Lethal photosensitization of wound-associated microbes using indocyanine green and near-infrared light.(Research article)

Authors: Ghada S Omar [1,2]; Michael Wilson (corresponding author) [1]; Sean P Nair [1]

Background

Wound infections are an ongoing problem not only for patients but also for healthcare providers worldwide. Infected wounds are responsible for significant morbidity and mortality, and an increase in the duration and the cost of hospital stay [1, 2, 3]. The growing resistance to conventional antibiotics among organisms that infect wounds and burns makes such infections difficult to treat [4, 5, 6]. Therefore there is a drive to develop novel antimicrobial strategies to which pathogens will not easily develop resistance.

One promising alternative is photodynamic therapy (PDT). Photodynamic therapy is the application of a non-toxic compound, termed a photosensitizer (PS) or light-activated antimicrobial agent (LAAA), which can be activated by light of an appropriate wavelength to produce reactive oxygen species (ROS) (i.e. singlet oxygen and free radicals) which can then exert a microbicidal effect [7, 8]. Light of the appropriate wavelength excites the PS molecule into a triplet state which reacts with either a substrate to produce radical ions which in turn react with oxygen to produce cytotoxic species such as superoxide and hydroxyl radicals (type I reaction), or reacts directly with molecular oxygen to produce singlet oxygen (

1 O2 ) (type II reaction). PDT has a number of advantages over conventional antibiotics. Firstly, as the mechanism of killing is non-specific, with reactive oxygen species causing damage to many bacterial components, resistance is unlikely to develop from repeated use [9, 10]. Secondly, both the PS and the light are applied locally to the target tissue; therefore reducing the risk of adverse systemic effects [11]. Cutaneous wound infections are particularly appropriate for treatment by PDT due to their easy accessibility to both a topical PS and light. The organisms most frequently responsible for infections of wounds and burns are Streptococcus pyogenes , staphylococci, such as methicillin-resistant Staphylococcus aureus (MRSA), and the Gram-negative bacterium Pseudomonas aeruginosa [1]. Staphylococcus aureus is a common wound-infecting organism which results in delayed epithelial closure of the wound, possibly due to its interaction with fibronectin and inhibition of keratinocyte migration [12]. The eradication of wound-infecting bacteria using lethal photosensitization has been reported in the literature, e.g. Staph. aureus [13, 14, 15], P. aeruginosa [16, 17, 18, 19], Strep. pyogenes [20].

In this study we have investigated whether common wound-associated organisms are sensitive to lethal photosensitization using the dye indocyanine green (ICG) coupled with light from a near-infrared (NIR) laser emitting at 808 nm. This combination of PS and light source has a number of desirable characteristics over those described in the literature [13, 14, 15, 16, 17, 18, 19, 20]. NIR laser light has a greater capacity to penetrate tissues than light of lower wavelengths whilst ICG is a NIR-absorbing water-soluble tricarbocyanine dye, which has been approved by the United States Food and Drug Administration (US FDA) for medical diagnostic studies. ICG has a very low toxicity and a high absorption at wavelengths around 800 nm. Recently, PDT with ICG has been used to treat tumors [21, 22]. However it has, as yet, not been used for the PDT of infections with the exception of its topical use for the treatment of acne vulgaris [23, 24, 25].

Methods

Bacteria

Two Gram-positive organisms were used in this study;

Staph. aureus strain 8325-4 and Strep. pyogenes strain 12202. In addition, the Gram-negative bacterium Pseudomonas aeruginosa strain PA01 was used. Gram-positive bacteria were maintained by weekly subculture on blood agar base (Oxoid Ltd, UK) supplemented with 5% horse blood whereas P. aeruginosa was subcultured on nutrient agar (Oxoid). Staph. aureus and P. aeruginosa were grown aerobically in nutrient broth (Oxoid) with shaking at 37[degrees]C for 16 h. Strep. pyogenes was grown in brain heart infusion broth (Oxoid) for 16 h at 37[degrees]C in an atmosphere of 5% CO2 /95% air. Cells were then harvested by centrifugation and resuspended in phosphate buffered saline (PBS) to an optical density of 0.05 at 600 nm which corresponded to approximately 106 -107 colony forming units per ml (CFU ml-1 ) for the Gram-positive bacteria and 108 -109 CFU ml-1 for P. aeruginosa .

Photosensitizer

4,5-benzoindotricarbocyanine (Indocyanine green) C

43 H47 N2 NaO6 S2 is a negatively-charged polymethine dye and was obtained from Sigma-Aldrich-UK. Fresh stock solutions were prepared immediately prior to each experiment in either PBS or sterile distilled water (H2 O) or deuterium oxide (D2 O) and kept in the dark.

Laser light

A 500 mW Gallium-aluminum-arsenide (Ga-Al-As) NIR-Laser (Thor International Ltd - UK) emitting light with a wavelength of 808 [+ -] 5 nm was used for irradiation. For experimental purposes, the distance of the laser probe to the plate surface was adjusted to give fluence rates of 1.37, 0.07 or 0.048 W cm

-2 with an actual power output of 470, 225 and 150 mW respectively.

Effect of photosensitizer concentration on lethal photosensitization

Aliquots (50 [mu]l) of a suspension of

Staph. aureus, Strep. pyogenes (containing approximately 106 -107 CFU ml-1 ) or P. aeruginosa (108 -109 CFU ml-1 ) in sterile PBS were transferred into a 96-well plate and an equal volume of ICG in PBS was added to each well to give final concentrations ranging from 25 [mu]g ml-1 to 250 [mu]g ml-1 . After addition of the ICG, the wells were left in the dark for 10 or 30 minutes (pre-irradiation time) and then exposed to a measured dose of high intensity laser light at a fluence rate of 1.37 W cm-2 . In this system, an exposure of 5 minutes corresponded to a light dose of 411 J cm-2 . Each experimental condition was tested in at least triplicate and each experiment was carried out on at least two occasions. The conditions tested were; 1) controls which contained neither ICG nor received irradiation (L- S-), 2) incubation with ICG in the dark (L- S+), 3) irradiation in the absence of ICG (L+ S-) and 4) the test which was irradiated in the presence of ICG (L+S+).

To enumerate the surviving bacteria, serial 10-fold dilutions were plated in duplicate either on blood agar (

Staph. aureus and Strep. pyogenes ) or nutrient agar (P. aeruginosa ).

Effect of low intensity laser light

To avoid any heating effect that may occur during high intensity irradiation, the experiments were repeated using a laser power output of 150 mW to irradiate the samples at a low fluence rate of 0.048 W cm

-2 . Aliquots (50 [mu]l) of a suspension of Staph. aureus or Strep. pyogenes (containing approximately 106 -107 CFU ml-1 ) in sterile PBS were transferred into a 96-well plate and an equal volume of ICG in PBS was added to each well to give final concentration of 25 [mu]g ml-1 . After addition of the ICG, the wells were exposed to a measured dose of low intensity laser light for 15 or 30 minutes corresponding to light doses of 43 and 86 J cm-2 respectively. Similar experiments were performed with P. aeruginosa …