Fundamentals and Applications of Biofilms Bacterial Biofilm Control: Photodynamic Therapy

Transcription

1 1 Fundamentals and Applications of Biofilms Bacterial Biofilm Control: Photodynamic Therapy Ching-Tsan Huang ( 黃慶璨 ) Office: Agronomy Building, Room 111 Tel: (02) cthuang@ntu.edu.tw

2 2 Common Methods in Microbial Control Prescott Microbiology 2008.

3 3 Antimicrobial Methods Chemotherapy Heavy metal Chemical agents Antibiotics Peptides Resistance! Photodynamic action

4 4 Photodynamic Therapy

5 5 Photodynamic Mechanism Nature reviews cancer, 2003 (3) 380-7

6 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

7 Photosensitizers (PS) 7 J Dent Res. 86(11): 1126, 2007.

8 8 Active oxygen Ref : Malik et al. 1990

9 9 Photosensitizers -Aminolaevulinic acid (ALA): Endogenous Merocyanine 540 (MC 540): Exogenous PpIX ALA

10 10 Light Small range of wavelength White light Halogen lamp LED (Light Emitting Diode) Laser (Light Amplification by Stimulated Emission of Radiation )

11 LED used for PDT 11

12 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 13 Biofilm Resistance Mechanism

14 Planktonic cells: S. aureus Relation between antimicrobial activity and excitation level of MC 540 Survival fraction (log (N/N0) : 1-fold : 2-fold : 5-fold : 10-fold Excitation level of MC 540 (ug/10 8 cells) Light dose (J/cm )

15 2 15 Planktonic cells: S. aureus MC540-mediated PDT on antimicrobial activity and excitation level of MC540 Survival fraction (log (N/N0)) : 0 µg/ml : 5 µg/ml : 10 µg/ml : 15 µg/ml : 20 µg/ml Excitationof MC 540 (ug/10 8 cells) Light dose (J/cm 2 )

16 16 Hypothesis A threshold value of photosensitizer excited is required to reach complete kill.

17 Biofilm Apparatus 17

18 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 ) Light dose (J/cm 2 )

19 19 Confocal Laser Scanning Microscopy 0 J/cm 2 30 J/cm 2 60 J/cm 2 20 m 150 J/cm J/cm J/cm 2

20 20 Reactor design Pump LED Material

21 21 Reactor design Step 1 Step 3 Step 2 Step 4

22 22 PACT (small scale 10 beads) Initiative conc.: 10 8 cells/ml Interaction conc.: 10 7 cells/ml Wild type MSSA MRSA log(survival fraction) log(survival fraction) g/ml 100 g/ml control g/ml 100 g/ml control incident dose (J/cm 2 ) incident dose (J/cm 2 )

23 23 Planktonic cells: P. aeruginosa Relation between antimicrobial activity and excitation level of -ALA With irradiation Without irradiation J cm log(survival fraction) J cm J cm -2 log(survival fraction) ALA concentration (mm) ALA concentration (mm)

24 24 Biofilm cells: P. aeruginosa Irradiation on excitation level of -ALA 10 Biofilm density log(cfu cm -2 ) J cm -2 Control ALA concentration (mm)

25 25 FIG. 3. Eradication of 3-h P. aeruginosa biofilms using MB-PAA-DNP Wu, J. et al Antimicrob. Agents Chemother. 53(7):

26 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 27 Biofilm cells: P. aeruginosa Regrowth after -ALA-mediated PDT Control Biofilm density log(cfu cm -2 ) st 2nd Time of regrowth (h)

28 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 29 Difference in Structure Gram-positive cell wall Gram-negative cell wall

30 PDT on Clinical Treatment 30 J Dent Res. 86(11): 1126, 2007.

31 PACT on Periodontitis 31 Photodynamic Antimicrobial ChemoTherapy Periodontol ;51:

32 The Periowave Photodisinfection Process 32