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.
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.
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.
1. Preparation of Bacterial Culture
2. Mouse Model of Burn Infection Caused by Bioluminescent A. baumannii
3. Antimicrobial Blue Light Therapy for A. baumannii Infection in Mice
4. Bioluminescence Imaging of Infections in Mice
5. Euthanasia of the Mice
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 after bacterial inoculation. A Gram-stain of the histological section of a representative mouse skin burn specimen (harvested at 24 h post-inoculation) demonstrated the presence of A. baumannii biofilms on the surface of the infected burn (Figure 1B). As shown in Figure 1A, the bacterial luminescence was almost eradicated after an exposure of 360 J/cm2 aBL was delivered (60 min of irradiation at an irradiance of 100 mW/cm2). Figure 1C is the dose-response curve of the mean bacterial luminescence from mouse burns infected with 5 x 106A. baumannii and treated with aBL at 24 h after bacterial inoculation (n = 10). To achieve a 3-log10 inactivation of A. baumannii in mouse burns, approximately 360 J/cm2 aBL was required. The bacterial luminescence of the mouse burns unexposed to aBL remained almost unchanged during an equivalent period of time (data not shown; P <0.001).
Figure 1: aBL Inactivation of Bacteria in Infected Mouse Burns. (A) Successive bacterial luminescence images from a representative mouse burn infected with 5 x 106 CFU of A. baumannii and exposed to 360 J/cm2 aBL at 24 h after bacterial inoculation. (B) Gram-stained histological section of a representative mouse skin burn showing the presence of A. baumannii biofilms (arrows) in the mouse burn. The skin sample was harvested at 24 h after bacterial inoculation. (C) Dose-response curve of mean bacterial luminescence of mouse burns infected with 5 x 106A. baumannii and treated with an exposure of 360 J/cm2 aBL at 24 h (n = 10) after bacterial inoculation. Bars: standard deviation. Please click here to view a larger version of this figure.
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 strain of MDR, A. baumannii. With the use of bioluminescent bacteria, the extent of infection can be non-invasively monitored in real time in living animals via bioluminescent imaging. The use of engineered bioluminescent strains of bacteria and the low-light imaging technique creates an efficient technique for monitoring infections in real-time during antimicrobial therapy. This method can also be used in the investigations of infections caused by other microbial species and located at other sites. Besides the efficacy assessment of antimicrobial approaches, this method can also be used to track the progress of infection.
By using this mouse model, we demonstrated that aBL (415 nm) successfully inactivated bacteria in established infections (Figure 1A and C). Prior to aBL therapy, clusters of bacteria were observed in the established infections (Figure 1B), which is a feature of biofilms. Biofilms are more tolerant of traditional antibiotics and host defense compared to their planktonic counterparts6,7 and are frequently associated with persistent infections8,9. The representative results are promising in that 415-nm aBL is biofilm-penetrating. In addition, together with previous reports29,30,31,32, our results demonstrate that the effectiveness of aBL persists regardless of the drug-resistance profile of bacteria.
The protocol described here involves three main procedures: (1) the development of a mouse model of burn infections, (2) aBL therapy, and (3) bioluminescence imaging. While developing a mouse model of burn infections, we noted that there were several factors that affect the extent of infection and the subsequent effectiveness of aBL: (1) The burning time affects the wound depth and the proliferation of bacteria. When the burning time was increased from 3 to 7 s, the bacterial luminescence was much stronger (indicating a higher extent of infection) at 24 h post-inoculation, and the eradication of infection required much higher aBL exposures (>360 J/cm2). (2) The inoculum of the bacteria is a key parameter for the development of infections. A higher bacterial inoculum usually results in a higher extent of infection, while a sufficiently low inoculum frequently fails to develop stable infections in mice. In the latter condition, bacterial luminescence usually becomes undetectable soon after bacterial inoculation. (3) The interaction between bacteria and hosts is dependent upon the bacterial species. We also used P. aeruginosa to develop an infection model. We found that, under the same conditions (i.e., burning time and bacterial inoculum), the infections caused by P. aeruginosa progressed much more rapidly than A. baumannii infections, and sepsis was always observed in mice within 48 h post-inoculation25.
For the execution of aBL therapy, there are several important points that need to be addressed: (1) Proper light irradiance is required for the maximized efficacy of aBL therapy. (2) The surface of the burn in the mice should be placed as horizontally as possible. A failure to appropriately position the burn surface can compromise the efficacy of aBL therapy. (3) During light exposure, it is suggested that the eyes of mice be protected with aluminum foil, especially when a laser is used as the light source. (4) During light exposure, care should be taken to monitor the mice in case they awaken from anesthesia. In this case, a small additional dose of anesthetics should be administrated to keep the animals anesthetized. (5) Both aBL-treated mice and untreated mice should be placed on a heating bed to maintain the body temperature when under anesthesia. During the process of bioluminescence imaging, the bioluminescence of bacteria could decrease when the burns become dry. Therefore, it is recommended to moisturize the mouse burns with PBS before imaging.
There are also some limitations of the techniques discussed in this protocol: (1) For the purpose of monitoring of the extent of infection in real time, bioluminescent bacterial strains must be used. Therefore, before a clinical strain can be tested in the animal model, it must be genetically modified by the transfection of the lux CDABE operon11. (2) The effectiveness of aBL is related to the wavelengths33 and bacterial species/strains34 used. The blue wavelengths, together with other parameters, should be further optimized for inactivating different bacterial species/strains. (3) We only investigated superficial infections in mice. For deep-seated infections, the topical delivery of aBL may not be able to reach the infections, so interstitial light delivery may be needed35. (4) There is a sensitivity limitation of the imaging system, especially when imaging deep infections19. As a result, even when the pixels of bioluminescence are completely eliminated, there might still be viable bacterial cells remaining, allowing bacterial regrowth to occur. An extended exposure to aBL is recommended after the elimination of bacterial luminescence in order to prevent bacterial regrowth.
The authors have nothing to disclose.
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.
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 15mL 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, 1-200µL | 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 |