Name: in vivo investigation of antimicrobial blue light therapy for multidrug-resistant acinetobacter baumannii burn infections using bioluminescence imaging
Uploaded: 2017-04-28T14:00:00
Description: 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

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. 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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. 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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. 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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. 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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. 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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.

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	<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 ...

Research

JoVE Journal

Immunology and Infection

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Basophil Activation Test for Investigation of IgE-Mediated Mechanisms in Drug Hypersensitivity

Hypersensitivity reactions against non-steroidal anti-inflammatory drugs (NSAIDs) like propyphenazone (PP) and diclofenac (DF) can manifest as Type I-like ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary ...