ENDODONTIC MICROBIOLOGYSECOND EDITION

ASHRAF F. FOUAD

Endodontic Microbiology

Endodontic Microbiology

Second Edition

Edited by

Ashraf F. FouadFreedland Distinguished Professor and ChairDepartment of EndodonticsSchool of Dentistry, University of North CarolinaChapel Hill, NC, USA

This edition first published 2017© 2017 by John Wiley & Sons, Inc.

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Library of Congress Cataloging-in-Publication DataNames: Fouad, Ashraf F., editor.Title: Endodontic microbiology / edited by Ashraf F. Fouad.Description: Second edition. | Hoboken, NJ : John Wiley & Sons Inc., 2017. | Includes bibliographical references and index.Identifiers: LCCN 2016042792 | ISBN 9781118758243 (cloth) | ISBN 9781118975497 (Adobe PDF) |

ISBN 9781118975503 (epub)Subjects: | MESH: Dental Pulp Diseases–microbiology | Dental Pulp Diseases–drug therapy | Periapical Diseases–microbiology |

Periapical Diseases–drug therapy | Anti-Infective Agents–therapeutic use | Root Canal TherapyClassification: LCC RK351 | NLM WU 230 | DDC 617.6/342–dc23 LC record available at https://lccn.loc.gov/2016042792

Cover images courtesy of the author

Set in 9.5/11.25pt TimesLTStd by Aptara Inc., New Delhi, India

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Dedication

To Amal, Fikry, Lori, Amani, George, Anthony Gade, and Edward; thank you forproviding me the opportunity, the inspiration, the motivation, and the love.

Ashraf F. Fouad

Contents

Contributors ixPreface xiPreface to the First Edition xiii

1 Microbial Perspectives in theTwenty-First Century 1William Wade

2 Diagnosis, Epidemiology, and GlobalImpact of Endodontic Infections 11Dag Ørstavik

3 Microbiology of Dental Caries andDentinal Tubule Infection 25Robert M. Love and Anne C.R. Tanner

4 Culture-Based Analysis of EndodonticInfections 51Gunnar Dahlen

5 Molecular Analysis of EndodonticInfections 81Jose F. Siqueira, Jr, and Isabela N. Rocas

6 Extraradicular Endodontic Infections 129Brenda P. F. A. Gomes and Ericka T. Pinheiro

7 Virulence of Endodontic BacterialPathogens 149Christine Sedgley

8 Viruses in Endodontic Pathosis 179Mohamed Sabeti

9 Fungi in Endodontic Infections 197Bilge Hakan Sen and B. Guniz Baksi

10 Severe Head and Neck Infections 231Jaime S. Brahim and Robert A. Ord

11 Endodontic Infections and Pain 251Anibal Diogenes and Ken M. Hargreaves

12 Systemic Antibiotics in EndodonticInfections 269Ashraf F. Fouad

13 Topical Antimicrobials in Endodontics 287Anil Kishen

14 Endodontic Infections in IncompletelyDeveloped Teeth 311George T.J. Huang, Domenico Ricucci,and Louis M. Lin

15 Prognosis of Healing in Treated Teethwith Endodontic Infections 341Shimon Friedman

16 Endodontic Infections and SystemicDisease 385Ashraf F. Fouad

Glossary 409Index 413

vii

Contributors

Editor

Ashraf F. Fouad, DDS, MSFreedland Distinguished

Professor and ChairDepartment of EndodonticsSchool of Dentistry,

University of NorthCarolina

Chapel Hill, NC, USA

Authors

B. Guniz Baksi, DDS, PhDProfessorDepartment of Oral Diagnosis

and RadiologySchool of DentistryEge UniversityIzmir, Turkey

Jaime S. Brahim, DDS, MSProfessor, Undergraduate DirectorOral and Maxillofacial Surgery

DepartmentUniversity of Maryland Dental

School and HospitalBaltimore, MD, USA

Gunnar Dahlen, DDS, PhD (DrOdont)

Professor and ChairmanDepartment of Oral Microbiology

and ImmunologyInstitute of OdontologySahlgrenska Academy, University

of GothenburgGothenburg, Sweden

Anibal Diogenes, DDS, PhDDiplomate, American Board of

EndodonticsDirector, Advanced Program in

EndodonticsDepartment of EndodonticsUniversity of Texas Health

Science Center at San AntonioDental School

San Antonio, TX, USA

Shimon Friedman, DMDProfessor, MSc Endodontics

ProgramUniversity of Toronto Faculty of

DentistryToronto, Ontario, Canada

Brenda P.F.A. Gomes, MSc, PhD,BDS

Professor, EndodonticsPiracicaba Dental SchoolState University of CampinasPiracicaba, SP, Brazil

Ken M. Hargreaves, DDS, PhDProfessor and ChairDepartment of EndodonticsUniversity of Texas Health

Science Center at San AntonioDental School

San Antonio, TX, USA

George T.J. Huang, DDS, MSD,DSc

ProfessorDirector for Stem Cells and

Regenerative Therapies

Department of BioscienceResearch

College of DentistryUniversity of Tennessee Health

Science CenterMemphis, TN, USA

Anil Kishen, PhD, MDS, BDSProfessor and HeadDiscipline of EndodonticsFaculty of Dentistry, University of

TorontoToronto, Ontario, Canada

Louis M. Lin, BDS, DMD, PhDProfessor of Department of

EndodonticsCollege of DentistryNew York UniversityNew York, NY, USA

Robert M. Love, BDS, MDS,PhD, FRACDS

Professor, Dean and Head ofSchool

School of Dentistry and OralHealth

Griffith UniversityQueensland, Australia

Robert A. Ord, DDS, MD, FRCS,FACS, MS

Chairman and ProfessorDepartment of Oral and

Maxillofacial SurgeryUniversity of Maryland Medical

Center

ix

x Contributors

University of Maryland School ofDentistry

Baltimore, MD, USA

Dag Ørstavik, Cand Odont, DrOdont

Professor and Head, Departmentof Endodontics

Institute for Clinical DentistryUniversity of OsloOslo, Norway

Ericka T. Pinheiro, MSc, PhD,BDS

Assistant ProfessorDepartment of DentistryUniversity of Sao PauloSao Paulo, Brazil

Domenico Ricucci, MD, DDSLaboratory HeadPrivate practiceCetraro, CS, Italy

Isabela N. Rocas, DDS, MSc,PhD

Professor, Department ofEndodontics;

Head, Molecular MicrobiologyLaboratory

Faculty of Dentistry, Estacio deSa University

Rio de Janeiro, Brazil

Mohamed Sabeti, DDS, MADiplomate, American Board of

EndodonticsAssociate ProfessorLoma Linda UniversityLos Angeles, CA, USA

Christine Sedgley, MDS, MDSc,FRACDS, MRACDS(ENDO),PhD

Professor and ChairDepartment of EndodontologySchool of Dentistry, Oregon

Health & Science UniversityPortland, OR, USA

Bilge Hakan Sen, DDS, PhDEge UniversityIzmir, Turkey

Jose F. Siqueira, Jr, DDS, MSc,PhD

Chairman and DirectorPostGraduate Program in

Endodontics

Faculty of Dentistry, Estacio deSa University

Rio de Janeiro, Brazil

Anne C.R. Tanner, BDS, PhDSenior Member of StaffDepartment of MicrobiologyThe Forsyth InstituteCambridge, MA, USA;Associate Clinical ProfessorHarvard School of Dental

MedicineBoston, MA, USA

William Wade, BSc, PhDProfessor of Oral MicrobiologyCentre for Immunology and

Infectious DiseaseBlizard InstituteBarts and The London School of

Medicine and DentistryQueen Mary University of

LondonLondon, UK

Preface

Much has happened in endodontic microbiology sincethe publication of the first edition of this book. Hun-dreds of important research studies and reviews havebeen added to the literature in this important field.We now have many better epidemiologic studies onthe prevalence of endodontic disease, its associationwith systemic disease, and its potential contributionsto major morbidity and mortality of patients. The areaof polymicrobial infections is now recognized as amajor public health problem. In the last decade it hasseen innovations in research methodologies as wellas the conceptual descriptions of how these infectionscan produce disease. In the field of endodontic micro-biology, next generation sequencing is now commonlyused in research, revealing hundreds if not thousands ofmicrobial taxa that are involved in endodontic patho-sis. The study of microbial virulence has also seenmajor advances. These include the interplay of differ-ent pathogens, such as bacteria, viruses, and fungi, toincrease the pathogenicity of either, the host–microbialinteractions, the differences in clinical presentations,and responses to treatment observed with differentgenomic and epigenetic variations in the host, bac-terial load issues, quorum sensing, and the keystonepathogen concept that describes how a pathogen caninduce host changes that converts a microbial commu-nity to become dysbiotic.

Endodontic microbiology research still has manyfrontiers that have not been adequately studied. Theseinclude the reasons why chronic infections can exac-erbate to produce severe and spreading infections, thedegree to which endodontic microflora travel to distant

sites in acute and chronic infections, the exact relation-ship between residual bacteria and healing, and theeffects of residual bacteria on the success of regener-ative therapies. The interaction of the microbial com-munity in the deep carious lesion or the necrotic pulpwith the host response can produce chronic asymp-tomatic disease or severe pain. The degree to whichthe composition of the microflora, the expressed vir-ulence factors, and the host’s innate susceptibility todisease interact to produce the resultant clinical mani-festation needs further elucidation.

The ability to eliminate microbial irritants isparamount to adequate healing in endodontics. Thereare still no clinical markers that can predict the long-term responses to vital pulp therapy or to endodon-tic treatment. Scientific explorations that utilizecutting edge technologies, such as shotgun sequenc-ing or metagenomics, transcriptomics, and proteomicshave not been sufficiently incorporated in endodonticresearch. The degree to which microbial eliminationis required to mediate the regeneration of the dentalpulp, or even just revitalization in the pulp space, isnot clear. Finally, we still do not have rapid molecu-lar methods of identifying antibiotic resistance, whichwould allow the efficient and effective selection ofthe right antibiotic. These and many other questionswill continue to inspire many studies and insights thatwould allow us to improve the success of our treat-ment modalities, save more teeth from extraction, andimprove the patients’ quality of life.

Ashraf F. Fouad

xi

Preface to the First Edition

Endodontic infections are very prevalent, because theymostly represent complications of dental caries and itstreatment, as well as traumatic injuries to teeth, whichare all very prevalent occurrences. Collectively, theyrepresent the majority of dental infections that presentwith significantly acute local and systemic signs andsymptoms. This is the first textbook devoted to thestudy of endodontic infections, which hitherto has beenlimited to isolated single chapters in endodontic text-books. This textbook is intended to provide a collec-tion of work showing the state of the knowledge inthis field. It is also intended to provide some researchquestions and hypotheses that, hopefully, will stimu-late more efforts to understand the disease process andidentify effective treatment methods.

The study of endodontic microbiology has beencomplicated by difficulty in epidemiological data inobtaining adequate endodontic diagnosis on largenumbers of nonpatient populations. In addition, sam-pling is a major challenge in endodontics. Contami-nation from the tooth surface, caries, or saliva mustfirst be avoided. Access to the potentially very com-plex root canal anatomy and disruption of biofilm onthe majority of canal walls in these areas is neces-sary. It is almost impossible to differentiate specimensobtained from the apical and coronal portions of theroot canals; thus, the effect of location of microflorawithin the canal is poorly understood, and can onlybe studied in teeth that are extracted. Finally, samplingafter completion of treatment to assess effectiveness oftreatment and determine the long-term outcome risk iscomplicated by the fact that only the areas that couldbe reached could be sampled.

The difference in sensitivity between tradi-tional culturing and modern molecular methods are

especially important in endodontic microbiology,because the endodontic specimen has so little material,and sensitivity, therefore, has a major role in microbialidentification. The description of traditional bacterialpathogens and their virulence factors represents mostof the available literature today. The contributions ofthe not-yet-cultivated bacteria and the bacteria ren-dered temporarily uncultivable by traditional treatmentmethods have not been adequately studied. Likewise,we are just beginning to understand some of the con-tributions of fungi and viruses to the pathogenesis ofendodontic infections.

The debate on viable versus dead microorgan-isms that are detected by molecular techniques mustbe resolved by using more accurate technologiesthat assess microbial counts, their viability, andtheir pathogenicity. Likewise, consistent and stringentmethodologies, including sequencing of amplificationproducts, are essential for assuring accurate results andenabling comparisons among studies.

Persistent endodontic pathosis may be due to per-sistent infection or new infection after treatment. Sam-pling of apical lesions during periapical surgery iscomplicated by the lack of sterility of the surgical field.Therefore, the microbiology of nonhealing endodonticcases is still in its infancy at this time.

It is clear that in order to determine effective treat-ment modalities, better sampling and identificationtechniques must be employed and more adequatelydesigned outcome studies need to be performed.

Finally, the relationship between endodontic patho-sis and systemic disease must be more comprehen-sively studied. Endodontic infections were historicallythought to contribute to numerous systemic diseases.While the potential for systemic spread of an acute

xiii

xiv Preface to the First Edition

endodontic infection is well-known and documented,earlier studies have failed to demonstrate that chronicendodontic infections contribute to systemic diseases.However, these hypotheses must be reexamined nowthat we have more accurate research tools. In addition,the creation of large patient databases for longitudinal

analysis of treatment outcomes, and their relationshipswith systemic disease will be imperative in future stud-ies that address this issue.

Ashraf F. Fouad

Chapter 1Microbial Perspectives in theTwenty-First CenturyWilliam Wade

1.1 Introduction1.2 Genomics1.3 Molecular microbial ecology and the

study of uncultivable bacteria1.4 Intraspecies variation1.5 Metagenomics and metatranscriptomics

1.6 Bacterial–bacterial communication1.7 Host–bacterial interactions1.8 Complex infectious diseases1.9 The future

1.10 References

1.1 Introduction

The final quarter of the nineteenth century wasarguably the golden age of medical microbiology. Theground-breaking work of Pasteur, Koch, and othersled to the development of broth and agar media thatwere able to support the growth in the laboratory of themajor bacterial pathogens affecting humans. The abil-ity to grow these organisms in pure culture led to theproduction of vaccines for many of the diseases theycaused. These advances, and the subsequent discoveryand development of antimicrobials, led to the mistakenbelief that infectious disease had been beaten.

Of course, it is now realized that this optimisticviewpoint is not justified, not least because of the rapidemergence of bacterial resistance to antimicrobials.Indeed, the consensus view is that the battle againstbacterial resistance is currently being lost, because ofboth the difficulty and costs associated with devel-oping new antimicrobials and indiscriminate use ofthose currently available. The predicted ultimate fail-ure of antimicrobial strategies has led to renewed inter-est in elucidating the pathogenic mechanisms used by

bacteria to cause disease, with the ultimate aim ofdevising new methods of disease prevention andtreatment.

At the same time, interest in the microbial popula-tions of the Earth has been intense and new techniqueshave become available to characterize the bacterialcommunities found in every ecosystem on the planet.These have revealed the quite astonishing diversity ofmicrobial life on Earth and the extreme complexity ofmost bacterial communities. Furthermore, the extentof subspecific diversity is only now being fully appre-ciated. Bacterial readily exchange DNA and can “shuf-fle” their own genomes to generate diversity with theultimate aim of responding and adapting to environ-mental change. As discussed later, bacteria in commu-nities communicate with each other and, in the caseof commensals living with plants and animals, theirhosts. These interactions operate at various levels andcan be remarkably sophisticated. The twenty-first cen-tury will be a period of tremendous advances in ourunderstanding of the microbial world.

The aim of this chapter is to review recent deve-lopments in microbiology and to highlight selected

Endodontic Microbiology, Second Edition. Edited by Ashraf F. Fouad.© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

1

2 Endodontic Microbiology

areas that are likely to change our conceptual view ofinfectious disease as a whole, and oral and endodon-tic infections in particular. Inevitably, a single shortchapter cannot provide a comprehensive overview ofan entire discipline, but the interlinked topics cov-ered are those that will undoubtedly change our viewof the microbial world and its relationship with thehuman host.

1.2 Genomics

The sequence of the human genome was publishedin 2001. The benefits of this outstanding achievementare now being realized with the identification of genesresponsible for or causing a predisposition for a largenumber of diseases (Wellcome Trust Case ControlConsortium 2007). At the same time, and largely pos-sible because of the technical advances made as partof the human genome sequencing effort, genomes ofother organisms are being sequenced, including thoseof bacteria.

As of February 2015, the sequencing of the genomesof 26 522 bacteria and 647 archaea had been com-pleted, while 15 800 and 424, respectively, were inprogress or available as a draft (for more informationsee www.genomesonline.org). As expected, the dataobtained have revealed the enormous genetic potentialcontained within bacterial genomes; in each genomesequenced, around one-third of the genes present havebeen novel and the function of a significant proportionremains unknown.

The availability of genome sequence data is allow-ing a far more robust bacterial classification to be con-structed than previously possible. Bacterial taxonomywas once based purely on phenotypic characters andwas very inexact because of the difficulties involvedin obtaining and interpreting such data compared toplants and animals where differences in phenotype arefar more obvious. In recent years, genetic informa-tion has been increasingly used, but on a limited scale,and typically only the sequences of the 16S riboso-mal RNA (rRNA) and other housekeeping genes havebeen used. New methods are now being introduced tomake use of the sequence data available for completegenomes (Konstantinidis and Tiedje 2005). In gen-eral, the results of using such methods have supportedthe 16S rRNA gene taxonomy at species and genuslevel but, in addition, have provided improved clar-ity of the relationships among the higher taxonomic

ranks, where substantial overlap between ranks hasbeen observed.

The results of the analysis of some genomic datahave been extremely surprising. A Gram-positivecoccus found in amoebae could not be identified bythe conventional molecular analysis of 16S rRNAgene sequencing because no ribosomal genes couldbe amplified for sequencing. Genomic data explainedthis difficulty by revealing that the organism wasactually a virus, the largest yet discovered. Nownamed Mimivirus, the large virus particles are upto 0.8 μm in diameter, the size of many bacteria. Itprimarily infects amoebae but has been implicatedas a cause of pneumonia on serologic grounds andhas caused a laboratory-acquired pneumonia in aresearcher (Raoult et al. 2007). At the other end of thebacterial scale, members of the genus Epulopiscium,found in the intestine of certain surgeonfish (Angertet al. 1993), have been discovered that are visible withthe naked eye.

In addition to correctly identifying evolutionaryoddities, genomic data have identified numerous novelbiochemical pathways with the potential for exploita-tion. Among these are some novel antimicrobialsalthough the range of targets within bacterial cells thathas arisen by natural evolution is rather narrow. Amore promising avenue to the development of novelantimicrobials is to use genomic data to identify noveltargets for antimicrobial treatments (Pucci 2006). Pre-dictions can be made from genome data as to howessential a given gene is to an organism and there-fore how disrupting the gene would affect the vitalityof the organism. These predictions can then be testedin an appropriate manner experimentally using a widerange of methods that have been developed in responseto the availability of genomic data. These include ran-dom mutagenesis mediated by transposons or inser-tion of plasmids, targeted gene disruption or in vivotechniques such as signature-tagged mutagenesis andin vivo expression technology. Structural genomics,where sequence data is used to predict the structureof essential bacterial proteins, is also being used toidentify potential targets for antimicrobials. Finally,comparative genomics can be used to identify com-mon features of pathogens affecting a particular bodysite to custom design antimicrobials for specific pur-poses, for example, respiratory tract infection.

Next generation sequencing technologies suchas the Roche 454 and Illumina systems have beenintroduced and have brought the ability to sequence

Microbial Perspectives in the Twenty-First Century 3

bacterial genomes within the reach of individuallaboratories. Accurate interpretation of the dataremains a challenge, however, although a number ofuseful software programs are now available (Edwardsand Holt 2013). The information obtained thus farhas been of extraordinary value in understandingthe role of pathogenic bacteria in disease and is thefundamental basis of other new technologies such astranscriptomics and proteomics. The next task will beto understand how gene products interact both withina bacterial cell and in response to external stimulifrom the environment and other organisms.

1.3 Molecular microbial ecology and thestudy of uncultivable bacteria

Almost without exception, oral infections are polymi-crobial in nature and difficult to study because aroundhalf of the bacteria present in the oral cavity cannot begrown using conventional culture media. It has longbeen recognized that not all bacteria from a given habi-tat can be cultured on artificial media in the laboratory.Indeed, it has been estimated that less than 2% of bac-teria on Earth can be cultured.

Methods for the characterization of complex bac-terial communities were developed as a consequenceof the use of DNA sequence data for the constructionof evolutionary trees. This was done by comparingthe sequences of genes encoding essential functions,the so-called housekeeping genes that are found in allcellular organisms. The gene most commonly used todate has been encoding the small subunit (16S) rRNAmolecule. Ribosomes have the essential function oftranslating messenger RNA (mRNA) into amino acidchains and, because of the need to preserve function,have evolved slowly. Some of the regions of the genehave changed very little over time and are therefore vir-tually identical in all bacteria. These regions are veryuseful for the design of universal polymerase chainreaction (PCR) primers that can amplify the gene froma wide range of different bacteria. Other regions aremore variable and can be used to discriminate betweenorganisms, almost to species level. Woese and col-leagues used small subunit rRNA comparisons to con-struct a tree of life (Figure 1.1), which showed thatbacteria had evolved into two domains, the Archaeaand Bacteria, while eukaryotic organisms fell into asingle third domain, the Eukarya (Woese 1987). It was

originally thought that organisms found in the domainBacteria were those found in normal environmentswhile the Archaea were present in extreme environ-ments such as the deep sea and associated with volca-noes and so on. However, these associations have sincebeen shown not to be true and members of Archaea arenow known to be widely distributed, and an archaealgenus, Methanobrevibacter, can be found in the humanmouth.

A major consequence of the availability of this treeis that unknown organisms of any type can be identi-fied simply by sequencing their rRNA gene and addingthe sequence to the tree or by directly comparing thesequence with the hundreds of thousands of bacte-rial sequences held in the sequence databases. Com-plex bacterial communities can be characterized bythe PCR, cloning, and sequencing of 16S rRNA. Suchstudies have been performed with samples from thehuman mouth in health and disease and are describedin more detail in Chapter 5 and throughout the text-book. A common finding of every study to date hasbeen to confirm that around half of the oral micro-biota is uncultivable. Around 700 species have beendetected, 95% of which belong to the phyla Fir-micutes, Bacteroidetes, Proteobacteria, Actinobacte-ria, Fusobacteria, and Spirochaetes (Dewhirst et al.2010). Other phyla consistently detected are Syn-ergistetes, Chloroflexi, and the un-named phylum-level divisions GN02, SR1, and TM7 (Camanochaand Dewhirst 2014). The Human Oral MicrobiomeDatabase (www.homd.org) lists the bacterial taxafound in the mouth and provides descriptions of theirphenotypes, where available, with links to genomesequence data as well as a 16S rRNA gene sequenceidentification tool (Chen et al. 2010).

Major efforts are now being made to improveour understanding of currently unculturable bacteria.These include the development of new culture mediathat better mimic the natural environment. Very often,laboratory culture media are far richer in nutrients thanthe natural habitat and the use of dilute media or filterednatural substrate has been successful in culturing pre-viously uncultured organisms. This approach has notbeen applied systematically to the study of oral uncul-turable bacteria, but should be possible. Around halfof oral bacteria cannot be cultivated in vitro. Thereappears to be no single reason for this but it hasbeen shown that because oral bacteria naturally livein a multispecies community, some species require thepresence of other bacteria to grow. The development

4 Endodontic Microbiology

Fig. 1.1 Phylogenetic tree showing representatives of the domains Eukarya, Archaea, and Bacteria.

of coculture methods linked with enrichment of tar-get organisms by in situ hybridization has enabled thecultivation of members of a previously uncultivatedlineage of the phylum Synergistetes (Vartoukian et al.2010). The commonly occurring division TM7 haslong been a target for cultivation and it has been foundthat it can be detected readily in mixed laboratorycultures but not cultured independently (Hugenholtzet al. 2001). The reason for this observation has beendetermined recently in that a TM7 phylotype has beenrevealed to be an obligate episymbiont of other bacte-ria, and also capable of entering the host bacterial cells(He et al. 2015). It has a small genome (approximately0.7 MB) and lacks genes for the synthesis of essentialamino acids and so presumably needs to obtain aminoacids from its associated bacterial host. TM7 is a largephylum widely distributed in the environment as wellas being a component of mammalian microbiomes andit will be interesting to determine if all representatives

of the division are episymbionts or if some have otherlifestyles.

The potential benefit of growing previously uncul-tivable organisms has been demonstrated recently inthe successful attempts to cultivate soil bacteria byproviding a natural community in contact with the cul-ture system via a permeable membrane which has ledto the discovery of a new antibiotic, teixobactin (Linget al. 2015).

1.4 Intraspecies variation

The majority of microbiological diagnostic methodsidentify the target organism to species level. However,it is now recognized that individual strains within aspecies often vary markedly in their virulence. Withina species, some strains may be pathogenic while othersare harmless. The extent of the genetic variation within

Microbial Perspectives in the Twenty-First Century 5

species, however, has only been fully realized by thesequencing of the genomes of multiple representativesof the same species.

In one such study, three strains of Escherichiacoli were compared: the well-known harmless lab-oratory strain K12, an enterohaemorrhagic serotypeO157 strain of the group associated with beef prod-ucts, and a uropathogenic strain. It was found that theyhad only 39% of their genes in common, a surpris-ingly small number (Welch et al. 2002). These com-mon genes encoded the functions that gave the strainstheir identity as members of the species E. coli, whilethe remaining genes gave them the ability to colonizeparticular body sites and/or damage the host by meansof a specialized set of virulence factors appropriatefor their natural habitat and lifestyle. Genes acquiredfrom other organisms by horizontal gene transfer canbe critical to that organism’s behavior, and in the pastmay have been the reason a species was given a partic-ular name. For example, if the mainly harmless envi-ronmental organism Bacillus cereus acquires plasmidspXO1 and pXO2, which carry genes coding for fourtoxins and the enzymes required to make a capsule,it becomes Bacillus anthracis, the causative agent ofanthrax.

This work has given rise to some new genomic con-cepts. The core genome is that shared by all strains ofthe species, while the peripheral or accessory genomeincludes genes found in some strains but not others, butwhich nonetheless may be important in pathogenesis.Some bacteria go further and have two chromosomes;in this case, one normally encodes housekeeping genesand the second genes that confer fitness for competi-tion in the environment.

The range of genes encoded by the peripheralgenome can be extensive. In a study of the genomesequences of eight Streptococcus agalactiae strains,the authors calculated the number of strains of thespecies that would have to be sequenced to reveal thefull genetic diversity of the species (Tettelin et al.2005). The result was infinity. In other words, S.agalactiae can incorporate DNA into its genome fromsuch a wide range of sources that all the possible genesthat could be found within this species will never beknown.

The implications of these findings are significant.Although much work has been invested in the develop-ment of rapid assays to detect the presence of specificorganisms in clinical samples, including those col-lected from oral diseases, the association of a species

with disease may be insufficient for diagnostic pur-poses. Detection of the presence (and expression) ofspecific virulence genes may be required to provide ameaningful microbiologic diagnosis. This will clearlybe difficult for diseases where the virulence determi-nants important in disease are currently unknown orwhere multiple virulence mechanisms are operating.

1.5 Metagenomics andmetatranscriptomics

As individual bacterial strains can vary greatly in theirgenetic composition and the assignation of an isolateto a species alone is likely to give a poor indication ofits pathogenicity, alternative methods of analysis needto be developed to determine the role of bacterial com-munities in human disease. New methods are particu-larly needed for complex diseases because the bacterialcommunities associated with the mucous membranes,where these diseases primarily occur, are so diversethat their routine characterization is not practicable. Anovel approach does not attempt to isolate and purifyall of the component species and strains, but, ratherit considers the whole community and all of its con-stituent genes as a whole. The bacterial communityfound at a habitat is termed the microbiome and allthe genetic material of the community members is themetagenome (Rondon et al. 2000).

The first stage in any such analysis is to extractDNA from all of the bacteria present in the sample.In early metagenomics studies, the DNA was clonedinto either small plasmid vectors for ease of sequenc-ing or into systems such as bacterial artificial chro-mosomes (BAC), which allowed DNA fragments upto 100 kb in length to be stably maintained in an E.coli host and genes expressed to seek functions ofinterest. These approaches have now been supersededby the use of next generation sequencing, which doesnot involve a cloning step. The cloning approach hasallowed a number of new antibiotics to be discov-ered from metagenomic analyses of soil and marineenvironments. For example, turbomycin A and B werediscovered in this way (Gillespie et al. 2002). Inter-estingly, a single gene was responsible for the activitywhich was mediated by an interaction between indole,normally produced by E. coli, and the gene product.The success rate in identifying novel antimicrobials inmetagenomic libraries has been low; typically, several

6 Endodontic Microbiology

hundred thousand clones have to be screened to findone new active compound. This relative lack of suc-cess partly reflects the methodology where E. coli isused as the host. Successful expression may requirethe presence of specific promoters or other accessorymolecules and factors such as the G+C content of theinsert and codon usage may adversely affect expres-sion. Thus, in general, cloned fragments of DNA areexpressed most easily in their natural host and the morephylogenetically distant the clone host, the less likelythat expression will be successful. To overcome this,new vector– host systems are being introduced for theexpression of metagenomic libraries to enable a bet-ter match between the insert and the host in which itis being expressed. These include Streptomyces andPseudomonas, two genera members of which natu-rally produce secondary metabolites with propertiesof interest. An alternative approach is to use bioin-formatic methods to screen for gene clusters encod-ing potentially useful compounds within metagenomicdata. This approach has been successfully applied toenvironmental samples (Owen et al. 2013).

Metagenomic sequence data are being used toassemble genomes of bacteria in natural communi-ties. This has been successfully performed for the rel-atively restricted microbiota found in a subterraneanacid mine drainage biofilm where near-complete bac-terial genomes were reconstructed for two previouslyuncultured bacteria (Tyson et al. 2004). More ambi-tiously, Venter et al. (2004) randomly sequenced themetagenome of water specimens collected from theSargasso Sea. Over one trillion base pairs of DNAwere sequenced which were found to be derived from1800 species including 148 not previously character-ized. More than one million novel genes were found.However, it proved difficult to reconstruct completegenomes from these data because of the number ofclosely related species present and the large num-bers of mobile elements with high levels of sequencesimilarity. A number of software tools are now avail-able for genome assembly from metagenomic data,both as stand-alone programs or as online resources(Hunter et al. 2014) although it remains a challengingtask.

Metagenome analyses allow an appreciation of thegenetic potential of a microbial community but noindication of actual activity. Analysis of the mRNAspresent in a sample shows which genes are currentlybeing expressed and thus the functional activity of the

microbiota. RNA is extracted directly from the sampleand then enriched for mRNA and reverse transcribed.The DNA is then fragmented and sequenced using nextgeneration methods. After removal of contaminatingand repetitive sequences, the reads are then mapped toreference sequences in nucleotide databases in order toidentify genes and operons which have been expressed(Carvalhais et al. 2012). This approach has been suc-cessfully used with oral samples from subjects withperiodontitis and caries (Duran-Pinedo et al. 2014;Simon-Soro et al. 2014).

1.6 Bacterial–bacterial communication

Originally thought of as simple dumb solitary crea-tures, it is now known that bacteria live together incommunities with a number of features in commonwith multicellular organisms. The basis for the cooper-ation of individual bacterial cells within a communityis communication. Communication is mediated by theproduction of signaling molecules often genericallydescribed as quorum-sensing molecules, after the firstbacterial signaling system to be described.

In many circumstances, the total number of bac-terial cells in a community is important to the over-all health of the community. In the environment, theavailability of nutrients and external stresses are fac-tors that cause the community to behave in a particularway. This may increase or decrease its overall rate ofgrowth, become more motile to move to a new habitatto obtain nutrients and, once there, switch to a biofilmmode of growth to colonize the new environment. Forpathogenic bacteria of exogenous source, bacterial–bacterial communication is particular important. Thefirst bacteria to colonize the host will necessarily bepresent in small numbers and will want to multiplywithout alerting the host to their presence in order toavoid the host’s immune system. As virulence factorssuch as protein toxins are typically highly antigenic,the pathogen will not produce them until the com-munity is sufficiently numerous to resist the host’sdefenses. Once this “quorum” has been achieved, themembers of the community will turn on the productionof their virulence genes in order to damage the hostand cause disease.

The molecular basis for quorum sensing was firstelucidated for the bioluminescent marine bacteriumVibrio fischeri. This organism is commonly found

Microbial Perspectives in the Twenty-First Century 7

as a symbiont in the light-producing organs ofluminescent fish or squid. The signaling mechanismis a two-component system; the luxI gene producesautoinducer 1 (AI-1), an acyl homoserine lactone.This is produced constitutively, but when sufficientnumbers of V. fischeri are present, the high con-centration of AI-1 enables binding to the receptor,the product of luxR, which activates transcription ofthe luciferase operon and leads to production of thelight-emitting compounds.

Following its discovery in V. fischeri, AI-1 analogshave been found in a wide range of Gram-negativebacteria. The AI-1 of a particular species is normallyspecific to that species so that cross-talk is avoidedwithin multispecies communities.

Gram-positive bacteria also produce signalingmolecules, but those so far described have all beenpeptides derived from larger precursors by posttrans-lation modification. An important group of signalingmolecules in Gram-positives is of those that inducecompetence. Competence is the ability of bacteria totake up DNA present in the environment. This is animportant mechanism of genetic exchange and is par-ticularly common among members of the oral micro-biota such as the oral streptococci. In many strepto-cocci, the precursor molecule is Com C which is mod-ified as it is transported out of the cell by the ComABtransporter. The signaling molecule itself is the C-terminal end of Com C and is termed the competence-stimulating peptide (CSP). When the bacterial num-bers reach their quorum, CSP binds to the receptor,the histidine kinase Com D, which then stimulates theresponse regulator Com E.

In addition to the species-specific quorum-sensingmechanisms described above, bacteria also make useof nonspecific systems that allow general communica-tion between bacteria in communities, across speciesbarriers. One such molecule, AI-2, is produced by, andcan be detected by, a wide range of both Gram-negativeand Gram-positive bacteria. AI-2 forms spontaneouslyfrom 4,5-dihydroxy-2,3-pentanedione (DPD). whichis a product of the Lux S enzyme in the catabolism of S-ribosylhomocysteine. A number of oral bacteria havebeen shown to produce AI-2 and it has been found tohave an important role in dental plaque formation, Forexample, Streptococcus oralis and Actinomyces naes-lundii are known to coaggregate early in the develop-ment of dental plaque biofilms and grow together in invitro models, forming a profuse plaque with physical

interaction between cells of the two species. A luxSmutant of S. oralis that did not produce AI-2, how-ever, did not form such biofilms with A. naeslundii,while the mutualistic activity was restored by luxScomplementation (Rickard et al. 2006). Quorumsensing is thus a central mechanism in bacterialmetabolism, with particular importance in biofilm for-mation. The use of quorum-sensing inhibitors haspotential for use as antibiofilm agents (Brackman andCoenye 2015).

Another group of bacterial cell-signaling moleculesare the family proteins related to resuscitation-promoting factors (Rpf). Originally discovered inMicrococcus luteus where they were able to reviveM. luteus cells that had entered a dormant phase, theywere subsequently found to be widespread amongmembers of the phylum Actinobacteria, the HighG+C Gram-positives (Mukamolova et al. 1998). Inter-estingly, the growth of Mycobacterium tuberculosis,which is normally extremely slow in vitro, is greatlystimulated by Rpf. Rpf is a protein, structurally similarto lysozyme, which can exert its effects at extremelylow concentrations. It has therefore been termed a bac-terial cytokine because of its resemblance to mam-malian cytokines that have similar properties. Themolecular basis of its action has yet to be determinedbut it would appear to cleave the peptidoglycan of dor-mant cells and either release a second messenger orphysically allow the cells to resume growth. Peptido-glycan fragments, muropeptides, have recently beenrecognized to be important mediators of communica-tion both between bacteria and between bacteria andeukaryotes (Dworkin 2014).

Novel mechanisms of bacterial communication arebeing discovered all the time, and it is extremely likelythat a network of sophisticated interactions existsamong the bacterial community in dental plaque. Manyof these are clearly relevant to endodontic infectionand the survival of bacteria under restorations andin the treated root canal. The realization that vege-tative bacterial cells can go into a dormant state, dis-tinct from endospore production, and survive for manyyears may explain how bacteria survive under restora-tions or despite calcium hydroxide treatment in the rootcanal. Furthermore, a change in the environment maystimulate the production of broad-range growth stim-ulation factors that cause the community to undergorapid growth, causing damage to the affected tooth andpain to the patient.

8 Endodontic Microbiology

1.7 Host–bacterial interactions

All plants and animals are colonized by bacteria. Mam-mals are born sterile but extremely quickly becomecolonized with the microbiota characteristic for theirspecies. The commensal microbiota associated withmammals has evolved over millions of years, and it ispossible to reconstruct the evolution of the commensalmicrobiota in parallel with each mammalian host, thephenomenon of cospeciation. Thus, for the majority ofbacteria found in the human mouth, there are versionsof that organism found in other animals. For example,among the mutans group streptococci, associated withdental caries, S. mutans and S. sobrinus are found inhumans, S. ferus and S. rattus in rats, S. cricetus inhamsters, and so on.

The recognition that human cells make up onlyaround one-third of all cells in the body, with the major-ity of the remainder Bacteria (American Academyof Microbiology 2014), led to initiatives to describethe microbial populations and their genomes at var-ious body sites, principally the National Institutesof Health-funded Human Microbiome Project (HMP)(Human Microbiome Project Consortium 2012a). Theprincipal findings of the HMP to date have beenthat each body site has its own characteristic micro-biota and that individuals have their own microbiome,which is relatively stable over time (Human Micro-biome Project Consortium 2012b; Ding and Schloss2014).

Our normal microbiota protects us from exogenousinfection via the phenomenon of colonization resis-tance. All external surfaces of the body are normallycovered in bacteria and thus potential binding sites forexogenous pathogens are blocked. In addition, mem-bers of the normal microbiota can produce antimi-crobial substances that inhibit the growth of otherorganisms. However, if the commensal microbiota isdisturbed then infection can result. For example, it iswell known that treatment with antibiotics can disruptthe normal microbiota to such a degree that oppor-tunistic infection with other organisms such as col-iform bacteria or the yeast Candida albicans can occur.Vaginal thrush and antibiotic sore tongue are examplesof such conditions.

The presence of the normal microbiota is essentialfor the proper development of the gut. The intesti-nal microbiota is highly diverse, with over 1000 bac-terial species present. A commonly found species,Bacteroides thetaiotaomicron, has profound effects on

the development of the blood supply to the gut. Ingerm-free mice, introduction of B. thetaiotaomicroninduced intestinal angiogenesis (Stappenbeck et al.2002). Interestingly, the genome of B. thetaiotaomi-cron includes an unusually high number of genesencoding signaling molecules of both the one- andtwo-component types (Xu et al. 2003). The mucin-degrading bacterial species Akkermansia muciniphilais associated with gut health and its numbers aredepleted in inflammatory bowel disease (Belzer andde Vos 2012). It is one of a number of species thathave found to be health associated and regarded asbeneficial.

1.8 Complex infectious diseases

“Classic” infectious diseases normally occur when apathogen infects a susceptible host and produces a spe-cific virulence factor that damages the host in a char-acteristic way, causing the signs and symptoms of thedisease. For many diseases, particularly those associ-ated with the mucous membranes, no single pathogenhas been identified, but instead the disease appears tobe the result of an aberrant interaction between the hostand its normal resident microbiota. These so-calledcomplex infectious diseases include the inflammatorybowel diseases and oral infections such as chronic peri-odontitis and, to some extent, endodontic infections.It has been suggested that a change in composition ofthe microbiome to one that is in a state of dysbiosismay have a role in obesity, diabetes, mental health,and other conditions (Devaraj et al. 2013; Clarke et al.2014). The question that remains to be answered iswhether a dysbiotic microbiome is a primary driverof disease or whether it is a result of the diseaseprocess.

Host susceptibility is of primary importance in thesediseases, but typically the susceptibility is conferredby multiple genes with no single genotype responsi-ble. For oral diseases such as periodontitis, the genesresponsible have yet to be discovered although thereis growing evidence that increased susceptibility iscaused by subtle differences in the immune and inflam-matory responses. For example, genetic polymor-phisms associated with cytokines such as interleukin-1 have been identified, which are associated withincreased cytokine secretion and severity of chronicinflammatory disease (Brett et al. 2005). Another keyfactor is the environment in its widest sense. Host

Microbial Perspectives in the Twenty-First Century 9

factors such as stress are known to contribute to theseverity of complex diseases, presumably by adverselyaffecting the immune system. Diet and social factorssuch as smoking can also be important, particularly inthe principal oral diseases such as dental caries and theperiodontal diseases.

It is likely that in the investigation of oral infectionsand diseases, we have clung too long to the classicinfectious disease model and have sought single infec-tious causes for them in the hope that antimicrobialscould be used in a targeted way to treat them. It mustbe remembered, however, that these diseases are bac-terial diseases and the presence of the normal micro-biota is required. By mechanisms as yet unknown,it appears that the communication and cooperationbetween the host and its commensal microbiota breaksdown, resulting in damage to the host. Much work onthese diseases is therefore currently being focused onbetter understanding health, the question being that ifthe human gut is colonized by so many bacteria withthe potential to cause disease, how do the majorityof individuals remain healthy? Better understandingof how this healthy balance is maintained will permitinsights into how disease arises when the homeostasisbreaks down. It may also be possible to influence thehost–microbiome interaction with probiotic bacteriaor by the administration of prebiotics to increase therelative proportions of beneficial bacteria (Claes et al.2014). A more extreme treatment for patients withsevere dysbiosis such as that seen in pseudomembra-nous colitis due to Clostridium difficile is a fecal trans-plant from a healthy donor, which has been shown toresult in an excellent clinical response (Rao and Young2015).

1.9 The future

We are still in the early years of the twenty-first cen-tury, so what do we have to look forward to in terms ofhow microbiology will impact on our understandingof infectious disease, including oral and endodonticinfections? The Human Microbiome Project is pro-viding an enormous bank of data on the compositionof the human-associated microbiota and its geneticpotential. The next challenge is to exploit these datato devise novel preventive and therapeutic strategies.It may be possible, for example, to construct a mix-ture of beneficial bacteria as an alternative to fecal

transplants, to be used to prevent, or reverse, dysbio-sis. From the host perspective, advances in geneticswill undoubtedly give us a far better understanding ofindividuals’ susceptibility to disease and the challengewill be put this into context with new knowledge ofthe genetic potential of the commensal microbiota inorder to predict and influence interactions among thehost, its microbiome, and the environment.

1.10 References

American Academy of Microbiology. 2014. FAQ: HumanMicrobiome.Angert ER, Clements KD, Pace NR. 1993. The largest bac-

terium. Nature 362: 239–241.Belzer C, de Vos WM. 2012. Microbes inside: from diversity

to function: the case of Akkermansia. ISME J 6: 1449–1458.

Brackman G, Coenye T. 2015. Quorum sensing inhibitors asanti-biofilm agents. Curr Pharm Des 21: 5–11.

Brett PM, Zygogianni P, Griffiths GS, et al. 2005. Functionalgene polymorphisms in aggressive and chronic periodon-titis. J Dental Res 84: 1149–1153.

Camanocha A, Dewhirst FE. 2014. Host-associated bacte-rial taxa from Chlorobi, Chloroflexi, GN02, Synergistetes,SR1, TM7, and WPS-2 Phyla/candidate divisions. J OralMicrobiol 6.

Carvalhais LC, Dennis PG, Tyson GW, Schenk PM. 2012.Application of metatranscriptomics to soil environments.J Microbiol Methods 91: 246–251.

Chen T, Yu WH, Izard J, Baranova OV, LakshmananA, Dewhirst FE. 2010. The Human Oral MicrobiomeDatabase: a web accessible resource for investigating oralmicrobe taxonomic and genomic information. Database(Oxford) 2010: baq013.

Claes IJ, Vargas Garcia CE, Lebeer S. 2014. Novel opportu-nities for the exploitation of host-microbiome interactionsin the intestine. Curr Opin Biotechnol 32c: 28–34.

Clarke G, O’Mahony SM, Dinan TG, Cryan JF. 2014. Prim-ing for health: gut microbiota acquired in early life regu-lates physiology, brain and behaviour. Acta Paediatr 103:812–819.

Devaraj S, Hemarajata P, Versalovic J. 2013. The humangut microbiome and body metabolism: implications forobesity and diabetes. Clin Chem 59: 617–628.

Dewhirst FE, Chen T, Izard J, et al. 2010. The human oralmicrobiome. J Bacteriol 192: 5002–5017.

Ding T, Schloss PD. 2014. Dynamics and associations ofmicrobial community types across the human body. Nature509: 357–360.

Duran-Pinedo AE, Chen T, Teles R, et al. 2014. Community-wide transcriptome of the oral microbiome in subjects withand without periodontitis. ISME J 8: 1659–1672.

Dworkin J. 2014. The medium is the message: inter-species and interkingdom signaling by peptidoglycan andrelated bacterial glycans. Annu Rev Microbiol 68: 137–154.

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Edwards DJ, Holt KE. 2013. Beginner’s guide to comparativebacterial genome analysis using next-generation sequencedata. Microb Inform Exp 3: 2.

Gillespie DE, Brady SF, Bettermann AD, et al. 2002. Isola-tion of antibiotics turbomycin a and B from a metagenomiclibrary of soil microbial DNA. Appl Environ Microbiol 68:4301–4306.

He X, McLean JS, Edlund A, Yooseph S, et al. 2015. Cul-tivation of a human-associated TM7 phylotype reveals areduced genome and epibiotic parasitic lifestyle. Proc NatlAcad Sci U S A 112: 244–249.

Hugenholtz P, Tyson GW, Webb RI, Wagner AM, Black-all LL. 2001. Investigation of candidate division TM7, arecently recognized major lineage of the domain Bacteriawith no known pure-culture representatives. Appl EnvironMicrobiol 67: 411–419.

Human Microbiome Project Consortium. 2012a. A frame-work for human microbiome research. Nature 486: 215–221.

Human Microbiome Project Consortium. 2012b. Structure,function and diversity of the healthy human microbiome.Nature 486: 207–214.

Hunter S, Corbett M, Denise H, et al. 2014. EBI metage-nomics: a new resource for the analysis and archiving ofmetagenomic data. Nucleic Acids Res 42: D600–606.

Konstantinidis KT, Tiedje JM. 2005. Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187: 6258–6264.

Ling LL, Schneider T, Peoples AJ, et al. 2015. A new antibi-otic kills pathogens without detectable resistance. Nature517: 455–459.

Mukamolova GV, Kaprelyants AS, Young DI, Young M,Kell DB. 1998. A bacterial cytokine. Proc Natl Acad SciU S A 95: 8916–8921.

Owen JG, Reddy BV, Ternei MA, et al. 2013. Mapping geneclusters within arrayed metagenomic libraries to expandthe structural diversity of biomedically relevant naturalproducts. Proc Natl Acad Sci U S A 110: 11797–11802.

Pucci MJ. 2006. Use of genomics to select antibacterial tar-gets. Biochem Pharmacol 71: 1066–1072.

Rao K, Young VB. 2015. Fecal microbiota transplantation forthe management of Clostridium difficile infection. InfectDis Clin North Am 29: 109–122.

Raoult D, La Scola B, Birtles R. 2007. The discovery andcharacterization of Mimivirus, the largest known virus andputative pneumonia agent. Clin Infect Dis 45: 95–102.

Rickard AH, Palmer RJ Jr, Blehert DS, et al. 2006. Autoin-ducer 2: a concentration-dependent signal for mutualis-tic bacterial biofilm growth. Mol Microbiol 60: 1446–1456.

Rondon MR, August PR, Bettermann AD, et al. 2000.Cloning the soil metagenome: a strategy for access-ing the genetic and functional diversity of unculturedmicroorganisms. Appl Environ Microbiol 66: 2541–2547.

Simon-Soro A, Guillen-Navarro M, Mira A. 2014. Metatran-scriptomics reveals overall active bacterial composition incaries lesions. J Oral Microbiol 6: 25443.

Stappenbeck TS, Hooper LV, and Gordon JI. 2002. Develop-mental regulation of intestinal angiogenesis by indigenousmicrobes via Paneth cells. Proc Natl Acad Sci U S A 99:15451–15455.

Tettelin H, Masignani V, Cieslewicz MJ, et al. 2005. Genomeanalysis of multiple pathogenic isolates of Streptococcusagalactiae: implications for the microbial “pan-genome.”Proc Natl Acad Sci U S A 102: 13950–13955.

Tyson GW, Chapman J, Hugenholtz P, et al. 2004. Com-munity structure and metabolism through reconstructionof microbial genomes from the environment. Nature 428:37–43.

Vartoukian SR, Palmer RM, Wade WG. 2010. Cultivation of aSynergistetes strain representing a previously uncultivatedlineage. Environ Microbiol 12: 916–928.

Venter JC, Remington K, Heidelberg JF, et al. 2004. Environ-mental genome shotgun sequencing of the Sargasso Sea.Science 304: 66–74.

Welch RA, Burland V, Plunkett G 3rd, et al. 2002. Exten-sive mosaic structure revealed by the complete genomesequence of uropathogenic Escherichia coli. Proc NatlAcad Sci U S A 99: 17020–17024.

Wellcome Trust Case Control Consortium; Australo-Anglo-American Spondylitis Consortium (TASC); Biologics inRA Genetics and Genomics Study Syndicate (BRAGGS)Steering Committee; Breast Cancer Susceptibility Collab-oration (UK). 2007. Association scan of 14,500 nonsyn-onymous SNPs in four diseases identifies autoimmunityvariants. Nat Genet 39(11): 1329–1337.

Woese CR. 1987. Bacterial evolution. Microbiol Rev 51: 221–271.

Xu J, Bjursell MK, Himrod J, et al. 2003. A genomic view ofthe human–Bacteroides thetaiotaomicron symbiosis. Sci-ence 299: 2074–2076.

Chapter 2Diagnosis, Epidemiology, and Global Impactof Endodontic InfectionsDag Ørstavik

2.1 Endodontic disease: irritation,inflammation, and infection of the pulpand periapical tissues

2.2 Primary diagnostic criteria: subjectivesymptoms and radiographic changes2.2.1 Pulpal involvement2.2.2 Periapical diagnosis

2.3 Pulpal inflammation and infection: publichealth consequences

2.4 Epidemiology of endodontic diseases2.4.1 Basic principles of epidemiologic

approaches to dental disease2.4.2 Infections with pulpal involvement2.4.3 Infections with periapical

involvement

2.4.4 Radiographic surveys ofasymptomatic apical periodontitis:methodology

2.4.5 Results of epidemiologic surveys ofasymptomatic apical periodontitis

2.5 Quality of root canal treatment and thedevelopment and persistence of apicalperiodontitis

2.6 Treatment strategies: prevention,treatment, and extraction

2.7 General oral health, oral health strategies,and tooth preservation as risk factors fororal infections

2.8 Conclusions2.9 References

2.1 Endodontic disease: irritation,inflammation, and infection of the pulpand periapical tissues

Endodontics deals with diseases of the pulp–dentinorgan and the periapical tissues. For practical pur-poses, these are infectious processes. Noninfectiousconditions affecting the pulp or apical periodontiumare much rarer and are seldom dealt with by specificendodontic treatment; however, they represent impor-tant differential diagnostic challenges.

The sources of pulpal and apical periodontal infec-tions are numerous. Traditionally, endodontic diseasehas been seen as a sequel to dental caries; however,

bacteria find their way to a vulnerable pulp in manyother instances as well. Dental trauma is one well-known situation, so is pulp damage and infection fol-lowing preparation and restoration of teeth. Low-gradeirritation of pulpal nervous elements can occur follow-ing attrition and erosion, sometimes developing intopulpal necrosis and infection.

Historically, the focus has been on the inflamma-tory reactions of the pulp and periapical tissues, asso-ciating clinical disease with the tissue response. Theinflammatory reactions have been related to infection,but at times they have been related to tissue damageduring treatment and to the toxic effects of medica-ments and materials. It is clearly an improvement in the

Endodontic Microbiology, Second Edition. Edited by Ashraf F. Fouad.© 2017 John Wiley & Sons, Inc. Published 2017 by John Wiley & Sons, Inc.

11

12 Endodontic Microbiology

concept of diagnosis and treatment planning that therehas been a shift towards stressing the level and extentof the infectious process, rather than wild-guessing thetype and degree of the inflammatory reaction. Inflam-mation of the pulp and periapical tissues is a sign ofinfection; clinically progressing disease is hardly evercaused by trauma or materials. This concept has beenproductive because virtually all successful therapeuticmeasures are directed towards combating or prevent-ing infection, with reduced or eliminated inflammationfollowing as a consequence. Moreover, the concept ofendodontic diseases as infections has implications forpublic oral health assessment in general, and placesthe association of local infectious disease in perspec-tive relative to local and regional (Ricucci and Bergen-holtz 2003) systemic health issues, particularly cardio-vascular disease (Caplan et al. 2006; Joshipura et al.2006; Cotti and Mercuro 2015). In this context, it isimportant to relate epidemiologic aspects of pulpal andperiapical disease to endodontic microbiology.

2.2 Primary diagnostic criteria: subjectivesymptoms and radiographic changes

2.2.1 Pulpal involvement

Initial pulpal infection is recognized primarily by clin-ical symptoms or through explorative excavation ofinvolved dentin. While conventional radiography maysuggest that a resorptive or carious process is imping-ing on the pulp, such methods do not allow definitiveassessment of pulpal involvement.

Traditionally, it has been held that there is only aweak association between clinical features and thehistologic characteristics of pulpitis (Cisneros-Cabelloand Segura-Egea 2005; Giuroiu et al. 2015). However,in a recent study, Ricucci et al. (2014) were quite suc-cessful in correlating pulpal history and symptomatol-ogy with histology and histobacteriology in 95 humanteeth. The diagnosis of pulpal infection/inflammationis therefore largely an operational one: based on expe-rience, and on knowledge of the underlying biologicprocesses, pulpitis is categorized as either reversibleor irreversible. This scheme sidesteps the need togive a precise description of the extent and severityof inflammation in the pulp, but it allows for treat-ment decisions based on the extent of microbial con-tamination or infection. It is assumed that in the case

of reversible pulpitis, pulp vitality may be preservedwith proper treatment; irreversible pulpitis implies thatno treatment short of pulp extirpation and root fill-ing can eliminate the disease. Briefly, reversible pul-pitis causes clinical symptoms of short duration (sec-onds) and only when irritated by external stimuli, andthe pulp proper is either not exposed or traumaticallyexposed for a short period only (< 2 days) (Heide andKerekes 1987). By contrast, irreversible pulpitis givesrise to symptoms of longer duration (minutes) that mayalso occur spontaneously, and an exposure of the pulpto the oral environment through caries, fractures, orcracks is suspected or confirmed. This concept is sup-ported by clinical experience and experiments (Roddand Boissonade 2000; Sigurdsson 2003; Iqbal et al.2007; Ricucci et al. 2014) and by experimental studieson the effects of pulpal inflammation on nerve activ-ity (Rodd and Boissonade 2000; Bletsa et al. 2006;Kokkas et al. 2007). Box 2.1 lists the salient clinicalsigns of irreversible pulpitis (i.e., infection of the pulpnecessitating endodontic treatment by root filling).

Box 2.1 Clinical characteristics ofirreversible pulpitis

Severe pain necessitating dental emergencytreatment

A history of repeated pain episodes Self-medication with analgesics Pain lingering after end of stimulus Sleep or work affected Supporting findings: positive, sometimes exag-

gerated pain on thermal or electrical stimula-tion; tooth localization difficult; percussion testlargely negative

Sometimes, a carious process may have reached thepulp without any symptoms. Traditionally, and in mostsettings in a dental office, this is considered an irre-versible pulpitis (i.e., the tooth will need endodon-tic treatment; Bjørndal et al. 2010). However, mod-ern materials and aseptic techniques may also providepredictable results from pulp-preserving approachesin these clinical situations (Bogen et al. 2008; Mar-ques et al. 2015), but such approaches can be highlyoperator-sensitive (Miles et al. 2010) and thereforemay not yet be recommended as standard practice.

Diagnosis, Epidemiology, and Global Impact of Endodontic Infections 13

Sensitivity testing by temperature or electricalpulses can give reasonably accurate assessment ofnerve tissue activity in the pulp, but relating suchrecordings to the degree of pulpal inflammation is dif-ficult considering the large variation in such measure-ments and their dependence on other clinical param-eters associated with the tooth (Fischer et al. 1991;Chen and Abbott 2009; Alomari et al. 2011; Mejareet al. 2012).

Radiography, including cone-beam radiographictechniques, is useful in special circumstances, such asfor detection of internal and external cervical resorp-tion (Celikten et al. 2014; Kalender et al. 2014; Ven-skutonis et al. 2014; Creanga et al. 2015; Dogramaciet al. 2015; Mavridou et al. 2016), which often affectsthe dental pulp. Pulp calcifications (diffuse and globu-lar) and obliteration as seen radiographically may giveindications of the physiologic state of the pulp, but lit-tle information about pulpal infection or inflammation.

In summary, a clinical pulpal diagnosis is most oftenmade based on anamnestic and subjective data sup-ported by sensitivity testing and caries excavation.

2.2.2 Periapical diagnosis

When the infection of the dental pulp affects the peri-odontium, apical periodontitis occurs. Pathologically,the inflammation is organized as a granuloma that mayor may not develop a radicular cyst as a sequela (Nair2008). Periapical disease also has a significant clinicalcomponent. In comparison with symptomatic pulpitis,symptomatic apical periodontitis is typically charac-terized by dull rather than sharp pain, and positivepercussion and palpation tests (Iqbal et al. 2007; Sig-urdsson 2008). Total infection of the pulp with virulentorganisms can give rise to acute apical abscess, a verypainful and potentially harmful condition (Antuneset al. 2013; Chunduri et al. 2013; Moazzam et al.2015), exemplifying a disease that historically definedthe dental profession. Longstanding pulp infectionswith chronic apical lesions can exacerbate with thesame symptomatic apical periodontitis or acute api-cal abscess. Apart from distinguishing such conditionsfrom marginal periodontal inflammation, and in par-ticular a periodontal abscess, they are seldom difficultto diagnose.

Asymptomatic apical periodontitis is, on the otherhand, dependent on radiographic signs for diagnosis.

In its early stages and during healing, this may be verydifficult, whereas a well-established, asymptomaticperiapical lesion is a simple condition to identify onradiographs (Ørstavik and Pitt Ford 2008). In teleo-logic terms, an infected root canal of a tooth is prob-ably perceived by the body as a risk zone for inva-sion by (life-threatening) microbes. A defense regionis then established in which the tissue architecture ischanged to prepare for the containment of invadingmicroorganisms (Ørstavik and Larheim 2008). Boneis gradually replaced by granulomatous tissue withvascular and cellular components mobilized for hostdefense. These initial events produce changes in bonestructure at the apex, which may be very hard to detectby periapical radiography (Brynolf 1967), and theymay occur with teeth that still have vitality or at leastneural activity in the pulp (Figure 2.1). When periapi-cal tissue remodeling has reached a state of completegranulomatous transformation, the lesion is very char-acteristic and easily diagnosed on the radiograph, par-ticularly when a cortical plate is affected (Figure 2.2).If such a tooth does not respond to sensitivity testing, adiagnosis of pulpal infection and apical periodontitis iscertain, and treatment options instantly available. Onthe other hand, there may be total pulp necrosis and noinfection or associated inflammation at the apex, suchas when the pulp is devitalized by traumatic injury(Sundqvist 1976).

The chronic development of apical periodontitismay be totally without symptoms, in which case theterm asymptomatic apical periodontitis is appropriate.However, symptoms may occur at any stage during theprocess, ranging from barely perceptible tenderness tothe acute symptoms described earlier.

In summary, chronic asympomatic apical periodon-titis needs radiography for detection; symptomatic andacute phases are diagnosed by clinical symptoms andsigns.

2.3 Pulpal inflammation and infection:public health consequences

The clinical aspects of endodontic diseases may beserious and with some consequences for individualand public health. Pulpitis and apical periodontitis aretraditionally categorized under “caries and its seque-lae,” and it is certainly true that deep carious lesions

14 Endodontic Microbiology

(a) (b)

Fig. 2.1 Minimal bone structural changes at the apex in conjunction with chronic pulpitis (a), necessitating endodontictreatment (b).

are indicators of pulpal and periapical inflammation.This is a confounding factor for assessments of therelative importance of these conditions in the overallincidence and prevalence of orofacial pain. Pulpitismay be very painful and lead to loss of quality of life(Constante et al. 2012). It may also cause absencefrom work and loss of income (Miotto et al. 2012).

It is unfortunate that pulpal pain is pooled with othertooth-related pain and often with the whole specterof orofacial pain conditions in surveys and screeningstudies. If one assumes that emergency treatment ofdental caries is initiated by pulpal pain and thus fallsunder the category of endodontic disease, symp-tomatic irreversible pulpitis and apical periodontitis