôças and Siqueira 2013
u Self-Adjusting File and Twisted File Adaptive for retreatment cases
Rodrigues et al. 2015
u Single Reciproc file and BioRaCe seriesNeves et al. 2016
Studies used real-time qPCR
Clinician challenges�Uninstrumented niches for biofilms
Peters 2004
Antimicrobials
Mechanical
removal
Mechanicaldisruption
Clinical management of infected root canals
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Clinician tactics and strategies �Antimicrobials
Ø Sodium hypochlorite
Ø Chlorhexidine
Ø EDTA
Ø Calcium hydroxide
Ø Antibiotics
Sodium hypochlorite (NaOCl)
Is sodium hypochlorite an effective antimicrobial agent?