Monitoring photodynamic therapy of localized infections by bioluminescence imaging of genetically engineered bacteria

Abstract

The increasing occurrence of multi-antibiotic resistant microbes has led to the search for alternative methods of killing pathogens and treating infections. Photodynamic therapy (PDT) uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that can kill mammalian and microbial cells. Although the photodynamic inactivation of bacteria has been known for over a hundred years, its use to treat infections has not been much developed. This may be partly due to the difficulty of monitoring the effectiveness of PDT in animal models of infection. In order to facilitate this monitoring process, we have developed a procedure that uses bioluminescent genetically engineered bacteria and a light sensitive imaging system to allow real-time visualization of infections. When these bacteria are treated with PDT in vitro, the loss of luminescence parallels the loss of colony-forming ability. We have developed several models of infections in wounds and soft-tissue abscesses in mice that can be followed by bioluminescence imaging. The size and intensity of the infection can be sequentially monitored in a non-invasive fashion in individual mice in real-time. When photosensitizers are introduced into the infected tissue followed by illumination with red light, a light-dose dependent loss of luminescence is seen. If the bacterium is invasive, the loss of luminescence correlates with increased survival of the mice, whilst animals in control groups die of sepsis within five days. Healing of the PDT treated wounds is not impaired and may actually be improved. This approach can allow many animal models of localized infections to be accurately monitored for efficacy of treatment by PDT.

Optical imaging of bacterial infection models

“…Open circular wounds were induced on the backs of rats, then infected with bioluminescent E. coli and irradiated at a fluence of 260 J/cm 2 which increased the temperature to 45° C. A complete loss of bacterial bioluminescence from the wound was noted after 48 h. The antibacterial effect was attributed to dessication of the wound rather than a direct photodynamic effect via excitation of bacterial chromophores [28]. A related study employed laser irradiation to induce Photodynamic Therapy (PDT) of an open wound infection [29]. In this case, the wound was pretreated with a photosensitizer, a non-toxic dye that can be excited by red light to produce reactive oxygen species that kill the neighboring cells.…”

Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection

“…Moreover, bioluminescent bacteria can be quantified over sequential procedures without destroying the sample [37]. Our laboratory has employed bioluminescent imaging as a convenient means to monitor the effectiveness of antimicrobial PDT in animal models of infections caused by several bioluminescent pathogens [38]. This methodology has been demonstrated in mouse models of infected wounds [39,40] burns [41] and abscesses [42] using both Gram-positive and Gram-negative bacteria.…”

Antibacterial photodynamic therapy: overview of a promising approach to fight antibiotic-resistant bacterial infections

“…The PSs that have been tested in vivo include phenothiaziniums (section 2.1) such as methylene blue [19,209], Rose Bengal [210], and EtNBS [188] (Table 1), ZnPc derivative RLP068/Cl [211], (Table 1), and C70 fullerene [60,64,68] (Table 1), among others. Animal models have been developed that mimic clinical infections, including a mouse model of skin abrasion caused by MRSA [212] and a guinea pig model of burn infections with S. aureus [213].…”

“…During in vivo APDT experiments, animals are often sacrificed following the intervention to determine the standard bacterial count (CFU/mL). Recent developments in these models entail the inoculation of the animals with endogenously luminescent bacteria so that the animals can be followed in real time after APDT [210]. The intensity of luminescence can be directly correlated to the extent of the infection [210,218].…”