- Email: [email protected]
Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin
Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57 www.elsevier.com/locate/jphotobiol
Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin Saskia A.G. Lambrechts a,b, Maurice C.G. Aalders a,*, Frank D. Verbraak a, Johan W.M. Lagerberg c,1, Jacob B. Dankert b, Johannes J. Schuitmaker c b
a Laser Center K01-225-5, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c PhotoBioChem, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Received 6 July 2004; received in revised form 21 October 2004; accepted 30 November 2004 Available online 19 January 2005
Abstract Background: Photodynamic inactivation (PDI) employs visible light and a photosensitizer to inactivate cells. The technique is currently clinically used for the treatment of several malignancies. However, the PDI of microorganisms still remains in the research phase. Purpose: To study the effect of human blood plasma and human serum albumin (HSA) on the PDI of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Methods: PDI experiments were performed using white light (30 mW cm 2) and the cationic 5-phenyl-10,15,20-tris(N-methyl-4pyridyl)porphyrin chloride (TriP[4]) as photosensitizer. Results: The microorganisms could be successfully photoinactivated by TriP[4] when suspended in phosphate buffered saline (PBS). In this medium, P. aeruginosa was the most resistant microorganism. Changing the suspending medium from PBS to human blood plasma reduced the PDI of all three microorganisms. In human blood plasma C. albicans was the most resistant microorganism. The same results were obtained with 4.5% and 7% HSA/PBS suspensions. Conclusions: Albumin inhibits the PDI of S. aureus, P. aeruginosa and C. albicans in a dose dependent manner. However, our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Albumin; Microorganisms; Human blood plasma; Photodynamic inactivation; Porphyrin
1. Introduction The growing resistance against antibacterial and antifungal agents has generated a search for alternative antimicrobial treatments. In particular, the use of topical antibiotics is under discussion since it has been *
Corresponding author. Tel.: +31 (0)20 566 3829; fax: +31 (0)20 697 5594. E-mail address: [email protected] (M.C.G. Aalders). 1 Present address: Sanquin research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. 1011-1344/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2004.11.020
suggested that such an approach induces antibiotic resistance faster than the use of oral antibiotics [1,2]. A potential alternative may be antimicrobial photodynamic inactivation (PDI). PDI employs visible light to activate a photosensitizer. The activated photosensitizer can react with molecules from its direct environment by electron or hydrogen transfer, leading to the production of radicals (type 1 reaction) or it can transfer its energy to oxygen, generating the highly reactive singlet oxygen (type 2 reaction). Both pathways can lead to cell death [3]. Despite the frequent clinical use
52
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
of PDI for the treatment of several malignancies [4], the use of PDI to inactivate microorganisms is still in the research phase. This is in part due to the fact that the PDI of Gram-negative bacteria with first generation photosensitizers such as the anionic haematoporphyrin had to be mediated by membrane permeabilizers such as ethylenediaminetetraacetic acid or polymyxin nonapeptide [5]. Later it was discovered that cationic porphyrins did not require these membrane permeabilizers in order to successfully inactivate Gram-negative bacteria [6]. It has been demonstrated that Gram-positive, Gram-negative bacteria and fungi can all be successfully photodynamically inactivated by a single cationic photosensitizer, e.g., 5-phenyl10,15,20-tris(N-methyl-4-pyridyl)porphyrin (TriP[4]) [7–9] or toluidine blue [10–12]. Furthermore, it has been shown that both antibiotic sensitive and resistant strains can be successfully photoinactivated [13,14] and so far repeated photosensitization of bacterial cells has not induced a selection of resistant strains [15]. In addition PDI has the advantage of dual selectivity, both the photosensitizer and the illumination can be targeted. All these qualities combined make PDI an interesting alternative antimicrobial treatment for superficial wound infections, which are easily reached by light. In this study, we investigated the PDI of three different clinically interesting microorganisms: a Grampositive bacterium, Staphylococcus aureus and a Gramnegative bacterium, Pseudomonas aeruginosa and the yeast Candida albicans. Staphylococcus aureus colonizes the nasal passage and axillae in 30–40% of the population. In most of such carriers S. aureus is harmless, but if it enters the body through a lesion in the skin it can cause a range of mild to severe infections [16]. Methicillin resistant S. aureus is now one of the most feared causes of nosocomial infections [16–19]. P. aeruginosa is a widely distributed environmental bacterium that is the cause of a substantial portion of nosocomial infections and is frequently found to be multiresistant [17,20]. A superficial wound infection in which both S. aureus and P. aeruginosa play an important role is the burn wound infection [18,20,21]. Burn wound infections can impede wound closure and are an important cause of death in burn wound patients [22,23]. Candida albicans is a common inhabitant of the mouth, throat, digestive tract and skin. In hosts with a compromised immune system it can become pathogenic [24,25]. Oropharyngeal candidiasis is one of the most common opportunistic infections accompanying AIDS patients. Resistance of C. albicans against fluconazole, an antifungal agent frequently used by such patients, has begun to appear [26]. Both burn wound infections and oral candidiasis are superficial lesions for which PDI could be a potential treatment.
With some exceptions, in most in vitro studies, buffer or broth (sometimes diluted) is used as suspending medium. It is known however that the consistency of the suspending medium strongly influences the efficacy of antimicrobial PDI [8,27]. Therefore in vivo circumstances, such as the presence of wound fluid are expected to influence the efficacy of antimicrobial PDI. In the present study, we used human blood plasma and various suspensions of human serum albumin (HSA) in phosphate buffered saline (PBS) to serve as a wound fluid model [28,29]. The cationic porphyrin TriP[4] was chosen as photosensitizer since it has proven to be a successful antimicrobial photosensitizer [7–9]. We investigated the feasibility to use TriP[4] mediated PDI to inactivate S. aureus, P. aeruginosa and C. albicans in the presence of human blood plasma and HSA. The variance in sensitivity to PDI of three wild type strains of each species studied was also investigated.
2. Materials and methods 2.1. Microorganisms Microorganisms, isolated from burn wound patients were kindly provided by the Central Bacteriological Laboratory, Haarlem, the Netherlands. These were S. aureus (strains 2000.1.46382.0, 2000.1.46384.3, 2000.1.47260.2), P. aeruginosa (strains 2000.1.47063.0, 2000.1.47260.2, 2000.1.49012.5) and C. albicans (strains 2000.1.40456.9, 2000.1.48525.7, 2000.1.48703.4). The microorganisms were grown aerobically in 10 ml brain heart infusion broth (Oxoid Ltd., Hampshire, UK) at 37 °C. After 18 h the stationary phase microorganisms were harvested by centrifugation at 3000 rpm for 10 min, washed twice with PBS (pH 7.4) and suspended in 2 ml PBS, human blood plasma or HSA suspension. 2.2. Photosensitizer A stock solution of the photosensitizer, 5-phenyl10,15,20-tris(N-methyl-4-pyridyl)-21,23H-porphyrin chloride (TriP[4]) (Mid-Century Chemicals, Posen, IL), was prepared in a 50 mM sodium phosphate buffer (pH 7.4) at a concentration of 1 mM (0.78 mg ml 1) and stored at 4 °C in the dark. Before use, the stock solution was allowed to warm up to room temperature. 2.3. Light source All illuminations were carried out with white light from a 500 W halogen lamp (Massive, the Netherlands). To avoid heating of the samples, the light was passed through a 1-cm thick water filter. The irradiance at the level of the microorganism samples was 30 mW cm 2 as measured with an IL 1400A power meter (Interna-
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
tional Light, Newburyport, MA). The temperature of the samples never exceeded 22 °C.
53
lM. Survival was determined after 15 min of illumination, which corresponds to a light dose of 27 J cm 2. One wild-type strain of each species was studied.
2.4. PDI studies 2.5. Statistics Samples of 1 ml were prepared, all contained 100 ll of microorganism suspension, resulting in a final cell concentration of 108–109 cells ml 1 for S. aureus and P. aeruginosa and of 106–107 cells ml 1 for C. albicans, the appropriate amount of photosensitizer stock solution (ranging from 3.1 to 50 ll) and PBS. Directly after addition of the photosensitizer, the suspensions were transferred to polystyrene culture dishes with a diameter of 3 cm (Greiner, Alphen a/d Rijn, the Netherlands) and illuminated. After illumination, microbial survival was determined with a modified version of the Miles and Misra method [30]. For this, the suspensions were serially 10-fold diluted with PBS. Then, drops of 10 ll of each dilution were applied onto Iso-sensitest agar in 5fold (Oxoid Ltd., Hampshire, UK) and incubated at 37 °C under aerobic conditions. After 18 h for S. aureus and P. aeruginosa and 48 h for C. albicans the number of colony forming units (CFU) was counted. Survival was expressed as a percentage relative to a control sample containing no photosensitizer, taken at the beginning of each experiment prior to illumination. Survival was also determined for samples after illumination in the absence of photosensitizer and for samples after incubation with the photosensitizer in the dark. 2.4.1. PDI in PBS The microorganisms were harvested as described above and suspended in PBS. The photosensitizer concentrations were; 3.1, 6.3, 12.5, 25 and 50 lM. Survival was determined after 15 min of illumination which corresponded to a light dose of 27 J cm 2. Three wild-type strains of each species were studied. 2.4.2. PDI in plasma The microorganisms were harvested as described above and suspended in human blood plasma obtained from buffy coat (Blood bank Leiden-Haaglanden, the Netherlands) by centrifugation at 2000 rpm for 10 min. A photosensitizer concentration of 50 lM was used in these experiments. Survival was determined after 15, 30, 45 and 60 min of illumination which corresponds to light doses of 27, 54, 81 and 108 J cm 2. Aliquots were derived from one dish after each additive light dose. One wild-type strain of each species was studied. 2.4.3. PDI in albumin suspensions The microorganisms were harvested as described above and suspended in PBS containing 1%, 4.5% or 7% (w/v) HSA. A commercially available 20% solution HSA suspension (CealbÒ, CLB, the Netherlands) was used. The final photosensitizer concentration was 25
Values are expressed as mean percent change from starting control ± standard deviation. Differences between means of multiple groups were analyzed using a StudentÕs t test. Statistical significance was assumed at the p < 0.05 level.
3. Results 3.1. PDI in PBS The photosensitizer concentration dependent inactivation of S. aureus, P. aeruginosa and C. albicans suspended in PBS is shown in Fig. 1. S. aureus was more sensitive to PDI than P. aeruginosa and C. albicans (Fig. 1(a)). Exposed to a TriP[4] concentration of 12.5 lM and a light dose of 27 J cm 2, P. aeruginosa showed a 1 log10-unit reduction in viable count. Under the same conditions S. aureus and C. albicans showed a 7 and 5 log10-unit reduction in viable count, respectively. These reductions in viable count were close to the minimal detection level (MDL) and thus close to the maximal inactivation that could be measured using the described assessment method. The minimal detection level of this method is the point at which no colony forming units can be observed in the undiluted sample. P. aeruginosa had not yet reached this limit at a TriP[4] concentration as high as 50 lM. The three wild type strains studied of each species showed some significant variation in their sensitivity to PDI. The statistical significant (p < 0.05) variations between strains are marked with an asterisks in Figs. 1(b)–(c). Small effects were observed in samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark. These effects were always smaller than 0.3 log10-unit reduction in viable count and were not significantly different from a zero (no change) interpretation for all controls. 3.2. PDI in plasma When the microorganisms were suspended in human blood plasma, the order in sensitivity to PDI changed (Fig. 2). S. aureus remained the most sensitive organism, but C. albicans became less sensitive than P. aeruginosa. Exposed to a TriP[4] concentration as high as 50 lM and a light dose 27 J cm 2, no significant (p > 0.05) reduction in viable count was observed for C. albicans.
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57 10
2
10
1
10
0
10
-1
10
-2
10
-3
P. aeruginosa
C. albicans
†
†
Survival %
Survival %
54
-4
10
-5
10 10
0
10
†
20
30
40
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
-5
10
-6
MDL
0
10
(c)
20
30
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
S. aureus
MDL
* 0
*
P. aeruginosa
10
10
10
20
40
TriP[4] µM
10
2
10
1
10
0
40
50
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
C. albicans
MDL
*
0
50
30
TriP[4] µM
(b)
Survival %
10
2
50
TriP[4] µM
(a)
Survival %
S. aureus
†
-6
10
(d)
*
10
20
30
40
50
TriP[4] µM
Fig. 1. (a) Survival curves of S. aureus (s), P. aeruginosa (n) and C. albicans (h) suspended in PBS exposed to different concentrations TriP[4] and a light dose of 27 J cm 2. Each value is the average of three wild-type strains, each of them performed in triplicate. The error bars indicate the standard deviation between the averages. , No CFU detected, below minimal detectable limit. (b)–(d) Separate survival curves of the three different wild-type strains S. aureus, strains 2000.1.46382.0 (h, j), 2000.1.46384.3 (s, d), 2000.1.47260.2 (n, m) (b), P. aeruginosa, strains 2000.1.49012.5 (h, j), 2000.1.47063.0 (s, d), 2000.1.47260.2 (n, m) (c) and C. albicans strains 2000.1.40456.9 (e, r), 2000.1.48703.4 (h, j), 2000.1.48525.7 (s, d) (d) suspended in PBS and exposed to different concentrations TriP[4]. Open symbols, samples exposed to a light dose of 27 J cm 2. Each point is the mean of at least three experiments ± standard deviation. Closed symbols, samples kept in the dark for 15 min. Each point is the mean of at least two experiments ± standard deviation. Asterisks, significant (p > 0.05) deviation from the average value. MDL, minimal detectable limit.
Under the same conditions a 3 log10-unit and <1 log10unit reduction in viable count was observed for S. aureus and P. aeruginosa, respectively. No significant (p > 0.05) effect was observed in the samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark.
effect was observed in the samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark.
3.3. PDI in albumin suspensions
Staphylococcus aureus, P. aeruginosa and C. albicans, suspended in PBS, could be successfully photoinactivated by TriP[4]. Of each species we studied three different wild type strains and no large significant differences in their sensitivity to PDI could be observed, We therefore assume that our results are representative for these species in general. To our knowledge, the PDI of a yeast by a meso-substituted cationic porphyrin has not yet been reported. A requisite for an effective antimicrobial treatment for superficial wound infections is its effectiveness in the inevitable presence of wound fluid. To create an in vitro situation more comparable to wound fluid than
The influence of albumin (HSA) on the PDI of the microorganism is shown in Fig. 3. Albumin exerted a dose dependent protective effect against the PDI of all three microorganisms. The strongest protection was observed for C. albicans. When suspended in a 4.5% albumin suspension, exposed to a TriP[4] concentration of 25 lM and a light dose 27 J cm 2, C. albicans showed a reduction in viable count smaller than 1 log10-unit. Under the same conditions a 5 log10-unit and 1 log10unit reduction in viable count was observed for S. aureus and P. aeruginosa, respectively. No significant (p > 0.05)
4. Discussion
Survival %
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
10
3
10
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
C. albicans
P. aeruginosa
S. aureus †
†
0
27
54
81
108
-2
Light dose (J.cm )
% Survival
Fig. 2. Survival curves of S. aureus 2000.1.46384.3 (s), P. aeruginosa 2000.1.49012.5 (n) and C. albicans 2000.1.48525.7 (h) suspended in human blood plasma exposed to 50 lM TriP[4] and different light doses. , No CFU detected, below minimal detectable limit. Each point is the mean of three experiments ± standard deviation.
10
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
S. aureus P. aeruginosa C. albicans
†
†
0
1
4.5
7
Albumin % Fig. 3. Survival histogram of S. aureus 2000. 2000.1.46384.3 (hatched), P. aeruginosa 2000.1.49012.5 (cross hatched) and C. albicans 2000.1.48525.7 (black) suspended in various PBS/HSA suspensions, exposed to 25 lM TriP[4] and a light dose of 27 J cm 2. , No CFU detected, below minimal detectable limit. Each bar is the mean of three experiments ± standard deviation.
PBS, the microorganisms were suspended in human blood plasma. Under this condition, a reduction in sensitivity to PDI was observed for all three microorganisms but especially C. albicans became less sensitive. Interestingly, the relative order in sensitivity to PDI changed from: S. aureus > C. albicans P.aeruginosa in PBS to: S. aureus > P.aeruginosa > C. albicans in human blood plasma. In 4.5% and 7% HSA suspensions the order in sensitivity to PDI was comparable to that found in human blood plasma. The total protein con-
55
centration of human blood plasma is 7.3% of which 4.5% is albumin, [31] therefore we hypothesize that albumin plays the key role in the protection of C. albicans. The inhibiting effect of albumin on the PDI of the microorganisms was not unexpected. The binding of porphyrins to albumin and the quenching and scavenging of reactive oxygen species by albumin are phenomena that have been described extensively [27,32]. However, the highly reduced sensitivity of C. albicans to PDI in an albumin rich suspending fluid was surprising when compared to the more moderate reduction in sensitivity of S. aureus and P. aeruginosa. The sensitivity of C. albicans suspended in PBS to PDI is an indication that its cell envelope does not provide a strong barrier against PDI. Its resistance in an albumin rich environment could be due to a poor binding of TriP[4] to C. albicans relative to the binding of TriP[4] to albumin and either bacterial species. In literature few data are available on the in vitro PDI of C. albicans. Most of the in vitro studies used haematoporphyrin (HP) or a derivative [33–38], toluidine blue or methylene blue [34,39,40] as photosensitizer. Taking into account the variation in experimental design, it appears that the PDI of C. albicans mediated by HP and its derivatives is strongly inhibited by proteins, which is comparable to our observations. Experiments performed in PBS using HP or its derivatives, resulted in the successful PDI of both C. albicans and S. aureus [35,37,38]. However, when the experiments were performed in protein containing broth, no PDI was observed for C. albicans while S. aureus remained sensitive [36]. Similarly, no PDI was observed for C. albicans suspended in broth using a dihaematoporphyrin ester as photosensitizer [34]. In contrast, no influence of the suspending fluid on the PDI of C. albicans was observed when toluidine blue was used as photosensitizer [39]. A factor that can influence the efficacy of PDI is the electronic charge of the photosensitizer, however in this case it does not seem to be of importance as TriP[4] and toluidine blue are both cationic and HP is anionic. The strongly reduced sensitivity of C. albicans to PDI in the presence of high concentrations of albumin is not expected to impose limitations to a potential future treatment of oropharyngeal candidiasis with PDI. The protein content of saliva is low and ranges from 0.1% to 0.3% [41,42] and the PDI of C. albicans suspended in a 1% HSA suspension does not differ significantly from that in PBS (p = 0.325) (Fig. 3). The next step, more comparable to the in vivo situation, would be to investigate wether C. albicans can be photoinactivated when it is present in a biofilm. Like bacterial biofilms, biofilm-grown C. albicans is highly resistant to antimicrobial agents [43]. Despite this increased resistance it has been shown by various groups that bacteria in biofilms can be successfully photoinactivated [44,45].
56
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
S. aureus was substantially less affected by the inhibiting effect of albumin, implying a sustainable sensitivity to PDI when present in wounds with protein rich exudate. We chose human serum albumin and blood plasma as a model since wound fluids are predominantly serum derived. However the protein content of wound fluid can be much lower than that of blood serum, according to Moseley et al. wound fluid from acute and chronic wounds contain as little as 1.476 (±0.123) mg ml 1 and 0.644 (±0.153) mg ml 1 protein, respectively [46]. This corresponds to 0.15 and 0.06 w/v%, respectively. Therefore the inhibition of wound fluid proteins will most likely not impose a problem for the in vivo PDI of the investigated species. However the use of antimicrobial PDI with TriP[4] for wounds containing a substantial amount of blood will most likely be ineffective due to the inhibiting effect of albumin. S. aureus was substantially less affected by the inhibiting effect of albumin, implying a sustainable sensitivity to PDI when present in wounds with protein rich exudate. In conclusion, our experiments have demonstrated that S. aureus, P. aeruginosa and C. albicans can be photoinactivated using TriP[4] as photosensitizer. However, the degree of microbial PDI strongly depended on the albumin content of the suspending fluid. The strongest inhibiting effect of albumin was observed for the PDI of C. albicans. Our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans
Acknowledgements We thank Threes G.M. Smijs (Leiden University Medical Centre, Leiden, the Netherlands, supported by the Dutch Technology Foundation STW LGN 4890) for the constructive discussions and reviewing the article. We thank the Central Bacteriological Laboratory, Haarlem, the Netherlands for providing the microorganisms.
References [1] J.I. Ross, A.M. Snelling, E. Carnegie, P. Coates, W.J. Cunliffe, V. Bettoli, G. Tosti, A. Katsambas, J.I. Galvan Perez Del Pulgar, O. Rollman, L. Torok, E.A. Eady, J.H. Cove, Antibiotic-resistant acne: lessons from Europe, Br. J. Dermatol. 148 (2003) 467–478. [2] P. Coates, C.A. Adams, W.J. Cunliffe, K.T. McGinley, E.A. Eady, J.J. Leyden, J. Ravenscroft, S. Vyakrnam, B. Vowels, Does oral isotretinoin prevent Propionibacterium acnes resistance?, Dermatology 195 (Suppl 1) (1997) 4–9. [3] M. Wainwright, Photodynamic antimicrobial chemotherapy (PACT), J. Antimicrob. Chemother. 42 (1998) 13–28. [4] C. Fritsch, G. Goerz, T. Ruzicka, Photodynamic therapy in dermatology, Arch. Dermatol. 134 (1998) 207–214.
[5] Z. Malik, H. Ladan, Y. Nitzan, Photodynamic inactivation of Gram-negative bacteria: problems and possible solutions, J. Photochem. Photobiol. 14 (1992) 262–266. [6] M. Merchat, G. Bertolini, P. Giacomini, A. Villanueva, G. Jori, Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positive and Gram-negative bacteria, J. Photochem. Photobiol. 32 (1996) 153–157. [7] T.G.M. Smijs, H.J. Schuitmaker, Photodynamic inactivation of the dermatophyte Trichophyton rubrum, Photochem. Photobiol. 77 (2003) 556–560. [8] S.A.G. Lambrechts, M.C.G. Aalders, D.H. Langeveld-Klerks, Y. Khayali, J.W.M. Lagerberg, Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins, Photochem. Photobiol. 79 (2003) 297–302. [9] M. Merchat, J.D. Spikes, G. Bertoloni, G. Jori, Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins, J. Photochem. Photobiol. 35 (1996) 149–157. [10] Z. Jackson, S. Meghji, A. MacRobert, B. Henderson, M. Wilson, Killing of the yeast and hyphal forms of Candida albicans using a light-activated antimicrobial agent, Lasers Med. Sci. 14 (1999) 150–157. [11] M.N. Usacheva, M.C. Teichert, M.A. Biel, Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms, Lasers Surg. Med. 29 (2001) 165–173. [12] N. Komerik, M. Wilson, Factors influencing the susceptibility of Gram-negative bacteria to toluidine blue O-mediated lethal photosensitization, J. Appl. Microbiol. 92 (2002) 618–623. [13] Z. Malik, S. Gozhanski, Y. Nitzan, Effects of photoactivated HPD on bacteria and antibiotic resistance, Microbios. Lett. 21 (1982) 103–112. [14] M. Soncin, C. Fabris, A. Busetti, D. Dei, D. Nistri, G. Roncucci, G. Jori, Approaches to selectivity in the Zn(II)-phthalocyaninephotosensitized inactivation of wild-type and antibiotic-resistant Staphylococcus aureus, Photochem. Photobiol. Sci. 1 (2002) 815– 819. [15] F.M. Lauro, P. Pretto, L. Covolo, G. Jori, G. Bertoloni, Photoinactivation of bacterial strains involved in periodontal diseases sensitized by porphycene–polylysine conjugates, Photochem. Photobiol. Sci. 1 (2002) 468–470. [16] S. Baron, Staphylococcus, in: R.C. Peake, D.A. James, M. Susman, C.A. Kennedy, M.J.D. Singleton, S. Schuenke (Eds.), Medical Microbiology, fourth ed., The University of Texas Medical Branch, Galveston, 1996. [17] J.L. Vincent, D.J. Bihari, P.M. Suter, H.A. Bruining, J. White, M.H. Nicolas-Chanoin, M. Wolff, R.C. Spencer, M. Hemmer, The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. EPIC International Advisory Committee, JAMA 274 (1995) 639–644. [18] N. Cook, Methicillin-resistant Staphylococcus aureus versus the burn patient, Burns 24 (1998) 91–98. [19] D.M. Livermore, Antibiotic resistance in staphylococci, Int. J. Antimicrob. Agents 16 (2000) S3–S10. [20] J.P. Pirnay, D. De Vos, C. Cochez, F. Bilocq, J. Pirson, M. Struelens, L. Duinslaeger, P. Cornelis, M. Zizi, A. Vanderkelen, Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone, J. Clin. Microbiol. 41 (2003) 1192–1202. [21] C.G. Mayhall, The epidemiology of burn wound infections: then and now, Clin. Infect. Dis. 37 (2003) 543–550. [22] B. Pruitt, A. McManus, S. Kim, C. Goodwin, Burn wound infections: current status, World J. Surg. 22 (1998) 135–145. [23] A.J. Singer, S.A. McClain, Persistent wound infection delays epidermal maturation and increases scarring in thermal burns, Wound Repair Regen. 10 (2002) 372–377.
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57 [24] G. Garber, An overview of fungal infections, Drugs 61 (Suppl. 1) (2001) 1–12. [25] S.K. Fridkin, W.R. Jarvis, Epidemiology of nosocomial fungal infections, Clin. Microbiol. Rev. 9 (1996) 499–511. [26] J.H. Rex, M.G. Rinaldi, M.A. Pfaller, Resistance of Candida Species to Fluconazole, Antimicrob. Agents Chemother. 39 (1995) 1–8. [27] Y. Nitzan, A. Balzam-Sudakevitz, H. Ashkenazi, Eradication of Acinetobacter baumannii by photosensitized agents in vitro, J. Photochem. Photobiol. 42 (1998) 211–218. [28] G. Birke, S.O. Liljedahl, L.O. Plantin, Distribution and losses of plasma proteins during the early stage of severe burns, Ann. N.Y. Acad. Sci. 150 (1968) 895–904. [29] G. Birke, S.O. Liljedahl, L.O. Plantin, P. Reizenstein, Studies on burns. IX. The distribution and losses through the wound of 131Jalbumin measured by whole-body counting, Acta Chir. Scand. 134 (1968) 27–36. [30] A.A. Miles, S.S. Misra, The estimation of the bactericidal power of the blood, J. Hygiene 38 (1938) 732–749. [31] A.C. Guyton, J.E. Hall, The Microcirculation and the Lymphatic System: Capillary Fluid Exchange, Interstitial Fluid, Lymph Flow, Textbook of Medical Physiology, nineth ed., W.B. Saunders Company, Philadelphia, 1996, pp. 183–196. [32] J. Moan, C. Rimington, A. Western, The binding of dihematoporphyrin ether (photofrin II) to human serum albumin, Clin. Chim. Acta 145 (1985) 227–236. [33] J.M. Bliss, C.E. Bigelow, T.H. Foster, C.G. Haidaris, Susceptibility of Candida species to photodynamic effects of photofrin, Antimicrob. Agents Chemother. 48 (2004) 2000–2006. [34] M. Wilson, N. Mia, Sensitisation of Candida albicans to killing by low-power laser light, J. Oral Pathol. Med. 22 (1993) 354–357. [35] G. Bertoloni, F. Zambotto, L. Conventi, E. Reddi, G. Jori, Role of specific cellular targets in the hematoporphyrin-sensitized photoinactivation of microbial cells, Photochem. Photobiol. 46 (1987) 695–698. [36] F.R. Venezio, C. DiVincenzo, R. Sherman, M. Reichman, T.C. Origitano, K. Thompson, O.H. Reichman, Bactericidal effects of
[37]
[38]
[39]
[40]
[41]
[42] [43]
[44]
[45]
[46]
57
photoradiation therapy with hematoporphyrin derivative, J. Infect. Dis. 151 (1985) 166–169. G. Bertoloni, B. Salvato, M. DallÕAcqua, M. Vazzoler, G. Jori, Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis., Photochem. Photobiol. 39 (1984) 811–816. G. Bertoloni, E. Reddi, M. Gatta, C. Burlini, G. Jori, Factors influencing the haematoporphyrin-sensitized photoinactivation of Candida albicans, J. Gen. Microbiol. 135 (1989) 957–966. M. Wilson, N. Mia, Effect of environmental factors on the lethal photosensitization of Candida albicans in vitro, Lasers Surg. Med. 9 (1994) 105–109. B. Zeina, J. Greenman, W.M. Purcell, B. Das, Killing of cutaneous microbial species by photodynamic therapy, Br. J. Dermatol. 144 (2001) 274–278. M.W. Dodds, D.A. Johnson, C.C. Mobley, K.M. Hattaway, Parotid saliva protein profiles in caries-free and caries-active adults, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 83 (1997) 244–251. C.A. Bonilla, Human mixed saliva protein concentration, J. Dent. Res. 51 (1972) 664. J. Chandra, D.M. Kuhn, P.K. Mukherjee, L.L. Hoyer, T. McCormick, M.A. Ghannoum, Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance, J. Bacteriol. 183 (2001) 5385–5394. M. Wilson, T. Burns, J. Pratten, Killing of Streptococcus sanguis in biofilms using a light-activated antimicrobial agent, J. Antimicrob. Chemother. 37 (1996) 377–381. N.S. Soukos, S.E. Mulholland, S.S. Socransky, A.G. Doukas, Photodestruction of human dental plaque bacteria: Enhancement of the photodynamic effect by photomechanical waves in an oral biofilm model, Lasers Surg. Med. 33 (2003) 161–168. R. Moseley, J.R. Hilton, R.J. Waddington, K.G. Harding, P. Stephens, D.W. Thomas, Comparison of oxidative stress biomarker profiles between acute and chronic wound environments, Wound. Repair Regen. 12 (2004) 419–429.
Effect of albumin on the photodynamic inactivation of microorganisms by a cationic porphyrin Saskia A.G. Lambrechts a,b, Maurice C.G. Aalders a,*, Frank D. Verbraak a, Johan W.M. Lagerberg c,1, Jacob B. Dankert b, Johannes J. Schuitmaker c b
a Laser Center K01-225-5, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands c PhotoBioChem, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
Received 6 July 2004; received in revised form 21 October 2004; accepted 30 November 2004 Available online 19 January 2005
Abstract Background: Photodynamic inactivation (PDI) employs visible light and a photosensitizer to inactivate cells. The technique is currently clinically used for the treatment of several malignancies. However, the PDI of microorganisms still remains in the research phase. Purpose: To study the effect of human blood plasma and human serum albumin (HSA) on the PDI of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Methods: PDI experiments were performed using white light (30 mW cm 2) and the cationic 5-phenyl-10,15,20-tris(N-methyl-4pyridyl)porphyrin chloride (TriP[4]) as photosensitizer. Results: The microorganisms could be successfully photoinactivated by TriP[4] when suspended in phosphate buffered saline (PBS). In this medium, P. aeruginosa was the most resistant microorganism. Changing the suspending medium from PBS to human blood plasma reduced the PDI of all three microorganisms. In human blood plasma C. albicans was the most resistant microorganism. The same results were obtained with 4.5% and 7% HSA/PBS suspensions. Conclusions: Albumin inhibits the PDI of S. aureus, P. aeruginosa and C. albicans in a dose dependent manner. However, our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans. Ó 2004 Elsevier B.V. All rights reserved. Keywords: Albumin; Microorganisms; Human blood plasma; Photodynamic inactivation; Porphyrin
1. Introduction The growing resistance against antibacterial and antifungal agents has generated a search for alternative antimicrobial treatments. In particular, the use of topical antibiotics is under discussion since it has been *
Corresponding author. Tel.: +31 (0)20 566 3829; fax: +31 (0)20 697 5594. E-mail address: [email protected] (M.C.G. Aalders). 1 Present address: Sanquin research at CLB, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. 1011-1344/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2004.11.020
suggested that such an approach induces antibiotic resistance faster than the use of oral antibiotics [1,2]. A potential alternative may be antimicrobial photodynamic inactivation (PDI). PDI employs visible light to activate a photosensitizer. The activated photosensitizer can react with molecules from its direct environment by electron or hydrogen transfer, leading to the production of radicals (type 1 reaction) or it can transfer its energy to oxygen, generating the highly reactive singlet oxygen (type 2 reaction). Both pathways can lead to cell death [3]. Despite the frequent clinical use
52
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
of PDI for the treatment of several malignancies [4], the use of PDI to inactivate microorganisms is still in the research phase. This is in part due to the fact that the PDI of Gram-negative bacteria with first generation photosensitizers such as the anionic haematoporphyrin had to be mediated by membrane permeabilizers such as ethylenediaminetetraacetic acid or polymyxin nonapeptide [5]. Later it was discovered that cationic porphyrins did not require these membrane permeabilizers in order to successfully inactivate Gram-negative bacteria [6]. It has been demonstrated that Gram-positive, Gram-negative bacteria and fungi can all be successfully photodynamically inactivated by a single cationic photosensitizer, e.g., 5-phenyl10,15,20-tris(N-methyl-4-pyridyl)porphyrin (TriP[4]) [7–9] or toluidine blue [10–12]. Furthermore, it has been shown that both antibiotic sensitive and resistant strains can be successfully photoinactivated [13,14] and so far repeated photosensitization of bacterial cells has not induced a selection of resistant strains [15]. In addition PDI has the advantage of dual selectivity, both the photosensitizer and the illumination can be targeted. All these qualities combined make PDI an interesting alternative antimicrobial treatment for superficial wound infections, which are easily reached by light. In this study, we investigated the PDI of three different clinically interesting microorganisms: a Grampositive bacterium, Staphylococcus aureus and a Gramnegative bacterium, Pseudomonas aeruginosa and the yeast Candida albicans. Staphylococcus aureus colonizes the nasal passage and axillae in 30–40% of the population. In most of such carriers S. aureus is harmless, but if it enters the body through a lesion in the skin it can cause a range of mild to severe infections [16]. Methicillin resistant S. aureus is now one of the most feared causes of nosocomial infections [16–19]. P. aeruginosa is a widely distributed environmental bacterium that is the cause of a substantial portion of nosocomial infections and is frequently found to be multiresistant [17,20]. A superficial wound infection in which both S. aureus and P. aeruginosa play an important role is the burn wound infection [18,20,21]. Burn wound infections can impede wound closure and are an important cause of death in burn wound patients [22,23]. Candida albicans is a common inhabitant of the mouth, throat, digestive tract and skin. In hosts with a compromised immune system it can become pathogenic [24,25]. Oropharyngeal candidiasis is one of the most common opportunistic infections accompanying AIDS patients. Resistance of C. albicans against fluconazole, an antifungal agent frequently used by such patients, has begun to appear [26]. Both burn wound infections and oral candidiasis are superficial lesions for which PDI could be a potential treatment.
With some exceptions, in most in vitro studies, buffer or broth (sometimes diluted) is used as suspending medium. It is known however that the consistency of the suspending medium strongly influences the efficacy of antimicrobial PDI [8,27]. Therefore in vivo circumstances, such as the presence of wound fluid are expected to influence the efficacy of antimicrobial PDI. In the present study, we used human blood plasma and various suspensions of human serum albumin (HSA) in phosphate buffered saline (PBS) to serve as a wound fluid model [28,29]. The cationic porphyrin TriP[4] was chosen as photosensitizer since it has proven to be a successful antimicrobial photosensitizer [7–9]. We investigated the feasibility to use TriP[4] mediated PDI to inactivate S. aureus, P. aeruginosa and C. albicans in the presence of human blood plasma and HSA. The variance in sensitivity to PDI of three wild type strains of each species studied was also investigated.
2. Materials and methods 2.1. Microorganisms Microorganisms, isolated from burn wound patients were kindly provided by the Central Bacteriological Laboratory, Haarlem, the Netherlands. These were S. aureus (strains 2000.1.46382.0, 2000.1.46384.3, 2000.1.47260.2), P. aeruginosa (strains 2000.1.47063.0, 2000.1.47260.2, 2000.1.49012.5) and C. albicans (strains 2000.1.40456.9, 2000.1.48525.7, 2000.1.48703.4). The microorganisms were grown aerobically in 10 ml brain heart infusion broth (Oxoid Ltd., Hampshire, UK) at 37 °C. After 18 h the stationary phase microorganisms were harvested by centrifugation at 3000 rpm for 10 min, washed twice with PBS (pH 7.4) and suspended in 2 ml PBS, human blood plasma or HSA suspension. 2.2. Photosensitizer A stock solution of the photosensitizer, 5-phenyl10,15,20-tris(N-methyl-4-pyridyl)-21,23H-porphyrin chloride (TriP[4]) (Mid-Century Chemicals, Posen, IL), was prepared in a 50 mM sodium phosphate buffer (pH 7.4) at a concentration of 1 mM (0.78 mg ml 1) and stored at 4 °C in the dark. Before use, the stock solution was allowed to warm up to room temperature. 2.3. Light source All illuminations were carried out with white light from a 500 W halogen lamp (Massive, the Netherlands). To avoid heating of the samples, the light was passed through a 1-cm thick water filter. The irradiance at the level of the microorganism samples was 30 mW cm 2 as measured with an IL 1400A power meter (Interna-
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
tional Light, Newburyport, MA). The temperature of the samples never exceeded 22 °C.
53
lM. Survival was determined after 15 min of illumination, which corresponds to a light dose of 27 J cm 2. One wild-type strain of each species was studied.
2.4. PDI studies 2.5. Statistics Samples of 1 ml were prepared, all contained 100 ll of microorganism suspension, resulting in a final cell concentration of 108–109 cells ml 1 for S. aureus and P. aeruginosa and of 106–107 cells ml 1 for C. albicans, the appropriate amount of photosensitizer stock solution (ranging from 3.1 to 50 ll) and PBS. Directly after addition of the photosensitizer, the suspensions were transferred to polystyrene culture dishes with a diameter of 3 cm (Greiner, Alphen a/d Rijn, the Netherlands) and illuminated. After illumination, microbial survival was determined with a modified version of the Miles and Misra method [30]. For this, the suspensions were serially 10-fold diluted with PBS. Then, drops of 10 ll of each dilution were applied onto Iso-sensitest agar in 5fold (Oxoid Ltd., Hampshire, UK) and incubated at 37 °C under aerobic conditions. After 18 h for S. aureus and P. aeruginosa and 48 h for C. albicans the number of colony forming units (CFU) was counted. Survival was expressed as a percentage relative to a control sample containing no photosensitizer, taken at the beginning of each experiment prior to illumination. Survival was also determined for samples after illumination in the absence of photosensitizer and for samples after incubation with the photosensitizer in the dark. 2.4.1. PDI in PBS The microorganisms were harvested as described above and suspended in PBS. The photosensitizer concentrations were; 3.1, 6.3, 12.5, 25 and 50 lM. Survival was determined after 15 min of illumination which corresponded to a light dose of 27 J cm 2. Three wild-type strains of each species were studied. 2.4.2. PDI in plasma The microorganisms were harvested as described above and suspended in human blood plasma obtained from buffy coat (Blood bank Leiden-Haaglanden, the Netherlands) by centrifugation at 2000 rpm for 10 min. A photosensitizer concentration of 50 lM was used in these experiments. Survival was determined after 15, 30, 45 and 60 min of illumination which corresponds to light doses of 27, 54, 81 and 108 J cm 2. Aliquots were derived from one dish after each additive light dose. One wild-type strain of each species was studied. 2.4.3. PDI in albumin suspensions The microorganisms were harvested as described above and suspended in PBS containing 1%, 4.5% or 7% (w/v) HSA. A commercially available 20% solution HSA suspension (CealbÒ, CLB, the Netherlands) was used. The final photosensitizer concentration was 25
Values are expressed as mean percent change from starting control ± standard deviation. Differences between means of multiple groups were analyzed using a StudentÕs t test. Statistical significance was assumed at the p < 0.05 level.
3. Results 3.1. PDI in PBS The photosensitizer concentration dependent inactivation of S. aureus, P. aeruginosa and C. albicans suspended in PBS is shown in Fig. 1. S. aureus was more sensitive to PDI than P. aeruginosa and C. albicans (Fig. 1(a)). Exposed to a TriP[4] concentration of 12.5 lM and a light dose of 27 J cm 2, P. aeruginosa showed a 1 log10-unit reduction in viable count. Under the same conditions S. aureus and C. albicans showed a 7 and 5 log10-unit reduction in viable count, respectively. These reductions in viable count were close to the minimal detection level (MDL) and thus close to the maximal inactivation that could be measured using the described assessment method. The minimal detection level of this method is the point at which no colony forming units can be observed in the undiluted sample. P. aeruginosa had not yet reached this limit at a TriP[4] concentration as high as 50 lM. The three wild type strains studied of each species showed some significant variation in their sensitivity to PDI. The statistical significant (p < 0.05) variations between strains are marked with an asterisks in Figs. 1(b)–(c). Small effects were observed in samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark. These effects were always smaller than 0.3 log10-unit reduction in viable count and were not significantly different from a zero (no change) interpretation for all controls. 3.2. PDI in plasma When the microorganisms were suspended in human blood plasma, the order in sensitivity to PDI changed (Fig. 2). S. aureus remained the most sensitive organism, but C. albicans became less sensitive than P. aeruginosa. Exposed to a TriP[4] concentration as high as 50 lM and a light dose 27 J cm 2, no significant (p > 0.05) reduction in viable count was observed for C. albicans.
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57 10
2
10
1
10
0
10
-1
10
-2
10
-3
P. aeruginosa
C. albicans
†
†
Survival %
Survival %
54
-4
10
-5
10 10
0
10
†
20
30
40
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
-5
10
-6
MDL
0
10
(c)
20
30
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
S. aureus
MDL
* 0
*
P. aeruginosa
10
10
10
20
40
TriP[4] µM
10
2
10
1
10
0
40
50
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
C. albicans
MDL
*
0
50
30
TriP[4] µM
(b)
Survival %
10
2
50
TriP[4] µM
(a)
Survival %
S. aureus
†
-6
10
(d)
*
10
20
30
40
50
TriP[4] µM
Fig. 1. (a) Survival curves of S. aureus (s), P. aeruginosa (n) and C. albicans (h) suspended in PBS exposed to different concentrations TriP[4] and a light dose of 27 J cm 2. Each value is the average of three wild-type strains, each of them performed in triplicate. The error bars indicate the standard deviation between the averages. , No CFU detected, below minimal detectable limit. (b)–(d) Separate survival curves of the three different wild-type strains S. aureus, strains 2000.1.46382.0 (h, j), 2000.1.46384.3 (s, d), 2000.1.47260.2 (n, m) (b), P. aeruginosa, strains 2000.1.49012.5 (h, j), 2000.1.47063.0 (s, d), 2000.1.47260.2 (n, m) (c) and C. albicans strains 2000.1.40456.9 (e, r), 2000.1.48703.4 (h, j), 2000.1.48525.7 (s, d) (d) suspended in PBS and exposed to different concentrations TriP[4]. Open symbols, samples exposed to a light dose of 27 J cm 2. Each point is the mean of at least three experiments ± standard deviation. Closed symbols, samples kept in the dark for 15 min. Each point is the mean of at least two experiments ± standard deviation. Asterisks, significant (p > 0.05) deviation from the average value. MDL, minimal detectable limit.
Under the same conditions a 3 log10-unit and <1 log10unit reduction in viable count was observed for S. aureus and P. aeruginosa, respectively. No significant (p > 0.05) effect was observed in the samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark.
effect was observed in the samples after illumination in the absence of photosensitizer and in samples that were incubated with the photosensitizer in the dark.
3.3. PDI in albumin suspensions
Staphylococcus aureus, P. aeruginosa and C. albicans, suspended in PBS, could be successfully photoinactivated by TriP[4]. Of each species we studied three different wild type strains and no large significant differences in their sensitivity to PDI could be observed, We therefore assume that our results are representative for these species in general. To our knowledge, the PDI of a yeast by a meso-substituted cationic porphyrin has not yet been reported. A requisite for an effective antimicrobial treatment for superficial wound infections is its effectiveness in the inevitable presence of wound fluid. To create an in vitro situation more comparable to wound fluid than
The influence of albumin (HSA) on the PDI of the microorganism is shown in Fig. 3. Albumin exerted a dose dependent protective effect against the PDI of all three microorganisms. The strongest protection was observed for C. albicans. When suspended in a 4.5% albumin suspension, exposed to a TriP[4] concentration of 25 lM and a light dose 27 J cm 2, C. albicans showed a reduction in viable count smaller than 1 log10-unit. Under the same conditions a 5 log10-unit and 1 log10unit reduction in viable count was observed for S. aureus and P. aeruginosa, respectively. No significant (p > 0.05)
4. Discussion
Survival %
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
10
3
10
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
C. albicans
P. aeruginosa
S. aureus †
†
0
27
54
81
108
-2
Light dose (J.cm )
% Survival
Fig. 2. Survival curves of S. aureus 2000.1.46384.3 (s), P. aeruginosa 2000.1.49012.5 (n) and C. albicans 2000.1.48525.7 (h) suspended in human blood plasma exposed to 50 lM TriP[4] and different light doses. , No CFU detected, below minimal detectable limit. Each point is the mean of three experiments ± standard deviation.
10
2
10
1
10
0
10
-1
10
-2
10
-3
10
-4
10
-5
10
-6
S. aureus P. aeruginosa C. albicans
†
†
0
1
4.5
7
Albumin % Fig. 3. Survival histogram of S. aureus 2000. 2000.1.46384.3 (hatched), P. aeruginosa 2000.1.49012.5 (cross hatched) and C. albicans 2000.1.48525.7 (black) suspended in various PBS/HSA suspensions, exposed to 25 lM TriP[4] and a light dose of 27 J cm 2. , No CFU detected, below minimal detectable limit. Each bar is the mean of three experiments ± standard deviation.
PBS, the microorganisms were suspended in human blood plasma. Under this condition, a reduction in sensitivity to PDI was observed for all three microorganisms but especially C. albicans became less sensitive. Interestingly, the relative order in sensitivity to PDI changed from: S. aureus > C. albicans P.aeruginosa in PBS to: S. aureus > P.aeruginosa > C. albicans in human blood plasma. In 4.5% and 7% HSA suspensions the order in sensitivity to PDI was comparable to that found in human blood plasma. The total protein con-
55
centration of human blood plasma is 7.3% of which 4.5% is albumin, [31] therefore we hypothesize that albumin plays the key role in the protection of C. albicans. The inhibiting effect of albumin on the PDI of the microorganisms was not unexpected. The binding of porphyrins to albumin and the quenching and scavenging of reactive oxygen species by albumin are phenomena that have been described extensively [27,32]. However, the highly reduced sensitivity of C. albicans to PDI in an albumin rich suspending fluid was surprising when compared to the more moderate reduction in sensitivity of S. aureus and P. aeruginosa. The sensitivity of C. albicans suspended in PBS to PDI is an indication that its cell envelope does not provide a strong barrier against PDI. Its resistance in an albumin rich environment could be due to a poor binding of TriP[4] to C. albicans relative to the binding of TriP[4] to albumin and either bacterial species. In literature few data are available on the in vitro PDI of C. albicans. Most of the in vitro studies used haematoporphyrin (HP) or a derivative [33–38], toluidine blue or methylene blue [34,39,40] as photosensitizer. Taking into account the variation in experimental design, it appears that the PDI of C. albicans mediated by HP and its derivatives is strongly inhibited by proteins, which is comparable to our observations. Experiments performed in PBS using HP or its derivatives, resulted in the successful PDI of both C. albicans and S. aureus [35,37,38]. However, when the experiments were performed in protein containing broth, no PDI was observed for C. albicans while S. aureus remained sensitive [36]. Similarly, no PDI was observed for C. albicans suspended in broth using a dihaematoporphyrin ester as photosensitizer [34]. In contrast, no influence of the suspending fluid on the PDI of C. albicans was observed when toluidine blue was used as photosensitizer [39]. A factor that can influence the efficacy of PDI is the electronic charge of the photosensitizer, however in this case it does not seem to be of importance as TriP[4] and toluidine blue are both cationic and HP is anionic. The strongly reduced sensitivity of C. albicans to PDI in the presence of high concentrations of albumin is not expected to impose limitations to a potential future treatment of oropharyngeal candidiasis with PDI. The protein content of saliva is low and ranges from 0.1% to 0.3% [41,42] and the PDI of C. albicans suspended in a 1% HSA suspension does not differ significantly from that in PBS (p = 0.325) (Fig. 3). The next step, more comparable to the in vivo situation, would be to investigate wether C. albicans can be photoinactivated when it is present in a biofilm. Like bacterial biofilms, biofilm-grown C. albicans is highly resistant to antimicrobial agents [43]. Despite this increased resistance it has been shown by various groups that bacteria in biofilms can be successfully photoinactivated [44,45].
56
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57
S. aureus was substantially less affected by the inhibiting effect of albumin, implying a sustainable sensitivity to PDI when present in wounds with protein rich exudate. We chose human serum albumin and blood plasma as a model since wound fluids are predominantly serum derived. However the protein content of wound fluid can be much lower than that of blood serum, according to Moseley et al. wound fluid from acute and chronic wounds contain as little as 1.476 (±0.123) mg ml 1 and 0.644 (±0.153) mg ml 1 protein, respectively [46]. This corresponds to 0.15 and 0.06 w/v%, respectively. Therefore the inhibition of wound fluid proteins will most likely not impose a problem for the in vivo PDI of the investigated species. However the use of antimicrobial PDI with TriP[4] for wounds containing a substantial amount of blood will most likely be ineffective due to the inhibiting effect of albumin. S. aureus was substantially less affected by the inhibiting effect of albumin, implying a sustainable sensitivity to PDI when present in wounds with protein rich exudate. In conclusion, our experiments have demonstrated that S. aureus, P. aeruginosa and C. albicans can be photoinactivated using TriP[4] as photosensitizer. However, the degree of microbial PDI strongly depended on the albumin content of the suspending fluid. The strongest inhibiting effect of albumin was observed for the PDI of C. albicans. Our results are encouraging towards the potential future application of PDI for the treatment of superficial wound infections caused by S. aureus, P. aeruginosa and C. albicans
Acknowledgements We thank Threes G.M. Smijs (Leiden University Medical Centre, Leiden, the Netherlands, supported by the Dutch Technology Foundation STW LGN 4890) for the constructive discussions and reviewing the article. We thank the Central Bacteriological Laboratory, Haarlem, the Netherlands for providing the microorganisms.
References [1] J.I. Ross, A.M. Snelling, E. Carnegie, P. Coates, W.J. Cunliffe, V. Bettoli, G. Tosti, A. Katsambas, J.I. Galvan Perez Del Pulgar, O. Rollman, L. Torok, E.A. Eady, J.H. Cove, Antibiotic-resistant acne: lessons from Europe, Br. J. Dermatol. 148 (2003) 467–478. [2] P. Coates, C.A. Adams, W.J. Cunliffe, K.T. McGinley, E.A. Eady, J.J. Leyden, J. Ravenscroft, S. Vyakrnam, B. Vowels, Does oral isotretinoin prevent Propionibacterium acnes resistance?, Dermatology 195 (Suppl 1) (1997) 4–9. [3] M. Wainwright, Photodynamic antimicrobial chemotherapy (PACT), J. Antimicrob. Chemother. 42 (1998) 13–28. [4] C. Fritsch, G. Goerz, T. Ruzicka, Photodynamic therapy in dermatology, Arch. Dermatol. 134 (1998) 207–214.
[5] Z. Malik, H. Ladan, Y. Nitzan, Photodynamic inactivation of Gram-negative bacteria: problems and possible solutions, J. Photochem. Photobiol. 14 (1992) 262–266. [6] M. Merchat, G. Bertolini, P. Giacomini, A. Villanueva, G. Jori, Meso-substituted cationic porphyrins as efficient photosensitizers of Gram-positive and Gram-negative bacteria, J. Photochem. Photobiol. 32 (1996) 153–157. [7] T.G.M. Smijs, H.J. Schuitmaker, Photodynamic inactivation of the dermatophyte Trichophyton rubrum, Photochem. Photobiol. 77 (2003) 556–560. [8] S.A.G. Lambrechts, M.C.G. Aalders, D.H. Langeveld-Klerks, Y. Khayali, J.W.M. Lagerberg, Effect of monovalent and divalent cations on the photoinactivation of bacteria with meso-substituted cationic porphyrins, Photochem. Photobiol. 79 (2003) 297–302. [9] M. Merchat, J.D. Spikes, G. Bertoloni, G. Jori, Studies on the mechanism of bacteria photosensitization by meso-substituted cationic porphyrins, J. Photochem. Photobiol. 35 (1996) 149–157. [10] Z. Jackson, S. Meghji, A. MacRobert, B. Henderson, M. Wilson, Killing of the yeast and hyphal forms of Candida albicans using a light-activated antimicrobial agent, Lasers Med. Sci. 14 (1999) 150–157. [11] M.N. Usacheva, M.C. Teichert, M.A. Biel, Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms, Lasers Surg. Med. 29 (2001) 165–173. [12] N. Komerik, M. Wilson, Factors influencing the susceptibility of Gram-negative bacteria to toluidine blue O-mediated lethal photosensitization, J. Appl. Microbiol. 92 (2002) 618–623. [13] Z. Malik, S. Gozhanski, Y. Nitzan, Effects of photoactivated HPD on bacteria and antibiotic resistance, Microbios. Lett. 21 (1982) 103–112. [14] M. Soncin, C. Fabris, A. Busetti, D. Dei, D. Nistri, G. Roncucci, G. Jori, Approaches to selectivity in the Zn(II)-phthalocyaninephotosensitized inactivation of wild-type and antibiotic-resistant Staphylococcus aureus, Photochem. Photobiol. Sci. 1 (2002) 815– 819. [15] F.M. Lauro, P. Pretto, L. Covolo, G. Jori, G. Bertoloni, Photoinactivation of bacterial strains involved in periodontal diseases sensitized by porphycene–polylysine conjugates, Photochem. Photobiol. Sci. 1 (2002) 468–470. [16] S. Baron, Staphylococcus, in: R.C. Peake, D.A. James, M. Susman, C.A. Kennedy, M.J.D. Singleton, S. Schuenke (Eds.), Medical Microbiology, fourth ed., The University of Texas Medical Branch, Galveston, 1996. [17] J.L. Vincent, D.J. Bihari, P.M. Suter, H.A. Bruining, J. White, M.H. Nicolas-Chanoin, M. Wolff, R.C. Spencer, M. Hemmer, The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection in Intensive Care (EPIC) Study. EPIC International Advisory Committee, JAMA 274 (1995) 639–644. [18] N. Cook, Methicillin-resistant Staphylococcus aureus versus the burn patient, Burns 24 (1998) 91–98. [19] D.M. Livermore, Antibiotic resistance in staphylococci, Int. J. Antimicrob. Agents 16 (2000) S3–S10. [20] J.P. Pirnay, D. De Vos, C. Cochez, F. Bilocq, J. Pirson, M. Struelens, L. Duinslaeger, P. Cornelis, M. Zizi, A. Vanderkelen, Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone, J. Clin. Microbiol. 41 (2003) 1192–1202. [21] C.G. Mayhall, The epidemiology of burn wound infections: then and now, Clin. Infect. Dis. 37 (2003) 543–550. [22] B. Pruitt, A. McManus, S. Kim, C. Goodwin, Burn wound infections: current status, World J. Surg. 22 (1998) 135–145. [23] A.J. Singer, S.A. McClain, Persistent wound infection delays epidermal maturation and increases scarring in thermal burns, Wound Repair Regen. 10 (2002) 372–377.
S.A.G. Lambrechts et al. / Journal of Photochemistry and Photobiology B: Biology 79 (2005) 51–57 [24] G. Garber, An overview of fungal infections, Drugs 61 (Suppl. 1) (2001) 1–12. [25] S.K. Fridkin, W.R. Jarvis, Epidemiology of nosocomial fungal infections, Clin. Microbiol. Rev. 9 (1996) 499–511. [26] J.H. Rex, M.G. Rinaldi, M.A. Pfaller, Resistance of Candida Species to Fluconazole, Antimicrob. Agents Chemother. 39 (1995) 1–8. [27] Y. Nitzan, A. Balzam-Sudakevitz, H. Ashkenazi, Eradication of Acinetobacter baumannii by photosensitized agents in vitro, J. Photochem. Photobiol. 42 (1998) 211–218. [28] G. Birke, S.O. Liljedahl, L.O. Plantin, Distribution and losses of plasma proteins during the early stage of severe burns, Ann. N.Y. Acad. Sci. 150 (1968) 895–904. [29] G. Birke, S.O. Liljedahl, L.O. Plantin, P. Reizenstein, Studies on burns. IX. The distribution and losses through the wound of 131Jalbumin measured by whole-body counting, Acta Chir. Scand. 134 (1968) 27–36. [30] A.A. Miles, S.S. Misra, The estimation of the bactericidal power of the blood, J. Hygiene 38 (1938) 732–749. [31] A.C. Guyton, J.E. Hall, The Microcirculation and the Lymphatic System: Capillary Fluid Exchange, Interstitial Fluid, Lymph Flow, Textbook of Medical Physiology, nineth ed., W.B. Saunders Company, Philadelphia, 1996, pp. 183–196. [32] J. Moan, C. Rimington, A. Western, The binding of dihematoporphyrin ether (photofrin II) to human serum albumin, Clin. Chim. Acta 145 (1985) 227–236. [33] J.M. Bliss, C.E. Bigelow, T.H. Foster, C.G. Haidaris, Susceptibility of Candida species to photodynamic effects of photofrin, Antimicrob. Agents Chemother. 48 (2004) 2000–2006. [34] M. Wilson, N. Mia, Sensitisation of Candida albicans to killing by low-power laser light, J. Oral Pathol. Med. 22 (1993) 354–357. [35] G. Bertoloni, F. Zambotto, L. Conventi, E. Reddi, G. Jori, Role of specific cellular targets in the hematoporphyrin-sensitized photoinactivation of microbial cells, Photochem. Photobiol. 46 (1987) 695–698. [36] F.R. Venezio, C. DiVincenzo, R. Sherman, M. Reichman, T.C. Origitano, K. Thompson, O.H. Reichman, Bactericidal effects of
[37]
[38]
[39]
[40]
[41]
[42] [43]
[44]
[45]
[46]
57
photoradiation therapy with hematoporphyrin derivative, J. Infect. Dis. 151 (1985) 166–169. G. Bertoloni, B. Salvato, M. DallÕAcqua, M. Vazzoler, G. Jori, Hematoporphyrin-sensitized photoinactivation of Streptococcus faecalis., Photochem. Photobiol. 39 (1984) 811–816. G. Bertoloni, E. Reddi, M. Gatta, C. Burlini, G. Jori, Factors influencing the haematoporphyrin-sensitized photoinactivation of Candida albicans, J. Gen. Microbiol. 135 (1989) 957–966. M. Wilson, N. Mia, Effect of environmental factors on the lethal photosensitization of Candida albicans in vitro, Lasers Surg. Med. 9 (1994) 105–109. B. Zeina, J. Greenman, W.M. Purcell, B. Das, Killing of cutaneous microbial species by photodynamic therapy, Br. J. Dermatol. 144 (2001) 274–278. M.W. Dodds, D.A. Johnson, C.C. Mobley, K.M. Hattaway, Parotid saliva protein profiles in caries-free and caries-active adults, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 83 (1997) 244–251. C.A. Bonilla, Human mixed saliva protein concentration, J. Dent. Res. 51 (1972) 664. J. Chandra, D.M. Kuhn, P.K. Mukherjee, L.L. Hoyer, T. McCormick, M.A. Ghannoum, Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance, J. Bacteriol. 183 (2001) 5385–5394. M. Wilson, T. Burns, J. Pratten, Killing of Streptococcus sanguis in biofilms using a light-activated antimicrobial agent, J. Antimicrob. Chemother. 37 (1996) 377–381. N.S. Soukos, S.E. Mulholland, S.S. Socransky, A.G. Doukas, Photodestruction of human dental plaque bacteria: Enhancement of the photodynamic effect by photomechanical waves in an oral biofilm model, Lasers Surg. Med. 33 (2003) 161–168. R. Moseley, J.R. Hilton, R.J. Waddington, K.G. Harding, P. Stephens, D.W. Thomas, Comparison of oxidative stress biomarker profiles between acute and chronic wound environments, Wound. Repair Regen. 12 (2004) 419–429.