Lasers Med Sci 1999, 14:54–61 © 1999 Springer-Verlag London Limited
Photodynamic Inactivation of Staphylococci with 5-Aminolaevulinic Acid or Photofrin S. Karrer, R.-M. Szeimies, S. Ernst, C. Abels, W. Ba¨umler and M. Landthaler Department of Dermatology, University of Regensburg, Regensburg, Germany
Abstract. Widespread use of antibiotics for bacterial infections is a reason for antibiotic resistance. Therefore, alternatives like photodynamic therapy (PDT) are studied for bacterial eradication. The viability of Staphylococcus (S.) aureus and S. epidermidis was studied following photosensitisation with di#erent concentrations of 5-aminolevulinic acid (5-ALA) or Photofrin and irradiation with a dye laser. After ALA without irradiation growth of S. epidermidis was reduced, surprisingly growth of S. aureus significantly increased. Using ALA and light a significant and concentration-dependent decrease of bacterial growth was measured in both strains. However, for S. epidermidis there was no di#erence between ALA with or without irradiation. Photofrin without light decreased bacterial growth significantly, indicating a high dark toxicity of the drug. Photofrin and light lead to a highly significant reduction of growth in both strains. While 5-ALA killed only up to 39% of Staphylococci, Photofrin was able to eradicate up to 93% of the bacteria. The poor response to 5-ALA may be due to an insu$cient synthesis of photodynamically active porphyrins in Staphylococci. Although topical PDT could be useful for superinfected wounds or leg ulcers, the in-vitro bactericidal e#ect of 5-ALA PDT on staphylococci was not satisfactory. Keywords: 5-Aminolaevulinic acid; Dye laser; Gram positive bacteria; Photodynamic therapy; Photofrin; Protoporphyrin IX; Wound infections
INTRODUCTION In 1904, Jodlbauer and von Tappeiner first described the photodynamic inactivation of bacteria with exogenously applied photosensitisers [1]. They treated Bacterium acidi lactici, Bacillus prodigiosus and Proteus vulgaris with eosin, erythrosin, phenosafranin, rose bengal or methylene blue and exposed the bacteria to daylight. Eosin showed hardly any e#ect on cell viability, whereas erythrosin and light killed bacteria within 2–4 days and rose bengal, methylene blue or phenosafranin killed the bacteria even within 1–2 days. Huber [2] exposed Streptococcus pyogenes and Bacillus diphtheriae to daylight after sensitisation with 1‰ eosin or 1‰ erythrosin. Within 2–3 h of illumination by daylight in the presence of oxygen, bacteria were killed and their virulence was lost when injected subcutaneously into the back of white mice.
Correspondence to: Rolf-Markus Szeimies, MD, Department of Dermatology, University of Regensburg, D-93042 Regensburg, Germany. Fax: +49 941 944 9628.
At the beginning of the twentieth century, the search for an e#ective antibacterial treatment was based on the lack of antibiotics to treat bacterial infections at that time. Although today various antibacterial drugs are available, the incorrect and extensive use of antibiotics has resulted in an increase of antibiotic-resistant strains of bacteria [3]. Therefore, there is growing interest in the application of alternative methods for the eradication of bacteria. Photodynamic therapy (PDT) involves the photo-oxidation of biological molecules in the presence of a sensitiser, light and oxygen. PDT using exogenous photosensitisers like haematoporphyrin (HP), haematoporphyrin derivative (HPD), deuteroporphyrin (DT) or AlPcS2 [4–8] has already proven its e$cacy in killing Gram-positive bacteria and diminishing their antibiotic resistance [5,9]. 5-Aminolaevulinic acid (5-ALA) is a precursor of endogenous porphyrins in the biosynthetic pathway for haem which induces the biosynthesis of photosensitising concentrations of protoporphyrin IX and other porphyrin metabolites within the cells [10,11]. It
Photodynamic Inactivation of Staphylococci
has been shown that the exogenous application of 5-ALA leads to accumulation of endogenous porphyrins, mainly coproporphyrin in Gram-positive and Gram-negative bacteria [12]. Staphylococcus (S.) aureus is the aetiological agent of a wide range of dermatologically relevant infections, e.g. abscess, furuncle, wound infections or impetigo contagiosa, and local or systemic antibiotics are often used for their prevention, control and treatment [13]. However, in most developed countries there has been an increase in colonisation and infection of patients by strains of S. aureus resistant to methicillin and other antibiotics which can cause bacteraemia with a poor prognosis [3]. Thus, in the present study we endeavoured to elucidate the e#ects of photosensitisation of staphylococci with the endogenous photosensitiser 5-ALA or the exogenous photosensitiser Photofrin (IPSEN Pharma GmbH, Ettlingen, Germany) and subsequent irradiation on their viability.
MATERIALS AND METHODS Bacterial Strains Both bacterial strains, S. aureus and S. epidermidis, were cultured from clinical material of the Dept. of Dermatology, University of Regensburg. Biological di#erentiation and evaluation of resistance against di#erent antibiotics were performed at the Institute of Microbiology, University of Regensburg.
Bacterial Growth An overnight culture in nutrient agar pH 7.0 (Difco, Becton-Dickinson, Heidelberg, Germany) was transferred into fresh nutrient broth medium, then bacteria were grown for 14 h at 37C with aeration. The number of colony forming units (CFU) was then counted. Absorbance was determined with a spectrophotometer (UVIKON 930, Kontron Instruments, Mailand, Italy). At the beginning of the experiments, cultures had an optical density of 0.4–0.5 at 528 nm. This optical density corresponded to a CFU count of 14.5107/ml100% for S. aureus and 17.1107/ml24% for S. epidermidis.
55
Photodynamic Treatment Since there was a temperature increase of about 4.1C during irradiation which might influence bacterial growth, the irradiation only group (without sensitiser) was used as control, particularly since there was no statistical di#erence in the viability of bacteria without any treatment and irradiated only. Three groups were formed: group A served as control and was irradiated only; group B received sensitiser only; group C was treated with sensitiser (5-ALA or Photofrin) and irradiation. For each concentration in each group eight experiments were performed. Cultures of the bacteria were incubated with the drugs in their exponential growth phase (log phase, 6–14 h of cultivation). It has been shown for exogenous photosensitisers that at this time bacteria are most sensitive to PDT; it is not known if this is also true for endogenous photosensitisers, such as 5-ALA [4]. Bu#ered 5-ALA solution (9.09 mg/ml, pH 7.5) or Photofrin (containing a mixture of ester- or ether-bound porphyrin monomers up to polymers) (2.5 mg/ml) were added to the bacteria. Sensitiser end-concentrations for 5-ALA were 10, 102 and 104 g/ml, and for Photofrin 12.5 and 200 /ml. Irradiation was performed 10 h after addition of the drug with an argon-ion pumped dye laser (model 375b, Spectra Physics Inc., Mt View, CA, USA) (=630 nm, total light dose 180 J/cm2, fluence rate 250 mW/cm2). Viable bacteria were monitored by counting automatically (ARTEK ACCUCOUNT, Artek Systems Corporation, New York, USA) the number of CFU grown on nutrient agar plates after incubation for 24 h at 37C. Survivor growth (SG) was calculated according to the equation N/N0 100=SG, where (N0) is the number of CFU of the only irradiated culture (group A, SG=100%) and (N) is the number of CFU of the treated cultures (groups B and C).
Statistics Statistical evaluation was performed with the statistical program Professional Calc (Gold Disk Inc., Ontario, Canada). Results were expressed as mean and standard error of the mean (SEM). Student’s t-test was used to compare the numbers of viable bacteria following treatment with the sensitiser and/or laser light (groups A, B and C). Descriptive
56
S. Karrer et al.
Table 1. Number of colony forming units (CFU) and survival growth (SG) in Staphylococcus aureus after PDT with 5-ALA, after incubation with 5-ALA only and after irradiation only 5-ALA (g/ml)
Groupa
101
A B C A B C A B C
102 104
a
CFUSEM
130.877.43 157.6311.66 116.54.74 79.113.43 111.259.73 74.673.93 1317.37 224.521.57 91.55.1
% of controlSEM (SG) 1005.68 1218.95 893.62 1004.34 14112.33 954.99 1005.63 17216.53 703.90
A, irradiation only; B, sensitiser only; C, sensitiser and irradiation.
statistics and the David, Pearson and Stephens test were used. Probability values smaller than 5% were considered statistically significant.
RESULTS 5-Aminolaevulinic acid S. aureus In group B, (sensitiser only, no irradiation) all 5-ALA concentrations led to a significant viability increase of S. aureus to an SG of 1219% when using 10 g/ml 5-ALA (p
Photodynamic Inactivation of Staphylococci with 5-Aminolaevulinic Acid or Photofrin S. Karrer, R.-M. Szeimies, S. Ernst, C. Abels, W. Ba¨umler and M. Landthaler Department of Dermatology, University of Regensburg, Regensburg, Germany
Abstract. Widespread use of antibiotics for bacterial infections is a reason for antibiotic resistance. Therefore, alternatives like photodynamic therapy (PDT) are studied for bacterial eradication. The viability of Staphylococcus (S.) aureus and S. epidermidis was studied following photosensitisation with di#erent concentrations of 5-aminolevulinic acid (5-ALA) or Photofrin and irradiation with a dye laser. After ALA without irradiation growth of S. epidermidis was reduced, surprisingly growth of S. aureus significantly increased. Using ALA and light a significant and concentration-dependent decrease of bacterial growth was measured in both strains. However, for S. epidermidis there was no di#erence between ALA with or without irradiation. Photofrin without light decreased bacterial growth significantly, indicating a high dark toxicity of the drug. Photofrin and light lead to a highly significant reduction of growth in both strains. While 5-ALA killed only up to 39% of Staphylococci, Photofrin was able to eradicate up to 93% of the bacteria. The poor response to 5-ALA may be due to an insu$cient synthesis of photodynamically active porphyrins in Staphylococci. Although topical PDT could be useful for superinfected wounds or leg ulcers, the in-vitro bactericidal e#ect of 5-ALA PDT on staphylococci was not satisfactory. Keywords: 5-Aminolaevulinic acid; Dye laser; Gram positive bacteria; Photodynamic therapy; Photofrin; Protoporphyrin IX; Wound infections
INTRODUCTION In 1904, Jodlbauer and von Tappeiner first described the photodynamic inactivation of bacteria with exogenously applied photosensitisers [1]. They treated Bacterium acidi lactici, Bacillus prodigiosus and Proteus vulgaris with eosin, erythrosin, phenosafranin, rose bengal or methylene blue and exposed the bacteria to daylight. Eosin showed hardly any e#ect on cell viability, whereas erythrosin and light killed bacteria within 2–4 days and rose bengal, methylene blue or phenosafranin killed the bacteria even within 1–2 days. Huber [2] exposed Streptococcus pyogenes and Bacillus diphtheriae to daylight after sensitisation with 1‰ eosin or 1‰ erythrosin. Within 2–3 h of illumination by daylight in the presence of oxygen, bacteria were killed and their virulence was lost when injected subcutaneously into the back of white mice.
Correspondence to: Rolf-Markus Szeimies, MD, Department of Dermatology, University of Regensburg, D-93042 Regensburg, Germany. Fax: +49 941 944 9628.
At the beginning of the twentieth century, the search for an e#ective antibacterial treatment was based on the lack of antibiotics to treat bacterial infections at that time. Although today various antibacterial drugs are available, the incorrect and extensive use of antibiotics has resulted in an increase of antibiotic-resistant strains of bacteria [3]. Therefore, there is growing interest in the application of alternative methods for the eradication of bacteria. Photodynamic therapy (PDT) involves the photo-oxidation of biological molecules in the presence of a sensitiser, light and oxygen. PDT using exogenous photosensitisers like haematoporphyrin (HP), haematoporphyrin derivative (HPD), deuteroporphyrin (DT) or AlPcS2 [4–8] has already proven its e$cacy in killing Gram-positive bacteria and diminishing their antibiotic resistance [5,9]. 5-Aminolaevulinic acid (5-ALA) is a precursor of endogenous porphyrins in the biosynthetic pathway for haem which induces the biosynthesis of photosensitising concentrations of protoporphyrin IX and other porphyrin metabolites within the cells [10,11]. It
Photodynamic Inactivation of Staphylococci
has been shown that the exogenous application of 5-ALA leads to accumulation of endogenous porphyrins, mainly coproporphyrin in Gram-positive and Gram-negative bacteria [12]. Staphylococcus (S.) aureus is the aetiological agent of a wide range of dermatologically relevant infections, e.g. abscess, furuncle, wound infections or impetigo contagiosa, and local or systemic antibiotics are often used for their prevention, control and treatment [13]. However, in most developed countries there has been an increase in colonisation and infection of patients by strains of S. aureus resistant to methicillin and other antibiotics which can cause bacteraemia with a poor prognosis [3]. Thus, in the present study we endeavoured to elucidate the e#ects of photosensitisation of staphylococci with the endogenous photosensitiser 5-ALA or the exogenous photosensitiser Photofrin (IPSEN Pharma GmbH, Ettlingen, Germany) and subsequent irradiation on their viability.
MATERIALS AND METHODS Bacterial Strains Both bacterial strains, S. aureus and S. epidermidis, were cultured from clinical material of the Dept. of Dermatology, University of Regensburg. Biological di#erentiation and evaluation of resistance against di#erent antibiotics were performed at the Institute of Microbiology, University of Regensburg.
Bacterial Growth An overnight culture in nutrient agar pH 7.0 (Difco, Becton-Dickinson, Heidelberg, Germany) was transferred into fresh nutrient broth medium, then bacteria were grown for 14 h at 37C with aeration. The number of colony forming units (CFU) was then counted. Absorbance was determined with a spectrophotometer (UVIKON 930, Kontron Instruments, Mailand, Italy). At the beginning of the experiments, cultures had an optical density of 0.4–0.5 at 528 nm. This optical density corresponded to a CFU count of 14.5107/ml100% for S. aureus and 17.1107/ml24% for S. epidermidis.
55
Photodynamic Treatment Since there was a temperature increase of about 4.1C during irradiation which might influence bacterial growth, the irradiation only group (without sensitiser) was used as control, particularly since there was no statistical di#erence in the viability of bacteria without any treatment and irradiated only. Three groups were formed: group A served as control and was irradiated only; group B received sensitiser only; group C was treated with sensitiser (5-ALA or Photofrin) and irradiation. For each concentration in each group eight experiments were performed. Cultures of the bacteria were incubated with the drugs in their exponential growth phase (log phase, 6–14 h of cultivation). It has been shown for exogenous photosensitisers that at this time bacteria are most sensitive to PDT; it is not known if this is also true for endogenous photosensitisers, such as 5-ALA [4]. Bu#ered 5-ALA solution (9.09 mg/ml, pH 7.5) or Photofrin (containing a mixture of ester- or ether-bound porphyrin monomers up to polymers) (2.5 mg/ml) were added to the bacteria. Sensitiser end-concentrations for 5-ALA were 10, 102 and 104 g/ml, and for Photofrin 12.5 and 200 /ml. Irradiation was performed 10 h after addition of the drug with an argon-ion pumped dye laser (model 375b, Spectra Physics Inc., Mt View, CA, USA) (=630 nm, total light dose 180 J/cm2, fluence rate 250 mW/cm2). Viable bacteria were monitored by counting automatically (ARTEK ACCUCOUNT, Artek Systems Corporation, New York, USA) the number of CFU grown on nutrient agar plates after incubation for 24 h at 37C. Survivor growth (SG) was calculated according to the equation N/N0 100=SG, where (N0) is the number of CFU of the only irradiated culture (group A, SG=100%) and (N) is the number of CFU of the treated cultures (groups B and C).
Statistics Statistical evaluation was performed with the statistical program Professional Calc (Gold Disk Inc., Ontario, Canada). Results were expressed as mean and standard error of the mean (SEM). Student’s t-test was used to compare the numbers of viable bacteria following treatment with the sensitiser and/or laser light (groups A, B and C). Descriptive
56
S. Karrer et al.
Table 1. Number of colony forming units (CFU) and survival growth (SG) in Staphylococcus aureus after PDT with 5-ALA, after incubation with 5-ALA only and after irradiation only 5-ALA (g/ml)
Groupa
101
A B C A B C A B C
102 104
a
CFUSEM
130.877.43 157.6311.66 116.54.74 79.113.43 111.259.73 74.673.93 1317.37 224.521.57 91.55.1
% of controlSEM (SG) 1005.68 1218.95 893.62 1004.34 14112.33 954.99 1005.63 17216.53 703.90
A, irradiation only; B, sensitiser only; C, sensitiser and irradiation.
statistics and the David, Pearson and Stephens test were used. Probability values smaller than 5% were considered statistically significant.
RESULTS 5-Aminolaevulinic acid S. aureus In group B, (sensitiser only, no irradiation) all 5-ALA concentrations led to a significant viability increase of S. aureus to an SG of 1219% when using 10 g/ml 5-ALA (p
