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

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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

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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)

Thank you

E: caerwynash@yahoo.co.uk

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