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 ...

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

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 generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

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 allergic reactions 1. In clinical practice, diagnosis of drug hypersensitivity is mainly performed by patient history, as skin testing is not reliable and oral provocation testing bears life-threatening risks for the patient 2. Hence, evidence for an underlying IgE-mediated pathomechanism is hard to obtain. 
Here, we present an in vitro method based on the use of human basophils derived from drug-hypersensitive patients that mimics the allergic effector reaction in vivo. As basophils of drug-allergic patients carry IgE molecules specific for the culprit drug, they become activated upon IgE receptor crosslinking and release allergic effector molecules. The activation of basophils can be monitored by the determination of the upregulation of CD63 surface expression using flow cytometry 3. 
In the case of low molecular weight drugs, conjugates are designed to enable IgE receptor crosslinking on basophils. As depicted in Figure 1, two representatives of NSAIDs, PP and DF, are covalently bound to human serum albumin (HSA) via a carboxyl group reacting with the primary amino group of lysine residues. DF carries an intrinsic carboxyl group and, thus, can be used directly 4, whereas a carboxyl group-containing derivative of PP had to be organochemically synthesized prior to the study 1.
The coupling degree of the low molecular weight compounds on the protein carrier molecule and their spatial distribution is important to guarantee crosslinking of two IgE receptor molecules. The here described protocol applies high performance-size exclusion chromatography (HPSEC) equipped with a sequential refractive index (RI) and ultra violet (UV) detection system for determination of the coupling degree.
As the described methodology may be applied for other drugs, the basophil activation test (BAT) bears the potential to be used for the determination of IgE-mediated mechanisms in drug hypersensitivity. Here, we determine PP hypersensitivity as IgE-mediated and DF hypersensitivity as non-IgE-mediated by BAT.

Basophil activation test is a potent tool for the detection of IgE-dependent allergies in vitro. Here, an optimized protocol for basophil activation test is used to investigate drug hypersensitivity. A method for the efficient production of covalent drug-protein conjugates and their physicochemical characterization is described.

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

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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 tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

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

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

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

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

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 (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

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

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

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

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

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

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

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

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

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, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load 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

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

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

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

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