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

Summary

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.

Abstract

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.

Introduction

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

Protocol

1. Preparation of Bacterial Culture

1. 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 °C) for 18 h.
2. Centrifuge the culture of cells at 3,500 × g for 5 min, remove the supernatant, and wash the pellets in phosphate-buffered saline (PBS).
3. Re-suspend the bacteria pellets in fresh PBS and thoroughly pipette the suspension.
4. Collect 100 µL of the bacterial suspension and make a 1:10 dilution using fresh PBS.
5. Transfer the dilution to a 1.5 mL semi-micro c....

Results

The A. baumannii strain that we used is an MDR clinical isolate, as reported previously^12,17. 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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IVIS	PerkinElmer Inc, Waltham, MA	IVIS Lumina Series III	Pre-clinical in vivo imaging
Light-emitting diode LED	VieLight Inc, Toronto, Canada	415 nm	Light source for illumination
Power/energy meter	Thorlabs, Inc., Newton, NJ	PM100D	Light irradiance detector
Mouse	Charles River Laboratories, Wilmington, MA	BALB/c	7-8 weeks age, 17-19 g weight
Acinetobacter baumannii	Brooke Army Medical Center, Fort Sam Houston, TX	Clinical isolate	Engineered luminescent strain
Insulin Syringes	Fisher Scientific	14-826-79	BD Lo-Dose U-100 Insulin Syringes for injection
Sodium Chloride	Fisher Scientific	721016	0.9% Sodium Chloride
Phosphate Buffered Saline, 1x Solution	Fisher Scientific	BP24384	A standard phosphate buffer used in many biomolecular procedures
Brain Heart Infusion	Fisher Scientific	B11059	Bacterial culture medium
Falcon 15 mL Conical Centrifuge Tubes	Fisher Scientific	14-959-70C	For bacterial suspension centrifuge
Benchtop Incubated Orbital Shakers	Laboratory Supply Network, Inc, Atkinson, NH	Incu-Shaker Mini	For culturing of bacteria
Inoculating Loops	Fisher Scientific	22-363-605	For smearing bacterial inoclum on burn surface of mice
Fisher Scientific Redi-Tip Pipet Tips, 1-200 µL	Fisher Scientific	02-707-502	Pipet Tips
Thermo Scientific Sorvall Legend X1 Centrifuge	Fisher Scientific	75-004-220	For bacterial suspension seperation
Brass Block	Small Parts, Inc., Miami, FL	10 mm by 10 mm	For creation of burns in mice
Extreme Dragon PBI/Kevlar High-Heat Gloves	Superior Glove Works Ltd, Cheektowaga, NY	PBI83514	Heat Resistant Gloves
Greiner dishes	Sigma-Aldrich Co. LLC	P5112-740EA	35 mm ×10 mm
Corning Digital Hot Plate	Cole-Parmer Instrument Company, LLC	UX-84301-65	10" x 10", 220 VAC, for boiling water
Mouse/Rat Thin Line Water Heated Surgical Bed	E-Z Systems	EZ-211	Prevents heat loss and hypothermia during surgery
Vet ointment	S&K Pharma, GmbH	Kerato Biciron 5%, Augensalbe	opthalmic ointment to prevent eye dryness