Biofilm –subject for

antimicrobial
photodynamic therapy

Dr Saji George

Dr Anil Kishen’s Group

Faculty of Dentistry
National University of
Singapore
 Presentation Outline

• Biofilm- definition, structure, occurrence, formation

and characteristics

• Biofilm mediated human infections

• Biofilm- resistance to antibiotics

• Antibiofilm concepts

• Antimicrobial photodynamic therapy, definition and

principle

• PDT for eradicating biofilm- model studies and result

• Discussion and Summary

Biofilm- definition, structure, occurrence, formation and
What is a
characteristics

Biofilm?
Fluid Phase

Protective Matrix
Micro Organisms
Micro
Colonies
Fluid Channels

Solid Substratum

Biofilm is a mode of microbial growth

where a community of
microorganisms adhere to a solid non-
shedding surfaces which are
embedded in a self made matrix .
Biofilm- definition, structure, occurrence, formation and
characteristics
Biofilm-often an ignored but omnipresent
microbial growth

Rivers
Pipelines
Food processing appliances
Ship hull
Biofilm- definition, structure, occurrence, formation and
characteristics
Stages of Biofilm Formation

Phenotypic EPS
variation. production,
Altered Gene
Coaggregation and
expressions.
Coadhesion of planktonic
cells

Planktonic cells

Formation of elevated mushroom like

structures of bacterial cells

Disperse Modification
bacterial by Mineral
cells to the accumulation
bulk fluid
Mature biofilm with new bacterial cells emerging
Biofilm- definition, structure, occurrence, formation and
characteristics

Main incentives behind the formation of

biofilms
(1)protection from
harmful conditions in
the host (defense)

(2)sequestration to a
nutrient-rich area
(colonization)

(3) utilization of
cooperative benefits
(community)

(4) biofilms as the

default mode of
growth (planktonic
cultures are an in
vitro artifact).
 Biofilm-City of
microbes
Together we stand, individual we fall…….

Share many
common features
Community of interacting
organism
Elevated tower like
structures
Voids acting as water
channel-primitive circulatory
system
Transport of materials
 Signalling
Biofilm- throughoccurrence, formation and
definition, structure,
Biofilm- definition, structure, occurrence, formation and
characteristics

Microbial Interactions
Coaggregatio
n and
Nutrient
Coadhession
exchange and
Facilitate

Biofilm
sharing Physical
Metaboli gene
Communicatio
c transfer
n
Determine
Spatial
relationship
Tolerance to
extremes of pH,
salinity, Nutrients Genetic
and Antibiotics

Increased
rate of Specific gene
Gene transfer
activation
transformatio
across MOs
n
Biofilm- definition, structure, occurrence, formation and
characteristics

Nutrient sharing
Eubacteria,
Peptostreptococci,
F.nucleatum
Fo
e s Hy rm
tid dr ate
p og o
Pe en r

Campylobacter rectus
P.intermedia, & P. gingivalis Hemin
Biofilm- definition, structure, occurrence, formation and
characteristics
Heterogeneity in biofilm
The status of nutrients,
oxygen,and pH vary in
different parts of biofilm

Nutrient and oxygen

concentrations decreases
towards the interior

The heterogeneity of
various physicochemical
factors facilitate the
coexistance of microbes
with varying physiological
Biofilm- definition, structure, occurrence, formation and
characteristics
Quorum sensing - cell-to-cell
signaling

Detect the cell density

and coordinate the
expression behavior

Gram-Negative
signaling transfer
molecules - acyl
homoserine lactone
(AHL)
Biofilm- definition, structure, occurrence, formation and
characteristics
Biofilm matrix

Extracellular polymeric
substances (EPS)
Carbohydrates, Proteins,
Lipids, Nucleic acids

Functions
Trap nutrients- micro
nutrients
Protection from
antimicrobial agents
Prevent from being washed
away
Protection from water loss
 Your Body Is a Planet: 90% of the cells
within us are not ours but microbes'.
(Josie Glausiusz)

Biofilm mediated human infections

Biofilm mediated human infections

Biofilm Paradox in medicine

-Antony van Leewenhoek
First observed biofilm bacteria from tooth

Rober Koch –
First isolated pure culture of bacteria…and
corelated to disease

“Until the late seventies, no one even knew biofilms existed. Scientists
thought most of the bacterial world was made up of free-floating
bacteria. They developed antibiotics and vaccines using bacteria
floating in a test tube. In many cases, the medicines just didn't work —
prostatitis, middle ear infections in children and periodontal disease to
name a few. As it turns out, scientists were targeting the wrong kind of
bacteria”.

Kate Dalke… Genome News Net work

Biofilm mediated human infections

Human infections involving

biofilm
More than 60% of all human
infections are mediated by
bacterial biofilm

Biofilm mediated diseases-

emphasizes community
theory of infection

Planktonic cells- emphasizes

germ theory of infection
Biofilm mediated human infections

Human infections involving

biofilm
Biofilm- resistance to antibiotics

Resistance mechanisms in biofilm

Bacteria in biofilm are resistant to

 Antibiotics (more than 1500 times resistant compared to
planktonic bacteria!!)

 Heat
 Quaternary ammonium compounds
 Iodine, Chlorine etc

Understanding various stages and method of bacterial

resistance mechanisms to antimicrobials forms the
1st step in developing a antibiofilm regime
Biofilm- resistance to antibiotics

Sites of Antimicrobial Matrix of the biofilm

Resistance
Constituents of Matrix

Enzymes present in
Matrix

Metabolic and genetic

alteration of bacteria

Metabolic state of bacteria

Enzyme
a.
Limited Mediated
SlowDiffusion
rate of growth resistance
through Matrix
Neutralization of
Glycocalyx
a. Active
b. Reduction of cations
antimicrobials
metabolism ofto
Ionic interaction
Metals
antibiotics
a. Constituents
Sieving effect of Glycocalyx
b. Altered
c. Action
Increased of detoxifying
protein
eg neutralization
viscosity profile
of I2
enzymes
d.
b. Multi
Act asdrug efflux
an ion pump resin
exchange
Antibiofilm concepts

Fight against
biofilm- antibiofilm
strategies
Antibiofilm concepts

Anti-biofilm coatings
Use of Furanones- ( Bavega et al, Givskov et al., J Bacteriol.
1996; 178:6618-6622).

 As anti bacterial coating on biomaterials

 Furanones are the compounds isolated from
sea weeds (Delisea pulchra-Australian red algae)
 Prevents Staphylococcus epidermis adhesion
and slime production on biomaterial

 Furanones target the quorum sensing agents.

Antibiofilm concepts

Surface modification
 Modify the solid surfaces to prevent bacterial
adhesion (antibacterial nano-particles)

Replacement therapy
 Replace potential pathogenic micro-organisms with
genetically modified organisms that are less virulent

Immunization
 The aim is to inhibit adhesion or reduce the virulence
of putative microbial etiologic agents.
Antibiofilm concepts

Use of laser irradiation- (Asta Richter et al)

 Pulsed nitrogen laser to the in vitro cultivated

biofilm
 Damage to the surface substrate at higher
power of laser
 Removing efficiency depends on the surface
 Substrate matrix enhances the susceptibility to
photodamage as seen in P. aeroginosa
Antimicrobial photodynamic therapy, definition
and principle

Photodynamic Therapy (PDT)

as an antimicrobial agent

Killing of microorganisms when

photosensitizer selectively
accumulated in the target is activated
by a visible light of appropriate
wavelength.

cc c c

c c

Sensitized bacterial cell Damaged cell Cell destruction

Antimicrobial photodynamic therapy, definition
and principle

Photodynamic Therapy (PDT) as an

antimicrobial agent
 Broad spectrum antimicrobial action-
suitable for eliminating microbial
community
 Less chance of bacteria resisting PDT
(singlet oxygen) - appropriate for
treating biofilms since the indwellers
are resistant to antimicrobials
 Photosensitization can even cause
EPS breakdown (Mark Wainwright)
Antimicrobial photodynamic therapy, definition
and principle
Mechanism of
photosensitization

The ability of a photosensitizer depends on the

proportion undergoing inter system crossing .

Highly fluorescent compound dissipating energy

as fluorescence are less efficient as PDT agents.

Aromatic compoudns with π system makes long

lived triplet state
(Journal of Antimicrobial Chemotherapy (1998) 42, 13–28)
 Mechanism of
photosensitization
Type I - Photosensitiser triplet state reacts first with a
substrate other than molecular oxygen.
Type II pathway- The photosensitiser triplet state reacts with
molecular oxygen to generate singlet oxygen
LIGHT
Type 1 mechanism
PS**

PS Biomolecules
PS

Type 2 mechanism (Photodynamic effect)

LIGHT

O**
PS**

PS PS O2 Biomolecules

Antimicrobial photodynamic therapy, definition

Antimicrobial photodynamic therapy, definition
and principle
Singlet oxygen and
bacteria
Both gram positive and
gram negative were killed on
exposure to singlet oxygen

Killing curves for gram

negatives were indicative of
multihit killing, whereas
curves for gram positive
exhibited single-hit kinetics

Direct action of singlet

oxygen on gram positive

Secondary radicals
production from the LPS of Dahl et al. Journal Of Bacteriology, Apr. 1989, p.
2188-2194
Antimicrobial photodynamic therapy, definition
and principle
Type of bacteria and
Susceptibility to PDT
Polysaccharides

LPS (Outer membrane)

Glycan layer (GlcNAc &

MurNAc)

Cell membrane

Gram positive cell wall Gram negative cell surface

Net negative charge on the

bacterial cell-due to LPS and
Polysaccharides
Outer membrane-reason for
resistance
Check entry of the chemicals into
Antimicrobial photodynamic therapy, definition
and principle
Mechanism of antibacterial
action Water

MIX

Cell wall damage due to PDT

Mark Con L+ WL- M L- WL+ M L+
Marker Con WL- ML- WL+ ML+

Outer membrane profile

DNA damage caused by PDT
before and after PDT
S. George and Kishen A. Photochemistry and Photobiology (in press)
Antimicrobial photodynamic therapy, definition
and principle
Is PDT a magic
bullets?
Bacteria associated with
fibroblast
y= 95.939e-
0.0764x

The rate of bacterial killing -

faster than loss of mammalian y= 137.48e-
cells.
1.0088x

(George and Kishen 2007)

Bacteria internalized by
THP1

100% elimination of
internalized bacteria at the
cost of 70% THP1 cells with
damaged cell membrane!!!
(unpublished data)
PDT for eradicating biofilm- model studies
and result

Antimicrobial Photodynamic
Therapy for root canal disinfection
Concept of APDT in Root Canal
Disinfection

Infected tooth Access cavity Sensitization Light treatment Restored tooth

PDT for eradicating biofilm- model studies and
result
Antibacterial efficacy of PDT depends
on the stage of biofilm maturation!!!!!

A. 1 week old biofilm A B

B. 4 weeks old biofilm

The structure and
composition of root canal
(in vitro) biofilm changes
along with the stage of
maturation 12

1 wee k
10
Antimicrobial 4 wee ks
log numebr of bacteria
treatment in four 8

weeks old root 6

canal biofilm is 4
less effective than
2
1 week old biofilm
0
Modified
Control Conventional ANILAD RCT
PDT
Lim et al. Communicated. PDT
PDT for eradicating biofilm- model studies
and result
The ultrastructure of matured root canal
biofilm –limited accessibility to the biofilm
core
Confocal microscopy

Biofilm

Dentine

Fluid phase
Photosensitiz
er

Bacteria
mediated
dentine
dissolution

Dentine
surface

A. Kishen, S. George and R. Kumar (2006). J Biomed Mat Res A. 77(2):406-415.

PDT for eradicating biofilm- model studies
and result
Matrix disrupting
Matrix disrupting agent
augment the antibiofilm
efficacy of PDT
The Laser Confocal A B

Scanning Microscopy of
biofilm subjected to PDT.

C D

PDT using a
photosensitizing
formulation containing
a matrix disrupting E F

agent (PF4) could

eradicate matured
biofilm A- biofilm receiving no treatment, B- biofilm subjected to
irradiation alone, C- biofilm subjected to sensitization with
100μM MB, D- biofilm subjected to sensitization with MB
followed by irradiation, E- biofilm subjected to PF4, F-
biofilm subjected to PF4 and irradiation.
PDT for eradicating biofilm- model studies
and result
Complete inactivation of root canal
biofilm is possible by modified
PDT!!!!
Treatment log10 after Number of tooth
groups four-hour specimens positive
enrichment for bacteria after
24-hour enrichment

Control 7.147 5/5

(±0.601)
Conventional-PDT 5.639 5/5
(±0.066)
Conventional RCT 0.0 3/5

PDT using PF4 0.0 0/5

Conventional 0.0 0/5
RCT+PDT using
PF4
Discussion and Summary

Discussion
 PDT can break down the matrix components of biofilm
(Wainwright et al., 2002).

 Bacterial killing during PDT - mostly confined to the outer

surface of biofilm (Zanin et al., 2005)

 PDT using matrix disrupting agent could breakdown the

biofilm matrix and completely inactivate bacteria.

 The complementing function of matrix disrupting agent

and enhanced molecular oxygen availability enhances the
antimicrobial potential of modified PDT
Discussion and Summary

Summary

• Biofilm can be formed on diverse materials

• Biofilm has definite structural and functional

properties that confer advantage over
planktonic mode of bacterial growth

• Treatment of biofilm mediated human infections

is a huge clinical task and newer therapeutic
strategies are being screened for preventing
and eradicating biofilm bacteria

• Photodynamic therapy involving the use of

photosensitizer and light has been tried as an
effective antimicrobial therapeutic strategy
Discussion and Summary

Summary

‘Matured’ bacterial biofilm, that is generally

resistant to antimicrobial agents, can be
eradicated by modified PDT
 Biofilm- the other side of
the story….
1. Green walls- lessons from
biofilm

2. Eco-friendly treatment of
toxic effluents Biofilm on clay
based substrate
for waste water
treatment

3. Biofilm technology in
solid state fermentation-
production of therapeutic
Acknowledgement
Faculty of Dentistry
Funding from the National University of Singapore
ARF, Grant No. FY 04 R-224-000-021-101

Dr Anil Kishen

n k
A/P Neo Chiew Lian, Jennifer
a
Dr Song Keang Peng (Monasch University

h
T ou
Malaysia)
Dr Raj Kumar (University of Florida)
Mrs Shibi George
Dr Sum Chee Peng
Mr Zhang Xu
Y
Dr Annie Shrestha
 Biofilm is formed on diverse materials

 Biofilm has definite structural and functional

properties that confer advantage over
planktonic mode of bacterial growth

t i
s
 Treatment of biofilm mediated human infections

e
is a huge clinical task and newer therapeutic
strategies are being screened for preventing

u s?
and eradicating biofilm bacteria

 Q n
Photodynamic therapy involving the use of

o
photosensitizer and light has been tried as an
effective antimicrobial therapeutic strategy