Priv.-Doz. Dr. rer. nat. Tim Maisch Department of Dermatology University Clinic Regensburg Franz-Josef-Strauss-Allee 11 D-93053 Regensburg, Germany Tel.: +49-941-944-8944 Fax: +49-941-944-8943 E-mail: firstname.lastname@example.org
The increasing resistance of bacteria against antibiotics is one of the most important clinical challenges of the 21st century. Within the gram-positive bacteria the methicillin-resistant Staphylococcus aureus and Enterococcus faecium represent the major obstacle to successful therapy. Apart from the development of new antibiotics it requires additional differently constituted approaches, like photodynamic inactivation in order to have further effective treatment options against bacteria available. Certain dyes, termed photosensitizers, are able to store the absorbed energy in long-lived electronic states upon light activation with appropriate wavelengths and thus make these states available for chemical activation of the immediate surroundings. The interaction with molecular oxygen, which leads to different, very reactive and thus cytotoxic oxygen species, is highlighted. In this review the application of the photodynamic inactivation of bacteria will be discussed regarding the possible indications in dermatology, like localized skin and wound infections or the reduction of nosocomial colonization with multi-resistant bacteria on the skin. The crucial advantage of the local application of photosensitizers followed by irradiation of the area of interest is the fact that independent of the resistance pattern of a bacterium a direct inactivation takes place similarly as with an antiseptic. In this review the physical-chemical and biological basics of photo-dynamic inactivation of bacteria (PIB) will be discussed as well as the possible dermatological indications.
In January 2009 the microbiologists Cesar Arias and Barbara Murray warned in the “New England Journal of Medicine” that bacteria might become the winners of evolution, as several strains have already adapted themselves so well, that they have become resistant to conventional therapies and thus may present a serious clinical challenge in the future . The pathogens termed “ESKAPE”, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter strains are of particular importance, as these pathogens can “escape” the effects of antibiotics .
At present in Germany 22.6 % of all S. aureus isolates are characterized as MRSA, in some other countries even over 50 % of isolates are MRSA-positive (e. g. USA 64.4 %) [3, 4]. In the time period from 1992 to 2003 the average increase of MRSA resistance was 3.1 % per year . The appearance of MRSA with additive resistance to vancomycin in 2000 compounds the problem, as for therapy of infections with MRSA naturally only a limited number of antibiotics are available . The MRSA problem in the treatment of superficial bacterial skin infections is clearly illustrated by the controversially discussed increase of resistance towards the topical antibiotic mupirocin (Turixin® ointment) which is specially used for the local decolonization of MRSA . Further, the topical application of antibiotics is associated with unwanted side effects such as the induction of contact sensitization or constitutes a risk factor for the selection of resistant pathogens from among the cutaneous flora. For example, neomycin induces contact sensitization in up to 30 % of patients with leg ulcers and in 15 % of patients with chronic otitis externa . Moreover, in the meantime the entire aerobic bacterial flora of the skin (especially S. epidermidis) is resistant towards topical antibiotic treatment e. g. with erythromycin. Thus these non-pathogens can serve as the sources for transmitting resistance plasmids to pathogens .
A further phenomenon is the occurrence of community-acquired MRSA isolates that can elicit skin and soft tissue infections in immunocompetent persons independent of hospitals or other nursing establishments.
The relatively rapid acquisition of antibiotic resistance is opposed by the relatively long time period needed for the development of antibiotics with new mechanisms of action that do not function according to the “lock-and-key” principle. For the fight against bacterial infections only three new antibiotics with novel mechanisms of action were approved in the past 10 years (Table 1), while all other newly licensed antibiotics were only modifications of already known antibiotics.
Table 1. List of antibiotics aproved between 1998 and 2008.
Year of approval
New mechanism of action
*Modified according to [47, 48] and the German Association of Research-based Pharmaceutical Companies 2008.
Simultaneously in the past 25 years the total number of newly approved antibacterial substances had dropped considerably (Figure 1). As it can be anticipated that the development of resistance will continue in coming years, it is only a question of time until resistance develops towards new antibiotics. For this reason the necessity exists for an immediate and continual search for alternative methods against bacteria towards which no resistance can develop or only very difficultly. Examples of such strategies are the utilization of bacteriophages  or synthetic antimicrobial peptides . One of the most promising and innovative methods in this respect is photodynamic inactivation of bacteria (PIB). This method is based on the photodynamic effect . Pathogenic gram-positive as well as gram-negative bacteria are killed with the help of a non-toxic dye or photosensitizer (PS) coupled with irradiation with light of suitable wavelengths in the presence of oxygen (Figure 2).
Photochemical and photophysical fundamentals of PIB
Two different molecular mechanisms play a central role in PIB. Prerequisite for both mechanisms is the sufficient presence of molecular oxygen and an attached and/or cellular photosensitizer (PS) that can be excited by light of the appropriate wavelength. In the type I mechanism, due to the interaction with the substrate, radicals are formed, which can react with the substrate and further oxygen to produce oxidation products. In the type II mechanism there is energy transfer form the triplet state of the PS excited by light to molecular oxygen (Figure 3). Here, highly reactive singlet oxygen is formed, which – on the one hand – can lead to the formation of further oxygen radicals and – on the other – directly react with appropriate molecules in its immediate vicinity.
Transferred to bacteria, this means that a PS, that accumulates intracellularly or at least close to the plasma membrane (even outside), after irradiation leads to irreversible damage of the immediate surroundings of the PS due to oxidative modification of important biomolecules. At present it is generally accepted that the production of singlet oxygen plays the key role in photosensitization .
Currently, PS from differing molecule classes with varying photodynamic activity towards gram-positive and gram-negative bacteria are already being utilized (Table 2).
Table 2. Overview of selected photoreactive substance classes.
Morphological effects on prokaryotes
n. d.: not yet determined, modified according to .
C60 fullerene coupled to polar diserinol groups or quaternary pyrrolidinium groups
Inhibition of protein kinase C
New methylene blue
Lipoxidation on membranes, protein-proteincross-linking
Due to structural differences of the outer bacterial cell wall of gram-positive and –negative bacteria differences naturally exist with respect to the efficacy of the various PS. The 40–80 nm thick outer cell wall of gram-positive bacteria with their up to 100 peptidoglycan layers do not represent an effective permeability barrier. In contrast, the outer cell membrane of gram-negative bacteria with a bi-lamellar membrane covering the only 3 nm thick peptidoglycan layer impedes diffusion considerably. Studies on penetration behavior of polysaccharides, glycopeptides or antibiotics have shown that hydrophilic substances with a molecular weight of < 0.6–0.7 kDa penetrate the outer membrane of E. coli via porin channels . It not only impedes numerous antibiotics, but also negatively charged or neutral PS. Various strategies have been developed to circumvent this barrier, such as pre-treatment with EDTA or polymyxin B, which make the outer bacterial wall more permeable to allow such PS to penetrate and accumulate on the cytoplasmic membrane [15, 16]. In contrast, variously positively charged PS (phenothiazines, phthalocyanines or porphyrins) are photodynamically active even without the addition of a penetration booster. In this manner both gram-positive as well as gram-negative bacteria have already been inactivated very successfully [17–19]. Not only resting or vegetative cells but also Bacillus spores have been successfully killed using the photodynamic effect . As a result of the high reactivity of singlet oxygen especially with proteins, its lifespan in a cellular environment is only of short duration (< 0.3 to 1.5 μs; depending on location), which results in a very short diffusion distance (< 30–100 nm). Therefore the effectiveness of a PS depends not only on the amount taken up, but especially on the location of the PS at the time point of irradiation . Due to the usually lipophilic nature of many PS they primarily are located in membranes consisting of lipid double layers. Irradiation therefore leads to oxidative damage to fatty acids in lipid double layers and particularly to proteins contained therein. Structural alterations are seen in the form of “elongated” bacterial cells without division of daughter cells, mesosome formation (membrane folding) of the cytoplasmic membrane and density differences in the composition of the cytoplasm and formation of what is termed “polyphosphates”. Although singlet oxygen possesses very high reactivity towards nucleic acid – in contrast to UV or ionizing radiation – damage of genomic DNA or plasmid DNA appears to be of subordinate significance. Thus Deinococcus radiodurans, a bacteria with a highly effective DNA repair mechanism and thus very tolerant to ionizing radiation, can effectively be killed by the photody-namic production of singlet oxygen . A particular advantage of PIB is that to date no mutagenic effect has been detected in photosensitized cells. Thus, no selection of photo-resistant bacteria is induced, even after repetitive treatment. This is a clear advantage in comparison to UV inactivation of bacteria, which can result in the development and selection of bacterial strains with new, unknown properties and to mutagenic effects in eukaryotic cells .
PIB in superficial skin infections
Superficial skin and soft tissue infections do not necessarily need to be treated with systemic antibiotics. Some significant facts on the use of PIB of bacteria are summarized in Table 3. Based on this list of current knowledge, PIB appears especially suited for the treatment of superficial skin and soft tissue infections, one-third of which are caused by S. aureus. For prevention of nosocomial colonization by MRSA, PIB might in the future also be employed as an alternative to hygienic hand disinfection.
Table 3. Important criteria for PIB.
Different chemical molecule classes act as photosensitizers, e. g. phenothiazines, tetrapyrroles (e. g. phthalocyanines, porphyrins, chlorines) and fullerenes.
Photosensitizers are after very short incubation periods (several minutes) under irradiation with applied energy doses of < 30 J/cm2 photodynamically active and induce an efficient reduction of vital pathogen numbers by ≥ 3–5 log10−Stufen.
Photodynamic inactivation is also effective towards mutiresistant bacterial strains. This is important with respect to repetitive use in chronic and/or recurrent bacterial infections.
Inactivation of bacteria growing in biofilms is possible.
Wound infections in vivo
The newest in vivo studies in animal models have shown that with the adequate choice of PS and light dose a significant reduction of bacteria in the treated area can be achieved without damage to the surrounding tissue with the advantage of preservation of the resident bacterial flora. In what is termed the infection animal model Hamblin et al. were able to show that topical application of a polycationic PS with subsequent irradiation reduces the number of vital pathogens in a cutaneous wound infected by E. coli. Here a chlorinee6 conjugate coupled to poly-L-lysin is employed to make the outer membrane more permeable and to make binding of the PS on its target structure possible. The healing of the infected and irradiated cutaneous wounds was equally good as the corresponding non-irradiated and uninfected control wounds. Berthiaume et al. examined the efficacy of antibody photosensitization against Pseudomonas aeruginosa in vivo with the use of a PS coupled to an antibody . Dorsal skin areas in the mouse model infected with P. aeruginosa were inoculated with a chlorinee6 antibody conjugate and subsequently irradiated with 160 J/cm2 at 630 nm. Here, a > 75 % reduction in vital bacterial colonies s observed. Untreated or control areas treated with an unspecific conjugate as well as treated but not irradiated areas displayed normal bacterial growth. In a further study efficient elimination of EMRSA (epidemic MRSA strain 16) with methylene blue (100 μg/ml) and 670 nm laser light (360 J/cm2) was achieved in an in vivo wound animal model .
Topical photodynamic therapy (PDT) with 5-ALA and visible light (550–700 nm) for acne leads to improvement of cutaneous findings as well as to a reduction in the development of new acne lesions . The bacterial colonization by Propionibacterium acnes in the follicles could be reduced significantly. Clinically, after a single PDT marked improvement of the affected sites was observed that lasted for 10 weeks and after repeated PDT even over 20 weeks . A normal skin state could be achieved. In irradiated skin areas a reduction of hair follicles and thus a reduced number of sebum-producing units was induced, though. In part serious side effects (pain, erythema, edema, blistering, purpura) appeared in association with photodynamic treatment. Further, oversensitivity to mechanical stimuli was observed or an acute flare of acneiform lesions occurred. With use of a 20 % ALA emulsion and a laser diode (635 nm; 25 mW/cm2; 15 J/cm2) for irradiation in 10 patients with acne grade 2–4 on a scale from 1 to 12 (Leeds grading) a marked improvement of clinical findings after two treatments could be achieved . Nevertheless, in this study no significant reduction of P. acnes could be detected. Recent results of Fabbrocini et al. showed that four weeks after the last PDT treatment with 5-ALA the “Global Acne Grading Score” was reduced by 50 %. The cyanoacrylate follicle biopsy also confirmed that the density of macrocomedones was reduced. The difference in results between these two studies might be due to the differing treatment schemes (once weekly vs. once every two weeks). As the P. acnes colonization as evaluated three weeks after the last PDT treatment, recolonization may have perhaps already occurred, so that no significant reduction could be detected.
On the whole, these results demonstrate that PDT not only has a positive effect on the treatment of acne by way of induced cytotoxic effects on seborrhea, but also acts antibacterially on Propionibacterium species. For routine use an optimization of treatment parameters, e. g. reduction of the 5-ALA concentration and/or intensity of irradiation is needed particularly to reduce the extent of side effects while, at the same time, to improve efficacy. Kim et al. first used ICG (indocya-nine green 0.06 %) as exogenous PS, that could be directly activated and a diode laser (805 nm, 12 J/cm2) in a pilot study on acne treatment in Asian patients resulting in an on the whole marked improvement of cutaneous findings four weeks after PDT treatment in comparison to the control area .
Possible uses of PIB
Acute and chronic stages of atopic dermatitis are currently being treated with conventional UVA/UVB therapy. Beyond the antiinflammatory components of UV irradiation the antibacterial properties also play a crucial role for treatment success. Jekler et al. were able to show in a study that the reduction of S. aureus leads to improvement of clinical findings . Interestingly, patients who failed to respond to light therapy displayed higher S. aureus colonization rates than those who responded to UV therapy . One risk in UV inactivation of S. aureus exists, as mentioned above, in the development of bacterial strains with unknown properties and further in the induction of mutagenic effects in eukaryotic cells, the keratinocytes . Exactly due to these long-term risks of UV therapy, PIB represents an alternative for treatment of atopic dermatitis.
Current therapy of choice for impetigo is the topical application of fusidic acid and mupirocin, as this has proven more effective in the eradication of the pathogen than oral administration of antibiotics [35, 36]. This is nonetheless no long-term solution as MRSA have also increasingly developed resistance to mupirocin. In 1998 already 4 % of MRSA populations studied (n = 104) had a high (MIC > 1 024 mg/l) and 32 % a low (MIC 8–24 mg/l) resistance . At present, in 10-year intervals no further increase of resistance towards mupirocin (2008: 4 %) is being observed, with this being dependent on country . A South African study showed that the resistance rate had increased to 7 %.
Discussion and outlook
Indications for PIB are first of all the treatment of local, superficial skin and soft tissue infections or its use for the reduction of nosocomial colonization of the skin by multi-resistant bacteria.
One advantage of topically applied PS with subsequent irradiation is the locally limited action of the photodynamic effect. The most severe side effects of conventional antibiotic use should thus be avoidable. In topical application there are allergic contact sensitization and development of resistance in the resident flora. Systemic administration can equally induce resistances in cutaneous bacteria. Hoiby et al. demonstrated that orally administered ciprofloxacin is excreted in sweat and that after 2.7 days on average resistances can be induced in S. epidermidis in the axillary vault .
A prerequisite for the effective use of PIB is the uptake/ binding of the PS on the bacterial cell wall or plasma membrane. Here the design of the functional side chains of the PS and the charge as well as what is termed “targeting”, i. e. the manner in which the PS is to be transported, are of crucial importance. One possibility is the use of antibody-coupled PS . The advantage of this “active” targeting is the specificity with which antibodies recognize selected proteins on the surface of bacteria and thus do not induce toxicity towards other cells, e. g. keratinocytes. A possible disadvantage could be the very high molecular weight of such PS complexes, which may inhibit penetration of the upper layers of the epidermis needed for effective treatment of superficial skin infections. Also alterations of the binding epitopes on the protein surface of the pathogens (resistance development) could result in a loss of antibody recognition and thus in a loss of photodynamic activity.
At the present time it is still questionable to what extent bacteria can develop resistances to PIB. Generally gram-negative bacterial are capable of controlling the uptake of PS through their outer membrane, which is why – as mentioned above – only positively charged PS are effective. A reduced negative charge of the LPS molecules of the outer membrane can undermine this. An adequate number of PS molecules could no longer bind on the LPS molecules and no killing be possible. This mechanism of reduced negatively charged LPS chains was shown for Samonella enterica serovar typhimurium . This isolate possesses resistance towards polymyxin B, a polycationic molecule. Therefore this mechanism might possibly play a role in use of polycationic PS. A further mechanism of resistance of gram-negative bacteria towards PS could be represented by porin-deficient mutations, as they are termed. These isolates possess resistance towards β-lactam antibiotics . This isolate lacks the OmpF protein (a tunnel protein of the outer membrane sheath), so that reduced penetration of antibiotics results. Also active outward transport of PS can prevent efficient photosensitization of bacteria. Alterations in the composition of the cell wall or in protein-protein cross-linking can result in altered penetration behavior of PS and thus block effective antibacterial PDT. Some isolates of gram-positive S. aureus with intermediate vancomycin resistance produce a much thicker outer peptidoglycan cell wall and thus inhibit vancomycin in displaying its effects .
Recently Grinholc et al. demonstrated that the photodynamic effect towards 40 clinical MRSA and 40 sensitive S. aureus isolates was very heterogeneous. And the inactivation efficacy ranged from 0 % to 99.0 %. Possibly resistance mechanisms such as e. g. “multi-drug” pumps that simply pump the PS out of the bacterium through specific efflux pumps could play a role. Nevertheless in this study no association between photodynamic inactivation efficacy and the antibiotic resistance pattern of the different MRSA strains or the antibiotic-sensitive, methicillin- sensitive S. aureus (MSSA) strains could be demonstrated. Overall the mechanism responsible for this phenomenon has not yet been definitively clarified.
The described forms of resistance development may in the future be elegantly avoided, if it succeeds to mediate the photodynamic effect via contact of the PS with the bacterial membrane without uptake by the bacterium. Intracellular uptake of the PS is not an absolutely mandatory prerequisite of a successful photodynamic effect, on the one hand due to the highly variable diffusion distance of singlet oxygen in water, on the other due to the presence of proteins and lipids. In pure water singlet oxygen can diffuse for about 1 μm, which corresponds to the size of most bacteria. In the vicinity of protein-rich lipid layers the diffusion distance is reduced to < 50 nm. This means that, when the PS is outside of the bacteria, the closest bacterium is attacked; when it is inside the cell, active oxygen species act exactly at the location of the PS .
It is questionable if bacteria are capable of developing resistance towards reactive oxygen species (ROS), e. g. singlet oxygen. To date possible mechanisms of the development of resistance to singlet oxygen are not described in the literature. This means, that the production of reactive oxygen species, particularly singlet oxygen, during irradiation occurs only precisely at the location of the PS. This assumption is supported by the fact that singlet oxygen is only short-lived in biological systems (e. g. in lipid membranes or cells) and in parallel possesses only a very limited diffusion distance .
In order to attain high antibacterial activity in the topical application of PS on the skin, a sufficient concentration of PS is needed. Therefore, not only must the physico-chemical properties of PS be optimized, but also the galenics must be tuned to the specific indication, e. g. in impetigo to reach bacteria in the upper layers of the epidermis. This property of galenics is essential to guarantee penetration through the stratum corneum and adequate accumulation of PS in the respective bacteria . The development of new PS that also provide for efficient inactivation of biofilm-forming pathogens is a subject of research activities. Here the aim is to better penetrate the extracellular slime and capsule of biofilm-forming pathogens on surfaces in order to attain what is termed “targeting of non-multiplying bacteria” for the efficient photodynamic inactivation of biofilms.
In conclusion, it can be said that the molecular structure of the respective PS as well as galenics decides if antibacterial PDT constitutes an alternative to topical antibiotic therapy. To what extent this new therapy approach will be established in clinical routine is determined by pharmacokinetics (incubation time) and the irradiation time.