mechanism of propionibacterium acne necrosis by initiation of reactive oxygen species (ros) by...
DESCRIPTION
Background and Objectives: This study provides scientific evidence to support the clinical effect of photodynamic therapy for acne clearance using 414 nm LEDs and filtered Intense Pulsed Light (IPL). The Propionibacterium acnes (P. acnes) was viewed using a scanning electron microscope (SEM) offering the potential to visualise the destructive mechanism of free radical generation by porphyrin absorption. The benefits of using light to treat acne vulgaris safely and effectively are well known. This study had two objectives, firstly to show the dose response for various wavelengths and energy, secondly to morphologically show microscopic changes in the bacteria due to the effect of wavelengths of light.Study Design/Materials and Methods: Cultured P. acnes NCTC 737 (HPA, Salisbury, UK) were diluted to 0.5 McFarland using 2 ml of Butterfield’s phosphate buffer solution. Following further dilution, this suspension was pipetted into a 15 mm well plate for a dosed illumination using four IPL and LED light sources, each with different cut-off filters. After illumination of the sample, the suspension was dispensed onto blood agar plates and incubated at 37ºC for 72 hours under anaerobic conditions. Quantitative assessment was performed by counting the total number of colony forming units (CFUs) on each agar plate. Three controls samples were produced per experiment.Results: At 2 J/cm2 the short wavelength 400 nm filtered IPL has a 15.6% reduction effect on CFUs compared to 5.7% reduction with a 414 nm LED and a 4.6% reduction with a 530 nm IPL. The unfiltered IPL had a 79% reduction at 2 J/cm2, although this output spectrum of the unfiltered IPL contained a significant amount of DNA-damaging wavelengths below 400 nm.Conclusions: This study showed the mechanism of photodynamic therapy of Propionibacterium at a cellular level. Therefore, illumination of the endogenous coproporphyrin with blue light (400-420 nm) plays a major role in P. acnes photo-inactivation.TRANSCRIPT
Caerwyn Ash PhD1, Llinos Harris PhD2, Thierry Maffeis PhD3,
Marc Clement PhD1, Gareth Stockman PhD1, Mike Kiernan PhD1,
Godfrey Town4 1. CyDen Institute of Light Therapy
2. Medical Microbiology & Infectious Diseases, Swansea University
3. School of Nanotechnology, Swansea University
4. University of Wales, Faculty of Applied Design & Engineering, Swansea Metropolitan, Swansea, SA1 6ED, UK
Mechanism of Propionibacterium Acne
Necrosis by initiation of Reactive Oxygen
Species (ROS) by Porphyrin Absorption
2011 Conference, Dallas Texas
Statement of Disclosure
The following potential conflict of interest
relationships are germane to my presentation:
▪ Salary,
▪ equipment,
▪ travel expenses
paid by CyDen Ltd, Swansea, UK
2011 Conference, Dallas Texas
Background
Collaboration:
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Background
Collaboration:
knowledge transfer
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Background
In-Vitro Dose response Scanning Electron
Microscope Various Fluence
Various Wavelengths Back scattered electrons
Transmission electron
microscopy
Investigative Study
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Absorption Coefficients
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Absorption Coefficients
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Inter-Cellular Porphyrin
In-Vitro Methodology
Stock syrup kept at -80, agar plated
and incubated for 72 hours at 37ºC
Grade 0.5 McFarland
solution using 2ml of
Butterfields buffer
10,000 fold dilution with
butterfield solution
illuminate 300µl in a
15mm well plate
(random sequence)
Count number of
Colony forming
units (CFU)
Incubate for 72hrs at 37ºC under
dark anaerobic conditions
Plate 50µl onto blood
agar petri dish using a
WASP
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Various wavelengths
and fluence values
In-Vitro Methodology
Counting plates was time
consuming
Custom software to count
total number of Colony
Forming units (CFU)
Also provided average,
minimum and maximum of
colony sizes. Information
key for studying effect of
metabolic changes
In-Vitro Results
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In-Vitro Results
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IPL covers Q-bands
414nm diode LED (soret)
IPL (soret & Q-band)
IPL (soret & Q-band also UV)
In-Vitro Results - Summary
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• Results of 400-1100nm IPL, 530-1100nm IPL, and 414nm
diode LED are linear wrt to increased fluence. Thus
suggesting a photochemical reaction proportional to
delivered photons.
• 330-1100nm IPL (unfiltered flashlamp) had large
clearance of bacteria in-vitro, but relationship clearly
different from other wavelengths. Colony size was
smaller on average, suggesting a metabolic change,
DNA Damage of short wavelengths is a hypotenuse.
Scanning Electron Microscope (SEM)
Scanning Electron Microscope • Back scattered electrons
• Transmission electron microscopy
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Bacteria Fixing for SEM
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Stock syrup kept at -80ºC, , agar plated
and incubated for 72 hours at 37ºC
Grade 2 McFarland
solution using 2ml of
Butterfield buffer
Illumination
• Control
• 414nm LED
• 330 – 1100nm IPL
• 400 – 1100nm IPL
Centrifuge at 4000rpm
for 5min
Wash with Butterfield
buffer
Centrifuge at 4000rpm
for 5min
Aspirate solution
Add 500µl of Butterfield
buffer
Add to well plate with
Aluminium slides
Attach to Aluminium
Stubs
SEM
2.5% glutaraldehyde
paraformaldehyde
SEM Results – 330-1100nm
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Asymmetrical
separation
SEM Results – 330-1100nm
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Asymmetrical
separation
SEM Results – 330-1100nm
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Asymmetrical
separation
SEM Results – 400-1100nm
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Elongated Cells due
to fluid loss
SEM Results - Transmission
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Samples was fixed with 1% osmium tetraoxide and uranyl
acetate. The cells were dehydrated with ethanol and
embedded in Epon resin. Thin sectioned slices of samples
were prepared using an ultratome LKB.
Unable to supply images!
Images show asymmetrical separation of treated cells and
cellular leakage
SEM Results - Summary
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This study has observed structural
damages to membranes in illuminated
bacteria's
• Morphological changes…
• Asymmetrical separation of treated
cells (budding phase)
• Elongation of cells and cellular
leakage
Discussion
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• This collaborative effort proved productive and
beneficial to all groups
• Although effective in-vitro penetration depth
in-vivo is an issue for short wavelengths
• The Soret band key in effective results in-vitro
• Consecutive treatments yield better results
(data not presented)
• Illumination did not induce heat into bacteria or
media.
Conclusions
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• This study bears relevance on current clinical
research and better understanding on Acne pathology.
• Illumination of porphyrin with photons in the Soret
region play a major role in P.acne cell necrosis.
• Benefits of using light therapy for acne treatment is
well known wrt current antibiotics.
• Light of 407-420nm is not phototoxic to human cells
• For a one fold decrease in viable bacteria
73J/cm2 for 530-1100nm IPL (Q-bands)
18.2J/cm2 for 414nm diode LED (Soret)
10J/cm2 for 400-1100nm IPL (Soret & Q-bands)