STUDY OF

ORAL BIOFILMS

MEDICAL DEPARTMENT DENTAID

DEFINITION

Bacteria in the oral cavity can be found in saliva or adhered to oral

surfaces (mucosa, tongue, teeth)

Oral biofilms: groups of bacteria embedded in matrices that attach to the

surface.

Composition: 15-20% microbial population

80-85% matrix containing extracellular polysaccharides,

proteins, mineral salts and cellular material

Location on teeth: supragingival

subgingival

interproximal

PATHOGENICITY

BIOFILMS

Supragingival and

Inteproximal

Cariogenic bacteria

Destruction of tooth

enamel

Subgingival

Inflammation and

destruction of the

periodontium

Periodontopathogenic

bacteria

CARIES GINGIVITIS PERIODONTITIS

Aggregatibacter actinomycetemcomitans

Porphyromonas gingivalis

Prevotella intermedia

Fusobacterium nucleatum

Tannerella forsythia

Campylobacter rectus

Eikenella corrodens

Parvimonas micra

Selenomonas spp

Eubacterium spp

Treponema spp

700 bacterial species are present in oral biofilm

BACTERIAL DIVERSITY IN THE ORAL CAVITY

BIOFILM FORMATION

Formation of acquired pellicle

with salivary proteins on the

enamel

1

Adhesion of primary colonisers:

gram-positive bacilli and cocci (S.

sanguis, S. oralis,…) and growth

2

Fusobacterium nucleatum

joins in: facilitating adhesion

by other bacteria (gram-

negative, anaerobes…)

3

4 Tertiary colonisers. Increased

complexity: gram-negative, strict

anaerobes

4 μm4 μm

BIOFILM PROPERTIES

When bacteria grow in the form of a biofilm, they work together as a

bacterial community, which gives them the following properties:

Physiological heterogeneity

Increased phenotypic resistance

Quorum sensing (interbacterial communication)

Adaptive capacity

Resistance to antimicrobial agents

IMPORTANCE OF THE STUDY OF BIOFILMS

Isolated bacteria (planktonic bacteria) behave differently and

have different properties than bacteria that are organised in

biofilms.

These different properties give them increased resistance to

antiseptics and greater pathogenicity.

It is necessary to study the behaviour of bacteria in biofilms

to therefore prevent the oral diseases they may cause.

In vitro Biofilm Development

Steady-state growth Growth under flow conditions

Mature biofilm

Culture plate Molecular techniques Microscopy techniques

PCR

PCR-RT

PCR-RT-PMA

Scanning electron microscopy

(SEM)

Confocal laser scanning

microscopy (CLSM)

AssaysARTIFICIAL MOUTH

IN VITRO BIOFILM DEVELOPMENT

Microtiter plate

Steady state growth

This is performed on microtiter plates. Oral inocula are used

(from saliva or subginvigal plaque) or a selection of supra and/or

subgingival bacterial.

The idea is to simulate the physiological conditions of the mouth

environment (pH, temp, anaerobiosis).

The mouth’s characteristic dynamism cannot be recreated:

saliva flow and crevicular flow to which bacteria are subjected.

This dynamism determines the structural properties of the

biofilm.

Studies show that a biofilm’s structure and properties vary

depending on whether growth is under steady state or flow

conditions.

Growth under flow conditions

- Study model for in vitro formation of multispecies oral biofilms under conditions similar to

reality, thanks to the flow system

- Similar growth to what we would observe in the oral cavity

ARTIFICIAL MOUTH

AFTER 4 DAYS

INCUBATION

to obtain a mature

oral biofilm

Bioreactor

IN VITRO BIOFILM DEVELOPMENT

Culture medium

(Food source)

6 bacterial

species

that tipically form a

subgingival biofilm

Hydroxyapatite discs

In conditions that simulate

real life:

Flow system,37ºC,anaerobiosis

ASSAYS

Different assays are performed on hydroxyapatite discs

containing the mature biofilms, in order to assess their

behaviour.

These assays mainly involve the use of antiseptics and

are aimed at evaluating their efficacy and/or biofilm

resistance.

Assays are also performed on the extracellular matrix

(80% of the biofilm), which has a great impact on the

biofilm properties such as resistance to antiseptics.

CULTURES

Anaerobic chamber

Culture plate

- The mature treated biofilm is plated on the culture media appropriate for its growth.

- Depending on the metabolic characteristics of the species, different culture media are used.

- Then, the microbial population that has grown is assessed (quantitatively and qualitatively).

MOLECULAR TECHNIQUES

PCR (polymerase chain reaction)

These are DNA extraction techniques. They make it possible

to amplify the bacterial DNA of a sample:

- PCR: Qualitative. Detects DNA through gel

eletrophoresis signalling

- PCR-RT (real time PCR): quantifies the product of this

amplification as it is synthesised with fluorochromes

- PCR-RT-PMA (PCR-RT with propidium monoazide).

When PMA is added, DNA from live and dead bacteria

can be distinguished to thereby assess antiseptic

efficacy

MICROSCOPY TECHNIQUES

Confocal Laser Scanning Microscopy (CLSM)

• Increased resolution

• Ability to penetrate biofilm

• Imaging with extremely thin optical sections.

• Single focal plane can be achieved .

• Live and dead bacterial populations can be identified and quantified:

ASSESSMENT OF ANTISEPTIC EFFICACY

Confocal Laser Scanning Microscopy images

CHX 0,12% + CPC 0,05%

PERIO·AIDNegative control

Confocal laser microscopy images of an oral biofilm developed in vitro. Syto9 marked microorganisms (live) are shown in green

and propidium iodide marked microorganisms (dead) are shown in red.

The image on the left is a negative control and the one on the right is the same bacterial population after treatment with 0.12%

Chlorhexidine + 0.05% CPC (Perio·Aid Tratamiento)

EVALUATING THE RESULTS–

MICROSCOPY TECHNIQUES

Scanning Electron Microscopy (SEM)

• Increased resolution to more than100,000 times.

• Electron beam (instead of light beam) that “sweeps the sample”, point by point.

• Realistic three-dimensional images on the surface of an object

• Large depth of field

• Obtaining images of the biofilm structure (bacterial morphology, interconnections, matrix,

etc.)

Scanning Electron Microscopy images

Different micrographs taken with SEM pertaining to mono- and multi-species oral biofilms grown in

the Microbiology Laboratory at Dentaid Research Center.

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