toluidine blue-mediated photodynamic effects on ...cthuang.bst.ntu.edu.tw/seminar/97slides/971i-lun...
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Speaker: I-Lun ChiangAdviser: Ching-Tsan Huang, PhDDate: 2008.12.9
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Mrinalini Sharma, Livia Visai, Francesca Bragheri, Haria Cristiani, Pradeep Kumar Gupta, and Pietro Speziale
Antimicrobial Agents And Chemotherapy, 52(1): 299-305, 2008
Toluidine Blue-Mediated Photodynamic Effects on Staphylococcal Biofilms
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Biofilms are everywhere!
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Biofilm on rock
Had you ever slipped on a rock?
Biofilm on pipe
cleaned a clogged drain pipe or toilet?
Biofilm in toilet
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What is Biofilm?a structured community of microorganismsMushroom-like conceptual model
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extracellular matrix
a living or nonliving surface.
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How do bacterial biofilms develop?
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(By Dr. Ghigo Jean-Marc)
pili
van der Waals forces
extracellular polymericsubstance(EPS)
quorum sensing
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Staphylococcal biofilms5
Staphylococcus aureus biofilm- Gram (+) bacteria
Staphylococcus epidermidis biofilm- Gram (+) bacteria
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CatheterInfection
Artificial hip implant
peridontal disease
Common Sites of Staphyloccocal Biofilm Infections
Mechanical heart valves
Wound infection
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Methicilin (1960)
Staphylococcus aureus ─ invade the wound after surgical procedure
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Penicillin (1940s)
Vancomycin
S. aureus Penicillin-resistantS. aureus2 years
Methicillin-resistant S. aureus (MRSA)
Vancomycin-intermediate S. aureus (VISA)
Vancomycin-resistant S. aureus (VRSA)
1 year
The discovery of new antibiotics might not catch up with the appearance of antibiotics resistance.
??
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Extracellular pulymeric substance (EPS) protect from phagocytosis
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Biofilm
Antibiotics resistance Antibiotics
resistanceExtracellularmatrix protection
resistant to phagocytosis
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Biofilm resistance mechanisms
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Strategies of Biofilm Control- 1. Prevention is better than cure 2. Removing or Killing
Traditional waysStop growth - by antimicrobial agentsBlock attachment - by changing surface materialPromote detachment - by surfactantsMechanical removal - by ultrasonic device Kill - by biocide, chemical agents
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But some ways cannot be carried out in hospitalized patients…
Toxicity ?
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Besides traditional ways…
• Is there any alternative way to inhibit the bacteria growth in biofilms?
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There is one way…
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Photodynamic Therapy (PDT)
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Light
Photosensitizer(excited state)
Photosensitizer(ground state)
Cellular toxicity
Fluorescence
Excitation
Three factors of PDT: Light , PS , Oxygen
3O2
1O2
singlet oxygen
e-
ROS( OH. H2 O2 O2
- )
Type I
Type II
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The difference of PDT effect between planktonic cells and biofilms
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S. aureus planktonic cells S. aureus biofilms
no MC 540
5 μ
g/ml MC 540
10 μ
g/ml MC 540
15 μ
g/ml MC 540
20 μ
g/ml MC 540
no MC 5405 μ
g/ml
10 μ
g/ml
15 μ
g/ml
20 μ
g/ml
(Lin, H. Y. et al. 2004)
Photosensitizer: Merocyanine 540 (MC540)
All-killedlight dose
All-killedlight dose
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Extracellur matrix in biofilms
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How to solve this problem?extracellular polymeric substance (EPS)
Hindrance of the penetration of light and
polysaccharide intercellular adhesion (PIA)
drugsthe uptake of photosensitizer
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Increase the permeability
Metal chelatorsEDTA and tetrasodium EDTA (TEDTA)
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carboxylate group
amine groupTEDTA
EDTA( salt form of EDTA, better solubility )
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How Metal Chelators dispersing the structure of a biofilm?• The sequestration of divalent cations, such as
Ca2+ and Mg2+, are important for maintaing the integrity of EPS in the biofilm.
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Mg2+
Mg2+
Mg2+
Ca2+Ca2+Ca2+
Ca2+
TEDTA
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The purpose of this study1. To examine the photodynamic effects of TBO on
the viability and structure of staphylococcal biofilms.
2. To investigate the effect of TEDTA pretreatment on the efficacy of the photodynamic inactivation of staphylococcal biofilms.
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The framework of this study
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Biofilm formation
No TEDTA pretreatment
TEDTA pretreatment
Survival Fraction
Confocal Laser Scanning Microscopy (CLSM)
Scanning electron Microscopy (SEM)
S. aureus LPS. epidermidis 1457
Photodynamic therapy with 40 μM TBO
Effect of TBO with light dose Effect of TEDTA treatment
CFU counting
Viability Morphology
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Materials and Methods19
Photosensitizer (TBO)Incubation time: 30 min
Stapylococcal biofilmsS. aureus
LP/ S. epidermidis 1457
+TEDTA( 1 hour )
TissueO2
CFU count (TSA)Survival Fraction
CLSM images
SEM images
640 nm diode laser
ROS
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Results20
S-L-
S+L-
S+L+
TEDTA+S-L-
TEDTA+S+L-TEDTA+S+L+
S:sensitizer (TBO:40 μM)L: light dose (100 J/cm2)TEDTA: 20 mM
TEDTA pretreatmentNo TEDTA
pretreatment
Survival fraction CFU of (S+ L+)
CFU of (S+ L-)
CFU of (TEDTA+S+L+)
CFU of (TEDTA+S-L-)or
TBO itself do no harm to staphylococcal biofilms
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Confocal laser scanning microscopy (CLSM)
• Use Live/Dead BacLight Bacterial Viability Kits-Two fluorescent nucleic acid stains:
SYTO9 : green(live)Propidium iodide(PI): red(dead)
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CLSM image
Cross section
Cross section
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Experiment conditions
CLSMimages
SEM images
CLSMimages
SEM images
TEDTA(-)TBO(40 μM)100 J/cm2
TEDTA(-)TBO(40 μM)200 J/cm2
TEDTA(+)Dark
TEDTA(+)TBO(40 μM)100 J/cm2
TEDTA(-)Dark
S. epidermidis
S. aureus
green(live)red(dead)
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23Staphylococcal biofilms
Photodynamic inactivation
No TEDTA pretreatment
TEDTA pretreatment
Ca2+Ca2+
Ca2+
Ca2+Ca2+
Mg2+
Mg2+ Mg2+
Mg2+
Mg2+
Photodynamic therapy
Antibiotics treatment Immune systemResistant toPhagocytosis
Antibiotics resistance
Mg2+
Mg2+
Mg2+
Ca2+Ca2+Ca2+
Ca2+
Hindrance of penetration
Pretreatment of Metal chelater
Enhance the efficacy of PDT
?
Photodynamic inactivation
The main cause of cell death Disperse biofilm structure
Effect of TBO with light dose Effect of TEDTA treatment
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The End
Thanks for your attention!!
Special thanks to Prof. Huang !
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Cell-cell communication• Cells in a biofilm “talk” to each other via
quorum sensing.
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Metal Chelator-Tetrasodium EDTA
• TEDTA is a novel central venous catheter lock solution against biofilm.
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(Infection Control and Hospital Epidemiology Vol.26 N0.6)
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Photodynamic therapy (PDT)Photodynamic therapy (PDT)
http://chemgroups.ucdavis.edu/~smith/PDT_Research/PDT.html
3P
S0
S1*
T1*
1O2
3O2
intersystem crossing
singlet oxygen
triplet state
Photosensitizer (ground state)
Photosensitizer (excited state)
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Photosensitizers used in PDT
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MC 540
Lipophilic , anionic
Amphiphilic , cationic
Hydrophobic– helps penetration in cellular membrane
Hydrophilic – helps diffusion.
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Wavelength Absorption of Photosensitizer
• Best if absorb at longer wavelengths• Longer wavelength emissions can penetrate
tissue farther.• Decreases probability of damage to surrounding
tissue.• Must have enough energy to excite O2
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Light source of PDT
• LED (Light-emitting diode):-coherent-long useful lifetime-high photoelectric conversion efficiency
• Laser-noncoherent-specific wavelength high intensity-low divergent effect
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Biofilm-associated infections
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PDT has better effect on Gram(+) bacteria due to the cell wall structure
Thick peptidoglycan cell wall integrated by severallipoteichoic acid forms many holes Photosensitizers can pass through Gram positive bacterial cell wall easier.
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barrier
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S. aureus v.s. S. epidermidisStaphylococcus aureus Staphylococcal epidermidis
Culture medium TSB (Tryptic soy broth) TSB (Tryptic soy broth)
Gram-staining Gram(+) Gram(+)
Biochemicalcharacteristics
catalase-positivecoagulase-positive
catalase-positivecoagulase-negative
habitat facultative anaerobe facultative anaerobe
color golden-yellow white