RESEARCH ARTICLE

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Antimicrobial photodynamic activity of hypericin against methicillin-susceptible and resistant Staphylococcus aureus biofilms Isabel García*,1,2, Sofía Ballesta1,2, Yolanda Gilaberte3, Antonio Rezusta4 & Álvaro Pascual1,2,5 ABSTRACT Aim: To evaluate the effectiveness of the photodynamic therapy using hypericin (HYP) against both planktonic and biofilm-forming Staphylococcus aureus. Materials & methods: HYP photoactivity was evaluated against methicillin-susceptible and resistant S. aureus. Bacterial suspension or biofilm were preincubated with HYP and subjected to LED illumination. Viable bacteria were determined by colony counting. Results: Preincubation with HYP (5 min) plus light exposure (10 min) showed bactericidal effect against planktonic methicillin-susceptible S. aureus and methicillin-resistant S. aureus. Longer preincubation times (24 h) and time light exposure (30 min) were required to reach HYP-photoactivity against S. aureus biofilms. HYP-photoactivity was correlated to the biofilm production. Conclusion: HYP could be a potential photosensitizer for the inactivation of staphylococcal biofilms forming on the surfaces accessible to visible light. Infections involving biofilms have significant clinical and economic impact. Implant-associated infections and many chronic infections, as cystic fibrosis, periodontitis, endocarditis and chronic wounds, are related to biofilms. Staphylococcus aureus is a pathogen commonly involved in many infections, including those related to medical devices and some chronic wounds, where the bacteria grows forming biofilm [1] . Biofilms are difficult to eradicate because microorganisms embedded in them are less susceptible to antimicrobial agents [2,3] . Treating such infections is further complicated by the emergence of antimicrobial-resistant strains. Since biofilms can resist currently available antibiotics, new strategies for treating these infections are needed [4] . Several studies have reported the efficacy of photodynamic therapy (PDT) against Gram-positive and Gram-negative bacteria, including multidrug-resistant pathogens such as methicillin-resistant S. aureus (MRSA). In general, Gram-negative bacteria are more resistant to PDT than Gram-positive because the cell wall of Gram-negative bacteria acts as a permeability barrier that impairs the penetration of many photosensitizers, especially hydrophobic compounds [5–7] . In addition, Grinholc et al. have reported differences in response to PDT between methicillin-susceptible S. aureus (MSSA) and MRSA [8] . PDT could be a novel antimicrobial treatment approach for treating infections associated to devices that can be reached by optical fibers or skin chronic infections. PDT involves the application

KEYWORDS 

• antimicrobial

photodynamic therapy • antimicrobial resistance • medical devices infections • Staphylococcus aureus

Department of Microbiology, School of Medicine, Universidad de Sevilla, Sevilla, Spain Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015), Instituto de Salud Carlos III, Madrid, Spain 3 Department of Dermatology, IIS Aragón, Hospital San Jorge, Huesca, Spain 4 Department of Microbiology, Hospital Universitario Miguel Servet, IIS Aragón, Universidad de Zaragoza, Zaragoza, Spain 5 Infectious Diseases & Clinical Microbiology Unit, Hospital Universitario Virgen Macarena, Sevilla, Spain *Author for correspondence: Tel.: +34 9545 52862; Fax: +34 9543 77413; [email protected] 1 2

10.2217/FMB.14.114 © 2015 Future Medicine Ltd

Future Microbiol. (2015) 10(3), 347–356

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Research Article  García, Ballesta, Gilaberte, Rezusta & Pascual of a photosensitizer, which is activated by visible light of an appropriate wavelength to generate reactive oxygen species, including singlet oxygen and hydroxyl radicals that are cytotoxic to target cells  [9] . To show high antibacterial photoactivity, the photosensitizer must cross the bacterial membrane and reach a high endocellular concentration. PDT has been effective against biofilms in dental plaque and oral implants [10,11] . Recent studies that use toluidine blue, methylene blue or porphyrins as the photosensitizer suggest that PDT could be a useful approach for the inactivation of staphylococcal biofilms [12–16] . Hypericin (HYP), a natural photosensitizer isolated from some plant species of the genus Hypericum, belongs to the new generation of PDT drugs. It has the potential to treat several types of cancer and some benign skin disorders [17,18] . Its photochemical and photobiological properties, such as a high singlet oxygen quantum yield and cytoplasmic membrane localization, make it suitable for use as a photosensitizer in antimicrobial PDT [19,20] . Previous in vitro studies have demonstrated that significant HYP-phototoxicity can be induced in cells, depending on the concentration and light dose. It is possible to preserve human cells by keeping the HYP concentration below 1 μM and the light dose below 38 J cm-2 [21,22] . HYP has shown in vitro antimicrobial photodynamic activity against planktonic Gram-positive bacteria, such as S. aureus and Enterococcus faecalis, and the fungus, Candida albicans [19,21,23] . The effectiveness of HYP-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake [19,20] . However, the antibacterial efficacy of HYP in biofilm-associated infections is currently unknown. The aim of this study was to evaluate PDT, using the photosensitizer HYP as a new strategy for treating infections related to biofilms, where S. aureus is a pathogen commonly involved. We have assayed different preincubation times and several light doses to optimize the HYPphotodynamic effect against planktonic and sessile methicillin-susceptible and resistant S. aureus. Materials & methods

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●●Biofilm production

Biofilm production was assessed by a previously described spectrophotometric method [24] . Overnight bacterial cultures were diluted at 1:100 in TSB containing 0.25% glucose (106 cells/ml) and 200 μl were added to the wells of a 96-well flat- bottom tissue culture plate (Cellstar®; Greiner bio-one, Sigma-Aldrich, Spain) incubated for 24 h at 37°C for biofilm growth (standard conditions). Briefly, biofilms formed on the plate were washed twice with phosphate-buffered saline (PBS) to remove planktonic cells, and adherent bacteria fixed with 95% ethanol for 10 min and stained with 0.1% crystal violet for 15 min. To measure biofilm formation, crystal violet was solubilized by adding 33% glacial acetic acid and the absorbance of the solubilized dye was measured at 590 nm (Infinite 200 PRO; Tecan Ibérica, Barcelona, Spain) . Strains were classified as weak, moderate or strong biofilm producers, in accordance with the Stephanovic classification [25] . ●●Characterization of biofilm production

The staphylococcal biofilm phenotype is tightly regulated and highly responsive to environ­mental conditions. To characterize the staphylococcal biofilm phenotype of both strains, overnight bacterial cultures were also diluted at 1:100 in TSB containing 1% glucose, which induces the formation of biofilm by icaADBC-independent mechanisms involving protein or 4% NaCl, which produces icaADBC-dependent, PIA/PNAG-mediated biofilm [26] . Biofilm formation was determined as described above. Data are compared with biofilm production in standard conditions (TSB containing 0.25% glucose). ●●Photosensitizer solutions

HYP stock (Merck KGaA, Darmstadt, Germany) solution (2 mM) was prepared in dimethyl sulfoxide, aliquoted and stored in the dark at -20°C. Stock solution was diluted in PBS (80 mM potassium phosphate-70 mM NaCl, pH: 7.2) to obtain the desired concentrations immediately prior to use. All solutions were prepared and handled under light-restricted conditions.

●●Bacterial strains & culture conditions

●●Antimicrobial photodynamic activity

The microorganisms used in this study were S. aureus ATCC 29213 (MSSA) and S. aureus ATCC 33591 (MRSA). Bacteria were routinely grown overnight in tryptic soy broth (TSB), under aerobic conditions at 37°C using a shaker incubator.

against planktonic bacteria

Future Microbiol. (2015) 10(3)

To test the antimicrobial photodynamic activity of HYP against planktonic bacteria, aliquots (200 μl) of the freshly prepared bacterial suspensions (overnight cultures diluted at 1:100 in TSB containing 0.25% glucose; 106 cells/ml) were

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Antimicrobial photodynamic activity of hypericin against Staphylococcus aureus biofilms  placed into sterile 96-well flat-bottom polystyrene microplates, preincubated in the dark for 5 min with concentrations of HYP ranging from 0.015 μM to 250 μM, then all the wells were subjected to simultaneous LED illumination (602 ± 10 nm; irradiance: 14 mW cm-2) for 10 and 30 min (fluence 8 and 25 J cm-2 respectively). The uniformness of the light spot was checked; the irradiance of the lamp was measured in the center (13.8 ± 0.43 mW cm-2) and 5 cm from the center (13.4 mW cm-2). This occurs in all no-coherent lights, therefore, there is a limitation in all the in vitro and clinical studies performed with these lamps. Moreover, the same strain (S. aureus ATCC 33591) was inoculated in different wells (center and four corners) with a HYP concentration of 0.12 μM and exposed to light (n = 4). The average values of surviving bacteria were 9.8 × 102 ± 0.7 × 102. Given the low variability of the results, we consider that all light spots are similar. Three controls were evaluated for each condition: a negative control (HYP-/light-); a light control (HYP-/light+) and a dark control (HYP+/light-). Afterwards, samples were serially diluted in PBS and viable bacteria determined by colony counting in Mueller Hinton Agar. Survival bacteria data are expressed as colony-forming unit/ml (CFU/ml). Detection limit: 80 CFU/ml. ●●Antimicrobial photodynamic activity

against biofilms

To investigate the effect of HYP-mediated photodynamic treatment against sessile bacteria, biofilms were formed as described above. Plates were washed three times with PBS, 100 μl HYP (0.015–250 μM) was added to each well and the plates were incubated in the dark for different periods of time (20 min and 2, 6 and 24 h). After exposure to LED illumination for 10 and 30 min (fluence 8 and 25 J cm-2 , respectively), the HYP was carefully removed; biofilms were washed three-times in PBS and adherent bacteria detached by sonication (40 kH, 2 min). Controls and bacterial survival were determined as previously described. Survival attached bacteria data are expressed as CFU/cm 2. The bactericidal activity of hypericin against biofilm-forming bacteria was evaluated using the same criterion as that for bactericidal activity against planktonic bacteria; HYP concentrations that reduced the original inoculum by 3-log10 (99.9%) were considered bactericidal [27] .

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●●Statistical analysis

All experiments were performed four-times. The results are expressed as mean ± standard deviation. Differences between groups were compared by analysis of variance with statistical significance at p ≤ 0.05. Results ●●Biofilm formation

Absorbance values in standard conditions (TSB containing 0.25% glucose) were 0.416 ± 0.035 and 0.290 ± 0.023 for the MSSA and MRSA strains respectively (limit value indicative of biofilm producing: 0.177). Based on the Stephanovic classification, the MSSA strain was considered as moderated biofilm producer and the MRSA strain like weak biofilm producer. Table 1 shows the biofilms production at different environmental conditions. MRSA biofilm production was unaffected by the presence of NaCl and slightly activated when the glucose concentration increased. By contrast, addition of NaCl to the growth medium triggered significantly the formation of biofilm by the MSSA strain. ●●Antimicrobial photodynamic activity

against planktonic bacteria

The antimicrobial photodynamic activity of different concentrations of HYP against bacterial suspensions preincubated for 5 min and LED illuminated for 10 min (fluence 8 J cm-2) is shown in Figure 1. Against both strains, a photodynamic bactericidal effect (a reduction in cell count of around 3-log10) was obtained with a HYP concentration of 0.06 μM. HYP 0.25 μM was enough to produce a complete photokilling of MSSA strain whereas this concentration was 1 μM for MRSA strain. The lowest concentration of HYP evaluated (0.015 μM) induced a complete photokilling of both strains using a fluence of 25 J cm-2 (reached after 30 min of LED illumination). Neither HYP nor light alone had an antibacterial effect on any of the tested bacterial strains (data not shown). ●●Antimicrobial photodynamic activity

against biofilms

HYP concentrations as high as 250 μM using a fluence of 8 J cm-2 did not show antimicrobial effect against staphylococcal biofilm. A higher light dose (25 J cm-2) was required to observe any photodynamic inactivation of this biofilm. Figure 2 shows the antimicrobial photodynamic activity of HYP against staphylococcal biofilms at different preincubation time intervals. Against the MRSA

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Research Article  García, Ballesta, Gilaberte, Rezusta & Pascual Table 1. Biofilm production by Staphylococcus aureus at different environmental conditions.  Staphylococcus aureus  

Absorbance values 0.25% Glucose standard conditions

ATCC 33591 (methicillin-resistant) 0.290 ± 0.031 ATCC 29213 (methicillin-susceptible) 0.416 ± 0.086

1% glucose

4% NaCl

0.406 ± 0.102 0.489 ± 0.127

0.245 ± 0.057 0.901 ± 0.224*

Data are expressed as absorbance values measured at 590 nm (n = 4). *p < 0.05 compared with absorbance value in 0.25% glucose (standard conditions).

strain (weak biofilm producing), a maximum 3.5log10 reduction was achieved with 0.125 μM HYP, preincubated for 24 h before irradiation. At shorter preincubation intervals, the reduction obtained ranged between 1.6- and 2.2-log10, preincubating 1 μM HYP for 6 h and 64 μM HYP for 20 min and 2 h (Figure 2A) . After 24 h of preincubation, the maximum reduction against MSSA (a moderate biofilm-producer strain) was 2.3-log10, obtained with 1 μM HYP. The maximum reduction at lower preincubation time intervals was also 2.3-log10, obtained with HYP concentrations of 250, 32 and 4 μM after 20 min, 2 and 6 h preincubation, respectively (Figure 2B) . HYP antimicrobial photodynamic activity against both staphylococcal strains at nontoxic concentrations (1 μM) and different preincubation time intervals is shown in Table 2. HYP 1 μM was not bactericidal against any of the two strains after preincubation of 20 min, 2 h or 6 h. A bactericidal effect was only observed against the weak biofilm-producing MRSA after 24 h of HYP preincubation. Discussion Infections related to biofilms are difficult to eradicate and new therapeutic strategies are required. Antimicrobial PDT has emerged as a promising therapeutic approach for treating these infections. In this study we evaluated the antimicrobial photodynamic activity of HYP against staphylococcal biofilms. HYP exerts a high antimicrobial photodynamic activity against planktonic S. aureus. HYP 0.06 μM was bactericidal against both strains. A HYP concentration 1 μM was enough to reach a complete photokilling. In vitro studies have demonstrated that keratinocytes and fibroblasts can be preserved by keeping the HYP concentration below 1 μM and the light dose less than 37 J cm-2 [21,22] . Our results showed that HYP was bactericidal against both strains evaluated at concentrations lower than those that induce a phototoxic effect. Previous studies have also reported the effectiveness of HYP-mediated photodynamic killing against

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planktonic S. aureus  [19,23] . HYP caused damage to the membrane, producing cell leakage and ultimately cell death. Loss of membrane integrity was dose-dependent and in good agreement with the cell-killing rate. Grinholc et al. evaluated the antimicrobial photodynamic activity of protoporphyrin against MRSA and MASA isolates and stated that MRSA seemed to be more resistant to photoinactivation than were MSSA strains [8,28] . According to these results, the photoactivity of HYP against the MRSA strain evaluated was slightly less than that showed against MSSA strain. Against bacterial biofilms, there was lower antimicrobial photodynamic activity of HYP than against planktonic bacteria. Longer preincubation intervals and higher concentrations of HYP and light dose were required to induce photodynamic killing in biofilm. Several factors could account for this, such as differences of cell wall composition between planktonic and sessile bacteria, growth rates and/or the presence of components in the biofilm that may affect HYP uptake and light penetration [13] . There was a clear correlation between both the HYP concentration–preincubation time and killing efficacy. The effect of preincubation times of other photosensitizers on antimicrobial photodynamic activity against bacterial biofilms has been previously described [16,29] . Pereira et al. observed that the effects of antimicrobial PDT occurred predominantly in the outermost layers of the biofilm [30] and suggested that a longer preincubation enabled deeper penetration into the biofilm layers, so leading to a greater reduction in metabolic activity. The photoactivity of other photosensitizers as gallium (III) phthalocyanine (GaPc) or toluidine blue O (TBO) against staphylococcal biofilms have been also lower than that showed against planktonic cultures. With a light dose of 50 J cm−2, a complete photoinactivation of planktonic S. aureus was observed, using with gallium(III) phthalocyanine (GaPc), while staphylococcal biofilms was only slightly inactivated (0.7–1.5 log10 )  [15] . The toluidine blue O concentration and light dose used for the

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Antimicrobial photodynamic activity of hypericin against Staphylococcus aureus biofilms  photoinactivation of biofilms were also considerably higher than those required to inactivate S. aureus suspensions. [12] . HYP photoactivity against S. aureus biofilms was higher than that observed with others photosensitizers. Light doses higher than that required with HYP were needed to obtain a reduction in surviving bacteria around 2 log10 with methylene blue, tetra-substituted N-methyl-pyridyl-porphine or TBO. [12–13,16] . HYP-photoactivity against sessile MRSA was higher than against sessile MSSA. This fact must be related to the biofilm production, weak in MRSA and moderate in MSSA, and not with the intrinsic HYP photoactivity, higher in the MSSA strain. More slime production may hinder the uptake of the photosensitizer and the penetration of light, so reducing the photosensitizing process  [31] . Moreover, MSSA biofilm was promoted by the osmotic stress induced by NaCl, data that points out an icaADBC-dependent phenotype, mediated by PIA/PNAG production, while the slight activation of MRSA biofilm production associated to addition of glucose to the growth medium indicates a phenotype icaADBCindependent  [32] . Abundant production of PIA has been related to obstruct the photosensitizer diffusion through the matrix [12] , which supports that in this strain the MSSA phenotype

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could contribute also to reduce the susceptibility of biofilm to photosensitization. Moreover, it is also known that, in addition to acting directly on bacterial cells, the products generated as a result of antimicrobial PDT also act on the extracellular matrix of biofilm, increasing photodynamic efficiency [33,34] . It is possible that structural differences in the extracellular matrix of the biofilms may regulate the photodynamic mechanism involving reactive oxygen species. Our results indicate that the antimicrobial photodynamic activity of HYP against staphylococcal biofilms is related to phenotype and biofilm production. At nonphototoxic concentrations, HYP showed antimicrobial photodynamic activity when it had been preincubated for 6 h or more. In this range of concentrations, a bactericidal effect was only obtained against the weak biofilm-producing S. aureus strain, after a preincubation of 24 h with a concentration as low as 0.125 μM. Regarding the moderate biofilm-producing S. aureus, a reduction of only 2-log10 was achieved after 24 h of preincubation with HYP concentration 1 μM. Against both S. aureus strains, increasing HYP concentrations did not result into a significant increase in the photokilling. At shorter preincubation times (20 min and 2 h), maxima HYP-antimicrobial photoactivity was reached at concentrations of

7 6

Log10 CFU/ml

5 4 3 2 1 0 0

0.015

0.03

0.06

0.12

0.25

0.5

1

2

4

8

Hypericin (µM) S. aureus ATCC33591

S. aureus ATCC 29213

Figure 1. Antimicrobial photodynamic activity of hypericin against planktonic methicillin-susceptible and resistant Staphylococcus aureus. Hypericin preincubation time 5 min, fluence 8 J cm-2 (n = 4). CFU: Colony-forming unit.

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Research Article  García, Ballesta, Gilaberte, Rezusta & Pascual

S. aureus ATCC 33591

A 8 7

Log10 CFU/m2

6

†

5 ‡

4 3 2 1 0 0

0.125

0.25

0.5

1

2 4 8 Hypericin (µM)

20 min

2h

16 6h

32

64

125

250

64

125

250

24h

S. aureus ATCC 29213 B 9 8

Log10 CFU/cm2

7 6 5 4 3 2 1 0 0

0.125

0.25

0.5

1

2

4 8 Hypericin (µM)

20 min

2h

16 6h

32 24 h

Figure 2. Antimicrobial photodynamic activity of hypericin against methicillin-susceptible and -resistant Staphylococcus aureus biofilms at different preincubation times. LED illumination 30 min; fluence 25 J cm-2. The dotted line indicates the limit of hypericin-phototoxic concentrations. (A) S. aureus ATCC 33591 (methicillin-resistant) and (B) S. aureus ATCC 29213 (methicillin-susceptible; n = 4). † Antimicrobial photodynamic activity compared with control (p < 0.05). ‡ Bactericidal effect: a >3-log10 reduction compared with control.

more than 1μM, showing also a saturable trend. There have been no previous reports of remarkable changes of phototoxicity against bacterial biofilms using increased concentrations of other photosensitizers  [12] . These results are consistent with the fact that HYP needs time to join/penetrate

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the bacterial cell. Once the maximum value for reduction in bacterial survival has been reached, increased concentrations and longer preincubation times do not yield any further increase in HYP photoactivity. Considering that the sub­ cellular localization of a photosensitizer is critical

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Antimicrobial photodynamic activity of hypericin against Staphylococcus aureus biofilms  to the therapeutic outcome of PDT and that HYP is capable of targeting cellular membrane systems very rapidly, factors related to the intracellular distribution of the photosensitizer could be implicated [20,21] . On the other hand, Kishen et al. found that an efflux pump inhibitor potentiated the photodynamic inactivation of biofilms [35] . In the same way that HYP efficacy is based on its ability to accumulate in the cytoplasmic membrane and generate cytotoxic singlet oxygen under light irradiation, so efflux mechanisms may also regulate the concentration of HYP. Conclusion HYP showed bactericidal photoactivity against MSSA and MRSA biofilm. The bactericidal effect was directly correlated with HYP concentration and preincubation time. The antimicrobial photodynamic activity of HYP against staphylococcal biofilm was related to the phenotype and biofilm production. Based on the present results, it may be concluded that HYP could be a potential photosensitizer for the inactivation of staphylococcal biofilms forming on the surfaces of medical devices or chronic infections that are accessible to visible light or related to skin. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, strategy for preventing the colonization or infection of medical devices related to biofilms as well as skin chronic infections. Future perspective S. aureus is a pathogen commonly involved in infections related to biofilm. Within the biofilm, the bacteria become inherently resistant to antibiotics and host immune defenses, which hinders their treatment and clearance. Treating such infections is further complicated by the emergence of methicillin-resistant strains (MRSA). In

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addition, there has been growing concern about the increasing resistance of MRSA to glycopeptides. It can be anticipated that the development of resistance will likely continue in the coming years. To overcome the antibiotic resistance of bacteria forming biofilm, more effective antimicrobial agents or new treatments are needed. PDT appears to be a promising and innovative approach to kill microbial cells that otherwise would hide in biofilms and escape from conventional antimicrobial agents. An advantage of PDT is fact that bacteria are not able to easily develop resistance, and multiresistant strains have shown to be as susceptible as their naive counterparts. Numerous reports have demonstrated that planktonic S. aureus including MRSA can be inactivated by a variety of photosensitizers. However, it has recently been described that the antimicrobial photoactivity seems to be straindependent and that the response of MRSA strains to PDT is slightly lower than that of MSSA. The mechanism underlying this phenomenon is still poorly understood. Factors as the presence of mec element, the ability to form biofilm, the efflux pumps, the antimicrobial susceptibility or the resistance mechanisms do not provide a full explanation of observed differences. Regarding to photoinactivation of S. aureus biofilms, non-slime-producing S. aureus strains appear more sensitive to PDT than their slimeproducing isolates. Biofilm production may impair the photosensitizer and/or light uptake, playing a role in the resistance to PDT. In recent years, differences in the biofilm phenotype of clinical isolates of MRSA and MSSA have been described. MSSA isolates are more likely to produce an icaADBC-dependent, PIA/PNAGmediated biofilm while some MRSA strains express mechanisms icaADBC-independent involving proteins. It is probable that differences in the extracellular slime composition contribute

Table 2. Antimicrobial photodynamic activity of hypericin against bacterial biofilms at different preincubation times (1 μM hypericin concentration and fluence of 25 J cm-2).  Preincubation time

Reduction in survival of staphylococcal in biofilm (log10)

 

Staphylococcus aureus ATCC 33591 (methicillin-resistant)

Staphylococcus aureus ATCC 29213 (methicillin-susceptible)

20 min 2h 6h 24h

No reduction No reduction 1.5 ± 0.4 3.5 ± 0.6†

No reduction 0.6 ± 0.2 1.2 ± 0.5 2.2 ± 0.5

Data are expressed as log10 reduction in survival rate, compared with the control (n = 4). † A reduction of ≥3-log10 was considered bactericidal.

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Research Article  García, Ballesta, Gilaberte, Rezusta & Pascual to varying degrees to reduce the effectiveness of PDT. In addition to production, other factors must be involved in biofilm sensibility to PDT, since some nonproducing strains also show resistance, and strains with a similar uptake possess significantly different susceptibility to PDT. The genetic background across S. aureus clinical isolates is highly variable. Over 40 virulence factors have been identified, which are involved in almost all processes from colonization to infection production. The virulence of some S. aureus strains has been reduced by PDT. To understand the factors that could influence S. aureus susceptibility to photodynamic treatment, it will be necessary to evaluate the photoactivity of different photosensitizers against a significant number of well-characterized MRSA/MSSA clinical isolates with different degrees of biofilm production and expressing different mechanisms. In spite of encouraging results about the use of antimicrobial PDT to inactivate MRSA in large in vitro studies, there are only few reports about their use to treat MRSA infection in vivo and all of them are confined to local MRSA infection or arthritis on rodent models. In vivo studies on infected wounds in rats, those treated with PDT show superior wound-healing potential, better epithelialization and keratinization of skin layers. Murine MRSA arthritis model demonstrated that an appropriate light dosimetry is necessary to simultaneous maximize bacterial inactivation and neutrophil accumulation into the infected site. The in vitro photodynamic activity showed by HYP against planktonic and sessile S. aureus strains evaluated in this manuscript suggests that HYP could be a promising candidate for treating

infections related to biofilms. We are currently evaluating the in vitro HYP-antimicrobial photoactivity against well-characterized clinical isolates of MRSA/MSSA. According to the results obtained in vitro, in vivo activity using an animal model will be assessed. If the results from these further tests are promising, clinical studies could also be carried out. The use of PDT in clinical infections related to biofilms still has a long way to progress. First of all, more effective delivery methods for both light and photosensitizers to the target site are necessary. It seems to be clear that this therapy can be effective in many clinical situations. The clinical trials in nonhealing leg ulcers, chronic sinusitis, decontamination of endotracheal tubes and nares decolonization of MRSA all show that clinical use of PDT in biofilm infections is in progress. Well-designed preclinical and clinical studies are needed to establish the optimal protocols and indications for antimicrobial PDT. Financial & competing interests disclosure This study was supported by Plan Nacional de I+D+i 20082011 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015) – cofinanced by European Development Regional Fund ‘A way to achieve Europe’ ERDF. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

EXECUTIVE SUMMARY Hypericin antimicrobial photodynamic activity against Staphylococcus aureus ●●

At nonphototoxic concentration (≤1 μM), HYP showed photoactivity both planktonic and biofilm-forming Staphylococcus aureus. The Hypericin (HYP) photoactivity was concentration and light dose dependent.

Hypericin antimicrobial photodynamic activity against planktonic methicillin-susceptible & -resistant S. aureus ●●

After 5 min of preincubation, HYP 0.06 μM plus a light dose of 8 J cm-2 showed bactericidal effect (a reduction of

3 log10) against methicillin-susceptible S. aureus and methicillin-resistant S. aureus strains evaluated. Methicillinresistant S. aureus strain was slightly more resistant to photoinactivation than methicillin-susceptible S. aureus strain. Hypericin antimicrobial photodynamic activity against methicillin-susceptible & -resistant S. aureus biofilms ●●

Longer preincubation times (24 h) and higher light doses (25 J cm-2) were required to reach HYP-photoactivity

against S. aureus biofilms. HYP-photoactivity was related to the biofilm production; at nontoxic concentrations a reduction of 3.5 log10 was reached against the weak biofilm producer strain while this value was only 2.2 against the moderate biofilm producer strain. It is probable that mechanisms involved in the biofilm composition (ica-dependent/independent) contribute also to the effectiveness of PDT.

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