1 Fundamentals and Applications of Biofilms Bacterial Biofilm Control: Photodynamic Therapy Ching-Tsan Huang ( 黃慶璨 ) Office: Agronomy Building, Room 111 Tel: (02) 33664454 E-mail: cthuang@ntu.edu.tw
2 Common Methods in Microbial Control Prescott Microbiology 2008.
3 Antimicrobial Methods Chemotherapy Heavy metal Chemical agents Antibiotics Peptides Resistance! Photodynamic action
4 Photodynamic Therapy http://www.biu.ac.il/ph/biophysics/pdt.jpg
5 Photodynamic Mechanism Nature reviews cancer, 2003 (3) 380-7
6 Photosensitizers (PS) Characteristics Selective adsorption or binding by cancer cells Different rates of metabolism between normal and cancer cells Excitation by an appropriate wavelength PS Porphyrins Cyanine dyes quinones
Photosensitizers (PS) 7 J Dent Res. 86(11): 1126, 2007.
8 Active oxygen Ref : Malik et al. 1990
9 Photosensitizers -Aminolaevulinic acid (ALA): Endogenous Merocyanine 540 (MC 540): Exogenous PpIX ALA
10 Light Small range of wavelength White light Halogen lamp LED (Light Emitting Diode) Laser (Light Amplification by Stimulated Emission of Radiation )
LED used for PDT 11
12 Why PDT Promising on Biofilm Control? Interact with cells only No adsorption or neutralization by EPS MW is generally small Less transport limitation Binding to cells Not related to physiology Already exist in cells Less chance to induce resistant strains
13 Biofilm Resistance Mechanism http://www.erc.montana.edu/multicellstrat/06-meeting_antimic-challenge/suscphypotheses.htm
Planktonic cells: S. aureus Relation between antimicrobial activity and excitation level of MC 540 Survival fraction (log (N/N0) 2 0-2 -4-6 -8-10 0 200 400 600 800 1000 14 : 1-fold : 2-fold : 5-fold : 10-fold Excitation level of MC 540 (ug/10 8 cells) 2.0 1.5 1.0 0.5 0.0 0 200 400 600 Light dose (J/cm 2 800 1000 )
2 15 Planktonic cells: S. aureus MC540-mediated PDT on antimicrobial activity and excitation level of MC540 Survival fraction (log (N/N0)) 0-2 -4-6 -8-10 0 200 400 600 800 1000 : 0 µg/ml : 5 µg/ml : 10 µg/ml : 15 µg/ml : 20 µg/ml Excitationof MC 540 (ug/10 8 cells) 2.0 1.5 1.0 0.5 0.0 0 200 400 600 800 1000 Light dose (J/cm 2 )
16 Hypothesis A threshold value of photosensitizer excited is required to reach complete kill.
Biofilm Apparatus 17
18 Biofilm cells: S. aureus Irradiation on antimicrobial activity and excitation level of MC 540 : 0 µg/ml : 5 µg/ml : 10 µg/ml : 15 µg/ml : 20 µg/ml Survival fraction (log (N/N0)) Excitation level of MC 540 (IOD/mm 2 ) 2 0-2 -4-6 -8 0 100 200 300 400 500 600 700 40 30 20 10 0 0 100 200 300 400 500 600 700 Light dose (J/cm 2 )
19 Confocal Laser Scanning Microscopy 0 J/cm 2 30 J/cm 2 60 J/cm 2 20 m 150 J/cm 2 300 J/cm 2 600 J/cm 2
20 Reactor design Pump LED Material
21 Reactor design Step 1 Step 3 Step 2 Step 4
22 PACT (small scale 10 beads) Initiative conc.: 10 8 cells/ml Interaction conc.: 10 7 cells/ml Wild type MSSA MRSA 1 1 0 0 log(survival fraction) -1-2 -3-4 -5-6 log(survival fraction) -1-2 -3-4 -5-6 -7-8 50 g/ml 100 g/ml control 0 20 40 60 80-7 -8 50 g/ml 100 g/ml control 0 20 40 60 80 incident dose (J/cm 2 ) incident dose (J/cm 2 )
23 Planktonic cells: P. aeruginosa Relation between antimicrobial activity and excitation level of -ALA With irradiation Without irradiation 1 1 0-1 120 J cm -2 0-1 log(survival fraction) -2-3 -4-5 -6-7 360 J cm -2 240 J cm -2 log(survival fraction) -2-3 -4-5 -6-7 -8-8 -9 0.0 2.5 5.0 7.5 10.0 12.5-9 0.0 2.5 5.0 7.5 10.0 12.5 ALA concentration (mm) ALA concentration (mm)
24 Biofilm cells: P. aeruginosa Irradiation on excitation level of -ALA 10 Biofilm density log(cfu cm -2 ) 8 6 4 2 120 J cm -2 Control 0 0 10 20 30 40 50 ALA concentration (mm)
25 FIG. 3. Eradication of 3-h P. aeruginosa biofilms using MB-PAA-DNP Wu, J. et al. 2009. Antimicrob. Agents Chemother. 53(7):3042-3048
26 Regrowth after antimicrobial treatment Regrowth (recovery) A rapid resumption of biofilms after antimicrobial treatment was terminated. The remaining biofilms contain enough viable organisms. The residual biofilm enhances transport and sorption of microbial cells and other compounds to the surface. Some oxidizing antimicrobial agents preferentially removes EPS and not biofilm cells. EPS is rapidly created by surviving organisms.
27 Biofilm cells: P. aeruginosa Regrowth after -ALA-mediated PDT 12 10 Control Biofilm density log(cfu cm -2 ) 8 6 4 2 1st 2nd 0-36 -24-12 0 12 24 36 48 60 Time of regrowth (h)
28 PDT against Microorganisms Microorganisms Photosensitizers Exogenous PS MC540, MB, HpD Endogenous PS -ALA Natural PS C-Phycocyanin Bacteria Gram + Gram - S. aureus P. aeruginosa S. epidermidis E. coli S. aureus P. aeruginosa S. epidermidis E. coli S. aureus P. aeruginosa S. epidermidis E. coli
29 Difference in Structure Gram-positive cell wall Gram-negative cell wall
PDT on Clinical Treatment 30 J Dent Res. 86(11): 1126, 2007.
PACT on Periodontitis 31 Photodynamic Antimicrobial ChemoTherapy Periodontol 2000. 2009;51:109-40.
The Periowave Photodisinfection Process 32