This work was supported in part by the Center for Integration of Medicine and Innovative Technology (CIMIT) under the U.S. Army Medical Research Acquisition Activity Cooperative Agreement (CIMIT No. 14-1894 to TD) and the National Institutes of Health (1R21AI109172 to TD). YW was supported by an ASLMS Student Research Grant (BS.S02.15). We are grateful to Tayyaba Hasan, PhD at the Wellman Center for her co-mentorship for YW.

1. Preparation of Bacterial Culture
<ol>
	<li>Add 7.5 mL of Brain Heart Infusion (BHI) medium to a 50 mL centrifuge tube. Seed A. baumannii cells in the BHI medium and then incubate the A. baumannii culture in an orbital incubator (37 &#176;C) for 18 h.</li>
	<li>Centrifuge the culture of cells at 3,500 &#215; g for 5 min, remove the supernatant, and wash the pellets in phosphate-buffered saline (PBS).</li>
	<li>Re-suspend the bacteria pellets in fresh PBS and thoroughly pipette the suspension.</li>
	...

<ol>
	<li>Gibran, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summary+of+the+2012+ABA+Burn+Quality+Consensus+conference.">Summary of the 2012 ABA Burn Quality Consensus conference.</a> J Burn Care Res. 34 (4), 361-385 (2013).</li><li>Sommer, R., Joachim, I., Wagner, S., Titz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+to+control+infections:+anti-biofilm+strategies+against+gram-negative+bacteria.">New approaches to control infections: anti-biofilm strategies against gram-negative bacteria.</a> Chimia (Aarau). 67 (4), 286-290 (2013).</li><li>Peleg, A. Y., Seifert, H., Paterson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acinetobacter+baumannii:+emergence+of+a+successful+pathogen.">Acinetobacter baumannii: emergence of a successful pathogen.</a> Clin Microbiol Rev. 21 (3), 538-582 (2008).</li><li>Uppu, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+side+chain+amphiphilic+polymers+disrupt+surface+established+bacterial+bio-films+and+protect+mice+from+chronic+Acinetobacter+baumannii+infection.">Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.</a> Biomaterials. 74, 131-143 (2016).</li><li>Schaber, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+forms+biofilms+in+acute+infection+independent+of+cell-to-cell+signaling.">Pseudomonas aeruginosa forms biofilms in acute infection independent of cell-to-cell signaling.</a> Infect Immun. 75 (8), 3715-3721 (2007).</li><li>Hoiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+resistance+of+bacterial+biofilms.">Antibiotic resistance of bacterial biofilms.</a> Int J Antimicrob Agents. 35 (4), 322-332 (2010).</li><li>Lebeaux, D., Ghigo, J. M., Beloin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-related+infections:+bridging+the+gap+between+clinical+management+and+fundamental+aspects+of+recalcitrance+toward+antibiotics.">Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics.</a> Microbiol Mol Biol Rev. 78 (3), 510-543 (2014).</li><li>Akers, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+and+persistent+wound+infections+in+United+States+military+trauma+patients:+a+case-control+analysis.">Biofilms and persistent wound infections in United States military trauma patients: a case-control analysis.</a> BMC Infect Dis. 14, 190 (2014).</li><li>Burmolle, 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=Biofilms+in+chronic+infections+-+a+matter+of+opportunity+-+monospecies+biofilms+in+multispecies+infections.">Biofilms in chronic infections - a matter of opportunity - monospecies biofilms in multispecies infections.</a> FEMS Immunol Med Microbiol. 59 (3), 324-336 (2010).</li><li>. National strategy on combating antibiotic-resistant bacteria Available from: <a target="_blank" href="https://www.whitehouse.gov/sites/default/files/docs/carb_national_strategy.pdf">https://www.whitehouse.gov/sites/default/files/docs/carb_national_strategy.pdf</a> (2014)</li><li>Dai, T. <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+for+Acinetobacter+baumannii+burn+infections+in+mice.">Photodynamic therapy for Acinetobacter baumannii burn infections in mice.</a> Antimicrob Agents Chemother. 53 (9), 3929-3934 (2009).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+blue+light+therapy+for+multidrug-resistant+Acinetobacter+baumannii+infection+in+a+mouse+burn+model:+implications+for+prophylaxis+and+treatment+of+combat-related+wound+infections.">Antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model: implications for prophylaxis and treatment of combat-related wound infections.</a> J Infect Dis. 209 (12), 1963-1971 (2014).</li><li>Dai, T., 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=Ultraviolet+C+light+for+Acinetobacter+baumannii+wound+infections+in+mice:+potential+use+for+battlefield+wound+decontamination?.">Ultraviolet C light for Acinetobacter baumannii wound infections in mice: potential use for battlefield wound decontamination?.</a> J Trauma Acute Care Surg. 73 (3), 661-667 (2012).</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light+for+infectious+diseases:+Propionibacterium+acnes,+Helicobacter+pylori,+and+beyond?.">Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond?.</a> Drug Resist Updat. 15 (4), 223-236 (2012).</li><li>Yin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+based+anti-infectives:+ultraviolet+C+irradiation,+photodynamic+therapy,+blue+light,+and+beyond.">Light based anti-infectives: ultraviolet C irradiation, photodynamic therapy, blue light, and beyond.</a> Curr Opin Pharmacol. 13 (5), 731-762 (2013).</li><li>Haisma, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+antimicrobial+responses+to+methicillin-resistant+Staphylococcus+aureus+in+an+in+vitro+wound+infection+model.">Inflammatory and antimicrobial responses to methicillin-resistant Staphylococcus aureus in an in vitro wound infection model.</a> PLoS One. 8 (12), e82800 (2013).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+Blue+Light+Inactivation+of+Gram-Negative+Pathogens+in+Biofilms:+In+Vitro+and+In+Vivo+Studies.">Antimicrobial Blue Light Inactivation of Gram-Negative Pathogens in Biofilms: In Vitro and In Vivo Studies.</a> J Infect Dis. 213 (9), 1380-1387 (2016).</li><li>Chen, D., Shen, Y., Huang, Z., Li, B., Xie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Emitting+Diode-Based+Illumination+System+for+In+Vitro+Photodynamic+Therapy.">Light-Emitting Diode-Based Illumination System for In Vitro Photodynamic Therapy.</a> Int J Photoenergy. 2012 (2), (2012).</li><li>Demidova, T. N., Gad, F., Zahra, T., Francis, K. P., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+photodynamic+therapy+of+localized+infections+by+bioluminescence+imaging+of+genetically+engineered+bacteria.">Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria.</a> J Photochem Photobiol B. 81 (1), 15-25 (2005).</li><li>Hamblin, M. R., Zahra, T., Contag, C. H., McManus, A. T., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+and+treatment+of+potentially+lethal+wound+infections+in+vivo.">Optical monitoring and treatment of potentially lethal wound infections in vivo.</a> J Infect Dis. 187 (11), 1717-1725 (2003).</li><li>Rowan, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+wound+healing+and+treatment:+review+and+advancements.">Burn wound healing and treatment: review and advancements.</a> Critical Care. 19, 243 (2015).</li><li>Marx, D. E., Barillo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+in+medicine:+The+basic+science.">Silver in medicine: The basic science.</a> Burns. 40 (Supplement 1), S9-S18 (2014).</li><li>Heyneman, A., Hoeksema, H., Vandekerckhove, D., Pirayesh, A., Monstrey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+silver+sulphadiazine+in+the+conservative+treatment+of+partial+thickness+burn+wounds:+A+systematic+review.">The role of silver sulphadiazine in the conservative treatment of partial thickness burn wounds: A systematic review.</a> Burns. 42 (7), 1377-1386 (2016).</li><li>Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individualised+antibiotic+dosing+for+patients+who+are+critically+ill:+challenges+and+potential+solutions.">Individualised antibiotic dosing for patients who are critically ill: challenges and potential solutions.</a> Lancet Infect Dis. 14 (6), 498-509 (2014).</li><li>Dai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+light+eliminates+community-acquired+methicillin-resistant+Staphylococcus+aureus+in+infected+mouse+skin+abrasions.">Blue light eliminates community-acquired methicillin-resistant Staphylococcus aureus in infected mouse skin abrasions.</a> Photomed Laser Surg. 31 (11), 531-538 (2013).</li><li>Uppu, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+side+chain+amphiphilic+polymers+disrupt+surface+established+bacterial+bio-films+and+protect+mice+from+chronic+Acinetobacter+baumannii+infection.">Amide side chain amphiphilic polymers disrupt surface established bacterial bio-films and protect mice from chronic Acinetobacter baumannii infection.</a> Biomaterials. 74, 131-143 (2016).</li><li>Donlan, R. M., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+survival+mechanisms+of+clinically+relevant+microorganisms.">Biofilms: survival mechanisms of clinically relevant microorganisms.</a> Clin Microbiol Rev. 15 (2), 167-193 (2002).</li><li>Olsen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+tolerance+and+resistance.">Biofilm-specific antibiotic tolerance and resistance.</a> Eur J Clin Microbiol Infect Dis. , (2015).</li><li>Song, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxic+effect+of+blue+light+on+the+planktonic+and+biofilm+state+of+anaerobic+periodontal+pathogens.">Phototoxic effect of blue light on the planktonic and biofilm state of anaerobic periodontal pathogens.</a> J Periodontal Implant Sci. 43 (2), 72-78 (2013).</li><li>Rosa, L. P., da Silva, F. C., Viana, M. S., Meira, 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=In+vitro+effectiveness+of+455-nm+blue+LED+to+reduce+the+load+of+Staphylococcus+aureus+and+Candida+albicans+biofilms+in+compact+bone+tissue.">In vitro effectiveness of 455-nm blue LED to reduce the load of Staphylococcus aureus and Candida albicans biofilms in compact bone tissue.</a> Lasers Med Sci. 31 (1), 27-32 (2015).</li><li>Guffey, J. S., Wilborn, J. <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+bactericidal+effects+of+405-nm+and+470-nm+blue+light.">In vitro bactericidal effects of 405-nm and 470-nm blue light.</a> Photomed Laser Surg. 24 (6), 684-688 (2006).</li><li>Enwemeka, C. S., Williams, D., Enwemeka, S. K., Hollosi, S., Yens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+470-nm+light+kills+methicillin-resistant+Staphylococcus+aureus+(MRSA)+in+vitro.">Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro.</a> Photomed Laser Surg. 27 (2), 221-226 (2009).</li><li>Bumah, V. V., Masson-Meyers, D. S., Cashin, S. E., Enwemeka, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelength+and+bacterial+density+influence+the+bactericidal+effect+of+blue+light+on+methicillin-resistant+Staphylococcus+aureus+(MRSA).">Wavelength and bacterial density influence the bactericidal effect of blue light on methicillin-resistant Staphylococcus aureus (MRSA).</a> Photomed Laser Surg. 31 (11), 547-553 (2013).</li><li>Maclean, M., MacGregor, S. J., Anderson, J. 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The authors declare that they have no competing financial interests.

aBL is a novel method for treating infections. Since its mechanism of action is completely different from that of chemotherapy, it is more of a physiotherapy. The agent that mediates the antimicrobial effect is blue light irradiation (400-470 nm). With the development of blue LEDs, we gained access to an effective and simple light-based antimicrobial approach for MDR infections.
In this protocol, we have described the development of a mouse model of burn infections caused by a bioluminescent s...

Burn infections, which are frequently reported because of cutaneous thermal injuries, continue to be an important cause of morbidity and mortality1. The management of burn infections has been further compromised by the increasing emergence of multidrug-resistant (MDR) bacterial strains2 due to the massive use of antibiotics. One important MDR Gram-negative bacteria is Acinetobacter baumannii, which is known to be associated with recent battle wounds and is resistant to almost all available antibiotics3. The presence of biofilms at the injured foci has been reported4,5 and is believed to exacerbate the tolerance to antibiotics and host defense6,7, causing persistent infections8,9. Therefore, there is a pressing need for the development of alternative treatments. In the recently announced National Strategy for Combating Antibiotic-Resistant Bacteria, the development of alternative therapeutics to antibiotics has been noted as an action by the government of the United States10.
Light-based antimicrobial approaches, as indicated by the name, require light irradiation with or without other agents. These approaches include antimicrobial photodynamic therapy (aPDT), ultraviolet-C (UVC) irradiation, and antimicrobial blue light (aBL). In previous studies, they have shown promising effectiveness in killing MDR bacterial strains11,12,13. Among the three light-based approaches, aBL has attracted increasing attention in recent years due to its intrinsic antibacterial properties without the use of photosensitizers14. In comparison to aPDT, aBL only involves the use of light, while aPDT requires a combination of light and a photosensitizer. Therefore, aBL is simple and inexpensive14. In comparison to UVC, aBL is believed to be much less cytotoxic and genotoxic to host cells15.
The goal of this protocol is to investigate the effectiveness of aBL for the treatment of burn infections caused by MDR A. baumannii in a mouse model. We use bioluminescent pathogenic bacteria to develop new mouse models of burn infections that allow the non-invasive monitoring of the bacterial burden in real time. Compared to the traditional method of body fluid/tissue sampling and subsequent plating and colony counting16, this technique provides accurate results. The process of tissue sampling could introduce another source of experimental error. Since the bacterial luminescence intensity is linearly proportional to the corresponding bacterial CFU17, we can directly measure the survival of bacteria after a certain dose of light irradiation. By monitoring the bacterial burden in living animals receiving the light treatment in real time, the kinetics of bacterial killing can be characterized using a significantly reduced number of mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IVIS&#160;</td>
			<td>PerkinElmer Inc, Waltham, MA</td>
			<td>IVIS Lumina Series III</td>
			<td>Pre-clinical in vivo imaging</td>
		</tr>
		<tr>
			<td>Light-emitting diode LED</td>
			<td>VieLight Inc, Toronto, Canada&#160;</td>
			<td>415 nm</td>
			<td>Light source for illumination</td>
		</tr>
		<tr>
			<td>Power/energy meter</td>
			<td>Thorlabs, Inc., Newton, NJ</td>
			<td>PM100D</td>
			<td>Light irradiance detector</td>
		</tr>
		<tr>
			<td>Mouse&#160;</td>
			<td>Charles River Laboratories, Wilmington, MA</td>
			<td>BALB/c</td>
			<td>7-8 weeks age, 17-19 g weight</td>
		</tr>
		<tr>
			<td>Acinetobacter baumannii&#160;</td>
			<td>Brooke Army Medical Center, Fort Sam Houston, TX</td>
			<td>Clinical isolate</td>
			<td>Engineered luminescent strain</td>
		</tr>
		<tr>
			<td>Insulin Syringes</td>
			<td>Fisher Scientific</td>
			<td>14-826-79</td>
			<td>BD Lo-Dose U-100 Insulin Syringes for injection</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>721016</td>
			<td>0.9% Sodium Chloride</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1X Solution</td>
			<td>Fisher Scientific</td>
			<td>BP24384&#160;</td>
			<td>A standard phosphate buffer used in many biomolecular procedures</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Fisher Scientific</td>
			<td>B11059</td>
			<td>Bacterial culture medium</td>
		</tr>
		<tr>
			<td>Falcon 15mL Conical Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-959-70C</td>
			<td>For bacterial suspension centrifuge</td>
		</tr>
		<tr>
			<td>Benchtop Incubated Orbital Shakers</td>
			<td>Laboratory Supply Network, Inc, Atkinson, NH&#160;</td>
			<td>Incu-Shaker Mini</td>
			<td>For culturing of bacteria</td>
		</tr>
		<tr>
			<td>Inoculating Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-605&#160;&#160;</td>
			<td>For smearing bacterial inoclum on burn surface of mice</td>
		</tr>
		<tr>
			<td>Fisher Scientific Redi-Tip Pipet Tips, 1-200&#181;L</td>
			<td>Fisher Scientific</td>
			<td>02-707-502</td>
			<td>Pipet Tips</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Sorvall Legend X1 Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75-004-220</td>
			<td>For bacterial suspension seperation</td>
		</tr>
		<tr>
			<td>Brass Block</td>
			<td>Small Parts, Inc., Miami, FL</td>
			<td>10 mm by 10 mm&#160;</td>
			<td>For creation of burns in mice</td>
		</tr>
		<tr>
			<td>Extreme Dragon PBI/Kevlar High-Heat Gloves</td>
			<td>Superior Glove Works Ltd, Cheektowaga, NY</td>
			<td>PBI83514&#160;</td>
			<td>Heat Resistant Gloves</td>
		</tr>
		<tr>
			<td>Greiner dishes</td>
			<td>Sigma-Aldrich Co. LLC</td>
			<td>P5112-740EA</td>
			<td>35 mm &#215;10 mm</td>
		</tr>
		<tr>
			<td>Corning Digital Hot Plate</td>
			<td>Cole-Parmer Instrument Company, LLC</td>
			<td>UX-84301-65</td>
			<td>10&#34; x 10&#34;, 220 VAC, for boiling water&#160;</td>
		</tr>
		<tr>
			<td>Mouse/Rat Thin Line Water Heated Surgical Bed</td>
			<td>E-Z Systems</td>
			<td>EZ-211</td>
			<td>Prevents heat loss and hypothermia during surgery</td>
		</tr>
	</tbody>
</table>

in vivo investigation of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii burn infections using bioluminescence imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

The A. baumannii strain that we used is an MDR clinical isolate, as reported previously12,17. The bacterial strain was made bioluminescent by the transfection of luxCDABE opera11. Figure 1A shows the successive bacterial luminescence images from a representative mouse burn infected with 5 x 106A. baumannii and exposed to a single aBL exposure at 24 h af...

Watch this Scientific Journal Video about In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging at JoV

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

The overall goal of this experiment is to observe the effectiveness of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii infections in mouse burns. This protocol can help us in answering key questions in the light based antimicrobial approach field such as whether antimicrobial blue light is effective in eliminating localized bacteria infections. Including those closer by multidrug-resistant strains.The main advantage of antimicrobial blue light is that it is a non-antibiotic approach without the involvement exogenous photosensitizer and is likely to be non-injurious to host cells and tissues. The implications of the technique extend towards therapy of multidrug-resistant bacterial infections, because antimicrobial blue light kills pathogenic bacteria regardless of their drug-resistant patterns. Though this method can provide insight into the antimicrobial effect of blue light on acinetobacter baumannii burn infections, it can also be applied to other types of localized infections caused by other bacterial species.We first had the idea for this method, when we were exploring the use of simple light-based antimicrobial approaches against multidrug-resistant localized infections. After anesthetizing a mouse, according to the text protocol, use a 50 blade hair clipper to shave the mouse on the back to expose as much skin as possible. Then, place the lid of a 35 millimeter petri dish underneath the mouse abdomen to keep the back in a relatively horizontal position.Use a 10 inch by 10 inch 220 VAC hot plate to boil water in a 150 milliliter beaker. Immerse a brass block into the beaker until thermal equilibration with the water is reached. Prior to creating the burn injury, administer a subcutaneous injection of preemptive analgesics for pain relief.10 minutes later, while wearing thermal gloves, gently press the heated brass block to the shaved area on the back of the mouse for seven seconds to induce burn wounds. To prevent hydration administer 0.5 milliliters of sterile saline through subcutaneous injections. Five minutes following the induction of the thermal injury, use a pipette to inoculate 50 microliters of bacterial suspension containing five times 10 to the sixth CFU and PBS onto the mouse burns.Then, by moving a sterile cotton swab in a zigzag motion on the skin, smear the aliquot on the burns to distribute the bacterial cells in the burned area as evenly as possible. Immediately following the inoculation, carry out bioluminescence imaging by starting the live imaging software. In the control panel, click initialize.Then wait until the color of the temperature box turns green, indicating that temperature of the stage in the specimen chamber has reached 37 degrees celsius. Place the anesthetized mouse on the warm stage in the specimen chamber with the infected burns directly under the camera. Next, in the control panel, put a check mark next to luminescent.Then select auto-exposure so that the exposure time for imaging will be optimized by the live imaging software based on the bioluminescence intensity. Select C from the field of view drop down list. Select the skin mid-range option to let the software determine the focal distance.Then put a check mark next to overlay. Now click acquire to capture the image. Then, in the edit image label box, click ok.An image window and tool palette will appear. Set the Auto-ROI parameters for auto-selection. 24 hours after bacterial infection, to carry out antimicrobial blue light therapy, use clip connectors to fix the LED to an optical support rod to allow the LED to move up and down.Once the power energy meter has been turned on, and the 415 nanometer wavelength has been selected, place the power energy meter right under the LED. While wearing blue light protective goggles, turn on the LED light and adjust the distance between the LED aperture and the light sensor of the power energy meter, so that the light spot covers the whole area of the light sensor. Carefully, tune the T-cube LED driver and record the reading of the power energy meter.Calculate the irradiance according to the reading. Adjust the irradiance of the LED to 100 milliwatt per centimeters squared, by tuning the T-cube LED driver. Then turn off the LED and measure the distance between the LED aperture and the light sensor of the power energy meter.After anesthetizing the mice, according to the text protocol, randomly divide the animals into an antimicrobial blue light or aBL treated group and an untreated control group. For the aBL treated group, use aluminum foil to cover the eyes of the mice to avoid overexposure to light. Place the mouse burns directly under the LED with a lid of a 35 millimeter petri dish underneath the mouse abdomen to keep the back in a horizontal position.Next, replace the power energy meter with a mouse on a square petri dish. Then adjust the height of the mouse back to a position where the distance between the LED aperture and the surface of the mouse burn is equal to the distance of between the LED aperture and the light sensor of the power energy meter. Irradiate the infected burns at an irradiance of 100 milliwatts per centimeters squared.Deliver aBL in aliquots of 72 joules per centimeters squared until the total dose of 360 joules per centimeters squared is reached. After each light dose, perform bioluminescence imaging of the mouse burns, as demonstrated earlier in this video. For the untreated control group, perform bioluminescence imaging of the mouse burns using the same time intervals as used for the ABL-treated group.The A.baumannii bacterial strain was made bioluminescent by the transfection of the luxCDABE opera. This figure shows successive bacterial luminescence images from a representative mouse burn infected with five times 10 to the sixth A.baumannii and exposed to 360 joules per centimeters squared of aBL, 24 hours after bacterial inoculation. As demonstrated in this panel, the bacterial luminescence was almost eradicated after an exposure of 360 joules per centimeters squared of aBL was delivered.Shown here is a gram stain of the histological section of a representative mouse skin burned specimen harvested at 24 hours post-inoculation, that demonstrates the presence of A.baumannii biofilms on the surface of the infected burn. This plot illustrates the dose response curve of the mean bacterial luminescence from mouse burns infected with five times 10 to the sixth A.baumannii and treated with aBL 24 hours after bacterial inoculation. To achieve a three log base 10 inactivation of A.baumannii in mouse burns, approximately 360 joules per centimeters squared aBL was required.The bacterial luminescence of the mouse burns unexposed to aBL remained almost unchanged during an equivalent period of time. Once mastered, this technique can be done in one hour if it's performed properly. While attempting this procedure, it's important to get the access to bioluminescence strains of bacteria.After it's development, this technique paved the way for researchers in the field of light based antimicrobial therapy to explore the use of antimicrobial blue light, without an exogenous photosensitizer, against localized bacterial infections, including those caused by multidrug-resistant strains. After watching this video, you should have a good understanding of how to use antimicrobial blue light to prevent and treat localized infectious diseases. Don't forget, that working with visible light and pathogenic bacteria can be extremely hazardous.And precautions such as wearing laboratory gloves and light protected goggles should always be taken while performing this procedure.

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Research

JoVE Journal

Immunology and Infection

9.2K vistas.  Massachusetts General Hospital, Harvard Medical School. The overall goal of this experiment is to observe the effectiveness of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii infections in mouse burns. This protocol can help us in answering key questions in the light based antimicrobial approach field such as whether antimicrobial blue light is effective in eliminating localized bacteria infections. Including those closer by multidrug-resistant strains.The main advantage of antimicrobial blue light is that i...

Video: In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging