BIOFILM

Aruna Rani
II-year PG
CONTENTS

PART I
• Introduction
• History
• Definitions
• Basic criteria for a biofilm
• Composition of biofilm
• Development of biofilm
• Characteristics of biofilm
• Classification of biofilm
• References
PART II

• Endodontic biofilm
• Detection and measurement of biofilms
• Strategies to manage biofilms
• Conclusion
• References
 INTRODUCTION

Microbes were Led to evolution of

struggling to survive, The oldest story post biofilm
grow and adapt of planktonic phenotype
“unity is strength”
taught
Moved to by the most Microbes formed
multispecies minute communities within
adaptation creatures biofilms to transfer to
different environments
on Earth

SINGLE START TO MINGLE TO FORM BETTER SINGLE

 HISTORY
Van Leeuwenhoek (1663)-discovery of microbial biofilms.
microscopes- first observed microorganisms on tooth
surfaces

Heukelekian and Heller (1940) - observed the “bottle effect”

for marine microrganisms.

Zobell (1950) - observed number of bacteria on surface

was higher than in surrounding medium (ex:seawater)

Jones et al. (1970) - used scanning & transmission

electron microscopy to examine biofilms
 Miller (1894) - observed many different microorganisms in the infected pulp
space and that the flora was different in the coronal, middle, and apical parts of
the canal system.

 Characklis (1973) - studied microbial slimes in industrial water systems and

showed - highly resistant to disinfectants such as chlorine.

 Costerton et al. (1978) - theory of biofilms that explained the mechanisms

whereby microorganisms adhere to living and non-living materials and the
benefits accrued by this ecologic niche.

 The first two medical reports using the word ‘biofilm’ were published in
1981 by dentists from the University of Lund, Sweden.

Hoiby N. A short history of microbial biofilms and biofilm Infections. APMIS 2017; 125: 272–
 DEFINITION OF BIOFILM
 Biofilm is defined as a community of microcolonies of microorganism in an
aqueous solution that is surrounded by a matrix of glycocalyx, which also
attaches the bacterial cells to a solid substratum - Grossman

 Biofilm is a mode of microbial growth where dynamic communities of

interacting sessile cells are irreversibly attached to a solid substratum, as well
as each other, and are embedded in a self-made matrix of extracellular
polymeric substance (EPS) - Ingle

 Biofilm is a microbially derived sessile community characterized by cells that

are irreversibly attached to a substratum or interface or to each other,
embedded in a matrix of extracellular polymeric substances that they have
produced, and exhibit an altered phenotype with respect to growth rate and
gene transcription - Donlan and Costerton
 BASIC CRITERIA FOR A BIOFILM

Biofilm is considered as community as it possesses following criteria: (Caldwell)

 
• Autopoiesis - Must possess the ability to self organize.
 
• Homeostasis - Should resist environmental perturbations
 
• Synergy - Must be more effective in association than in isolation
 
• Communality - Should respond to environmental changes as a unit rather
than as single individuals.

Ingle’s Endodontics : 6th Edition

 COMPOSITION OF BIOFILM

 Composed primarily of microbial cells and glycocalyx like matrix

(Extracellular polymeric substance)

 Basic structural unit of a biofilm is the micro colonies or cell cluster formed by
the surface adherent bacterial cells.

 85% : - Matrix (polysaccharides, proteins, nucleic acid & salts)

 15% : - Bacterial cells Ingle’s Endodontics : 6th Edition
  Viable, fully hydrated biofilm appears as ‘tower’ or ‘mushroom’ shaped structure adherent
to the substrate .

 Water channels : regarded as circulatory system in a biofilm : establish connection between

micro colonies.

- Facilitates efficient exchange of materials

between bacterial cells and fluid

- Coordinate functions in a biofilm community

Matrix: (House of biofilm cells)
 A glycocalyx matrix made up of extracellular polymeric substance (EPS)
surrounds the micro colonies and anchors the bacterial cell to the substrate.

EPS defined as “organic polymers of microbial origin which in biofilm systems

are frequently responsible for binding cells and other particulate materials
together (cohesion) and to the substratum (adhesion)”.

 EPS synthesized extracellularly or intracellularly and secreted into the outside

environment by the organisms 

 The EPS matrix is typically composed of polysaccharides, proteins, lipids, and

extracellular DNA (eDNA).
Hans-Curt Flemming, EPS-Then and Now, Microorganisms 2016, 4, 41
Hans-Curt Flemming, EPS-Then and Now, Microorganisms 2016, 4, 41
 Microbial cells:
1. Early colonizers - Actinomyces species and oral streptococci 
- These bacteria not only adhere to tooth surface but also interact
with each other and lay foundation for attachment of bridging
colonizer.

2. Secondary colonizers - Prevotella intermedia, P. loescheii,

Capnocytophaga spp and Fusobacterium nucleatum
Adhere to bacteria already present in the biofilm matrix.  

3. Late colonizers - Porphyromonas gingivalis,, Tannerella

forsythia and Treponema denticola
Aruni et al, The Biofilm Community-Rebels with a Cause, Curr Oral Health Rep. 2015 Mar 1; 2(1): 48–56. 
 DEVELOPMENT OF BIOFILM

• Bacteria can form biofilm on

any surface that is bathed in a
nutrition containing fluid.

• 3 major components involved

in biofilm formation
• Bacterial cells
• A solid surface
• A fluid medium

Ingle’s Endodontics : 6th Edition

STAGE 1

• Adsorption of inorganic and organic molecules to the solid surface, forming a thin layer
termed as conditioning layer.

• During dental plaque formation, the tooth surface is conditioned by the saliva pellicle.
STAGE 2

• Adhesion of microbial cells to the conditioning layer.

• Phase 1 - Transport of microbes to surface.
• Phase 2 - Initial non specific microbial-substrate adherence phase.
• Phase 3 - Specific microbial-substrate adherence phase.

• Pioneer organisms : Early colonizers - streptococci (major population)

STAGE 2 : Phase 1 : Transport of microbes to surface

• Physicochemical properties such as surface energy and charge density

determine the nature of initial bacteria-substrate interaction

• Microbial adherence to a substrate is mediated by bacterial surface

structures such as fimbriae, pili, flagella, and EPS (glycocalyx).

• Bacterial surface structures form bridges between the bacteria and the
conditioning film.
STAGE 2 : Phase 1 : Transport of microbes to surface

• Several factors that affect bacterial attachment to a solid substrate.

• pH
• Temperature
• Surface energy of the substrate
• Flow rate of the fluid passing over the surface
• Nutrient availability
• Length of time the bacteria is in contact with the surface
• Bacterial growth stage
• Bacterial cell surface charge
• Surface hydrophobicity
STAGE 2 : Phase 2 : Initial non specific microbial-substrate
adherence phase
• Bridges formed are a combination of
• Electrostatic attraction
• Covalent and hydrogen bonding
• Dipole interaction
• Hydrophobic interaction

• Bacteria with surface structures:

• P. gingivalis, S. mitis, Streptococcus salivarius, P.
intermedia, P. nigrescens, S. mutans and A. naeslundii
STAGE 2 : Phase 3 : Specific microbial-substrate adherence
phase
• Initial bonds : not strong
• With time these bonds gain in strength,
making the bacteria-substrate attachment
irreversible.
• A specific bacterial adhesion with a substrate is adhesin or ligand formation

produced via polysaccharide adhesin or ligand

formation - bind to receptors on the substrate.
• Specific bacterial adhesion is less affected by
many environmental factors such as
electrolyte, pH, or temperature.
STAGE 3

• Bacterial growth and expansion.

• Monolayer of microbes attracts secondary colonizers forming microcolony,
and the collection of microcolonies gives rise to the final structure of
biofilm.
• The lateral and vertical growth of indwellers gives rise to microcolonies
similar to towers.
• Two types of microbial interactions occur at
the cellular level

• Co-adhesion
• The process of recognition between
a suspended cell and a cell already
attached to substratum.

• Co-Aggregation
• Genetically distinct cells in suspension
recognize each other and clump together.
STAGE 4 : Detachment / Dispersion

• During detachment, the biofilm transfer particulate constituents from the

biofilm to the fluid bathing the biofilm.

• Detachment plays an important role in shaping the morphological

characteristics and structure of mature biofilm.
STAGE 4 : Detachment / Dispersion
• Because of flow effects, biofilm cells may be dispersed either by

 Shedding of daughter cells from actively growing cells

 Detachment as a result of nutrient levels
 Quorum sensing
 Shearing of biofilm aggregates
STAGE 4 : Detachment / Dispersion

• Brading et al have emphasized the importance of physical forces in detachment, stating

that the three main processes for detachment are

• Erosion or shearing (continuous removal of small portions of the

biofilm)
• Sloughing (rapid and massive removal), and
• Abrasion (detachment due to collision of particles from the bulk fluid
with the biofilm)
CHARACTERISTICS OF BIOFILM

 Protection of biofilm bacteria from environmental threats

 Nutrient Trapping And Metabolic Cooperativity In A Biofilm

 Biofilm structure displays organized internal

compartmentalization.
 Quorum Sensing

Ingle’s Endodontics : 6th Edition

Protection of Biofilm Bacteria from Environmental Threats

• Bacteria residing in a biofilm experiences certain degree of protection and homeostasis.

• Bacteria can produce polysaccharides, either as cell surface structures (eg. Capsules)
or as extracellular excretions (eg. EPS).

• EPS creates microniche.

• Protects from environmental stresses - UV radiation, pH shift, Osmotic shock
& desiccation.
• Sequestration of metallic cations and toxins.
• Physically prevents diffusion of certain compounds by acting as ion
exchanger.
Nutrient Trapping And Metabolic Cooperativity In A Biofilm

• An important characteristics of biofilm growing in a nutrient deprived

ecosystem is its ability to concentrate trace elements and nutrients by
physical trapping or by electrostatic interaction.

• Highly permeable and interconnected water channels provide an excellent

means of material exchange.
Nutrient Trapping And Metabolic Cooperativity In A Biofilm

• Juxta positioning of various microorganism provides cross feeding and metabolic

cooperativity between different species.

• Production of growth factors

• Production of different arrays of lytic enzymes

 Studies have reported the production of essential growth factors such as hemin by
W. recta to support the growth of fastidious organisms such as P. gingivalis

 Bacterial species possessing proteolytic enzymes make nutrients available to all

other bacteria in a protein-rich environment
Internal Compartmentalization
• Environmental niches that supports the physiological requirementof different
bacterial species in a biofilm.

• A mature biofilm structure displays gradients in the distribution of nutrition, pH, oxygen,
metabolic products and signaling molecules within the biofilm.

• This would create different microniches that can accommodate diverse bacterial species
within a biofilm.

• In a multispecies biofilm involving aerobic and anaerobic bacteria, oxygen is consumed

by the aerobic and facultative anaerobic species, making the environment rich in
carbon dioxide and other gases.
 Quorum sensing: Bacterial talk in biofilms
 Communications between bacterial cells residing in a biofilm is attained
through signaling molecules by a process called as quorum sensing

 Quorum sensing is mediated by low molecular weight molecules-

autoinducers

 Qs leads to
• Exchange of genetic materials between species
antibiotic resistance
• Nutrient breakdown
• Xenobiotic metabolism
• Coordinated behaviour of biofilm
 MOLECULES USED AS SIGNALS
• In gram positive bacteria- oligopeptides
• In gram negative bacteria- Acyl Homoserine lactones -AHL
• Both types of bacteria- AI-2
• In combination of bacteria and fungi- 3-oxo-C12 homoserine lactone

 These biochemicals diffuse through water channels seen in matrix.

 Regulate the transcription of various genes as per the requirement of the

community to produce various proteins, enzymes, etc.

 Serves as a major means of communication that allows the sessile organisms to

survive and grow in the presence of unsuitable environmental conditions and
antibiotics or detergents.
Resistance of Microbes in the Biofilm to Antimicrobials

• The nature of biofilm structure and physiological characteristics of resident

microorganisms offer an inherent resistance to antimicrobial agents, such as antibiotics,
disinfectants, or germicides.

• Mechanism responsible for resistance:

1. Resistance associated with extracellular polymeric matrix.
2. Resistance associated with growth rate and nutrient availability.
3. Resistance associated with adoption of resistance phenotype.

Ingle’s Endodontics : 6th Edition

Schematic diagram showing different methods by which bacteria in a biofilm gain resistance to antimicrobials (AM).

A. Kishen. Advanced Therapeutic Option for Endodontic biofilms. Endodontic Topics 2012, 22, 99–123.
Resistance associated with EPS :

• Diffusion barrier
• Direct neutralization of antimicrobials
• Inactivation by modified enzymes produced by bacteria (Beta-
lactamase enzymes , formaldehyde lyase and dehydrogenase)

 EPS with its highly charged and interwoven structure deters penetration of
antimicrobials by ionic or electrostatic interactions.
 Because the antimicrobial agents are usually positively charged and the EPS contains
negatively charged or neutral polysaccharides.
 Antibiotics such as aminoglycosides, which are hydrophilic and positively charged
molecules, are retarded by the biofilm matrix for the above reason.
Resistance associated with growth rate and nutrient availability

• Susceptibility towards antimicrobial is directly proportional to

growth rate.
• Resistance increases as thickness of biofilm increases
starved bacteria entering the stationary phase in biofilms

• Most antibiotics require at least some degree of cellular activity to

be effective.
• Therefore, bacterial cells in stationary phase might represent a
general mechanism of antibiotic resistance in the biofilm.
Benefits Of Biofilm To Microbes

• Helps the bacteria to survive in unfavorable environmental and nutritional conditions.

• Resistance to antimicrobial agents.

• Increase in local concentration of nutrients.

• Opportunity of genetic material exchange.

• Ability to communicate between bacterial population of same and or different species.

• Produce growth factors across species boundaries.

CLASSIFICATION OF BIOFILM

1. Based on habitats

 Percolating filters
 Rhizosphere
 Mammalian gut
 Human environment
 Ship fouling
 Dental plaque
2. Taxonomic diversity
 

• Gram positive species

• Gram negative species
• Bacteria
• Archaea
• Fungi
• Algae
• Multispecies
3. Based on field
• In medicine – infectious diseases, dentistry
• In industry
• Food industry
• In aquaculture

 
Dental biofilm

 The formation of oral cavity microbiota begins at the moment of birth through
the contact between the surface of the new born’s skin and mucous membrane
of its mouth with mother’s vaginal microbiota.
Classification of dental biofilm

1. On basis of location
 Teeth - Dental plaque
 Mucosa
 Tongue
 Gums
 Dental restorations
 Dental operatories

2. On basis of its location in teeth

 Supragingival - Present coronal to the gingival margin – coronal and marginal

 Subgingival - Present apical to the gingival margin – attached (tooth associated/tissue

associated) and unattached
3. On basis of pathogenicity
• Cariogenic - Generally acidogenic and gram-positive
• Perio-pathogenic - Mostly basophilic and gram-negative

4. On basis of Endodontic bacterial biofilms

• Intracanal biofilms
• Extra-radicular biofilms
• Periapical biofilms
• Biomaterial‑centred infections.
Teeth - Dental plaque

 One of the best known and most reported type of biofilm in the human
body
 More than 1000 different species of bacteria forming plaque have been
identified so far
 The studies conducted using molecular biology methods demonstrated five
dominant phylum of bacteria in dental plaque: Firmicutes, Actinobacteria,
Bacteroides, Fusobacteria, and Proteobacteria. They constitute 80–95% of
the oral cavity microflora.

Carranza’s Clinical Periodontology, 11th edition

• Oral bacteria have the capacity to form biofilms on distinct surfaces ranging from hard
to soft tissues.

• Oral biofilms are formed in three basic steps :

• Pellicle formation
• Bacterial colonization
• Biofilm maturation

• Composition :
• 80% : Water
• 20% : Inorganic & organic (80% bacteria)

Carranza’s Clinical Periodontology, 11th edition

• Organic : Carbohydrates, proteins, and lipids.

• Carbohydrates : produced by bacteria : glucans, fructans, or levans.

• Contribute to the adherence of microorganisms to each other and are the

stored form of energy in biofilm bacteria.
• Proteins (supragingival biofilm) : derived from saliva

• Proteins (subgingival biofilm) : derived from gingival sulcular fluid.

• Lipid : Endotoxins (LPS) from Gram-negative bacteria.

• Inorganic : Calcium, phosphorus, magnesium and fluoride.

• Saliva contains proline-rich proteins : aggregate together to form micelle
like globules called salivary micelle-like globules (SMGs).

• SMGs : adsorbed to the clean tooth surface to form acquired enamel

pellicle, which acts as a ‘‘foundation’’ for the future multilayered biofilm.

• Presence of calcium facilitates the formation of larger globules by bridging.

• Acquired Pellicle attracts Gram-positive cocci (S. mutans and S. sanguis :
pioneer organisms)

• Filamentous bacterium (F. nucleatum and slender rods) adheres to primary

colonizers.

• Gradually, the filamentous form grows int the cocci layer and replaces many
of the cocci.

• Vibrios and spirochetes appear as the biofilm thickens.

• Coaggregation of F. nucleatum with coccoid bacteria gives rise to “CORNCOB” structure,
which is unique in plaque biofilms.

• Presence of these bacteria makes it possible for other non-

aggregating bacteria to coexist in the biofilm, by acting as
coaggregating bridges.

• The existence of anaerobic bacteria in an aerobic environment is

made possible by the coexistence of aerobic and anaerobic
bacteria.
 • A decline in the host defense mechanisms caused by disease or immuno-suppressive
medications may render generally ‘‘harmless commensals’’ to become ‘‘opportunistic
pathogens.’’

• Ex. diseases caused by biofilm community:

• Dental caries
• Gingivitis
• Periodontitis
• Peri-implantitis
• Apical periodontitis

Carranza’s Clinical Periodontology, 11th edition

Carranza’s Clinical Periodontology, 11th edition
Oral mucosal biofilm

 Oral mucosal Candida infections are prevalent among individuals with

weakened, suppressed, or underdeveloped immune systems.

 Approximately 5% of newborns, 10% of elderly patients and almost

90% of HIV infected individuals develop oral pseudo membranous
candidiasis.

Ingle’s Endodontics : 6th Edition

Tongue biofilm: tongue coating

 The dorsal surface of the tongue colonized by large amounts of bacteria in the
presence of fissures, crypts and high mucosal papillae.

 These anatomical niches may create an environment where microorganisms are

well-protected from the flushing action of the saliva and where oxygen levels
are low, thus promoting the development of anaerobic microbiota

 Mucous membranes, tonsils and the tongue can frequently harbour different
periodontal pathogens such as Actinomycetem comitans, P. gingivalis,
or P. intermedia

Ingle’s Endodontics : 6th Edition

Dental restorations

 Dental restorations affect the composition of the biofilm in many

ways.
 There will be steps, gaps or groves between tooth and restoration.
 These will complicate mechanical biofilm removal and alter the
chemical balance in the biofilm in the region.
 Restorations differ from enamel regarding surface roughness,
surface energy and chemical composition.

Ingle’s Endodontics : 6th Edition

Factors related to restorations associated biofilms

1. Surface Energy
 Surfaces with a low surface energy usually display lower adherence to biofilms
than similar surfaces with higher surface energy.
 Most dental materials, except for ceramics, have a higher surface energy than
enamel and have thus a greater risk of biofilm accumulation.

2. Surface Roughness
 Increased surface roughness and complicated topography shows higher affinity
to microbes than smoother surfaces and subsequently increased difficulty in
complete removal of the biofilm by mechanical brushing.

Ingle’s Endodontics : 6th Edition

3. Chemical Composition
 The chemical composition of the dental material will affect the bacterial
adhesion since both proteins and microorganisms can chemically attach or attract
to components in the material, by van der Waal forces, acid-base reactions or
electrostatic interactions
 
4. Type of Dental Restorations
 Ceramic materials have a smooth, polished surface which is easily cleaned. This
helps in daily removal of the biofilm.
 Biofilms on gold restorations - have low viability.
 Some microbes are affected by the eluates from the metals. The mercury
leaching in very small amounts from fresh amalgam restorations has
bacteriostatic effect.
Dental operatories

 Biofilm formation in aqueous environments in a dental setting, such as in pipes,

tubing, tanks, wash basins and sinks, often begins with the formation of a
conditioning pellicle of inorganic compounds from the environment.

 Wet or humid parts of medical devices are favourable to the establishment and
growth of microbial biofilms and have been particularly associated with cross-
infection and cross-contamination ( Lemaitre et al, 1996 ; Hsueh et al, 1998 ;
Perola et al, 2002 ).
DUWLs and associated biofilms

 Numerous studies - untreated DUWLs harbour resident microbial biofilms that

result in the presence of high densities of microorganisms, in DUWL output
water (reviewed by Coleman et al, 2009).

 Dental handpieces and ultrasonic scalers generate aerosols and fine droplets
containing planktonic microorganisms, their endotoxins and pieces of biofilm
can be inhaled into the lungs both of patients and dental healthcare staff (Fotos
et al, 1985 ; Reinthaler et al, 1988 ; Atlas et al, 1995 ; Putnins et al, 2001 ;
Pankhurst et al, 2005 ; Dutil et al, 2007 ).
ENDODONTIC BIOFILM

• Less diverse compared to the oral microbiota

• Progression of infection alters the nutritional and environmental status within the root
canal.
• Root canal environment : more anaerobic and depleted nutritional levels
• Tough ecological niche for the surviving microorganisms.

• Microbes : anatomical complexities (isthmuses and deltas and in the apical portion of
RCS) shelter the adhering bacteria from cleaning and shaping procedures.

• Bacterial activities not confined to intracanal spaces, but also beyond the apical
foramen.
Ingle’s Endodontics : 6th Edition
• Endodontic bacterial biofilms can be categorized as

1. Intracanal biofilms
2. Extra radicular biofilms
3. Periapical biofilms
4. Biomaterial centered infections.

Ingle’s Endodontics : 6th Edition

INTRACANAL BIOFILMS
• Microbial biofilms formed on the root canal dentine of an endodontically
infected tooth.

• First documented by Nair et al in 1987

• Biofilm : Cocci, rods, and filamentous bacteria.

• Monolayer and/or multi-layered bacterial biofilms

were found to adhere to the dentinal wall of the root
canal.
 Nutrient-deprived Nutrient-deprived
condition after 1 week condition after 4 week

Nutrient-rich Nutrient-rich
condition after 1 week condition after 4 week
• Stage I : Adherence and micro colonies
• Stage II : Dissolution of dentin and
• Stage III : Mineralization of biofilm
- Carbonated apatite structure

• Obvious signs of dentine surface degradation under

nutrient - deprived environment
• Interaction of bacteria and their metabolic products on
dentine.
EXTRA-RADICULAR BIOFILM
• Biofilms formed on the root (cementum) surface adjacent to the root apex
of endodontically infected teeth.

• Reported in
• Asymptomatic periapical periodontitis
• Chronic apical abscesses associated with sinus tracts

• F.nucleatum, P.gingivalis, T.forsythenis

• Calcified biofilms was also reported by Riccuci et al.

PERIAPICAL MICROBIAL BIOFILMS

• Isolated biofilms found in the periapical region of an endodontically infected

teeth.

• May or may not be dependent on the root canal.

• Actinomyces and P. propionicum : Asymptomatic

periapical lesions
BIOMATERIAL-CENTERED INFECTION (BCI)

• Caused when bacteria adheres to an artificial

biomaterial surfaceand forms biofilm structures.

• BCI is one of the major complications associated with prosthesis and/or

implant-related infections.
BCI IN ENDODONTICS
• BC biofilms : formed on root canal obturating materials.

• Intraradicular

• Extra radicular

- depending upon whether the obturating material is within the

root canal space or has it extruded beyond the root apex.

• Filaments, long rods and spirochete-shaped bacteria were predominant

in the biofilm formed on guttapercha.
 BCI IN ENDODONTICS

• A study investigated the initial biofilm-forming ability of root canal isolates on gutta-percha points
in vitro.

• E. faecalis and S. sanguinis biofilms were significantly thicker than others.

• P. gingivalis, and P. intermedia did not form biofilms on gutta-percha.

• Suggests that Gram-positive facultative anaerobes have the

ability to colonize and form extracellular matrices on gutta-
percha points
• Serum plays a crucial role in biofilm formation.
- (Takemura et al. Eur J Oral Sci 2004)
Filamentous or spirochete shaped
bacteria on surface of GuttaPercha
Swimberghe et al. ,Biofilm model systems in
endodontology, International Endodontic Journal,
52, 604–628, 2019
Planktonic vs Biofilm
REFERENCES

• Ingle’s Endodontics : 6th Edition

• Endodontic Practice : Grossman : 13th Edition
• Pathways of the Pulp : Cohen : 11th Edition
• Endodontic Microbiology : Fauad : 2nd Edition
• Carranza’s Clinical Periodontology, 11th edition
• Hans-Curt Flemming, EPS-Then and Now, Microorganisms 2016, 4, 41
• Swimberghe et al. ,Biofilm model systems in endodontology, International Endodontic
Journal, 52, 604–628, 2019
• A. Kishen. Advanced Therapeutic Option for Endodontic biofilms. Endodontic Topics
2012, 22, 99–123.