Name: an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm
Uploaded: 2018-04-16T15:00:00
Description: Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

This work was funded by National Nature Science Foundation of China for Young Scholars (No. 81300810), Shanghai Young Doctor Training Program (No. 20141057), and National Natural Science Foundation of China (81671982, 81271791 and 81571955). We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.

1. Biofilm Formation
<ol>
	<li>Biofilm formation in 96-well microplates
	<ol>
		<li>Retrieve the S. aureus strain USA300 and 3 biofilm-forming clinical strains (C1 - C3) stored at -80 &#176;C. 
		NOTE: The ability of the clinical strains to form biofilms was determined by the microtiter plate assay described previously15.</li>
		<li>Inoculate the bacterium in 5 mL tryptone soya broth (TSB) medium, and cultivate in an incubator with shaking at 37 &#176;C overnight t...

<ol>
	<li>Lewis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riddle+of+biofilm+resistance.">Riddle of biofilm resistance.</a> Antimicrob Agents Chemother. 45 (4), 999-1007 (2001).</li><li>Rabin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+mechanisms+and+targets+for+developing+antibiofilm+agents.">Biofilm formation mechanisms and targets for developing antibiofilm agents.</a> Future Med Chem. 7 (4), 493-512 (2015).</li><li>Mah, T. F., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+biofilm+resistance+to+antimicrobial+agents.">Mechanisms of biofilm resistance to antimicrobial agents.</a> Trends Microbiol. 9 (1), 34-39 (2001).</li><li>Sharma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toluidine+blue-mediated+photodynamic+effects+on+staphylococcal+biofilms.">Toluidine blue-mediated photodynamic effects on staphylococcal biofilms.</a> Antimicrob Agents Chemother. 52 (1), 299-305 (2008).</li><li>Rosa, L. P., da Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effectiveness+of+antimicrobial+photodynamic+therapy+(APDT)+using+a+660+nm+laser+and+malachite+green+dye+in+Staphylococcus+aureus+biofilms+arranged+on+compact+and+cancellous+bone+specimens.">In vitro effectiveness of antimicrobial photodynamic therapy (APDT) using a 660 nm laser and malachite green dye in Staphylococcus aureus biofilms arranged on compact and cancellous bone specimens.</a> Lasers Med Sci. 29 (6), 1959-1965 (2014).</li><li>Rosa, L. P., Silva, F. C., Nader, S. A., Meira, G. A., Viana, M. 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C., Buzzola, F., Casas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+photodynamic+inactivation+of+Staphylococcus+aureus+biofilms+by+disruptive+strategies.">Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.</a> Lasers Med Sci. 32 (8), 1757-1767 (2017).</li><li>Baltazar, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+photodynamic+therapy:+an+effective+alternative+approach+to+control+fungal+infections.">Antimicrobial photodynamic therapy: an effective alternative approach to control fungal infections.</a> Front Microbiol. 6, 202 (2015).</li><li>Fernandes, T., Bhavsar, C., Sawarkar, S., D'Souza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+novel+approaches+for+control+of+dental+biofilm.">Current and novel approaches for control of dental biofilm.</a> Int J Pharm. 536 (1), 199-210 (2017).</li><li>De Sordi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Photodynamic+Antimicrobial+Chemotherapy+(PACT)+for+Clostridium+difficile.">Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.</a> PLoS One. 10 (8), e0135039 (2015).</li><li>Harris, F., Pierpoint, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+based+on+5-aminolevulinic+acid+and+its+use+as+an+antimicrobial+agent.">Photodynamic therapy based on 5-aminolevulinic acid and its use as an antimicrobial agent.</a> Med Res Rev. 32 (6), 1292-1327 (2012).</li><li>Donnelly, R. F., McCarron, P. A., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+photodynamic+therapy.">Antifungal photodynamic therapy.</a> Microbiol Res. 163 (1), 1-12 (2008).</li><li>Shi, H., Li, J., Zhang, H., Zhang, J., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+5-aminolevulinic+acid+photodynamic+therapy+on+Candida+albicans+biofilms:+An+in+vitro+study.">Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study.</a> Photodiagnosis Photodyn Ther. 15, 40-45 (2016).</li><li>Zhang, Q. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-aminolevulinic+acid-mediated+photodynamic+therapy+and+its+strain-dependent+combined+effect+with+antibiotics+on+Staphylococcus+aureus+biofilm.">5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.</a> PLoS One. 12 (3), 0174627 (2017).</li><li>Chang, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+single+cell+detection+of+Staphylococcus+aureus+by+aptamer-conjugated+gold+nanoparticles.">Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.</a> Sci Rep. 3, 1863 (2013).</li><li>Barra, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+and+Antibiotic+Therapy+in+Combination+to+Fight+Biofilms+and+Resistant+Surface+Bacterial+Infections.">Photodynamic and Antibiotic Therapy in Combination to Fight Biofilms and Resistant Surface Bacterial Infections.</a> Int J Mol Sci. 16 (9), 20417-20430 (2015).</li><li>St Denis, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+you+need+is+light:+antimicrobial+photoinactivation+as+an+evolving+and+emerging+discovery+strategy+against+infectious+disease.">All you need is light: antimicrobial photoinactivation as an evolving and emerging discovery strategy against infectious disease.</a> Virulence. 2 (6), 509-520 (2011).</li><li>O'Neill, J. F., Hope, C. K., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+bacteria+in+multi-species+biofilms+can+be+killed+by+red+light+in+the+presence+of+toluidine+blue.">Oral bacteria in multi-species biofilms can be killed by red light in the presence of toluidine blue.</a> Lasers Surg Med. 31 (2), 86-90 (2002).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+5-aminolevulinic+acid-mediated+photodynamic+therapy+on+antibiotic-resistant+staphylococcal+biofilm:+an+in+vitro+study.">Effects of 5-aminolevulinic acid-mediated photodynamic therapy on antibiotic-resistant staphylococcal biofilm: an in vitro study.</a> J Surg Res. 184 (2), 1013-1021 (2013).</li><li>Hall-Stoodley, L., Costerton, J. W., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms:+from+the+natural+environment+to+infectious+diseases.">Bacterial biofilms: from the natural environment to infectious diseases.</a> Nat Rev Microbiol. 2 (2), 95-108 (2004).</li><li>Elias, S., Banin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-species+biofilms:+living+with+friendly+neighbors.">Multi-species biofilms: living with friendly neighbors.</a> FEMS Microbiol Rev. 36 (5), 990-1004 (2012).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+Proof+of+Programmed+5-Aminolevulinic+Acid+Prodrug+Nanocarriers+for+Targeted+Photodynamic+Cancer+Therapy.">Design and Proof of Programmed 5-Aminolevulinic Acid Prodrug Nanocarriers for Targeted Photodynamic Cancer Therapy.</a> ACS Appl Mater Interfaces. 9 (17), 14596-14605 (2017).</li></ol>

PDT has been a well-studied therapy for the treatment of cancer since it was invented more than 100 years ago18. Over the last decade, PDT has been applied as an antimicrobial strategy and has shown effectiveness against some antibiotic-resistant pathogenic bacteria12. Compared to the planktonic state, bacterial biofilms appear to be more resistant to antibiotic treatment3, while the effect of ALA-PDT on biofilms has not been fully investigated yet.<...

S. aureus is an important Gram-positive pathogen that colonizes the skin and mucosa of human hosts. Its ability to form biofilms is considered an important aspect of its pathogenesis1. Bacterial biofilms are a community of bacteria embedded in a self-produced matrix, which is composed of extracellular polymeric substances, including polysaccharide, DNA, and protein. This matrix plays a significant role in the persistence of bacterial infections, contributing to a high degree of resistance to the human immune system and current anti-microbial therapies2. Antibiotics are still the major treatment for biofilm infections, although the effects of antibiotics on biofilms are limited. It has been shown previously that cells in biofilms are 10 - 1,000 times more resistant to antibiotics compared to their planktonic counterparts3. Thus, alternative strategies are needed to conquer this issue.
PDT, an alternative treatment for bacterial infections, uses the light of an appropriate wavelength to activate photosensitizers. This leads to the production of reactive oxygen species (ROS), which are lethal to target cells by disrupting the cell wall, inactivating enzymes, and damaging DNA4. This multi-target characteristic makes it difficult for bacteria to develop resistance to the PDT treatment.
The antimicrobial effect of PDT on bacterial and fungal biofilms, with multiple photosensitizers, such as toluidine blue, malachite green, methylene blue, chlorine e6, and porphyrins, has been studied in previous reports5,6,7,8,9,10,11,12,13. 5-ALA, a prodrug of the actual photosensitizer, PpIX, is characterized by its small molecular weight and rapid clearance12,14. These advantages give ALA-PDT major potential as a therapeutic application. Although the effect of ALA-PDT on planktonic bacteria has been studied by many groups12, the antimicrobial effect of ALA-PDT on bacterial biofilms has not yet been elucidated. Meanwhile, it is difficult to compare the results between previous studies. One of the reasons is that the different protocols are used by diverse groups. Thus, this protocol describes an in vitro model of an ALA-PDT system based on our previous work15. The effect of this model was confirmed by CFU calculation and viability staining with CLSM.

<table>
	<tbody>
		<tr>
			<td>Tryptone Soya Broth (TSB)</td>
			<td>OXOID</td>
			<td>CM0129B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone Soya Agar (TSA)</td>
			<td>OXOID</td>
			<td>CM0131</td>
			<td></td>
		</tr>
		<tr>
			<td>SYTO9</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Thermo Fisher Scientific</td>
			<td>L7012</td>
			<td>The LIVE/DEAD BacLight Bacterial Viability Kits&#160;</td>
		</tr>
		<tr>
			<td>Pancreatin</td>
			<td>Sigma-Aldrich</td>
			<td>P3292</td>
			<td></td>
		</tr>
		<tr>
			<td>5-aminolevulinic acid (ALA)</td>
			<td>Fudan Zhangjiang Bio-Pharm</td>
			<td>3.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus strain USA300</td>
			<td>/</td>
			<td>/</td>
			<td>The source of USA 300 references &#8220;Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6&#8221;.</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus clinical strains (C1-C3)</td>
			<td>/</td>
			<td>/</td>
			<td>All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].</td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Corning Inc</td>
			<td>3599</td>
			<td>Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile</td>
		</tr>
		<tr>
			<td>Fluorodish</td>
			<td>NEST Biotechnology</td>
			<td>801001</td>
			<td>Glass bottom, Non-pyrogenic</td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5417</td>
			<td>Eppendorf</td>
			<td>Z365998 |&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>SHKE4000</td>
			<td>MaxQ 4000 Benchtop Orbital Shakers</td>
		</tr>
		<tr>
			<td>Light emitting diode (LED)</td>
			<td>Wuhan Yage Optic and Electronic Technique CO</td>
			<td>LED-IB</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 confocal laser-scanning microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS AF software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IMARIS software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

The viability of the bacteria in the biofilms was decreased after ALA-PDT treatment when compared to the controls (ALA-LED-, ALA+LED-, and ALA-LED+) in both USA300 and the three clinical strains (Figure 1).
To confirm the results from the CFU assay and observe the antibacterial effect of ALA-PDT on the S. aureus biofilm in situ, the USA300 biofilms were visualized by CLSM with viab...

Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

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