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

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.