Monitoring of bactericidal action of laser by in vivo imaging of bioluminescent E. coli in a cutaneous wound infection

Abstract

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. This study aimed to evaluate the bactericidal action of an 810-nm diode laser in a cutaneous wound infection. An Escherichia coli strain was transformed with a shuttle vector (pRB474) containing firefly luciferase gene from Photinus pyralis resulting in a bioluminescent phenotype. Because firefly luciferase is an enzyme and as such is prone to inactivation at elevated temperature, the first phase has consisted in evaluating in vitro the effect of temperature elevation (30, 40, 50, and 60°C for 2 min) on bacteria bioluminescence. The second phase was performed in vivo. Two full-thickness circular, 14-mm diameter wounds (control and laser-irradiated) were induced on rats. Wound infection was carried out using a suspension (50 μl PBS) containing 5x10⁷ cells of bioluminescent E. coli (10⁹ cells/ml). Thirty minutes later, light irradiation was performed with an 810-nm diode laser (P=10 W, ∅=1.4 cm, fluence: 130, 195, and 260 J/cm²). Temperature was measured within each wound with a noncontact infrared thermometer. Light emission of the bioluminescent bacteria was monitored in vivo by a bioluminescence imaging system before and at 4, 8, 24, and 48 h after laser irradiation. In vitro, bacteria bioluminescence is not affected when temperature is maintained at 50°C for 2 min. In vivo, bioluminescence imaging showed that at 4 h, the viability of E. coli was reduced when compared to the control (CTRL) group (p<0.01). This observation was confirmed at 8 h (p<0.001), at 24 h (p<0.001), and finally at 48 h (p<0.001). Loss of viability of E. coli depends on laser fluence. At 48 h, bioluminescent bacteria were not detected (100% loss of viability) in the wound irradiated at 260 J/cm². For this fluence, the temperature reached 45°C at the end of the irradiation. This study confirms previous observations on the bactericidal effect of diode lasers. Because a progressive desiccation of the superficial dermis is usually observed when using laser irradiation, the hypothesis that laser irradiation dries out the wound making the wound an inhospitable place for bacteria is much more relevant than a direct effect of infrared light on chromophores inside bacteria. This is confirmed by the fact that in this latter case, one would expect an immediate drop in luminescence followed by an increase as the surviving bacteria started to divide and repopulate the wound. However, the exact mechanism deserves further studies. This study points out the advantage of using bioluminescence imaging to evaluate laser for the treatment of acute infections in vivo, nondestructively, and noninvasively.