Mechanism of Propionibacterium Acne Necrosis by initiation of Reactive Oxygen Species (ROS) by Porphyrin Absorption

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

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:

2011 Conference, Dallas Texas

Background

Collaboration: knowledge transfer

2011 Conference, Dallas Texas

Background
Investigative Study
In-Vitro Dose response
Various Fluence Various Wavelengths

Scanning Electron Microscope
Back scattered electrons Transmission electron microscopy

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Absorption Coefficients

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Absorption Coefficients
Inter-Cellular Porphyrin

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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) Plate 50µl onto blood agar petri dish using a WASP Incubate for 72hrs at 37ºC under dark anaerobic conditions Count number of Colony forming units (CFU) Various wavelengths and fluence values

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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

IPL covers Q-bands 414nm diode LED (soret) IPL (soret & Q-band) IPL (soret & Q-band also UV)

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In-Vitro Results - Summary
• 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.

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Scanning Electron Microscope (SEM)
Scanning Electron Microscope
• Back scattered electrons • Transmission electron microscopy

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Bacteria Fixing for SEM
Stock syrup kept at -80ºC, , agar plated and incubated for 72 hours at 37ºC Grade 2 McFarland solution using 2ml of Butterfield buffer SEM Attach to Aluminium Stubs Add to well plate with Aluminium slides

Illumination • Control • 414nm LED • 330 – 1100nm IPL • 400 – 1100nm IPL
Centrifuge at 4000rpm for 5min Aspirate solution Wash with Butterfield buffer

2.5% glutaraldehyde paraformaldehyde
Add 500µl of Butterfield buffer Centrifuge at 4000rpm for 5min

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SEM Results – 330-1100nm

Asymmetrical separation

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SEM Results – 330-1100nm

Asymmetrical separation

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SEM Results – 330-1100nm

Asymmetrical separation

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SEM Results – 400-1100nm

Elongated Cells due to fluid loss

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SEM Results - Transmission
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

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SEM Results - Summary 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

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Discussion
• 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.

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Conclusions
• 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 18.2J/cm2 for 414nm diode LED 10J/cm2 for 400-1100nm IPL
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(Q-bands) (Soret) (Soret & Q-bands)

Thank you
E: caerwynash@yahoo.co.uk

2011 Conference, Dallas Texas

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