We report on the use of optical techniques to monitor and treat Pseudomonas aeruginosa wound infections in mice. Bioluminescent bacteria transduced with a plasmid containing a bacterial lux gene operon allow the infection in excisional mouse wounds to be imaged by use of a sensitive charge-coupled device camera. Photodynamic therapy (PDT) targeted bacteria, by use of a polycationic photosensitizer conjugate, which is designed to penetrate the gram-negative cell wall and was topically applied to the wound and was followed by red-light illumination. There was a rapid light dose-dependent loss of luminescence, as measured by image analysis, in the wounds treated with conjugate and light, a loss that was not seen in untreated wounds, wounds treated with light alone, or wounds treated with conjugate alone. P. aeruginosa was invasive in our mouse model, and all 3 groups of control mice died within 5 days; in contrast, 90% of PDT-treated mice survived. PDT-treated wounds healed significantly faster than did silver nitrate-treated wounds, and this was not due to either inhibition of healing by silver nitrate or stimulation of healing by PDT.

The emergence of antibiotic resistance among pathogenic bacteria has led to efforts to find alternative antimicrobial therapeutics to which bacteria will not be easily able to develop resistance. One of these may be the combination of nontoxic dyes (photosensitizers [PS]) and visible light, known as photodynamic therapy, and we have reported its use to treat localized infections in animal models. While it is known that gram-positive species are generally susceptible to photodynamic inactivation (PDI), the factors that govern variation in degrees of killing are unknown. We used isogenic pairs of wild-type and transposon mutants deficient in capsular polysaccharide and slime production generated from Staphylococcus epidermidis and Staphylococcus aureus to examine the effects of extracellular slime on susceptibility to PDI mediated by two cationic PS (a polylysine-chlorin e6 conjugate, pL-c e6 , and methylene blue [MB]) and an anionic molecule, free c e6 , and subsequent exposure to 665-nm light at 0 to 40 J/cm 2 . Free c e6 gave more killing of mutant strains than wild type, despite the latter taking up more PS. Log-phase cultures were killed more than stationary-phase cultures, and this correlated with increased uptake. The cationic pL-c e6 and MB gave similar uptakes and killing despite a 50-fold difference in incubation concentration. Differences in susceptibility between strains and between growth phases observed with free c e6 largely disappeared with the cationic compounds despite significant differences in uptake. These data suggest that slime production and stationary phase can be obstacles against PDI for gram-positive bacteria but that these obstacles can be overcome by using cationic PS.

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.

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. Although many workers have used photodynamic therapy (PDT) to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections by Gram-(-) bacteria in living mice. However, these infected wound models involved a short timespan between infection (30 min) and treatment by PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. We used Staphylococcus aureus stably transformed with a Photorhabdus luminescenslux operon (luxABCDE) that was genetically modified to be functional in Gram-(+) bacteria. These engineered bacteria emitted bioluminescence, allowing the progress of the infection to be monitored in both space and time with a low light imaging charge-coupled device (CCD) camera. One million cells were injected into one or both thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four hours later, the bacteria had multiplied more than one hundredfold; poly-L-lysine chlorin e6 conjugate or free chlorin e6 was injected into one area of infected muscle and imaged with the CCD camera. Thirty minutes later, red light from a diode laser was delivered as a surface spot or by interstitial fiber into the infection. There was a light dose dependent loss of bioluminescence (to <5% of that seen in control infections) not seen in untreated infections or those treated with light alone, but in some cases, the infection recurred. Treatment with conjugate alone led to a lesser reduction in bioluminescence. Infections treated with free chlorin e6 responded less well and the infection subsequently increased over the succeeding days, probably due to PDT-mediated tissue damage. PDT-treated infected legs healed better than legs with untreated infections. This data shows that PDT may have applications in drug-resistant soft-tissue infections.

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. Although many workers have used photodynamic therapy (PDT) to kill bacteria in vitro, the use of this approach has seldom been reported in vivo in animal models of infection. We have previously described the first use of PDT to treat excisional wound infections by Gram-negative bacteria in living mice. However these infected wound models used a short time after infection (30 min) before PDT. We now report on the use of PDT to treat an established soft-tissue infection in mice. We used Staphylococcus aureus stably transformed with a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in Gram-positive bacteria. These engineered bacteria emitted bioluminescence allowing the progress of the infection to be monitored in both space and time with a lowlight imaging charged couple device (CCD) camera. One million cells were injected into one or both thigh muscles of mice that had previously been rendered neutropenic by cyclophosphamide administration. Twenty-four hours later the bacteria had multiplied more than one hundred-fold, and poly-L-lysine chlorin(e6) conjugate or free chlorin(e6) was injected into one area of infected muscle and imaged with the CCD camera. Thirty-minutes later red light from a diode laser was delivered as a surface spot or by interstitial fiber into the infection. There was a lightdose dependent loss of bioluminescence (to < 5% of that seen in control infections) not seen in untreated or light alone treated infections, but in some cases the infection recurred. Conjugate alone led to a lesser reduction in bioluminescence. Infections treated with free chlorin(e6) responded less and the infection subsequently increased over the succeeding days, probably due to PDT-mediated tissue damage. PDT-treated infected legs healed better than legs with untreated infections. This data shows that PDT may have applications in drug-resistant soft-tissue infections.

Inflammation plays an important role in the pathophysiology of atherosclerotic disease. We have previously shown that the targeted photosensitizer chlorin (e(6)) conjugated with maleylated albumin (MA-ce6) is taken up by macrophages via the scavenger receptor with high selectivity. In a rabbit model of inflamed plaque in New Zealand white rabbits via balloon injury of the aorto-iliac arteries and high cholesterol diet we showed that the targeted conjugate showed specificity towards plaques compared to free ce6. We now show that an intravascular fiber-based spectrofluorimeter advanced along the -iliac vessel through blood detects 24-fold higher fluorescence in atherosclerotic vessels compared to control rabbits (p < 0.001 ANOVA). Within the same animals, signal derived from the injured iliac artery was 16-fold higher than the contralateral uninjured iliac (p < 0.001). Arteries were removed and selective accumulation of MA-ce6 in plaques was confirmed using: (1) surface spectrofluorimetry, (2) fluorescence extraction of ce6 from aortic segments, and (3) confocal microscopy. Immunohistochemical analysis of the specimens showed a significant correlation between MA-ce6 uptake and RAM-11 macrophage staining (R = 0.83, p < 0.001) and an inverse correlation between MA-ce6 uptake and smooth muscle cell staining (R = -0.74, p < 0.001). MA-ce6 may function as a molecular imaging agent to detect and/or photodynamically treat inflamed plaques.

We have previously shown that a conjugate (MA-ce6) between maleylated serum albumin and the photosensitizer chlorin(e6) (ce6) is targeted in vitro to macrophages via class A scavenger receptors. We now report on the ability of this conjugate to localize in macrophage-rich atherosclerotic plaques in vivo. Both the conjugate and the free photosensitizer ce6 are studied after injection into New Zealand White rabbits that are rendered atherosclerotic by a combination of aortic endothelial injury and cholesterol feeding into normal rabbits. Rabbits are sacrificed at 6 and 24 h after injection and intravascular fluorescence spectroscopy is carried out by fiber-based fluorimetry in intact bloodfilled arteries. Surface spectrofluorimetry of numbered excised aortic segments together with injured and normal iliac arteries is carried out, and quantified ce6 content by subsequent extraction and quantitative fluorescence determination of the arterial segments and also of nontarget organs. There is good agreement between the various techniques for quantifying ce6 localization, and high contrast between arteries from atherosclerotic and normal rabbits is obtained. Fluorescence correlates with the highest burden of plaque in the aorta and the injured iliac artery. The highest accumulation in plaques is obtained using MA-ce6 at 24 h. Free ce6 gives better accumulation at 6 h compared to 24 h. The liver, spleen, lung, and gall bladder have the highest uptake in nontarget organs. Macrophagetargeted photosensitizer conjugates may have applications in both detecting and treating inflamed vulnerable plaque.

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