Oral Biofilms
247 pages
English

Vous pourrez modifier la taille du texte de cet ouvrage

Oral Biofilms , livre ebook

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus
247 pages
English

Vous pourrez modifier la taille du texte de cet ouvrage

Obtenez un accès à la bibliothèque pour le consulter en ligne
En savoir plus

Description

Oral Biofilms Monographs in Oral Science Vol. 29 Series Editors A. Lussi Bern M.A.R. Buzalaf São Paulo   Oral Biofilms Volume Editor Sigrun Eick Bern 52 figures, 39 in color, and 23 tables, 2021 _______________________ Sigrun Eick University of Bern Department of Periodontology Freiburgstrasse 7 CH–3010 Bern (Switzerland) This volume received generous financial support from E.M.S. Electro Medical Systems S.A., Nyon, Switzerland. Library of Congress Cataloging-in-Publication Data Names: Eick, Sigrun, editor. Title: Oral biofilms / volume editor, Sigrun Eick. Other titles: Monographs in oral science ; v. 29. 0077-0892 Description: Basel ; Hartford : Karger, 2021. | Series: Monographs in oral science, 0077-0892 ; vol. 29 | Includes bibliographical references and indexes. | Summary: “Biofilms are the most obvious and essential etiologic factors for all oral diseases with the exception of trauma and malignancies. This volume on “biofilms” looks at this structure from various angles. It provides analysis on various cariologic, endodontologic, periodontal, and peri-implant aspects, as well as those of dental unit water lines. The book is recommended for all dental students, graduate students, and biologically oriented dental clinicians”-- Provided by publisher. Identifiers: LCCN 2020041450 (print) | LCCN 2020041451 (ebook) | ISBN 9783318068511 (hardcover ; alk.

Sujets

Informations

Publié par
Date de parution 21 décembre 2020
Nombre de lectures 1
EAN13 9783318068528
Langue English
Poids de l'ouvrage 1 Mo

Informations légales : prix de location à la page 0,053€. Cette information est donnée uniquement à titre indicatif conformément à la législation en vigueur.

Exrait

Oral Biofilms
Monographs in Oral Science
Vol. 29
Series Editors
A. Lussi Bern
M.A.R. Buzalaf São Paulo
 
Oral Biofilms
Volume Editor
Sigrun Eick Bern
52 figures, 39 in color, and 23 tables, 2021
_______________________ Sigrun Eick University of Bern Department of Periodontology Freiburgstrasse 7 CH–3010 Bern (Switzerland)
This volume received generous financial support from E.M.S. Electro Medical Systems S.A., Nyon, Switzerland.

Library of Congress Cataloging-in-Publication Data
Names: Eick, Sigrun, editor.
Title: Oral biofilms / volume editor, Sigrun Eick.
Other titles: Monographs in oral science ; v. 29. 0077-0892
Description: Basel ; Hartford : Karger, 2021. | Series: Monographs in oral science, 0077-0892 ; vol. 29 | Includes bibliographical references and indexes. | Summary: “Biofilms are the most obvious and essential etiologic factors for all oral diseases with the exception of trauma and malignancies. This volume on “biofilms” looks at this structure from various angles. It provides analysis on various cariologic, endodontologic, periodontal, and peri-implant aspects, as well as those of dental unit water lines. The book is recommended for all dental students, graduate students, and biologically oriented dental clinicians”-- Provided by publisher.
Identifiers: LCCN 2020041450 (print) | LCCN 2020041451 (ebook) | ISBN 9783318068511 (hardcover ; alk. paper) | ISBN 9783318068528 (ebook)
Subjects: MESH: Tooth Diseases | Biofilms | Mouth Diseases
Classification: LCC RK305 (print) | LCC RK305 (ebook) | NLM W1 MO568E v.29 2021 | DDC 617.6/3--dc23
LC record available at https://lccn.loc.gov/2020041450
LC ebook record available at https://lccn.loc.gov/2020041451
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents ® and Index Medicus.
Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
© Copyright 2021 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)
www.karger.com
Printed in Germany on acid-free and non-aging paper (ISO 9706)
ISSN 0077–0892
e-ISSN 1662–3843
ISBN 978–3–318–06851–1
e-ISBN 978–3–318–06852–8
 
Contents
List of Contributors
Foreword
Lang, N.P. (Bern)
Biofilm in General
Biofilms
Eick, S. (Bern)
Biofilms in Dental Unit Water Lines
Dahlen, G. (Gothenburg)
The Impact of the pH Value on Biofilm Formation
Schultze, L.B.; Maldonado, A. (Bern); Lussi, A. (Bern/Freiburg); Sculean, A.; Eick, S. (Bern)
Biofilm Models
Biofilm Models to Study the Etiology and Pathogenesis of Oral Diseases
Thurnheer, T.; Paqué, P.N. (Zurich)
Biofilm Models for the Evaluation of Dental Treatment
Eick, S. (Bern)
Supragingival Biofilm
Cariogenic Biofilms and Caries from Birth to Old Age
Astasov-Frauenhoffer, M.; Kulik, E.M. (Basel)
Supragingival Biofilm: Toothpaste and Toothbrushes
Cvikl, B. (Vienna); Lussi, A. (Bern/Freiburg)
Actual Concepts for Individual Interdental Biofilm Removal
Jentsch, H.F.R. (Leipzig)
Arginine: A Weapon against Cariogenic Biofilm?
Eick, S. (Bern); Lussi, A. (Bern/Freiburg)
Oral Mouth Rinses against Supragingival Biofilm and Gingival Inflammation
Arweiler, N.B. (Marburg)
Subgingival and Peri-Implant Biofilm
Peri-Implant Diseases: Characteristics of the Microbiota and of the Host Response in Humans – A Narrative Review
Schmid, E.; Eick, S.; Sculean, A.; Salvi, G.E. (Bern)
Mechanical Removal of the Biofilm: Is the Curette Still the Gold Standard?
Bastendorf, K.-D.; Strafela-Bastendorf, N. (Eislingen); Lussi, A. (Bern/Freiburg)
Antibiotics against Periodontal Biofilms
Guentsch, A. (Milwaukee, WI)
Effectiveness of Photodynamic Therapy in the Treatment of Periodontal and Peri-Implant Diseases
Sculean, A. (Bern); Deppe, H. (Munich); Miron, R. (Bern); Schwarz, F. (Frankfurt); Romanos, G. (Stony Brook, NY); Cosgarea, R. (Bonn/Marburg/Cluj-Napoca)
Other Oral Biofilms
New Bacterial Combinations in Secondary Endodontic Infections of Patients with a Recent Systematic Antibiotic Therapy
Al-Ahmad, A. (Freiburg); Elamin, F. (London/Khartoum); Gärttner, R.; Anderson, A.; Wittmer, A. (Freiburg); Mirghani, Y. (Khartoum); Hellwig, E. (Freiburg)
Biofilms on Restorative Materials
Schmalz, G. (Regensburg/Bern); Cieplik, F. (Regensburg)
Halitosis
Ortiz, V.; Filippi, A. (Basel)
Biofilm and Orthodontic Therapy
Müller, L.K. (Mainz); Jungbauer, G. (Straubing/Bern); Jungbauer, R. (Regensburg); Wolf, M. (Aachen); Deschner, J. (Mainz)
In vitro Activity of Oral Health Care Products on Candida Biofilm Formation
Katagiri, H. (Bern/Niigata); Stuck, N.-J. (Bern); Arakawa, I. (Niigata/Bern); Nietzsche, S. (Jena); Eick, S. (Bern)
Author Index
Subject Index
 
List of Contributors
Ali Al-Ahmad
Albert-Ludwigs-University
Faculty of Medicine
Department of Operative Dentistry and
Periodontology
Hugstetter Straße 55
DE–79106 Freiburg i.Br. (Germany)
E-Mail ali.al-ahmad@uniklinik-freiburg.de
Annette Anderson
Albert-Ludwigs-University
Faculty of Medicine
Department of Operative Dentistry and
Periodontology
Hugstetter Straße 55
DE–79106 Freiburg i.Br. (Germany)
E-Mail annette.anderson@uniklinik-freiburg.de
Itsuka Arakawa
The Nippon Dental University Niigata Hospital
Comprehensive Dental Care,
1-8, Hamauracho, Chuo-ku
Niigata 951-8580 (Japan)
E-Mail itsuka@ngt.ndu.ac.jp
Nicole Birgit Arweiler
Philipps-University Marburg
Department of Periodontology
Georg-Voigt-Str. 3
DE–35039 Marburg (Germany)
E-Mail arweiler@med.uni-marburg.de
Monika Astasov-Frauenhoffer
University of Basel
Department Research, University Center for Dental
Medicine (UZB)
Mattenstrasse 40
CH–4058 Basel (Switzerland)
E-Mail m.astasov-frauenhoffer@unibas.ch
Klaus-Dieter Bastendorf
Zahnarzt
Logauweg 7
DE–73054 Eislingen (Germany)
E-Mail info@bastendorf.de
Fabian Cieplik
University Hospital Regensburg
Department for Conservative Dentistry and
Periodontology
Franz-Josef-Strauß-Allee 11
DE–93053 Regensburg (Germany)
E-Mail Fabian.Cieplik@klinik.uni-regensburg.de
Raluca Cosgarea
University of Bonn
Department of Periodontology, Cariology and
Preventive Dentistry
Welschnonnenstrasse 17
DE -53111 Bonn (Germany)
E-Mail ralucacosgarea@gmail.com
Barbara Cvikl
Sigmund Freud University
Department of Conservative Dentistry
Freudplatz 3
AT–1020 Vienna (Austria)
E-Mail barbara.cvikl@med.sfu.ac.at
Gunnar Dahlen
Sahlgrenska Academy, University of Gothenburg
Department of Oral Microbiology and Immunology
Institute of Odontology
Box 450
SE–405 30 Gothenburg (Sweden)
E-Mail gunnar.dahlen@odontologi.gu.se
Herbert Deppe
Technical University Munich
Clinic and Policlinic for Oral and Maxillofacial Surgery
Arcisstraße 21
DE–80333 Munich (Germany)
E-Mail herbert.deppe@tum.de
James Deschner
University Medical Center of the Johannes Gutenberg
University
Department of Periodontology and Operative
Dentistry
Augustusplatz 2
DE–55131 Mainz (Germany)
E-Mail james.deschner@uni-mainz.de
Sigrun Eick
University of Bern
Department of Periodontology
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail sigrun.eick@zmk.unibe.ch
Fadil Elamin
Khartoum Centre for Research and Medical Training
4th/5th Floor, Islamic Bank Building, Qasr Street
Khartoum (Sudan)
fadilelamin@yahoo.co.uk
Andreas Filippi
University Center for Dental Medicine Basel UZB
Clinic of Oral Surgery
Mattenstrasse 40
CH–4058 Basel (Switzerland)
E-Mail andreas.filippi@unibas.ch
Rebecca Gärttner
Albert-Ludwigs-University
Faculty of Medicine
Department of Operative Dentistry and
Periodontology
Hugstetter Straße 55
DE–79106 Freiburg i.Br. (Germany)
E-Mail rebeccagaerttner@gmail.com
Arndt Guentsch
Marquette University School of Dentistry
Department of Surgical Sciences
P.O. Box 1881
Milwaukee, WI 53201-1881 (USA)
E-Mail arndt.guentsch@marquette.edu
Elmar Hellwig
University of Freiburg
Center for Dental Medicine
Department of Operative Dentistry and
Periodontology
Hugstetter Strasse 55
DE–79106 Freiburg i.Br. (Germany)
E-Mail elmar.hellwig@uniklinik-freiburg.de
Holger Jentsch
Universitätsklinikum Leipzig
Leiter Funktionsbereich Parodontologie der Poliklinik
für Zahnerhaltung und Parodontologie
Liebigstr. 12, Haus 1
DE–04103 Leipzig (Germany)
E-Mail holger.jentsch@medizin.uni-leipzig.de
Gert Jungbauer
Private Dental Practice
Ludwigsplatz 14/16
DE–94315 Straubing (Germany)
E-Mail gert.jungbauer@zmk.unibe.ch
Rebecca Jungbauer
Universitätsklinikum Regensburg
Poliklinik für Kieferorthopädie
Franz-Josef-Strauß-Allee 11
DE–93053 Regensburg (Germany)
E-Mail Rebecca.jungbauer@ukr.de
Hiroki Katagiri
The Nippon Dental University Graduate School of Life
Dentistry at Niigata
Clinical Oro-Maxillofacial Bone Research and
Application
1-8, Hamauracho, Chuo-ku
Niigata 951-8580 (Japan)
E-Mail katagiri@ngt.ndu.ac.jp
Eva M. Kulik
Universitäres Zentrum für Zahnmedizin Basel
Leitung Labor
Mattenstrasse 40
CH–4058 Basel (Switzerland)
E-Mail eva.kulik@unibas.ch
Niklaus P. Lang
University of Bern
Scheuermattweg 33
CH–3043 Uettingen/Bern
E-Mail nplang@switzerland.net
Adrian Lussi
School of Dental Medicine, University of Bern
Freiburgstrasse 7
CH–3010 Bern (Switzerland) and
Center for Dental Medicine, Department of Operative
Dentistry and Periodontology, Hugstetter Strasse 55
DE–79106 Freiburg i.Br. (Germany)
E-Mail adrian.lussi@zmk.unibe.ch
Alejandra Maldonado
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail alejandra.maldonado@students.unibe.ch
Yousra Mirghani
Khartoum Center for Research and Medical Training
4th/5th Floor, Islamic Bank Building
Qasr Street, Khartoum (Sudan)
E-Mail yousramirghani@hotmail.com
Richard Miron
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail richard.miron@zmk.unibe.ch
Lena Katharina Müller
University Medical Center of the Johannes Gutenberg
University
Department of Periodontology and Operative
Dentistry
Augustusplatz 2
DE–55131 Mainz (Germany)
E-Mail Lenakatharina.Mueller@gmail.com
Sandor Nietzsche
Universitätsklinikum Jena
Elektronenmikroskopisches Zentrum
Ziegelmühlenweg 1
DE–07743 Jena (Germany)
E-Mail Sandor.Nietzsche@med.uni-jena.de
Virginia Ortiz
University Center for Dental Medicine Basel UZB
Mattenstrasse 40
CH–4058 Basel (Switzerland)
E-Mail virginia.ortiz@unibas.ch
Pune Nina Paqué
University of Zurich
Clinic of Conservative and Preventive Dentistry, Center
of Dental Medicine
Plattenstrasse 11
CH–8032 Zurich (Switzerland)
E-Mail puneninapaque@zzm.uzh.ch
Georgios Romanos
Stony Brook University, School of Dental Medicine
Department of Periodontology
106 Rockland Hall
Stony Brook, NY 11794-8700 (USA)
E-Mail georgios.romanos@stonybrook.edu
Giovanni E. Salvi
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail giovanni.salvi@zmk.unibe.ch
Gottfried Schmalz
University Hospital Regensburg
Department for Conservative Dentistry and
Periodontology
Franz-Josef-Strauß-Allee 11
DE–93053 Regensburg (Germany)
E-Mail Gottfried.Schmalz@klinik.uni-regensburg.de
Eric Schmid
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail eric.schmid@zmk.unibe.ch
Lara B. Schultze
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail Lara.Schultze@gmx.ch
Frank Schwarz
Johann Wolfgang Goethe University
Policlinic for Dental Surgery
Theodor-Stern-Kai 7
DE–60596 Frankfurt am Main (Germany)
E-Mail f.schwarz@med.uni-frankfurt.de
Anton Sculean
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail anton.sculean@zmk.unibe.ch
Nadine Strafela-Bastendorf
Independent Dentist
Gairenstrasse 6
DE–73054 Eislingen (Germany)
E-Mail praxis@Strafela-Bastendorf.de
Neil-Jérôme Stuck
University of Bern
Department of Periodontology
School of Dental Medicine
Freiburgstrasse 7
CH–3010 Bern (Switzerland)
E-Mail neil-jerome.stuck@students.unibe.ch
Thomas Thurnheer
University of Zurich
Division of Oral Microbiology and Immunology
Plattenstrasse 11
CH–8032 Zurich (Switzerland)
E-Mail Thomas.Thurnheer@zzm.uzh.ch
Annette Wittmer
Universitätsklinikum Freiburg
Department für Medizinische Mikrobiologie, Virologie
und Hygiene, Institut für Immunologie
Institut für medizinische Mikrobiologie
Hermann-Herder-Straße 11
DE–79104 Freiburg i.Br. (Germany)
E-Mail annette.wittmer@uniklinik-freiburg.de
Michael Wolf
Universitätsklinikum Aachen, AöR
Klinik für Kieferorthopädie
Pauwelsstraße 30
DE–52074 Aachen (Germany)
E-Mail michwolf@ukaachen.de
 
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp X–XI (DOI: 10.1159/000510204)
______________________
Foreword
Fifty-five years ago, a landmark article was published by a group of clinical researchers at the Royal Dental College of Aarhus, Denmark. With this clinical experiment entitled “Experimental Gingivitis in Man” a cause-and-effect relationship between “dental plaque” and the host response in the form of a gingivitis was established. Hence, Harald Löe and his coworkers had established the basic paradigm for the prevention as well as therapy of periodontal diseases. Some years later, van der Fehr and coworkers established a similar paradigm with their clinical study entitled “Experimental Caries in Man.” “Dental plaque” had become the major etiologic factor for the most frequently encountered oral diseases.
At that time, dental plaque was believed to represent a biomass of bacteria, and the quantity of bacteria was considered to reflect the degree of host response observed. Dental plaque was non-specifically inducing the disease process. In the 1980s, researchers were challenged to find specific pathogens for both caries and periodontal diseases, and it was realized that dental plaque could very well contain specific bacteria responsible for the initiation and promotion of oral diseases. This search for specific pathogens indeed identified groups of micro-organisms with high pathogenic potential and other groups associated with healthy conditions.
In the 1990s and up to the turn of the century, an opportunistic concept of dental plaque gained a lot of attention, and it was realized that the microbial environment substantially influenced the composition of dental plaque, thereby determining its pathogenicity. This paradigm shift in the understanding of the nature of dental plaque culminated in the research of William Costerton and coworkers from the Montana State University in Bozeman, USA, who – at the beginning of the 21st century – provided unequivocal evidence that the bacteria that cause device-related and other chronic infections grow in matrix-enclosed biofilms. The diagnostic and therapeutic strategies that have served in the partial eradication of acute epidemic bacterial diseases have not yielded accurate data or favorable outcomes when applied to these biofilm diseases. The potential benefits of the application of the methods and concepts developed by biofilm science and engineering to the clinical management of infectious diseases are obvious.
Hence, in recent years the term “dental plaque” has been replaced by “oral biofilms.” As biofilms form on all hard, non-shedding surfaces in a fluid system, it is of striking importance to realize that teeth, implants, and prosthetic devices are all affected by biofilm formation.
Biofilms are the most obvious and essential etiologic factors for all oral diseases with the exception of trauma and malignancies. It is, therefore, not surprising to find a chapter dealing with biofilms in each modern dental textbook. However, only few such chapters may deal comprehensively with the issue. The present volume on “biofilms” looks at this interesting structure from various angles. It sheds light on the life in a biofilm beyond the sheer enumeration of single bacterial species. The book is a multi-author production which assures a competent analysis on various cariologic, endodontologic, periodontal, and peri-implant aspects, as well as those of dental unit water lines. The presentation of the life in the biofilms provides a profound understanding of this very interesting and most important structure. The book is recommended for all dental students, graduate students, and biologically oriented dental clinicians.
Niklaus P. Lang Bern, Switzerland
Biofilm in General
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 1–11 (DOI: 10.1159/000510184)
______________________
Biofilms
Sigrun Eick
Department of Periodontology, Laboratory of Oral Microbiology, School of Dental Medicine, University of Bern, Bern, Switzerland
______________________
Abstract
In reality, most microorganisms are not free floating. They exist in biofilms, a community of many of them from the same species or from other genera and attached to surfaces. Microorganisms undergo a transition from free-floating, planktonic microorganisms to a sessile, surface-attached one. Contact with a surface induces changes in gene expression, and a strong attachment of microcolonies occurs only after a few hours. The maturation of a biofilm is associated with matrix formation. The matrix is of importance as it provides stability and protects against environmental insults, it consists of polysaccharides, water, lipids, proteins, and extracellular DNA. Biofilms can be found everywhere – in the environment, in water systems – and they play an important role in medicine and dentistry. In medicine, infections of chronic wounds, of the respiratory tract in cystic fibrosis infections, or when linked with incorporated biomaterial are mostly biofilm associated. In the oral cavity, the most prevalent oral diseases, dental caries, and periodontitis are multi-species biofilm-associated diseases. Although not acting alone, key pathogens drive the development of the microbial shift. Microorganisms metabolize sugar and create an acidic environment where aciduric bacteria (including mutans streptococci) become dominant, which leads to the demineralization of enamel and dentine. Porphyromonas gingivalis causes biofilm dysbiosis in the development of periodontal disease. Biofilm-associated infections are extremely difficult to treat. The matrix serves as a barrier to antimicrobial agents and there are subpopulations of dormant bacteria resistant to antimicrobials requiring metabolically active cells. Approaches to treat biofilm-associated infections include the modification of the biofilm composition, inhibitors of quorum-sensing molecules, or interfering with matrix constituents.
© 2021 S. Karger AG, Basel
Biofilm: A Major Form of Microorganism Living
The term bacterium suggests a free-floating microorganism. Medical microbiology nearly always analyses dispersed single bacteria growing on agar plates or within a liquid to determine their susceptibility to antimicrobials. However, in reality, most microorganisms are not free floating. Instead, they exist together with others from the same species or from other genera attached to surfaces.


Fig. 1. Biofilm formation.
Biofilms are present nearly everywhere. They occur in the environment and they may cause medical problems. Dental plaque as a specialized oral biofilm was investigated relatively early. The following overview deals first with biofilms in general, and then with oral biofilms in particular.
Biofilms in General
The attachment of microorganisms to surfaces is one criterion for a biofilm. Going back in the literature, J.W. Costerton [ 1 ] was one of the pioneers in biofilm work. In 1978 he claimed that bacteria are mostly surrounded by extracellular polysaccharides, a “glycocalyx” which may act as a barrier against antibiotics, the bacteria form organized communities. Thus, biofilms can be simply defined as communities that are attached to a surface [ 2 ]. They exist as single- and multi-species biofilms [ 2 ], with the latter form being the most dominant.
Formation of Biofilms
The biofilm formation is split into several steps, including the initiation, the maturation, the maintenance, and the dispersion of the biofilm [ 2 ] ( Fig. 1 ). Microorganisms undergo a transition from a free-floating, planktonic microorganism to a sessile, surface-attached one. They must be able to attach to surfaces, to move on them, and to form a three-dimensional structure [ 2 ].
Environmental signals such as pH, nutrients, temperature, oxygen, and others trigger the early attachment of microorganisms to surfaces [ 2 ]. In that process, many bacterial structures, like pili or lipopolysaccharides, are involved [ 2 ]. Contact with the surface may induce changes in the bacteria gene expression, for example the synthesis of extracellular polysaccharides is upregulated [ 2 ]. Also, within minutes microorganisms upregulate the secretion of intercellular signaling molecules; however, a strong attachment of microcolonies occurs only after a few hours [ 3 ].
The maturation of a biofilm is associated with the complex process of matrix formation and is dependent on nutrient availability, shear forces, and the influence of other microorganisms [ 4 ]. The maturation is complete only after 2–4 days following the initial attachment [ 3 ].
In the maintenance phase, there is relative stability within the community. However, several activities exist. For example, the biofilms continuously respond to desiccation by synthesis of extracellular polysaccharides molecules [ 4 ], and microorganisms (planktonic or microcolonies) are also shed into the environment [ 3 ]. The dispersion of biofilms might be mediated by ending the synthesis of matrix production, matrix degradation, and disruption of interactions between matric components [ 5 ].
Interactions between Microorganisms
When bacteria undergo the transition from a planktonic lifestyle to biofilm community, they must interact with microorganisms in close proximity [ 2 ]. Over a few years it has become clear that a communication among the microbial cells exists and that it plays an essential role in biofilm formation. Bacterial cells and also fungi produce certain molecules to which another microorganism can respond. The molecules produced by Gram-negative bacteria are acyl-homoserine lactones. Gram-positive bacteria may generate autoinducing peptide I and double-tryptophan signal peptide pheromone. Both Gram-positive and Gram-negative bacteria synthesize autoinducer 2, which is of importance in particular in interspecies communication [ 6 ]. Quorum sensing is controlled by the spatial distribution and the density of the bacterial cells [ 6 ]. In the process of biofilm formation, quorum sensing regulates the attachment of cells to surfaces [ 6 ]. Later, it regulates many cell processes, such as cell density in biofilms, exchange of genes, synthesis of bacteriocins, and biofilm dispersion. Synergistic and antagonistic behaviors occur among the microorganisms within a biofilm, for example microorganisms utilize the byproducts of others or compete on nutrients, and they coagregate or blanket the surface to compete for others [ 7 , 8 ]. The close proximity of many different microorganisms is an important place for genetic exchange. It is of importance to note that it provides a reservoir for transferring antibiotic resistance genes [ 9 ].
Biofilm Matrix
Mature biofilms have a complex structure, consisting of a polysaccharide matrix and fluid-filled channels [ 2 ]. A fully hydrated biofilm is composed of about 15% microbial cells and 85% matrix material [ 3 ]. The matrix is of importance as it provides stability and protects against environmental insults; it consists of polysaccharides, water, lipids, proteins, and considerable amounts of extracellular DNA [ 4 ]. The exopolysaccharides mask bacterial ligands and decrease the ability of phagocytes to recognize and to phagocytose bacteria in a biofilm [ 10 ]. Microorganisms synthesize different exopolysaccharides. For example, for Pseudomonas aeruginosa three different polysaccharides have been described, an alginate being a mucoid negatively charged polymer, a glucose-rich polysaccharide being of importance at the liquid-air interface, and a pentasaccharide acting as scaffold for other bacterial cells [ 10 ]. The proteins of the matrix originate from bacterial outer membrane vesicles and from cytoplasmic proteins; after being recycled, they polymerize into amyloid-like structures like curli fibers and flagella [ 5 ]. Proteins may also form a hydrophobic layer around a mature biofilm [ 5 ] which protects the biofilm against environmental insults. The extracellular DNA in the biofilm matrix originates from lysed bacterial cells or is actively released from membrane vesicles at bacterial cell surfaces; interestingly, not all bacterial DNases are able to degrade it [ 11 ]. Its functions in a biofilm is discussed as a nutrient reservoir, the dissemination of genes between microorganisms, and a strengthening of the biofilm structure [ 11 ].


Fig. 2. Specimens of PMMA loaded with gentamicin and contaminated in vitro with an S. aureus strain for 2 days (photograph: Center for Electron Microscopy, University Hospital of Jena, Jena, Germany).
Biofilms in Medicine
Biofilms are omnipresent.In mountain lakes, they may serve as indicators of environmental influences [ 12 ]. Biofilms are found in water drinking distributors [ 13 ] and in water hydraulic systems [ 14 ]. Biofilms might be a reservoir for pathogens, water systems may distribute Chlamydiales, Legionella sp. [ 15 ], and drains are a source of Pseudomonas aeruginosa [ 16 ].
After being neglected for several decades, biofilms are gaining more and more interest in medicine. There are infections associated with biofilm formation on incorporated materials or medical devices used for treatment. Furthermore, other infections are clearly associated with biofilms, such as chronic wound infections and lung infections in cystic fibrosis. The diagnostic criteria for identifying a biofilm-associated infection are a localized chronic or foreign infection, or a medical history of predisposing factors for biofilm formation, such as cystic fibrosis, a recurrent infection, and antibiotic failure, among others [ 3 ].
Chronic wound infections, such as diabetic food ulcera, develop slowly and are difficult to eradicate. The majority of these infections are biofilm associated, the healing time is prolonged as microorganisms in biofilms are protected against host response, and furthermore, the biofilm may act as a barrier against re-epithelialization [ 17 ]. Cystic fibrosis is a congenital disorder and affects many organs, clinically characteristic is the production of mucous and repeated respiratory infections. Often the lower respiratory tract is infected with P. aeruginosa as a biofilm-growing species, which means the host response is unable to clear the chronic infection [ 18 ]. The gastrointestinal tract is highly colonized by different microorganisms. Within the mucous layer, the close contact enables biofilm formation. Also, the ability of Clostridium difficile to form biofilms hinders treatment with antibiotics [ 19 ].
Modern medicine incorporates biomaterial into the human body, either temporary as central venous catheters or permanently as a joint arthroplasty. These artificial surfaces are immediately coated by conditioning layers and predestined to be colonized by microbial biofilms. Gram-negative rods such as Pseudomonas sp. and Acinetobacter sp., and also oral bacteria such as streptococci and Neisseria sp. are frequently found in biofilms formed in endotracheal tubes [ 20 ]. Prosthetic joint infections are the main reason for periprosthetic joint infection; they are the most common reason for revision of total knee arthroplasties and the third most common reason for revision of total hip arthroplasties [ 21 ]. The infections occur up to 24 months after surgery as a possible contamination during surgery and thereafter as a hematogenous spreading of microorganisms from other parts of the body. Most identified microorganisms are Staphylococcus aureus and gram-negative bacilli [ 22 ].
Antibiofilm Therapy
Biofilm-associated infections are extremely difficult to treat. For example, in the case of an orthopedic infection a two-stage revision is made. The infected artificial joint is removed and intermediately replaced by a spacer made of PMMA (polymethylmethacrylate) bone cements highly loaded with antibiotics, which should prevent renewed biofilm formation ( Fig. 2 ).
The biofilm matrix serves as a barrier not only to antimicrobial agents but also to immune cells. Even if phagocytes can penetrate they are ineffective, and the release of intracellular compounds increases the density and integrity of the biofilm matrix [ 3 ]. In biofilms, bacteria may become more virulent through the exchange of genes, including resistance genes [ 3 ]. Most importantly, there are subpopulations of dormant bacteria with metabolically low activity that are non-subdividing and, following resistance against antimicrobials, require metabolically active cells [ 3 ].
According to the increased awareness of biofilm-associated infections, different approaches are under investigation. For several years, attempts have been made to prevent or to retard biofilm formation on medical devices. Whilst taking the side effects into account, antibiotics are prophylactically used to kill planktonic bacteria before they are able to form biofilms [ 23 ]. Antiadhesive biomaterials are developed by modification of the surface with biosurfactants, cold plasma, or the incorporation of antimicrobial-acting agents which might be in part released into the environment; however, the effectiveness is generally very limited [ 23 ]. Promising approaches seem to be the inhibition of quorum sensing or interfering with matrix constituents. Inhibitors of quorum-sensing molecules have been developed, and inhibitors of acyl-homoserine lactones significantly reduced P. aeruginosa biofilm formation in vitro [ 24 ]. Other possibilities are the dispersion of the biofilm matrix and targets are alginate, extracellular DNA, and proteins which might be degraded by polysaccharides, DNases, or proteinases [ 24 ].
Oral Biofilms
As mentioned above, Costerton [ 1 ] was the first who defined the term “biofilm.” Among others, he described the formation of a glycocalyx by Streptococcus mutans and he published a graph of several oral bacteria in dental plaque [ 1 ], establishing that dental plaque is a biofilm. In PubMed, the term “dental plaque” gives the earliest articles dating from 1946. Interestingly, the very first one showed an association of dental plaque with both periodontal disease and caries in an animal model [ 25 ]. In the years thereafter the major focus was the association of dental plaque with caries. In 1965, Löe et al. [ 26 ] demonstrated that plaque formation induces gingivitis in man. Nowadays, it is well known that oral biofilms are formed not only on natural teeth but also on restorative materials, fixed and removable prosthetic constructions, dental implants, as well as to a certain extent on epithelium.
Dental Biofilm Formation
First, before bacteria attach to the tooth surface, a pellicle is formed. The adsorption of proteins to the enamel surface is selective, very fast saliva proteins (acidic proline-rich protein, cystatin, statherin, and protein S100-A9 proteins) attach, while serum proteins attach more slowly [ 27 ]. In the subgingival region, more serum-derived proteins are attached to the root surface [ 28 ], which can be seen in association with the different surface (cementum) or the flow of the gingival crevicular fluid.
Colonization of microorganisms in the oral cavity and in particular the biofilm formation on teeth depends on many factors, including age, diet, oral hygiene, and the immune response [ 29 ]. A few Gram-positive bacteria are able to adhere to pellicle-coated surfaces via specific adhesion-receptor interactions; other microorganisms subsequently attach to them and drive biofilm formation [ 29 ]. Analysis of early stages of dental biofilm formation by microarray analysis showed oral streptococci to be most prominent, together with Gemella haemolysans , Haemophilus parainfluenzae , Actinomyces sp., Rothia sp., Neisseria sp., Kingella orali s, Slackia exigua , and Veillonella sp. [ 30 ]. Fusobacterium sp. and Parvimonas micra were also present, whereas Porphyromonas gingivalis was not detected [ 30 ]. A low presence of Filifactor alocis and of the TM7 complex was linked with a potential role of these bacteria in developing periodontal disease, and the detection of S. mutans in about 25% of individuals can be correlated with potential caries development [ 30 ].
Already decades ago, attempts were made to characterize the microbial composition of mature dental biofilms in more detail. In 1993, Kolenbrander and London [ 31 ] published a scheme of organization of microorganisms in a subgingival biofilm. The later modified scheme shows that the first oral streptococci, among them S. oralis , S. sanguinis, and S. gordonii , and a few actinomyces ( A. oris , A. naeslundii ) adhere to the salivary pellicle-coated tooth surface. Then, several other Gram-positive rods and certain Gram-negative bacteria, such as F. nucleatum and Veillonella spp., can attach and provide receptors for late colonizers, including Aggregatibacter actinomycetemcomitans , Treponema denticola , and Tannerella forsythia [ 32 ]. A hallmark to describe networking in subgingival biofilm was the creation of different colored complexes by Socransky et al. [ 33 ]. By using a checkerboard technique allowing the determination of 40 different bacterial species, bacteria were grouped according to their joined presence, the red complex ( P. gingivalis , T. forsythia , and T. denticola ) characterized bacteria strongly associated with periodontal disease, the orange complex ( F. nucleatum , P. micra ,and others) represent the core bacteria, certain oral streptococci were grouped in the yellow complex, V. parvula and A. odontolyticus formed the purple complex, and the green complex consisted of unrelated species [ 33 ]. The latest sequencing technologies allow deeper insights into the microbial composition of oral biofilms, revealing that a mature supragingival biofilm is dominated by Streptococcus sp. and Neisseria sp. Furthermore, Gracilibacteria , TM7, Bacteroides , Catonella , Porphyromonas , Tannerella , and others were identified, but not Archaea, yeasts, protozoa, or viruses [ 34 ].
The matrix of an oral biofilm contributes to the structural integrity and protects the biofilm against environmental insults [ 35 ]. Molecules penetrate through channels and pores, and microorganisms metabolize nutrients, resulting in very different conditions (e.g., pH, redox potential) in short distances, which allows the co-existence of different microorganisms [ 35 ]. However, the composition of the oral biofilm matrix has not been well studied. Glycoconjugates binding to 10 lectins were identified in pooled supragingival biofilm samples [ 36 ]. Recently, extracellular DNA was visualized in vivo in dental biofilms as an important component for biofilm stability [ 37 ]. Among the extracellular polysaccharides, synthesis and the role of glucans are best described, but how other bacterial-produced polysaccharides (polymers being rich in mannose and capsular polysaccharides) contribute to the extracellular matrix remains to be determined [ 38 ].
A synergistic interaction with other bacteria is also essential in the oral biofilms. Several periodontal pathogens can grow and multiply only in the presence of other bacteria [ 39 ]. Communication is of importance in biofilm formation and maintenance. Oral bacteria produce two major classes of signaling molecules, the competence-stimulating peptides (CSP) and autoinducer-2 [ 39 ]. CSP are synthesized by Gram-positive bacteria, they promote biofilm formation, DNA release, and may inhibit bacteriocins produced by S. mutans [ 39 ]. Autoinducer-2 production seems to be of importance in the communication of periodontopathogens [ 39 ].
Dental Biofilm and Diseases
Microbial homeostasis can break down and result in restructuring of the biofilm with a different composition [ 35 ]. The most prevalent oral diseases, dental caries, and periodontitis are multi-species biofilm-associated diseases.
For many years, dental caries has been strongly associated with S. mutans , which utilizes many carbohydrates to produce acids and to synthesize extracellular polysaccharides, an important constituent of the matrix of cariogenic biofilm [ 29 ]. Nowadays, caries is seen as a result of a dysbiosis induced by environmental factors [ 40 ]. Microorganisms metabolize sugar supplied by food and create an acidic environment where aciduric bacteria (including mutans streptococci) become dominant, which leads to the demineralization of enamel and dentine [ 41 ]. Then, the bacterial-induced acidification also activates host-derived proteases, which contribute to the degradation of the organic matrix of dentine and of the root surface [ 42 ]. Using the Shannon index as a measure of diversity and evenness in the microbiome, a higher diversity is found in healthy individuals than in those with caries disease in general, but streptococci were enriched in the cohort with caries [ 34 ]. In caries-positive adolescence, the presence of S. mutans is associated with a lower diversity, while if S. mutans is not present many other saccharolytic species occur [ 43 ]. Functional profiles detect more sugar uptake systems and more systems associated with antimicrobial resistance in the microbiomes of caries-positive than in healthy individuals [ 34 ]. When comparing samples with active dentine caries with enamel caries and healthy samples, diversity values were higher for dentine caries, although in a few samples only a very low diversity was found [ 44 ].
The pathogenesis of periodontitis is thought to be an inflammatory response to a dysbiotic microbiota in the subgingival biofilm [ 45 ]. P. gingivalis has been postulated as being a keystone pathogen, it drives the development of the microbial shifts, and causes dysbiosis and inflammation by modulating the host response [ 46 ]. A systematic review summarizing the published data of complex analyses of the periodontal microbiota found high evidence for P. gingivalis , T. forsythia , T. denticola, F. alocis , TM7 spp., and Desulfobulbus spp. being more abundant in periodontal disease than in periodontal health [ 47 ]. Shannon index results did not reveal clear differences between periodontal health and disease [ 48 ]. Functional analysis found a higher abundance of genes involved with bacterial motility, lipopolysaccharide biosynthesis, and peptidase in the subgingival metagenome of subjects with periodontitis [ 48 ].
In the oral cavity, biofilms are formed not only on natural teeth but also on restorative materials, prosthetic constructions, and dental implants. The biofilm formation is very similar, although in detail distinct differences occur. For example, a biofilm at an implant is less diverse than one at a tooth, and abundances of the species differ between both biofilms [ 49 ].
Candida Biofilms
Microorganisms in the biofilms mentioned above are mostly bacteria. However, Candida sp. may also be an important member of the oral biofilm. Candida biofilms both affect soft and hard tissue, they are complex biofilms, and Candida sp. interact with bacteria and host factors [ 50 ]. Candida might be an active member of a cariogenic biofilm and acts synergistically with S. mutans in biofilm matrix formation. The low pH generated by oral streptococci enables Candida albicans to grow in yeast form, and C. albicans lowers oxygen tension, which promotes streptococcal growth [ 50 ]. C. albicans is the most frequent yeast isolated in denture stomatitis, the hyphae form is more present and seems to be the more invasive form, while proteolytic and lipolytic enzymes induce inflammation at the palatum [ 50 ]. Also on dentures and at the palatum, the combination of C. albicans with oral streptococci strengthens the pathogenicity. The interaction of S. oralis with C. albicans increases the biomass of the biofilm and the inflammatory response [ 51 ]. Furthermore, it activates µ-calpain, an enzyme targeting E-cadherin that is an important epithelial cell adhesion molecule [ 51 ]. The biofilm formation on oral surfaces is summarized in Figure 3 .


Fig. 3. Simplified diagram of oral biofilm formation.
Novel Treatment Approaches against Oral Biofilms
Oral biofilms are also more tolerant to the action of antimicrobials than planktonic bacteria. For example, a 50-fold MIC (minimal inhibitory concentration) of doxycycline and a 100-fold MIC of metronidazole against planktonic bacteria are required to be active against single species biofilms of P. gingivalis [ 52 ]. Still, mechanical removal of an oral biofilm associated with an oral disease is the gold standard in treatment, hence scaling and root planing as an anti-infective and anti-inflammatory therapy of periodontitis [ 53 ], or toothbrushing twice a day or more to prevent caries [ 54 ]. Still, there is a need for better methods to remove existing biofilms. In recent years new instrumentation treatment options, including certain lasers (e.g., Er:YAG), ultrasonication, or air polishing with special powders, have been introduced for the dentist. These options may combine more efficiency in biofilm removal with less damage of the natural tooth surface and better patient acceptance [ 55 – 57 ].
Novel approaches to overcome the antimicrobial inactivity against biofilms might be the targeted destruction of biofilm matrix molecules or the modification of the oral biofilm. Focusing on the dispersion of the biofilm matrix, enzymes might be of interest to destroy proteins or glucans, but their use is limited by a sensitivity to proteolysis or by limited penetrating ability [ 58 ]. A promising target seems to be eDNA; for example, sodium hypochlorite was shown in vitro to destroy eDNA as a component of the biofilm matrix ( Fig. 4 ) [ 59 ]. Also, hydrogen peroxide simultaneously degraded the extracellular matrix and killed bacteria within an S. mutans biofilm [ 60 ].


Fig. 4. DNA staining of an in vitro-formed multi-species biofilm without ( a ) and with ( b ) exposure to 0.1% chlorhexidine digluconate solution, or 1% sodium hypochlorite solution ( c ) for 1 min.
Modification of the oral biofilm can be achieved by the application of probiotics – viable bacteria which are beneficial. Probiotics were shown to reduce early childhood caries when applied to milk [ 61 ] and in periodontal therapy they may help to reduce deep periodontal pockets; however, a long-term colonization within biofilms does not seem to occur [ 62 ]. Biofilms can also be modified by applying substances affecting the metabolism of bacteria; arginine, which is utilized by certain oral streptococci, increases the pH in a cariogenic biofilm, thus suppressing mutans streptococci [ 63 ].
In conclusion, biofilms represent a major form of microbial life in the oral cavity. Although knowledge has increased enormously over the last decade, more research is needed about the composition of the biofilm matrix in oral diseases and the functionality of the microbiome to develop efficient strategies to combat pathogenic biofilms.
Conflict of Interest Statement
The author has no conflict of interest to declare.
Funding Sources
The author has no funding to declare.
References
1 Costerton JW, Geesey GG, Cheng KJ: How bacteria stick. Sci Am 1978;238:86–95.
2 O’Toole G, Kaplan HB, Kolter R: Biofilm formation as microbial development. Annu Rev Microbiol 2000;54:49–79.
3 Hall MR, McGillicuddy E, Kaplan LJ: Biofilm: basic principles, pathophysiology, and implications for clinicians. Surg Infect 2014;15:1–7.
4 Flemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S: Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 2016;14:563–575.
5 Solano C, Echeverz M, Lasa I: Biofilm dispersion and quorum sensing. Curr Opin Microbiol 2014;18:96–104.
6 Li YH, Tian X: Quorum sensing and bacterial social interactions in biofilms. Sensors 2012;12:2519–2538.
7 Rendueles O, Ghigo JM: Multi-species biofilms: how to avoid unfriendly neighbors. FEMS Microbiol Rev 2012;36:972–989.
8 Elias S, Banin E: Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev 2012;36:990–1004.
9 Dupin C, Tamanai-Shacoori Z, Ehrmann E, Dupont A, Barloy-Hubler F, Bousarghin L, Bonnaure-Mallet M, Jolivet-Gougeon A: Oral Gram-negative anaerobic bacilli as a reservoir of beta-lactam resistance genes facilitating infections with multiresistant bacteria. Int J Antimicrob Agents 2015;45:99–105.
10 Maunders E, Welch M: Matrix exopolysaccharides; the sticky side of biofilm formation. FEMS Microbiol Lett 2017;364.
11 Jakubovics NS, Shields RC, Rajarajan N, Burgess JG: Life after death: the critical role of extracellular DNA in microbial biofilms. Lett Appl Microbiol 2013;57:467–475.
12 Schmeller DS, Loyau A, Bao K, Brack W, Chatzinotas A, De Vleeschouwer F, Frie­sen J, Gandois L, Hansson SV, Haver M et al: People, pollution and pathogens – global change impacts in mountain freshwater ecosystems. Sci Total Environ 2018;622–623:756–763.
13 Wingender J, Flemming HC: Contamination potential of drinking water distribution network biofilms. Water Sci Technol 2004;49(11–12):277–286.
14 Soini SM, Koskinen KT, Vilenius MJ, Puhakka JA: Effects of fluid-flow velocity and water quality on planktonic and sessile microbial growth in water hydraulic system. Water Res 2002;36:3812–3820.
15 Lienard J, Croxatto A, Gervaix A, Levi Y, Loret JF, Posfay-Barbe KM, Greub G: Prevalence and diversity of Chlamydiales and other amoeba-resisting bacteria in domestic drinking water systems. New Microbes New Infect 2017;15:107–116.
16 Lalancette C, Charron D, Laferriere C, Dolce P, Deziel E, Prevost M, Bedard E: Hospital drains as reservoirs of Pseudomonas aeruginosa : multiple-locus variable-number of tandem repeats analysis genotypes recovered from faucets, sink surfaces and patients. Pathogens 2017;6:36.
17 Gompelman M, van Asten SA, Peters EJ: Update on the role of infection and biofilms in wound healing: pathophysiology and treatment. Plast Reconstr Surg 2016;138(3 Suppl):61S–70S.
18 Ciofu O, Tolker-Nielsen T, Jensen PO, Wang H, Hoiby N: Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Adv Drug Deliv Rev 2015;85:7–23.
19 Vuotto C, Donelli G, Buckley A, Chilton C: Clostridium difficile biofilm. Adv Exp Med Biol 2018;1050:97–115.
20 de Oliveira Ferreira T, Koto RY, Leite GF, Klautau GB, Nigro S, Silva CB, Souza AP, Mimica MJ, Cesar RG, Salles MJ: Microbial investigation of biofilms recovered from endotracheal tubes using sonication in intensive care unit pediatric patients. Braz J Infect Dis 2016;20:468–475.
21 Kamath AF, Ong KL, Lau E, Chan V, Vail TP, Rubash HE, Berry DJ, Bozic KJ: Quantifying the burden of revision total joint arthroplasty for periprosthetic infection. J Arthroplasty 2015;30:1492–1497.
22 Gbejuade HO, Lovering AM, Webb JC: The role of microbial biofilms in prosthetic joint infections. Acta Orthop 2015;86:147–158.
23 Rodrigues LR: Inhibition of bacterial adhesion on medical devices. Adv Exp Med Biol 2011;715:351–367.
24 Blackledge MS, Worthington RJ, Melander C: Biologically inspired strategies for combating bacterial biofilms. Curr Opin Pharmacol 2013;13:699–706.
25 Keyes PH, Likins RC: Plaque formation, periodontal disease, and dental caries in Syrian hamsters. J Dent Res 1946;25:166.
26 Löe H, Theilade E, Jensen SB: Experimental gingivitis in man. J Periodontol 1965;36:177–187.
27 Heller D, Helmerhorst EJ, Oppenheim FG: Saliva and serum protein exchange at the tooth enamel surface. J Dent Res 2017;96:437–443.
28 Rudiger SG, Dahlen G, Carlen A: Protein and bacteria binding to exposed root surfaces and the adjacent enamel surfaces in vivo. Swed Dent J 2015;39:11–22.
29 Bowen WH, Burne RA, Wu H, Koo H: Oral biofilms: pathogens, matrix, and polymicrobial interactions in microenvironments. Trends Microbiol 2018;26:229–242.
30 Heller D, Helmerhorst EJ, Gower AC, Siqueira WL, Paster BJ, Oppenheim FG: Microbial diversity in the early in vivo-formed dental biofilm. Appl Environ Microbiol 2016;82:1881–1888.
31 Kolenbrander PE, London J: Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol 1993;175:3247–3252.
32 Kolenbrander PE, Palmer RJ, Jr., Periasamy S, Jakubovics NS: Oral multispecies biofilm development and the key role of cell-cell distance. Nat Rev Microbiol 2010;8:471–480.
33 Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr.: Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134–144.
34 Espinoza JL, Harkins DM, Torralba M, Gomez A, Highlander SK, Jones MB, Leong P, Saffery R, Bockmann M, Kuelbs C et al: Supragingival plaque microbiome ecology and functional potential in the context of health and disease. mBio 2018;9:e01631–18.
35 Marsh PD, Moter A, Devine DA: Dental plaque biofilms: communities, conflict and control. Periodontol 2000 2011;55:16–35.
36 Tawakoli PN, Neu TR, Busck MM, Kuhlicke U, Schramm A, Attin T, Wie­demeier DB, Schlafer S: Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis. J Oral Microbiol 2017;9:1345581.
37 Schlafer S, Meyer RL, Dige I, Regina VR: Extracellular DNA contributes to dental biofilm stability. Caries Res 2017;51:436–442.
38 Cugini C, Shanmugam M, Landge N, Ramasubbu N: The role of Exopolysaccharides in oral biofilms. J Dent Res 2019;98:739–745.
39 Jakubovics NS: Talk of the town: interspecies communication in oral biofilms. Mol Oral Microbiol 2010;25:4–14.
40 Burne RA: Getting to know “the known unknowns”: heterogeneity in the oral microbiome. Adv Dent Res 2018;29:66–70.
41 Nyvad B, Crielaard W, Mira A, Takahashi N, Beighton D: Dental caries from a molecular microbiological perspective. Caries Res 2013;47:89–102.
42 Takahashi N, Nyvad B: Ecological hypothesis of dentin and root caries. Caries Res 2016;50:422–431.
43 Eriksson L, Lif Holgerson P, Esberg A, Johansson I: Microbial complexes and caries in 17-year-olds with and without Streptococcus mutans . J Dent Res 2018;97:275–282.
44 Richards VP, Alvarez AJ, Luce AR, Bedenbaugh M, Mitchell ML, Burne RA, Nascimento MM: Microbiomes of site-specific dental plaques from children with different caries status. Infect Immun 2017;85.
45 Silva N, Abusleme L, Bravo D, Dutzan N, Garcia-Sesnich J, Vernal R, Hernandez M, Gamonal J: Host response mechanisms in periodontal diseases. J Appl Oral Sci 2015;23:329–355.
46 Hajishengallis G, Darveau RP, Curtis MA: The keystone-pathogen hypothesis. Nat Rev Microbiol 2012;10:717–725.
47 Patini R, Staderini E, Lajolo C, Lopetuso L, Mohammed H, Rimondini L, Rocchetti V, Franceschi F, Cordaro M, Gallenzi P: Relationship between oral microbiota and periodontal disease: a systematic review. Eur Rev Med Pharmacol Sci 2018;22:5775–5788.
48 Kirst ME, Li EC, Alfant B, Chi YY, Walker C, Magnusson I, Wang GP: Dysbiosis and alterations in predicted functions of the subgingival microbiome in chronic periodontitis. Appl Environ Microbiol 2015;81:783–793.
49 Dabdoub SM, Tsigarida AA, Kumar PS: Patient-specific analysis of periodontal and peri-implant microbiomes. J Dent Res 2013;92(12 Suppl):168S–175S.
50 O’Donnell LE, Millhouse E, Sherry L, Kean R, Malcolm J, Nile CJ, Ramage G: Polymicrobial Candida biofilms: friends and foe in the oral cavity. FEMS Yeast Res 2015;15:fov077.
51 Koo H, Andes DR, Krysan DJ: Candida -streptococcal interactions in biofilm-associated oral diseases. PLoS Pathog 2018;14:e1007342.
52 Eick S, Seltmann T, Pfister W: Efficacy of antibiotics to strains of periodontopathogenic bacteria within a single species biofilm – an in vitro study. J Clin Periodontol 2004;31:376–383.
53 Berezow AB, Darveau RP: Microbial shift and periodontitis. Periodontol 2000 2011;55:36–47.
54 Kumar S, Tadakamadla J, Johnson NW: Effect of toothbrushing frequency on incidence and increment of dental caries: a systematic review and meta-analysis. J Dent Res 2016;95:1230–1236.
55 Krishna R, De Stefano JA: Ultrasonic vs. hand instrumentation in periodontal therapy: clinical outcomes. Periodontol 2000 2016;71:113–127.
56 Mizutani K, Aoki A, Coluzzi D, Yukna R, Wang CY, Pavlic V, Izumi Y: Lasers in minimally invasive periodontal and peri-implant therapy. Periodontol 2000 2016;71:185–212.
57 Cobb CM, Daubert DM, Davis K, Deming J, Flemmig TF, Pattison A, Roulet JF, Stambaugh RV: Consensus conference findings on supragingival and subgingival air polishing. Compend Contin Educ Dent 2017;38:e1–e4.
58 Pleszczynska M, Wiater A, Bachanek T, Szczodrak J: Enzymes in therapy of biofilm-related oral diseases. Biotechnol Appl Biochem 2017;64:337–346.
59 Jurczyk K, Nietzsche S, Ender C, Sculean A, Eick S: In-vitro activity of sodium-hypochlorite gel on bacteria associated with periodontitis. Clin Oral Investig 2016;20:2165–2173.
60 Gao L, Liu Y, Kim D, Li Y, Hwang G, Naha PC, Cormode DP, Koo H: Nanocatalysts promote Streptococcus mutans biofilm matrix degradation and enhance bacterial killing to suppress dental caries in vivo. Biomaterials 2016;101:272–284.
61 Rodriguez G, Ruiz B, Faleiros S, Vistoso A, Marro ML, Sanchez J, Urzua I, Cabello R: Probiotic compared with standard milk for high-caries children: a cluster randomized trial. J Dent Res 2016;95:402–407.
62 Tekce M, Ince G, Gursoy H, Dirikan Ipci S, Cakar G, Kadir T, Yilmaz S: Clinical and microbiological effects of probiotic lozenges in the treatment of chronic periodontitis: a 1-year follow-up study. J Clin Periodontol 2015;42:363–372.
63 He J, Hwang G, Liu Y, Gao L, Kilpatrick-Liverman L, Santarpia P, Zhou X, Koo H: l-arginine modifies the exopolysaccharide matrix and thwarts Streptococcus mutans outgrowth within mixed-species oral biofilms. J Bacteriol 2016;198:2651–2661.
Sigrun Eick University of Bern Department of Periodontology Freiburgstrasse 7 CH–3010 Bern (Switzerland) sigrun.eick@zmk.unibe.ch
Biofilm in General
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 12–18 (DOI: 10.1159/000510195)
______________________
Biofilms in Dental Unit Water Lines
Gunnar Dahlen
Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
______________________
Abstract
Biofilm formation has become a significant problem in dental unit water lines (DUWLs). The formation of biofilms and microbial growth in DUWLs leads to an unacceptably high number of microorganisms in the water used for spraying, cooling, and ultrasonication procedures. These procedures form aerosols which can be inhaled by the patients, and consequently dentistry constitutes an area of specific concern for patient safety. In particular, older and immunocompromised patients are at risk of serious respiratory tract infections if the water contains pathogens such as Legionella pneumophila and Pseudomonas spp. In the EU it is recommended that the water in DUWLs should not exceed 200 colony-forming units (CFU) of heterotrophic bacteria (bacteria living on organic material) per milliliter of water to be acceptable in dental work. A number of efficient products are available on the market that can be applied onto dental units. New dental units are nowadays equipped with “inbuilt” systems. Such measures have resulted in an acceptable standard of water in 95% of the 1,200 dental units in the Public Dental Health Service of the Västra Götalands region of Sweden that were followed yearly for 4 years. For the majority of the remaining DUWLs with an unacceptable standard this is due to neglect or inappropriate routines for water-cleaning procedures. It is the ability to follow instructions rather than the cleaning procedure itself that is decisive if clinics and dental units are to have an appropriate standard of water in their systems.
© 2021 S. Karger AG, Basel
Biofilm formation has become a significant problem in dental unit water lines (DUWLs) and the problem has been the subject of numerous investigations to determine the magnitude and risks, and its practical control [ 1 – 4 ]. The microorganisms of the biofilms are predominantly non-pathogenic heterotrophic environmental bacteria, although pathogenic bacteria such as Legionella pneumophila and Pseudomonas species can frequently be found [ 2 , 4 ]. The variations from one country or clinic to another can also be substantial [ 3 ]. Contaminated unit water has become a major concern for dentistry due to the potential risk of causing respiratory tract infections, especially in elderly and immunocompromised patients. This review considers biofilm formation in DUWLs, why and how biofilms are formed, what risks are connected with the biofilms and microorganisms in the water lines used for dental treatment procedures, and what can be done to minimize the hazard.
Biofilm Formation
Biofilms are complex and heterogeneous formations on most surfaces in nature and in man-made water systems where there is a slow streaming water flow [ 5 ]. All water systems contain minute amounts of organic materials and microorganisms unless the water is distilled and sterilized. Biofilms are a natural habitat for microorganisms to live and grow because they offer them many advantages in comparison to the free-living planktonic stage. The adherence to a surface and a biofilm makes it possible to build complex communities that can withstand flushing, dehydration, and antimicrobial measures. More importantly, due to nutrient limitation, most microbial cells are slow growing or even dormant. Also, the production of extracellular polysaccharides that build up the biofilm, constituting over 90% of a biofilm’s dry weight, forms a barrier that protects the microorganisms in deeper layers of the biofilm from antimicrobials. Other advantages are cooperation for nutrients (food web), which is important for the microorganisms living in the biofilms of DUWLs, where the level of nutrients is extremely low. In established biofilms there is a constant detaching of microbial cells, which can be measured in the output water. More than 100,000 colony-forming units (CFU)/mL have been reported, which illustrates that the water lines in DUWLs constitute an ideal environment for biofilm formation and bacterial growth [ 3 , 6 – 8 ].
There are several reasons why biofilms are easily formed in DUWLs. The main reason is the low water flow, estimated to be 0.5 mL/s, and the total volume of water in a dental unit is around 100 mL. In addition, the units are standing still most of the day, which makes it possible for the microorganisms in the biofilm to reorganize, cooperate, and multiply, increasing the biofilm’s size and thickness. During weekends and vacations there could be several days without use. The comparatively higher temperature (room temperature) than the incoming community water makes it possible for the attachment and growth of more thermophile and pathogenic bacteria. The incoming water contains minute amounts of organics (proteins, polysaccharides) as well as dead and viable microorganisms that easily attach to the inner surface of the lines. In the periphery of the lines, immediately adjacent to the surface and liquid interface, the flow velocity is negligible compared to that of the center where the flow is the highest. The surface roughness and hydrophobicity increase the attachment, which mainly occurs between hydrophobic areas of organic molecules and bacteria on the one hand, and the surface of the water lines on the other, today usually made of hydrophobic non-polar plastic material (polyvinyl or polyurethane). Once the first layer (a pellicle) has become established more microorganisms attach to this layer. Established bacteria will multiply and the growth rate depends on available nutrients. Heterotrophic (requiring organic material for growth) environmental bacteria, mostly Gram-negative aerobic or facultative but low fastidious microorganisms, will grow in the biofilm inside the DUWLs. The established biofilm structure comprises microbial cells and extracellular polymeric substances (EPS). The biofilms are highly complex, with microcolonies of bacterial cells, EPS matrix, cell communication, exchange of genes, quorum sensing, predation, and competition representing an ecological community that differs from one unit to another. Each unit has its own “inner life.”
During periods of no flow, the microbial growth and biofilm formation can be substantial and can even cause total stagnation of the flow. The problem is obvious when the flow is turned on and bigger masses are loosened, potentially causing the lines and valves to become clogged [ 4 ]. Filters have been attached to dental units but have the drawback of reducing the flow rate significantly [ 9 ]. The recommendation to flush the system for 3 min in the morning to reduce the microbial level in the output water only has a marginal and temporary effect [ 1 ]. A number of commercially available treatment products for DUWLs have been marketed that efficiently reduce the number of bacteria in the output water or reduce/eliminate the biofilms in the water lines [ 4 , 7 ].
Aerosols
The combination of biofilm formation and the use of the contaminated water are of high concern during patient treatment in health care and the dental environment [ 10 ]. The presence of a biofilm constantly delivers a number of detached microorganisms in a planktonic stage to the output water used for treatment procedures, such as spraying, cooling, and ultrasonication, which all form aerosols. Aerosols are easily inhaled by patients as well as the dentist and nurse. The personnel can protect themselves with a mask that should cover both the mouth and nose to fulfil its purpose. However, the patients cannot be protected and may be in a more vulnerable situation since the aerosols are formed in and around their mouths and they are exposed to a higher degree to the aerosols by inhalation. It should be pointed out that the contaminated water from DUWLs is not hazardous to drink, since gastrointestinal pathogens such as coliforms are extremely uncommon in DUWLs [ 2 – 4 ]. However, high numbers of microorganisms should be regarded as poor quality and in the worst cases the water can even be discolored and smell and taste bad, and unacceptable for used for irrigation in the mouth. With a high number of microorganisms in DUWLs, the potential risk is entirely connected to the risk of inhalation and development of respiratory tract infections. This is especially important in immune and medically compromised patients, the elderly, and other individuals more susceptible to developing respiratory tract infections, who can even develop infection from normally innocent microorganisms or their products (e.g., endotoxins) if they are exposed to a high number of microorganisms. The risk of complications increases when respiratory pathogens are present (see below).
The risk with aerosols is well known from other similar systems, such as air conditioners, air moisteners, water towers, and showers. In fact, the first well-recognized incidence with respiratory tract infection occurred in 1976 with the outbreak of pneumonia caused by Legionella among veterans who had assembled in Philadelphia (PA, USA) and were exposed to an air condition system containing L. pneumophilia [ 11 ]. Since then, a number of similar outbreaks have been reported in hotels, old people’s home, and hospitals [ 11 ]. Although only a few case reports are documented in dentistry [ 12 ], the theoretical risk of gaining respiratory tract infection from DUWLs is considered to be high. However, the actual frequency of respiratory tract infections caused by aerosols from dental unit water is unknown due to difficulties in tracing the origin of the bacteria.
Microorganisms in DUWLs
The microbiota of the water and biofilms in DUWLs can be very complex [ 2 , 4 ]. If the biofilms are not regularly removed they can build up complex microbial communities composed of heterotrophic environmental Gram-negative bacteria in the water. Opportunistic pathogens such as Legionella and Pseudomonas species have gained the most attention, but other heterotrophic species such as mycobacteria, staphylococci (S. aureus, S. epidermidis) , and a number of Gram-negative rods and fungi may also be present [ 2 , 4 , 7 ]. Potential pathogens grow rapidly (within 2 days) on ordinary solid bacteriological media in 36 °C. Samples could be incubated at room temperature (22 °C) but grow slower [ 8 ]. A longer incubation time (1 week) will increase the number of CFU due to the slow-growing nature of most heterotrophic bacteria for which the water lines are their natural habitat [ 4 ]. High numbers of slow-growing bacteria therefore indicate poor water quality, the presence of a biofilm, and a rich inner life of the DUWL.
A special concern relating to biofilms and the microbiota in water lines such as in DUWLs is the presence of protozoa/amoebae [ 13 ]. There are various examples, with the most recognized being Acanthamoeba species. Protozoans are of special interest in Legionella pathogenicity (see below).
Specific Pathogens in DUWLs
Although numerous microorganisms have been found in the water of DUWLs, some opportunistic pathogens are of special concern for patients and personnel, for example aerobic Gram-negative bacilli such as Pseudomonas spp. and Legionella spp. [ 4 , 13 ].
Pseudomonas spp. are regularly present in water, soil, and moist environments, and should be considered an indicator of poor water quality. Pseudomonas should not be present in drinking water and therefore not in the water of DUWLs. Pseudomonas spp. typically cause opportunistic infections, and respiratory tract infections in particular. Patients with respiratory diseases such as COPD and cystic fibrosis are a particular risk for Pseudomonas infections [ 14 – 17 ], with P. aeruginosa being the most common species.
Legionella infections are typically spread through aerosols from water lines containing various Legionella spp. [ 3 , 18 , 19 ]. L. pneumophila is responsible for most Legionella infections and can be serologically classified, which makes it possible to trace the origin [ 11 ]. Some outbreaks can be endemic, when many compromised patients can be exposed at the same time, for example in hotels, hospitals, and public baths. An aggravating circumstance of Legionella in water lines is their tendency to hide intracellularly in protozoa/amoebae residing in the biofilms [ 20 , 21 ]. This makes the action of some disinfectants, especially chlorine, less efficient [ 19 ].
Methods to Reduce Bacterial Counts in DUWLs
A number of solutions have been introduced on the market, but it is beyond the purpose of this paper to review the various products that are available. Principally, the methods are of two approaches, those that disinfect/reduce bacteria from the output water, and those that attack and eliminate/prevent the biofilm in the DUWLs [ 4 , 22 ]. The first is attractive since it is based on a continuous delivery of the disinfectant, needs less manipulation and care, but has the disadvantage that it may expose patients to antimicrobials that are still present in the water when it is used. A continuous delivery, such as using hydrogen peroxide, may also efficiently prevent biofilm formation in the DUWLs once they have been eliminated [ 23 ]. Another disadvantage is that disinfection is only performed at use – and bacterial growth and biofilm formation may take place in units that are only sporadically used or over the weekend and during vacations. The principle of non-continuous delivery is based on the disinfection being active for longer periods of time, such as overnight or at weekends, which allows for a more efficient elimination of the biofilm using chlorine products. An established biofilm is much more difficult to eradicate. It is sometimes necessary to expose the entire DUWL system to a strong disinfectant (shock treatment) in order to remove the biofilm. Sodium hydroxide 0.1 M (0.8%) or sodium hypochlorite 0.5% has been suggested [ 3 , 24 , 25 ]. However, it should only be used when necessary due to the risk of damage to metal pieces (e.g., valves and connections) within the DUWL by corrosion if used frequently and for longer periods of time. Importantly, these strong solutions should be stained (e.g., methylene blue) to make it possible to check that the chemicals have been washed off. In severe cases, when there is a total blockage in the water lines, the radical solution is to exchange the lines with new ones, although this is expensive [ 3 ].
The threshold for a sufficient water-cleaning system within the EU has been recommended to be <200 CFU/mL and the water in a DUWL should be checked yearly. The American Dental Association (ADA) has recommended the same, while the recommendation of the Center for Disease Control (CDC) for drinking water is 500 CFU/mL [ 4 ]. The Swedish recommendations, which were set in 2006 [ 26 ] and before the EU recommendation was decided, used the threshold of 100 CFU/mL and is still in use [ 27 , 28 ]. In practice it means that we accept, although with attention, up to 500 CFU/mL for heterofermentative fast-growing (<2 days) bacteria at room temperature (22 °C). In order to get a better picture of the “internal life” and presence of a biofilm, in our lab we practice incubation for 7 days for slow-growing bacteria, which should not exceed 5,000 CFU/mL according to the drinking water standard for tap water in Sweden [ 3 ]. Such high levels indicate the presence of biofilms in a DUWL and should be shock treated. However, when regular antimicrobial treatment of each DUWL in a clinic is performed, such high numbers of bacteria are rare.
Reasons for Failure
While most DUWLs with functioning water-cleaning systems show few or no bacteria, others still harbor bacteria to an unacceptable level. A 4-year follow-up study [ 28 ] showed that the installation of water-cleaning systems (added systems or inbuilt systems) leads to a significant improvement in output water quality. For 1,200 DUWLs at 140 clinics followed between 2013 and 2016, a 95% acceptance of the water quality was attained. The Alpro/Bilpron system was used in the majority of DUWLs (81.6%), but also Oxygenal (2.0%), Unit-Clean (5.2%), Sterilox (0.6%), as well as inbuilt systems (4.8%) were included in 2016. This study did not show any significant differences between the systems. In the remaining 5% of DUWLs that did not reach an acceptable level, the failures could be divided into problems related to the unit or to the clinic. Even if the unit is equipped with a water-cleaning system there is no guarantee of successful results. The most common reason for failure is neglect to run the water-cleaning system regularly. If the bacterial level in the output water is not acceptable, additional cleaning steps have to be taken. It is of outmost importance that the personnel are sufficiently informed and instructed and that necessary precautions are taken for servicing each unit.
Clinic-related problems can also prevail. Examples could be if the building in which the clinic is situated, such as a hospital, has a complex water system, or if the water supply to the clinic is accidentally contaminated with bacteria as a result of renovations and repair. It is recommended that the water in DUWLs is specifically checked after such accidents. Another clinic-related problem is when the clinic has insufficient routines or has chosen an insufficient method for water cleaning.
Conclusions
Biofilm formation and a high number of microorganisms in the output water of DWULs has been a main concern in dental practice for several decades. Today, most brands of dental units are equipped with in-built methods for reducing the number of microorganisms and/or eliminating biofilms. For older units, means of cleaning the water are available on the market and have a good efficiency. Ultimately, it is the handling and compliance with instructions rather than the cleaning procedure itself that determines whether a clinic maintains an appropriate standard of water in its dental unit systems.
Acknowledgements
Mrs. Susanne Blomqvist is gratefully acknowledged for fruitful discussions during preparation of the manuscript.
Conflict of Interest Statement
The author has no conflicts of interest to declare.
Funding Sources
No funding was raised or used for preparing this paper.
References
1 Pankhurst CL, Philpott-Howard JN: The microbiological quality of water in dental chair units. J Hosp Infect 1993;23:167–174.
2 Walker JT, Bradshaw DJ, Finney M, Fulford MR, Frandsen ER, Östergaard E, ten Cate JM, Moorer WR, Schel AJ, Mavridou A, Kamma JJ, Mandilara G, Stösser L, Kneist S, Araujo R, Contrers N, Goroncy-Bermes P, O’Mullane D, Burke F, Forde A, O’Sullivan M, Marsh PD: Microbial evaluation of dental unit water systems in general dental practice in Europe. Eur J Oral Sci 2004;112:412–418.
3 Dahlen G, Alenäs-Jarl E, Hjort G: Water quality in water lines of dental units in the Public Dental Health service in Göteborg, Sweden. Swe Dent J 2009;33:161–172.
4 Coleman DC, O´Donell MJ, Shore AC, Russell RJ: Biofilm problems in dental unit water systems and its practical control. J Appl Microbiol 2009;106:1424–1437.
5 Donlan RM: Biofilms: microbial life on surfaces. Emerg Infect Dis 2002;8:881–890.
6 Williams JF, Johnston AM, Johnson B, Huntington MK, McKenzie CD: Microbial contamination of dental unit water lines: prevalence, intensity and microbiological characteristics. J Am Dent Assoc 1993;124:59–65.
7 Walker JT, Bradshaw DJ, Bennett AM, Fulford MR, Martin MV, Marsh PD: Microbial biofilm formation and contamination of dental-unit water systems in general practice. Appl Environment Microbiol 2000;66:3363–3367.
8 Smith AJ, McHugh S, McCormick L, Stansfield R, McMillan A, Hood J: A cross-sectional study of water quality from dental water unit lines in dental practices in the West of Scotland. Br Dent J 2002;192:645–648.
9 Pankhurst CL, Philpott-Howard JN, Hewitt JH, Casewell MW: The efficacy of chlorination and filtration in the control and eradication of Legionella from dental chair water systems. J Hosp Infect 1990;16:9–18.
10 10 Zemouri C, de Soet H, Crielaard W, Laheij A: A scoping review on bio-aerosols in health care and the dental environment. PLoS One 2017;12:e0178007.
11 Mercante JW, Winchell JM: Current and emerging Legionella diagnostics for laboratory and outbreaks investigations. Clin Microbial Rev 2015;28:95–133.
12 Ricci ML, Fontana S, Pinci F, Fiumana E, Pedna MF, Farolfi P, et al: Pneumonia associated with a dental unit water line. Lancet 2012;61:1208–1213.
13 Lau HY, Ashbolt NJ: The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol 2009;107:368–378.
14 Vento S, Cainelli F, Temesgen Z: Lung infections after cancer chemotherapy. Lancet Oncol 2008;9:982–992.
15 Williams BJ, Dehnbostel J, Blackwell TS: Pseudomonas aeruginosa : host defence in lung diseases. Respiratology 2010;15:1037–1056.
16 Al-Hiyasat AS, Maáyeh SY, Hindiyeh MY, Khader YS: The presence of Pseudomonas aeruginosa in the dental unit waterline systems of teaching clinics. Int J Dent Hyg 2007;5:36–44.
17 Mainz JG, Gerber A, Lorenz M, Michl R, Hentschel J, Nader A, Beck JF, Pletz MW, Mueller AH: Pseudomonas aeruginosa acquisition in cystic fibrosis patients in context of otorhinolaryngological surgery or dental attendance: case series and discussion of preventive concepts. Case Rep Infect Dis 2015;2015:438517.
18 Challacombe SJ, Fernandes LL: Detecting Legionella pneumophila in water systems: a comparison of various dental units. J Am Dent Assoc 1995;126:603–608.
19 Zanetti F, Stampi S, De Luca G, Fateh-Moghadam P, Bucci Sabattini MA, Checchi L: Water characteristics associated with the occurrence of Legionella pneumophila in dental units. Eur J Oral Sci 2000;108:22–28.
20 Trabelsi H, Sellami A, Dendena F, Sellami H, Cheikh-Rouhou F, Makni F, Ben DS, Ayadi A: Free-living amoebae (FLA): morphological and molecular identification of Acanthamoeba in dental unit water. Parasite 2010;17:67–70.
21 21Hsu BM, Huang CC, Chen JS, Chen NH, Huang JT: Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments. Water Res 2011;45:5171–5183.
22 Wirthlin MR, Marshall GW Jr, Rowland RW: Formation and decontamination of biofilms in dental unit waterlines. J Periodontal 2003;74:1595–609.
23 Walker JT, Bradshaw DJ, Fulford MR, Marsh PD: Microbial evaluation of a range of disinfectant procedures to control mixed-species biofilm contamination in a laboratory model of a dental unit water system. Appl Environment Microbiol 2013;69:3327–3332.
24 Claesson R: Umeå har en metod för desinfektion av unitar. Tandläkartidningen 2006;98:56–58.
25 Möller Å, Dahlen G: The cleaning and disinfection of water systems in dental units according to the UnitClean method. Tandläkartidningen 2007;99:52–56.
26 Socialstyrelsen: Att förebygga vårdrelaterade infektioner. Stockholm, SOS, 2006.
27 Dahlen G, Hjort G, Spencer I: Water cleaning systems improves the water quality in dental unit water lines (DUWL). A report from the Public Dental Health of Västra Götaland region, Sweden. Swe Dent J 2013;37:171–177.
28 Dahlen G, Hjort G: The standard of water in dental units is increasingly better – a report from Public Dental Health Service of VG-region, Sweden, during 2013–2016. Tandläkartidningen 2017;109:50–53.
Gunnar Dahlen Department Oral Microbiology and Immunology, Institute of Odontology Sahlgrenska Academy, University of Gothenburg, Box 450 SE–405 30 Gothenburg (Sweden) dahlen@odontologi.gu.se
Biofilm in General
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 19–29 (DOI: 10.1159/000510196)
______________________
The Impact of the pH Value on Biofilm Formation
Lara B. Schultze a Alejandra Maldonado a Adrian Lussi b , c Anton Sculean a Sigrun Eick a
a Department of Periodontology, Laboratory of Oral Microbiology, School of Dental Medicine, University of Bern, Bern, Switzerland; b School of Dental Medicine, University of Bern, Bern, Switzerland; c Department of Operative Dentistry and Periodontology, University Medical Center, Freiburg, Germany
______________________
Abstract
The pH value of a biofilm influences the pathogenesis and therapy of oral diseases such as caries and periodontitis. This study aimed to investigate the influence of different initial pH values on the microbial composition, bacterial counts, metabolic activity, and quantity of three defined biofilms representing oral health, caries, and periodontal disease. Respective bacterial suspensions in the nutrient broth were initially adjusted to pH values between 5 and 8. Then biofilms were cultured on polystyrene surfaces coated with a proteinaceous solution for 2 h (“healthy” biofilm), 6 h (“healthy,” and “cariogenic” biofilms), 24 h (“cariogenic,” and “periodontitis” biofilms), and 48 h (“periodontitis” biofilm). In all biofilms, total bacterial counts were lower at an initial pH of 5 or 5.5 than at higher pH values. In the biofilm representing caries, the percentage of cariogenic bacteria ( Streptococcus mutans , S. sobrinus , Lactobacillus acidophilus ) was higher at a low pH, the metabolic activity was highest at pH 6–6.5, and biofilm mass was greatest at pH 7–7.5. In the biofilm representing periodontitis, the percentage of Porphyromonas gingivalis increased with the pH. Also, the metabolic activity was highest at pH 8, whereas mass had the highest value at pH 7. In conclusion, the initial pH value influences biofilm formation. In particular, metabolic activity and the amount of bacteria associated with disease correlated with the respective pH known to be of importance in the development of caries (relatively low pH) and periodontitis (higher pH). Modifying the pH level in oral biofilms might be an alternative concept in (primary) prevention and treatment, not only of caries but also of periodontitis.
© 2021 S. Karger AG, Basel
Two major oral diseases, caries and periodontal disease, are associated with biofilm formation on teeth. Caries is a disease affecting dental hard tissues. A collection of different microorganisms in a biofilm produces acids, decreases the pH, and moves the balance between demineralization and remineralization towards demineralization. The acidogenic stage is followed by an aciduric stage where microorganisms (including mutans streptococci) are still able to survive at low pH values [ 1 ]. Treatment options that increase the pH within supragingival biofilms are currently under discussion. For example, arginine as a supplement of toothpaste aims to modify the microbial biofilm composition to be less acidogenic [ 2 ].

Table 1. Bacterial strains used in the experimental biofilms

Periodontitis is a disease characterized by the destruction of the tissue surrounding teeth and, if untreated, ultimately leads to tooth loss. Among the many different bacteria found in subgingival biofilm, Porphyromonas gingivalis was postulated as being a keystone pathogen in the development of periodontitis by modifying the host response and finally the composition of the biofilm [ 3 ]. A recent analysis in adolescents showed that the severity of periodontitis was inversely associated with enamel caries, whereas the extent of periodontitis (numbers of affected sites) showed a positive association with dentin carious lesions [ 4 ]. After initial periodontal therapy, periodontopathogenic bacteria decrease while Streptococcus mutans increases [ 5 ]. This suggests a dependency of the composition of the oral biofilm on the surrounding micromilieu. One important factor might be the pH value.
The aim of this study was to investigate the influence of initially different pH values on biofilm formation. For this, nutrient media containing buffers were adjusted to pH values in a range from 5 to 8 in increments of 0.5. Defined microbial strains representing oral health, caries, and periodontal disease were studied for their ability to form biofilms regarding bacterial counts, microbial composition, biofilm mass, and metabolic activity.
Materials and Methods
Defined laboratory strains were used for the different biofilms. Early colonizers represent the biofilm associated with oral health, mutans streptococci belong to the five-species biofilm associated with caries, and P. gingivalis , Tannerella forsythia , and Treponema denticola are additional members of the eight-species biofilm representing periodontal disease ( Table 1 ).
The strains were passaged 48–24 h before the experiments on tryptic-soy agar plates with 5% sheep blood. For T. forsythia, N-acetylmuramic acid (10 mg/L) was added. T. denticola was cultivated in modified mycoplasma broth (BD, Franklin Lakes, NJ, USA) added with 5 mg/mL of cocarboxylase in anaerobic conditions.
Thereafter, the bacteria were suspended in 0.9% w/v NaCl to McFarland 4. The mixed suspension for the “healthy” biofilm consisted of two parts S. gordonii and three parts Actinomyces naeslundii. The cariogenic biofilm was mixed with one part S. gordonii and S. mutans, two parts A. naeslundii and S. sobrinus , and three parts Lactobacillus acidophilus . For the periodontal biofilm, the respective mixture was prepared with one part S. gordonii and three parts each of the other seven bacteria. These mixed suspensions were added 1:20 to the nutrient broth (Wilkins-Chalgren Broth; Oxoid, Basingstoke, UK) with 5 mg/L β-NAD (Sigma-Aldrich, Buchs, Switzerland) which had been adjusted to an approximate pH with two different buffers (citrate buffer and phosphate buffer) and precisely with NaOH or HCl until pH values of 5, 5.5, 6, 6.5, 7, 7.5, and 8 were achieved.
The biofilms were formed on 96-well plates. First the surface was coated with a proteinaceous solution (25% human serum, 0.27% mucin; both Sigma-Aldrich) for 1 h, before 250 µL/well of the bacteria/nutrient broth suspension was added. Thereafter, the plates were incubated at 37 °C with 10% CO 2 or anaerobically (periodontitis biofilm) for different lengths of time. The “healthy” biofilm was analyzed after 2 and 6 h, the “cariogenic” biofilm after 6 and 24 h, and the “periodontal” biofilm after 24 and 48 h. For the “periodontal” biofilm, 10 µL of microbial suspension consisting of one part each of T. denticola , T. forsythia , and P. gingivalis were added again after 24 h. Each of the three different plates were used in one experimental setting and per time point.
At the respective times, the nutrient broth was removed and the biofilms were careful washed once with 0.9% w/v NaCl. From the first plate, the biofilms were scraped from the surface and suspended in 0.9% w/v NaCl. After intensive mixing by pipetting and vortexing, a serial 10-fold dilution series was made. Each 25 µL were spread on tryptic-soy agar plates with 5% sheep blood (and 10 mg/L NAM). After incubation at 37 °C with 10% CO 2 or anaerobically (the “periodontitis” biofilm) for about 7 days, the total bacterial counts (log 10 colony-forming units; CFU) as well as the percent of the different bacteria used were determined. The identification was based on the colony morphology. As this was in part difficult or impossible ( T. denticola does not grow on the agar plates), P. gingivalis , T. forsythia , and T. denticola , were counted using real-time PCR as described previously [ 6 ].
The second 96-well plate was used for the determination of metabolic activity with the use of Alamar blue reagent as a redox indicator [ 7 ]. A total of 5 µL of Alamar blue (alamarBlue®, Thermo Fisher Scientific Inc., Waltham, MA, USA) was mixed with 100 µL of the nutrient media and added to the biofilm. After extensive mixing with the biofilm and an incubation for 1 h at 37 °C, absorbances were measured at 570 nm against 600 nm.
The biofilm mass was quantified from the third 96-well plate according to recently published protocols [ 8 ]. First, the biofilms were fixed at 60 °C for 60 min. Thereafter, each 50 µL of 0.06% crystal violet (Sigma-Aldrich) was added. The plate was incubated at 37 °C for 10 min, then washed 3 times with 200 µL of dH 2 O. Finally, the plate was read at 600 nm.
Each experiment was performed in quadruplicate in two independent series, resulting in at least eight single values each. ANOVA with post hoc Bonferroni was used for the statistical analysis. The level of significance was set to p = 0.05 and SPSS v.24.0 software (IBM SPSS Statistics, Chicago, IL, USA) was used.
Results
We present the total bacterial counts, the percentages of the respective bacteria (or groups), the metabolic activity, and the biofilm masses. Post hoc statistical analysis was restricted to the differences from pH 7.
Biofilm Representing Oral Health
Total counts of bacteria within each biofilm (log 10 CFU) differed significantly between the initial pH values at 2 and 6 h (each p < 0.001; Fig. 1a ). At 2 h, the lowest values were counted at pH 5 and 5.5 with approximately 5.8 log 10 CFU, while the highest counts were detected at pH 7 with a mean of 6.6 log 10 CFU. Post hoc analysis showed differences between pH 5, 5.5, 7.5, and 8 on the one hand, and pH 7 on the other (each p < 0.01). At 6 h, the lowest CFU counts remained at 5.7 log 10 when the initial pH was 5, the highest value increased to 7.3 log 10 (pH 7.5). Also at 6 h, the CFU count differences were each statistically significant between pH 5, 5.5, and 7 (each p < 0.01).


Fig. 1. Total bacterial counts (mean and SD; a ), bacterial composition ( b ), metabolic activity (mean and SD; c ), and biofilm mass (mean and SD; d ) of the two-species biofilm representing oral health in relation to the initial pH. Differences versus pH 7 are presented: * p < 0.05, ** p < 0.01 (the asterisk colors correspond with the composition of biofilms in b ).
At 2 h, the quantity of the two species did not differ significantly between the different pH levels. At 6 h, there was a significantly higher percentage of A. naeslundii and a lower percentage of S. gordonii at pH 5 in comparison with pH 7 (each p < 0.001; Fig. 1b ).
The metabolic activity differed between the different pH values at 2 and 6 h (each p < 0.001; Fig. 1c ). At both time points, the lowest activities were at pH 5 and 5.5, each with a statistically significant difference to pH 7 at 2 h (each p < 0.05) and at 6 h (each p < 0.01).
Regarding biofilm mass, no statistically significant difference from the initial pH values was found, either at 2 or 6 h ( Fig. 1d ). The pH measurements performed at 6 h still showed a gradient from pH 5.15 (initially pH 5) to pH 7.05 (initially pH 8; Table 2 ).

Table 2. Initial pH values and values (mean ± SD) in the surrounding media after 6 h of formation of the two-species biofilm representing “oral health”


Fig. 2. Total bacterial counts (mean and SD; a ), bacterial composition ( b ), metabolic activity (mean and SD; c ), and biofilm mass (mean and SD; d ) of the five-species biofilm representing caries in relation to the initial pH. Differences versus pH 7 are presented: * p < 0.05, ** p < 0.01 (the asterisk colors correspond with the composition of biofilms in b ).
Biofilm Representing Caries
In the biofilms representing caries, total counts of bacteria within each biofilm (log 10 CFU) differed significantly between the initial pH values at 6 and 24 h (each p < 0.001; Fig. 2a ). At 6 h, the lowest values were counted at pH 5 with approximately 5.8 log 10 CFU, while the highest counts were recorded at pH 6 with a mean of 7.7 log 10 CFU. Post hoc analysis showed differences between each pH 5 and 5.5 on the one hand, and pH 7 on the other (each p < 0.01). At 24 h, the lowest CFU counts were 6.0 log 10 when the initial pH was 5; the highest value was 9.7 log 10 (pH 7 and 8). At 24 h, the CFU count differences were each statistically significant between pH 5, 5.5, 6, and 7 (each p < 0.01).
At 6 h, there were no statistically significant differences in the percentage of S. gordonii , A. naeslundii , and S. mutans/S. sobrinus between the pH levels. The percentage of L. acidophilus was higher at pH 5 and 5.5 versus pH 7 ( p < 0.001, p = 0.017). At 24 h, the percentage of L. acidophilus remained high at pH 5 ( p < 0.001 vs. pH 7). Furthermore, there was a significantly higher percentage of S. mutans/S. sobrinus at pH 5 ( p < 0.001) and pH 6 ( p = 0.016), and a lower percentage of S. gordonii at pH 5, 5.5, and 6 (each p < 0.001) versus pH 7 ( Fig. 2b ).
The metabolic activity differed between the different pH values at 6 and 24 h (each p < 0.001; Fig. 2c ). At both time points, the lowest activities were at pH 5 and 5.5, with each being statistically significantly different to pH 7 (each p < 0.01). However, at 24 h the biofilm metabolic activity was higher for the initial pH level of 6 versus pH 7 ( p < 0.001).


Fig. 3. Total bacterial counts (mean and SD; a ), bacterial composition ( b ), metabolic activity (mean and SD; c ), and biofilm mass (mean and SD; d ) of the eight-species biofilm representing periodontitis in relation to the initial pH. Differences versus pH 7 are presented: * p < 0.05, ** p < 0.01 (the asterisk colors correspond with the composition of biofilms in b ).
At 6 h, the biofilm mass was higher at the initial pH level of 6 versus pH 7 ( p < 0.001). At 24 h, there was a decreased biofilm quantity at pH 5 and 5.5 in comparison with pH 7 ( p = 0.012, p = 0.007; Fig. 2d ).
The pH measurements were performed at 6 and 24 h. The gradients were from pH 5.15 and 5.10 (initially pH 5) to pH 7.04 and 7.05 (initially pH 8) at 6 and 24 h. The initial pH value of 7.5 decreased to 6.9 ( Table 3 ).

Table 3. Initial pH values and values (mean ± SD) in the surrounding media after 6 and 24 h of formation of the five-species biofilm representing “caries”

Biofilm Representing Periodontitis
In the biofilms representing periodontitis, the total counts of bacteria (log 10 CFU) differed significantly between the initial pH values at 24 and 48 h (each p < 0.001; Fig. 3a ). At 24 h, the lowest values were counted at pH 5 and 5.5 with approximately 5.8–5.9 log 10 CFU, while the highest counts were detected at pH 7–8 with a mean of 9.3–9.5 log 10 CFU. At 48 h, the lowest CFU counts were 6.3 log 10 when the initial pH was 5, and the highest values were 9.3–9.4 log 10 (pH 7–8). At both time points, post hoc analysis showed differences between each pH 5, 5.5, 6, and 6.5 on the one hand, and pH 7 on the other (each p < 0.01).
At 24 h, the percentages of T. forsythia and T. denticola were higher at pH 5 and 5.5 in comparison with the initial pH 7 (each p < 0.001). At 48 h, the percentage of both species remained high at pH 5 (both p < 0.001). In contrast, the percentage of P. gingivalis increased with increasing pH. The differences for pH 5–6.5 were all statistically significant versus pH 7 ( p < 0.001). Apart from P. gingivalis , T. forsythia , and T. denticola , the other bacteria were present at higher percentages at pH 5 ( p = 0.004), pH 5.5 ( p = 0.017), pH 6 ( p = 0.002), and pH 6.5 ( p = 0.012) in comparison with the initial pH 7 ( Fig. 3b ).
The metabolic activity also showed a dependency to the pH level, increasing with elevated pH values at 24 and at 48 h ( Fig. 3c ). At both time points, the activities were lowest at pH 5 and 5.5, with each being statistically significantly different to pH 7 (each p < 0.001). At 24 h, the biofilm metabolic activity was higher for the initial pH level of 8 versus pH 7 ( p < 0.001).
At 24 h, the biofilm mass was higher for the initial pH level of 6.5 versus pH 7 ( p < 0.001). At 48 h, there was a decreased mass for pH 5 and 5.5 in comparison with pH 7 (both p < 0.001; Fig. 3d ).
The pH measurements were performed at 24 and 48 h. The gradients were from pH 5.21 and 5.24 (initially pH 5) to pH 7.45 and 7.48 (initially pH 8) at 6 and 24 h. It is of interest to note that the initial pH 7 changed to 7.08 ( Table 4 ).

Table 4. Initial pH values and values (mean ± SD) in the surrounding media after 24 and 48 h of formation of the eight-species biofilm representing “periodontitis”

Discussion
In this in vitro study the influence of an initially defined pH on biofilm formation was investigated. The pH was adjusted on a scale from 5 to 8 by using two buffers. In each case the predefined conditions allowed biofilm formation and bacterial growth but to a differing extent. The follow-up time of the biofilm formation was limited and adjusted according to the clinical situation. In oral health only a thin, young biofilm exists, whereas subgingival biofilm contains several bacterial species that are present only at later stages. Static biofilms were cultivated, which might be a limitation of the study as a certain continuous flow and supply of nutrients and a removal of metabolic bacterial products occurs in vivo. Furthermore, the initial buffer system remained, and in vivo a wider range of buffering of the pH can be assumed. Saliva contains three major buffer systems (bicarbonate, phosphate, and protein); the bicarbonate buffer is most active at pH 6.25–7.25, whereas at a lower pH the protein buffer system contributes most to the buffer capacity [ 9 ]. In our study clearly defined laboratory strains were used, allowing a standardized comparison. However, in vivo oral biofilms consist of hundreds of species [ 10 ]. Both periodontopathogenic and cariogenic bacteria might be present in distinct oral environments in the same oral cavity [ 11 ], where an easy transfer can occur in the case of a changing micromilieu.
It is of interest to note that the initially adjusted pH gradient was only slightly weakened at the end of the experiments. Although a certain gradient remained, there were differences between the three biofilm models which underlines the influence of the included microorganisms on the pH levels. In the “healthy” and “caries” biofilm models, an initially low pH remained (increase from pH 5 to pH 5.15 and 5.10). In these models, an initially alkaline pH dropped, thus only the media initially adjusted to pH 8 were slightly above pH 7 (pH 7.05). In the “periodontitis” biofilm, an initial pH of 5 increased to 5.24, while the pH of the media initially adjusted to pH 7–8 were all above pH 7 at 48 h (with the highest being pH 7.48).
All biofilms at an initial pH of 5 and 5.5 resulted in the lowest quantities of bacteria, CFU counts, and metabolic activities. A low pH may prevent biofilms from growing but could cause other problems. This includes the dissolution of dental hard tissue without the involvement of microorganisms: dental erosion and the combination with mechanical wear. Erosive tooth wear is defined as a chemical-mechanical process which leads to a cumulative loss of hard dental tissue that it is not caused by bacteria [ 12 ]. A large study analyzing more than 3,000 young adults aged between 18 and 35 years found a prevalence of erosive tooth wear in 29.4%, which was clearly associated with a high consumption of fresh fruits and juice [ 13 ]. Beverages, in particular soft drinks, may have very low pH values (as low as pH 2.4), and the critical pH with respect to hydroxyapatite was calculated at between pH 4.9 and 6.5 for soft drinks [ 14 ]. However, it should be noted that although the pH level is the most important factor accounting for the erosive potential of a beverage, other factors, such as the addition of calcium, may counteract this and be protective [ 15 ].
Our “healthy” biofilm consisted only of the species S. gordonii and A. naeslundii . At an initial pH of 5, the percent of A. naeslundii increased in that biofilm. A. naeslundii seemed to be less sensitive to pH 5, but this was probably a short pH window that allowed multiplication of that species. At a lower pH (below pH 4) it was shown that A. naeslundii was killed, but demonstrated greater acid resistance when existing within a biofilm [ 16 ]. Interestingly, an initial pH of 5 or 5.5 also decreased the formation of the “cariogenic” biofilm. It can be assumed that biofilm formation begins at slightly higher pH values and the low pH leading to dissolution of enamel is a result of the bacterial metabolism in the deep layer of an advanced biofilm. This supports the different stages of the ecological caries theory [ 1 ]. In our study pH was only determined in the surrounding media, while differences within a biofilm are likely. pH values were measured both inside and outside of a single-species biofilm. With pH control (buffering of the media), the pH within the biofilm was about 6.1 and the pH in the surrounding media was 6.7 [ 17 ]. Thus, a “cariogenic” biofilm does not have a consistent pH. A recent study measuring pH values in different areas of an in vitro biofilm after sucrose-mediated biofilm formation found acidic regions (below pH 5.5) only in the interior of microcolonies, and in vivo analysis confirmed a spatial heterogeneity of pH, with acidic pH values only close to the enamel surface [ 18 ]. The metabolic activity of bacteria depends on the pH value. The expression of glucosyltransferases, which are major genes of S. mutans involved in biofilm formation, was demonstrated to be higher without pH control than in controlled conditions [ 17 ]. Also, the competence of S. mutans to internalize DNA from the environment depends on the environmental pH – it is only possible at a pH of around 7 [ 19 ]. The biofilm model focused only on enamel caries, and did not consider either root or dentine caries, which involve proteolytic bacteria that contribute to the proteolytic stage of the diseases [ 20 ].
A “periodontitis” biofilm is more metabolically active and has a higher quantity at slightly alkaline pH values. The pH of the gingival crevicular fluid increases in vivo from 6.9 to higher values with the severity of inflammation [ 21 ]. It was measured to be around pH 8.4 at inflamed sites and it was shown that the bicarbonate buffer system contributes most at that pH value [ 22 ]. Measuring the pH in periodontal pockets found levels below 7.0, with lower values in the case of acute inflammation, and alkaline values when the inflammation became chronic [ 23 ]. P. gingivalis growth is reduced at an acidic pH [ 24 ], which is in accordance with our results showing lower total counts and percentages in the in vitro biofilm assays. P. gingivalis is associated with periodontitis, whereas T. forsythia and T. denticola are present both in gingivitis and periodontitis [ 25 ]. In the present in vitro study, the “periodontitis” biofilm had a higher percentage of T. forsythia and T. denticola at lower pH values.


Fig. 4. Effect of different initial pH values on biofilm formation (quantity and metabolic activity), microbial composition, and the pH tendency in the environment of oral biofilms in general (grey), “caries” biofilms (blue), and “periodontitis” biofilms (purple), as well as possible associations with oral disease.
The dependency of biofilm formation on pH offers therapeutic options, and an increase of pH is of interest for preventing caries [ 26 ]. In recent years a major focus has been on arginine deiminase, an enzyme synthesized by several oral streptococci which utilizes arginine for alkali production [ 27 ]. Conversely, in therapy of periodontal disease a decrease of pH may be beneficial. In general, a relatively low pH seems to be favorable for wound healing by promoting an immune response [ 28 ]. However, this should be carefully balanced in periodontal therapy. In monkeys it was shown that short-term etching of root surfaces with a 37% orthophosphoric acid led to more connective tissue and a shorter epithelial junction, whereas a long-term etching for 3 min clearly impaired periodontal healing [ 29 ]. Also, the activity of antimicrobials can be affected by the environmental pH. For example, chlorhexidine is more active at an alkaline pH and hypochlorite at a more acidic pH [ 28 ]. Thus, investigating the influence of the pH value in periodontal therapy in more detail might be an interesting topic for further research.
Future in vitro research into the influence of pH values on oral biofilm formation should consider more complex models with more microorganisms and a different nutrient supply. More knowledge is needed about the influence of different pH values on the expression of important genes involved in biofilm formation and virulence.
In conclusion ( Fig. 4 ), an initially low pH (pH 5 and 5.5) may suppress biofilm formation and could be associated with the development of erosive tooth wear. A slightly higher pH (around pH 6) might favor the development of caries if respective nutrients (sugar) are available. A pH slightly less than 7 appears to be associated with a higher percentage of Tannerella sp. and Treponema sp., an acute gingivitis, and an increase in the percentage of aciduric bacteria. An initial slightly basic pH contributes to a biofilm associated with periodontitis, and in particular P. gingivalis . Therapeutics leading to a high pH may be helpful in the prevention of caries. In periodontitis, modulation of the pH could be an alternative option in therapy but should also include caries-preventive measures.
Acknowledgements
The authors acknowledge the technical support by Anna Magdoń and Prashanthnj Sivapatham, Department of Periodontology, Laboratory of Oral Microbiology, School of Dental Medicine, University of Bern.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
There was no funding in relation to this work.
Author Contributions
S.E. was responsible for the study design. L.B.S. and A.M. acquired and analyzed the data. S.E., A.S., and A.L. interpreted the data. S.E., L.B.S., and A.M. drafted the work. A.L. and A.S. revised it critically for important intellectual content. All authors approved to the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
References
1 Takahashi N, Nyvad B: Caries ecology revisited: microbial dynamics and the caries process. Caries Res 2008;42:409–418.
2 Nascimento MM, Browngardt C, Xiaohui X, Klepac-Ceraj V, Paster BJ, and Burne RA: The effect of arginine on oral biofilm communities. Mol Oral Microbiol 2014;29:45–54.
3 Darveau RP, Hajishengallis G, Curtis MA: Porphyromonas gingivalis as a potential community activist for disease. J Dent Res 2012;91:816–820.
4 Nascimento GG, Baelum V, Dahlen G, Lopez R: Methodological issues in assessing the association between periodontitis and caries among adolescents. Community Dent Oral Epidemiol 2018;46:303–309.
5 De Soete M, Dekeyser C, Pauwels M, Teughels W, van Steenberghe D, Quirynen M: Increase in cariogenic bacteria after initial periodontal therapy. J Dent Res 2005;84:48–53.
6 Eick S, Straube A, Guentsch A, Pfister W, Jentsch H: Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment. Diagn Microbiol Infect Dis 2011;69:12–20.
7 Pettit RK, Weber CA, Kean MJ, Hoffmann H, Pettit GR, Tan R, Franks KS, Horton ML: Microplate Alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother 2005;49:2612–2617.
8 Kwasny SM, Opperman TJ: Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery. Curr Protoc Pharmacol 2010;50:13A.8.1–23
9 Bardow A, Moe D, Nyvad B, Nauntofte B: The buffer capacity and buffer systems of human whole saliva measured without loss of CO 2 . Arch Oral Biol 2000;45:1–12.
10 Verma D, Garg PK, Dubey AK: Insights into the human oral microbiome. Arch Microbiol 2018;200:525–540.
11 Van der Reijden WA, Dellemijn-Kippuw N, Stijne-van Nes AM, de Soet JJ, van Winkelhoff AJ: Mutans streptococci in subgingival plaque of treated and untreated patients with periodontitis. J Clin Periodontol 2001;28:686–691.
12 Carvalho TS, Colon P, Ganss C, Huysmans MC, Lussi A, Schlueter N, Schmalz G, Shellis RP, Tveit AB, Wiegand A: Consensus report of the European Federation of Conservative Dentistry: erosive tooth wear – diagnosis and management. Clin Oral Investig 2015;19:1557–1561.
13 Bartlett DW, Lussi A, West NX, Bou­chard P, Sanz M, Bourgeois D: Prevalence of tooth wear on buccal and lingual surfaces and possible risk factors in young European adults. J Dent 2013;41:1007–1013.
14 Lussi A, Carvalho TS: Erosive tooth wear: a multifactorial condition of growing concern and increasing knowledge. Monogr Oral Sci 2014;25:1–15.
15 Barbour ME, Lussi A: Erosion in relation to nutrition and the environment. Monogr Oral Sci 2014;25:143–154.
16 Burne RA, Marquis RE: Biofilm acid/base physiology and gene expression in oral bacteria. Methods Enzymol 2001;337:403–415.
17 Li Y, Burne RA: Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology 2001;147:2841–2848.
18 Xiao J, Hara AT, Kim D, Zero DT, Koo H, Hwang G: Biofilm three-dimensional architecture influences in situ pH distribution pattern on the human enamel surface. Int J Oral Sci 2017;9:74–79.
19 Son M, Ghoreishi D, Ahn SJ, Burne RA, Hagen SJ: Sharply tuned pH response of genetic competence regulation in Streptococcus mutans : a microfluidic study of the environmental sensitivity of comX. Appl Environ Microbiol 2015;81:5622–5631.
20 Takahashi N, Nyvad B: Ecological hypothesis of dentin and root caries. Caries Res 2016;50:422–431.
21 Bickel M, Cimasoni G: The pH of human crevicular fluid measured by a new microanalytical technique. J Periodontal Res 1985;20:35–40.
22 Bickel M, Munoz JL, Giovannini P: Acid-base properties of human gingival crevicular fluid. J Dent Res 1985;64:1218–1220.
23 Watanabe T, Soeda W, Kobayashi K, Nagao M: The pH value changes in the periodontal pockets. Bull Tokyo Med Dent Univ 1996;43:67–73.
24 Xu X, Tong T, Yang X, Pan Y, Lin L, Li C: Differences in survival, virulence and biofilm formation between sialidase-deficient and W83 wild-type Porphyromonas gingivalis strains under stressful environmental conditions. BMC Microbiol 2017;17:178.
25 Scapoli L, Girardi A, Palmieri A, Martinelli M, Cura F, Lauritano D, Carinci F: Quantitative analysis of periodontal pathogens in periodontitis and gingivitis. J Biol Regul Homeost Agents 2015;29:101–110.
26 Bradshaw DJ, Lynch RJ: Diet and the microbial aetiology of dental caries: new paradigms. Int Dent J 2013;63(Suppl 2):64–72.
27 Liu YL, Nascimento M, Burne RA: Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J Oral Sci 2012;4:135–140.
28 Percival SL, McCarty S, Hunt JA, Woods EJ: The effects of pH on wound healing, biofilms, and antimicrobial efficacy. Wound Repair Regen 2014;22:174–186.
29 Blomlof J, Jansson L, Blomlof L, Lindskog S: Long-time etching at low pH jeopardizes periodontal healing. J Clin Periodontol 1995;22:459–463.
Sigrun Eick University of Bern Department of Periodontology Freiburgstrasse 7 CH -3010 Bern (Switzerland) sigrun.eick@zmk.unibe.ch
Biofilm Models
Published online: December 21, 2020
Eick S (ed): Oral Biofilms. Monogr Oral Sci. Basel, Karger, 2021, vol 29, pp 30–37 (DOI: 10.1159/000510197)
______________________
Biofilm Models to Study the Etiology and Pathogenesis of Oral Diseases
Thomas Thurnheer Pune Nina Paqué
Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland
______________________
Abstract
More than 700 microbial species inhabit the complex environment of the oral cavity. For years microorganisms have been studied in pure cultures, a highly artificial situation because microorganisms in natural habitats grow as complex ecologies, termed biofilms. These resemble multicellular organisms and are characterized by their overall metabolic activity upon multiple cellular interactions. Microorganisms in biofilms express different genes than their planktonic counterparts, resulting in higher resistance to antimicrobials, different nutritional requirements, or creation of a low redox potential allowing the growth of strictly anaerobic bacteria in the presence of oxygen. Multiple in vitro biofilm models have been described in the literature so far. The main emphasis here will be on multispecies biofilm batch culture models developed in Zurich. The standard 6-species supragingival biofilm model has been used to study basic aspects of oral biofilms such as structure, social behavior, and spatial distribution of microorganisms, or diffusion properties. Numerous parameters related to the inhibition of dental plaque were tested illustrating the high reliability of the model to predict the in vivo efficiency of antimicrobials. Modifications and advancements led to a 10-species subgingival model often combined with human gingival epithelial cells, as an integral part of the oral innate immune system, eliciting various cell responses ranging from cytokine production to apoptosis. In conclusion, biofilm models enable a multitude of questions to be addressed that cannot be studied with planktonic monocultures. The Zurich in vitro biofilm models are reproducible and reliable and may be used for basic studies, but also for application-oriented questions that could not be addressed using culture techniques. Oral biofilm research will certainly lead to a more realistic assessment of the role of microorganisms in the oral cavity in health and disease. In this respect, substantial progress has been made, but there is still more to explore.
© 2021 S. Karger AG, Basel
The oral cavity is a complex environment and home to more than 700 microbial species [ 1 ]. For many years, the oral ecosystem was studied using planktonically growing organisms in order to investigate and understand all the different components of this ecosystem. Although Zobell [ 2 ] reported in 1936 that microorganism

  • Accueil Accueil
  • Univers Univers
  • Ebooks Ebooks
  • Livres audio Livres audio
  • Presse Presse
  • BD BD
  • Documents Documents