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
characteristics

What is a 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 nonshedding 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
EPS production, Altered Gene expressions.

Phenotypic variation.
Coaggregation and Coadhesion of planktonic cells

Planktonic cells Formation of elevated mushroom like structures of bacterial cells

Disperse bacterial cells to the bulk fluid
Mature biofilm with new bacterial cells emerging

Modification by Mineral accumulation

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).

Together we stand, individual we fall…….

Biofilm-City of microbes

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

Biofilm- definition, structure, occurrence, formation and

characteristics

Microbial Interactions
Nutrient exchange and sharing Communicatio n Coaggregatio n and Coadhession Facilitate gene transfer Determine Spatial relationship

Biofilm

Metaboli c

Physical

Tolerance to extremes of pH, salinity, Nutrients and Antibiotics

Genetic

Increased Specific gene rate of Gene transfer activation transformatio across MOs n

Biofilm- definition, structure, occurrence, formation and

characteristics

Nutrient sharing
Eubacteria, Peptostreptococci, F.nucleatum Fo s Hy rm de i dr ate pt og o Pe en r P.intermedia, & P. gingivalis Hemin Campylobacter rectus

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 diseasesemphasizes 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 Heat

(more than 1500 times resistant compared to

planktonic bacteria!!)
  

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 Resistance

Matrix of the biofilm Constituents of Matrix Enzymes present in Matrix Metabolic and genetic alteration of bacteria

Metabolic state of bacteria Enzyme Mediated resistance a. Slow rate of through Limited Diffusion growth Matrix Neutralization of Glycocalyx a. Reduction of cations to b. Active antimicrobials metabolism of Ionic interaction Metals antibiotics a. Constituents of Glycocalyx Sieving effect b. Action of detoxifying 2 c. Altered protein of I eg neutralization Increased viscosity profile
enzymes d. Multi drug efflux pump resin b. Act as an ion exchange

Antibiofilm concepts

Fight against biofilm- antibiofilm strategies

Antibiofilm concepts

Anti-biofilm coatings
Use of Furanones1996; 178:6618-6622). ( Bavega et al, Givskov et al., J Bacteriol.

 

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 adhesion (antibacterial nano-particles)

bacterial

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 actionsuitable 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)

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

Mechanism of photosensitization

Type 1 mechanism
PS**

PS

PS

Biomolecules

Type 2 mechanism (Photodynamic effect)
LIGHT

PS**

O**

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 Water action

MIX

Cell wall damage due to PDT
Marker Con WL- ML- WL+ ML+ Mark Con L+ WLM LWL+ M L+

DNA damage caused by PDT

Outer membrane profile 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?
with

Bacteria associated fibroblast

y= 95.939e-

The rate of bacterial killing faster than loss of mammalian cells. (George and Kishen 2007) Bacteria THP1 internalized by

0.0 4 76 x

y= 137.48e1 0 8x .0 8

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 B

A. 1 week old biofilm B. 4 weeks old biofilm
The structure and composition of root canal (in vitro) biofilm changes along with the stage of maturation

12 10 log numebr of bacteria 8 6 4 2 0 Control 1 wee k 4 wee ks

Antimicrobial treatment in four weeks old root canal biofilm is less effective than 1 week old biofilm
Lim et al. Communicated.

Conventional ANILAD PDT PDT

Modified

RCT

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
A B

The Laser Confocal Scanning Microscopy of biofilm subjected to PDT.

C

D

PDT using a photosensitizing formulation containing a matrix disrupting agent (PF4) could eradicate matured biofilm

E

F

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, Fbiofilm 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 groups log10 after Number of tooth four-hour specimens positive enrichment for bacteria after 24-hour enrichment
7.147 (±0.601) 5/5 5/5 3/5 0/5 0/5

Control

Conventional-PDT 5.639 (±0.066) Conventional RCT 0.0 PDT using PF4 Conventional RCT+PDT using PF4 0.0 0.0

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

1. Green walls- lessons from biofilm

Biofilm- the other side of the story….

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

3. Biofilm technology in solid state fermentationproduction 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 A/P Neo Chiew Lian, Jennifer
Dr Song Keang Peng (Monasch University Malaysia) Dr Raj Kumar (University of Florida) Mrs Shibi George Dr Sum Chee Peng Mr Zhang Xu Dr Annie Shrestha

k n a h u T o Y

Biofilm is 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

i t s e u s? Q n o