Genetic and Physiological Effects of Noncoherent Visible Light - - PowerPoint PPT Presentation

Genetic and Physiological Effects of Noncoherent Visible Light Combined with Hydrogen Peroxide on Streptococcus mutants in Biofilm

Doron Steinberg, Daniel Moreinos, John Featherstone, Moshe Shemesh and Osnat Feuerstein Antimicrobial Agents and Chemotherapy, July 2008, p. 2626-2631, Vol. 52, No. 7

Speaker: Jee-Ling Ng Advisor: Ching-Tsan Huang, Ph.D. Date: 2008/12/02

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What are biofilms?

Biofilm in pipe section Dental plaque Biofilm in acidic pools at Yellowstone National Park.

3 Resource: The Center for Biofilm Engineering at Montana State University-Bozeman

Biofilm formation

Planktonic bacteria extracellular polymeric substances or (EPS)

Dynamic Genetic expression is different in biofilm Less sensitive to antimicrobial agents

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Characteristic of biofilms

5 Resource: The Center for Biofilm Engineering at Montana State University-Bozeman

How do biofilms impact our w orld?

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Dental plaque

Periodontal disease Caries Gingivitis

TOOTH DECAY

Bacterial Biofilm

The Center for Biofilm Engineering at Montana State University-Bozeman

Gram-positive, facultative

anaerobic bacteria

Utilize sucrose to produce

polysaccharide

Relies on biofilm lifestyle

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Streptococcus mutants

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Antimicrobial Methods

Antibiotics

Resistance!

Chemical photosensitizer Chemical photosensitizer Higher energy excited states Higher energy excited states

Reactive

• xygen species

(ROS) Reactive

• xygen species

(ROS) Free radicals Free radicals

Visible light / UV

Photodynamic action

Pros

• Capable to penetrate the deeper layers of

biofilm than any chemical agents.

• No bacteria resistant to light irradiation
• Can be targeted very precisely
• Intensity of the irradiation can be monitored.

Cons

• Can only treat areas where light can reach

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Pros and Cons of light irradiation for biofilm treatment

These results support the assumption that the phototoxic effect of blue light on the periopathogenic bacteria is oxygen dependent and that hydroxyl radicals play an important role in this process.

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H 2 O2

Blue light Mechanism of Visible Light Phototoxicity

• n

Porphyromonas gingivalis and Fusobacterium nucleatum

Photochemistry and Photobiology, 2005, 81: 1186–1189

Planktonic

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Noncoherent blue light + H 2 O2

Streptococcus mutants Bacterial viability Gene expression

Aim of this study Aim of this study

Microbial assays Confocal Scanning Laser

Microscopy (CSLM)

ATP analysis

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Bacterial Viability

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Synergistic effect

Bacterial biofilm growth after exposure to blue light in combination with H2 O2

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More effective against bacterial biofilm than against planktonic bacteria

Bacterial biofilm growth versus planktonic bacteria growth after blue light and 30 mM H2 O2 treatment

Time exposure Bacterial biofilm Planktonic bacteria 30 s 2.12 log 0.72 log 60 s 2.6 log 1.19 log

Fe2+ + H2 O2 Fe3+ + .OH + OH-

Phototoxic effect of blue light and H 2 O2

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H 2 O2

Fenton reaction Hydroxyl radical

Oxidative stress cell damage

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(a) Immediately after exposure

• No effect on bacterial viability

through all layers of the biofilm (b) 3 hours after exposure

• Significant bacteria death in the

middle layers (35~65μm) of the biofilm (c) 6 hours after exposure

• Most of the bacteria death through

all layers of the biofilm

Green: Live bacteria Red : Dead bacteria Yellow : Both live and dead bacteria Green: Live bacteria Red : Dead bacteria Yellow : Both live and dead bacteria

CSLM Images of different layers in biofilm

Live/dead BacLight bacterial viability kit

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Significant bacteria death in the middle layers (35~65μm) of the biofilm

Top of biofilm Deepest layer of biofilm

CSLM image of biofilm 3 hours after light exposure for 60 seconds

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CSLM image of biofilm 6 hours after light exposure for 60 seconds

Top of biofilm Deepest layer of biofilm

Most of the bacteria death through all layers of the biofilm

H2O2 are small compare with

the photosensitizers molecules

The disruption of the biofilm by

photo-oxidation may enhance the penetration into the biofilm

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The combination of light and H2 O2 has an advantage in penetrating the deep layers of the biofilm

H 2 O2 molecules Surface Deep layer Middle layer

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ATP levels of bacteria in biofilm light exposure for 60 seconds and 30 mM H2 O2

Significant effect on bacterial metabolic activity in biofilm

Accumulation of ROS in the biofilm creates

environmental conditions stressful to the exposed bacteria

In response, bacteria in biofilm enhance ATP

production to adapt the stressful environment

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Why bacterial ATP levels sharply increase after light and H2 O2 treatment ?

gtfB (GTFB, glucan production)

• Play a pivotal role in sucrose-dependent bacterial adhesion

brpA (Biofilm-regulating protein) smu630 (Biofilm formation hypothetical protein) comDE (Competence-stimulating peptide)

• Histidine

kinase receptor (com D)

• Response regulator (com E)

relA (Guanosine tetra (penta)-phosphate synthetase) ftf (FTF, fructan production)

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Several genes association with biofilm formation of S. mutants

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Relative expression of several genes related to biofilm formation 24 hours after treatment

X5 X6 X8 X4

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Conclusion

Synergistic effect

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• S. mutants

Gene Expression CSLM Image ATP Level CFU counts

Check Check

THANK YOU FOR YOUR ATTENTION !

Special Gratitude to Prof. Huang

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Photosensitizers (PS)

Characteristics

• Absorb light
• Cause photochemical reaction

More than 400 compounds are PS

• Dyes, drugs and natural substances
• Examples:

Natural Substances Dyes Flavins Chlorophyll Polyacetylenes Methylene blue Toluidine blue O Phthalocyanines

Low cost Generally recognized as safe as an

antimicrobial agent

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Hydrogen peroxide