Staphylococcus aureus reference strains were generously provided by Dr Joanna Empel from the National Medicines Institute (NMI), Warsaw, Poland. These strains were analyzed for toxin genes and genetic background ( Table 1 ). S. aureus was cultured in 5 mL tryptic soy broth (TSB, bioMérieux, France) under aerobic conditions at 37°C with shaking (150 rpm, Innova 40, New Brunswick Scientific, Sweden) for 16–20 h.

The eukaryotic cell lines used in the study were (1): shFLG HaCaT, cells with reduced expression of the filaggrin gene (a line transduced with lentiviral particles containing short RNA with a “hairpin” structure (sc-43364-V, Santa Cruz) and (2), shC HaCaT, cell line with an empty vector introduced by transfection, served as control (sc-108080, Santa Cruz) (40).

Ethical approval was granted by the Medical University of Gdańsk (NKBBN/621-574/2020). PBMCs were isolated from buffy coats of healthy donors via Lymphoprep gradient centrifugation. Cells from three donors were stained with CellTrace™ Far Red and seeded in 96-well plates (2 × 10⁵ cells/well) in RPMI-1640 with 10% FBS and antibiotics.

A mix of PBS, photosensitizer (RB: 0.5 or 5 µM; NMB: 5 µM), and staphylococcal toxin (3.2 µg/mL) was prepared. Toxins were irradiated with green (515 nm, 10–40 J/cm²) or red light (632 nm, 25 J/cm²). Controls included toxin + light, toxin + photosensitizer (dark), untreated toxin, and heat-inactivated toxin. Treated toxins (5 µL, final 80 ng/mL) were added to PBMCs; unstimulated cells served as negative controls.

After six days, cells were stained with CD3-PE, fixed, and analyzed by flow cytometry (InCyte) to assess T-cell proliferation.

ROS generation was assessed using HPF fluorescence in response to hydroxyl radicals. Toxins (SEA, SEB, SEC, SED, TSST-1; 3.2 µg/mL) were combined with PBS, HPF (5 µM), and photosensitizer (RB or NMB, 5 µM) in black 96-well plates. After dark incubation (RB: 10 min; NMB: 15 min), samples were irradiated (green: 515 nm, 40 J/cm²; red: 632 nm, 32.5 J/cm²). Fluorescence was recorded at 490/515 nm using an EnVision Plate Reader.

shFLG HaCaT and shC HaCaT cells (40) were seeded at 1 × 10⁴ cells/well in two 96-well plates (light and dark conditions) and incubated for 24 h in standard conditions. Cells were treated with RB/NMB (10–15 min, dark), washed and irradiated with:

After 24 h, cell viability was assessed using the MTT assay. Formazan absorbance (550 nm) was measured using a Victor Multilabel Plate Reader (PerkinElmer, USA). Viability (%) was expressed as a ratio of treated to untreated samples. Experiments were performed in triplicate, with four technical replicates per condition.

Statistical analyzes were performed using GraphPad Prism 8 (GraphPad Software, Inc., USA, 2019). One-way analysis of variance (AVOVA) followed by Dunnett’s test for multiple comparisons was used. For all statistical tests, a p-value of < 0.05 was considered statistically significant.

The impact of antimicrobial photodynamic inactivation (aPDI) on the expression of toxin genes was assessed in four S. aureus reference strains, each carrying genes for common staphylococcal toxins: 10798/11 (sea), 140/05 (seb), 1947/05 (sec), and 1005/05 (sed and tst). Two aPDI protocols were employed: (i) rose bengal (RB) with 515 nm light and (ii) new methylene blue (NMB) with 632 nm light. The study aimed to determine whether the observed effects depend on the photosensitizer, light wavelength, or photodynamically induced oxidative stress. Bacteria were exposed to sublethal aPDI conditions, corresponding to a reduction in viable counts of approximately 0.5 log₁₀ CFU/mL (please see Supplementary data for the details), and gene expression was quantified via qPCR.

We first determined the sublethal doses of the compound and light combination ( Supplementary Figure S1 , Supplementary Table S1 ). A significant decrease in the expression of sea, seb, sec and sed genes was observed as early as 20 minutes after the treatment, persisting for at least 40 minutes, regardless of the aPDI approach ( Figure 1 ). For RB + green light, sea, seb, and sec expression decreased due to both aPDI and the individual impact of either RB or light, with statistical significance observed only for sea. In contrast, NMB + red light significantly downregulated sea, seb, and sec expression, though red light alone produced variable effects, including both increases (sec) and decreases (seb, sed).

A distinct expression pattern was observed for tst. Unlike the other toxin genes, tst expression increased following aPDI, independent of the photosensitizer or light source. Notably, neither RB nor green light alone altered the tst expression, while both red light and NMB alone significantly reduced its expression ( Figure 1 ). This suggests that tst upregulation is directly linked to the photodynamic action rather than to the individual treatment components.

The results demonstrate that aPDI significantly influences toxin gene expression, with effects varying by gene and, to a lesser extent, by the aPDI conditions. This suggests that photogenerated oxidative stress differentially affects gene expression. However, the underlying mechanisms driving these gene-specific responses to photooxidative stress under different aPDI conditions require further investigation.

We then analyzed the effect of aPDI on SEs at the protein level ( Supplementary Figure S2 , Supplementary Table S2 ). Within the limits of our semi-quantitative Western blot analysis, aPDI treatment did not reveal consistent changes in SE protein levels at any time point ( Supplementary Figure S2 ). Therefore, we shifted our focus to evaluating its impact on the biological activity of the staphylococcal toxins, particularly their superantigen (SAg) function, which induces T-cell proliferation and cytokine release. The activity of the toxin was assessed using the T-cell proliferation assay. The optimal concentration of the toxin for effective T-cell stimulation was determined to be 80 ng/mL, inducing proliferation in 77% of T-cells ( Figure 2A ). Toxins were treated with: RB and green light or NMB and red light under sublethal (0.5 - 5 µM, 12–32 J/cm²) or lethal (5 µM, 40 J/cm²) conditions. aPDI-treated toxins were then incubated with PBMCs, and T-cell proliferation was measured with light or photosensitizers alone serving as controls.

The impact of aPDI on toxin activity varied depending on the photosensitizer and treatment conditions. RB + green light demonstrated potent toxin inactivation under lethal conditions, reducing T-cell proliferation to near-background levels (SEA: 14.8%, SEB: 14.5%, SEC: 16.4%, SED: 25.3%, TSST-1: 15.2%) ( Figure 2C ). In contrast, sublethal RB treatment had no discernible effect, and neither RB nor green light alone altered toxin activity ( Figure 2B ).

NMB + red light under sublethal conditions, in contrast, was less effective:. Only TSST-1 showed partial reduction (42.7%) while other toxins remained active ( Figure 2D ). Stronger NMB-aPDI conditions were not tested due to phototoxicity toward eukaryotic cells.

To test ROS generation in our experimental conditions following aPDI treatment, specific probes were employed to detect hydroxyl radicals (•OH). As anticipated, both aPDI treatments; RB + green light and NMB + red light resulted in significant ROS production ( Figures 3A, B ). The fluorescence signal intensity was higher for the NMB + red treatment (~80,000 relative units, RU) compared to RB + green (~50,000 RU) combination. Notably, we did not detect any ROS generation with purified enterotoxins alone ( Figures 3A, B ). Similarly, the results obtained from the dark incubation for both conditions confirmed no ROS production ( Supplementary Figure S3 , Supplementary Figure S4 ).

Next, we evaluated the impact of aPDI on human epidermal keratinocytes (HaCaT). The analysis included HaCaT cells with a knockdown of the FLG gene, which encodes profilaggrin, a crucial protein involved in the maintenance of the epidermal barrier (shFLG HaCaTs). The filaggrin insufficient keratinocytes used in this study served as an in vitro model of AD, since filaggrin insufficiency (either on genetic background or acquired, including a vicious loop between filaggrin reduction and S. aureus colonization) leads to the impairment in epidermal barrier quality is one of the disease hallmarks (50). As a control cell line transduced with an empty vector was used (shC HaCaT). The primary objective of this experiment was to determine whether the FLG knockdown significantly affects the survival of aPDI-treated eukaryotic cells.

MTT assay results demonstrated that the combination of RB with green light (λ_max = 515 nm) exhibited no cytotoxic effects on any of the tested cell lines. Cell viability remained high even at the highest RB concentration (10 μM) in the combination with light treatment: 89.3% for shC HaCaT and 93% for shFLG HaCaT cells. Furthermore, treatment with RB alone did not affect cell survival, with the viability values of 99.1% for shC HaCaTs and 106% for shFLG HaCaTs at 10 μM RB ( Figure 4A ).

A very different picture was observed for the combination of NMB with red light (λ_max = 632 nm). Exposure to red light with 1 μM NMB resulted in significant cell death, with survival rates of only 10.7% (shC HaCaT) and 7.1% (shFLG HaCaT). In contrast, cells incubated with NMB in the dark showed no cytotoxicity (shC HaCaT – 94.8%, shFLG HaCaT – 91.4%). A strong photo- and cytotoxic effect was observed for both tested HaCaT cell lines at 5 μM NMB ( Figure 4B ). The observed cytotoxic and/or phototoxic effect or lack thereof depends on the PS used in the photodynamic reaction.

We next verified the results obtained in vitro in an ex vivo model, using porcine skin, The primary objectives of this experiment were to assess SEA toxin production at the transcript and protein levels under aPDI. Porcine skin was chosen due to its structural and physiological similarity to human skin and its availability ( Supplementary Figure S5 ) (51). A higher RB concentration (35 µM) was used compared to in vitro studies due to the limited photosensitizer penetration into the skin and bacterial aggregates hindering binding (49). To evaluate the safety of this approach, we analyzed the cyto- and phototoxicity of RB at higher concentrations and observed some cytotoxic effects ( Supplementary Figure S6 ). Nevertheless, higher concentrations were employed because, in ex vivo and in vivo settings, bacteria are expected to form more resistant biofilm structures that are typically less susceptible to aPDI. The experiment involved colonizing porcine skin with S. aureus Xen40 strain, followed by RB application (35 µM) for 30 min, and subsequent green light irradiation (λ_max = 530–535 nm, 6.36 J/cm², 10 min). S. aureus Xen40 has the sea gene and produces active SEA protein.

Survival of the Staphylococcus aureus Xen40 strain was evaluated at 40 minutes and 24 hours following aPDI. Quantification of bacterial viability revealed a time-dependent reduction in colony-forming units, with decreases of 0.56 log₁₀ CFU/mL and 1.22 log₁₀ CFU/mL, respectively ( Figure 5B ), indicating a sustained bactericidal effect. The presence of staphylococcal enterotoxin A was confirmed in skin swabs by both PCR and Western blot analysis. Transcriptomic analysis showed a significant downregulation of sea gene expression following aPDI treatment, with a reduction of 2.92 log₂ units ( Figure 5C ). Interestingly, exposure to green light alone also resulted in a notable decrease in sea expression (2.14 log₂ units), suggesting a potential sub-lethal stress response. In contrast, treatment with RB alone led to a significant upregulation of sea gene expression, indicating that RB in the absence of light may act as a stressor that induces virulence factor expression. Interestingly, a different effect of gene expression was observed after treatment with RB in an in vitro planktonic culture and in an ex vivo porcine skin colonization model. In the in vitro environment, RB treatment resulted in decreased expression of sea, suggesting a possible antimicrobial or stress suppressive effect in a simplified, nutrient-controlled environment. However, in a more complex ex vivo model, RB treatment resulted in increased sea expression, suggesting that the interaction between bacterial cells and the host-like environment can modulate the expression of virulence genes in different ways.

To further assess the impact of aPDI on enterotoxin production, SEA protein levels were quantified at 40 minutes and 24 hours post-treatment using Western blot analysis. aPDI significantly reduced SEA concentrations compared to untreated controls, with levels decreasing from 5.59 µg/mL to 3.48 µg/mL at 40 minutes, and from 6.10 µg/mL to 2.41 µg/mL at 24 hours ( Figure 5D ). In contrast, treatment with either AD or green light alone did not result in a significant reduction in SEA protein levels, suggesting that the photodynamic activation of RB is essential for the observed effect. Importantly, results from the porcine skin colonization model corroborated the in vitro findings, demonstrating consistent reductions in SEA expression at both the transcript and protein levels following aPDI treatment. These data reinforce the potential of aPDI as an effective approach not only for bacterial inactivation but also for mitigating toxin-mediated virulence.

In this study, a tape-stripping model was used, this model effectively induces local inflammation by disrupting skin barrier, mimicking atopic skin conditions (52, 53). Previously published in vivo studies on aPDI efficacy involved skin wounding and bacterial application to the deeper layers, which does not accurately reflect natural colonization (54). Here, we colonized mechanically damaged mouse skin with bioluminescent S. aureus Xen40 to follow its fate after aPDI and identify the presence of staphylococcal enterotoxin A during colonization.

Schematic representation of in vivo experimental setup is shown in Supplementary Figure S7 (Supplementary data). In group 1 (tape-stripping only), skin redness and irritation were observed on day 1, followed by visible healing by day 3 and complete recovery by day 5 ( Figure 6A ). However, in S. aureus-colonized skin (group 2), no such healing occurred, indicating that bacterial colonization negatively affects skin condition and delays the healing process ( Supplementary Figure S8 ). Both single (group 4) and repeated (group 5) aPDI treatments resulted in a statistically significant reduction in S. aureus presence on the skin, as evidenced by decreased bioluminescence signals ( Figure 6A, B ). Although RB alone (without light activation) produced a transient reduction in S. aureus signal at day 3, this effect was not sustained over time. Such acute, short-term decreases in bacterial load are occasionally observed in aPDI studies, but durable clearance or inhibition of regrowth typically requires both the photosensitizer and light.

Histopathological evaluation of mouse skin revealed distinct tissue responses across experimental groups. In the tape-stripping group ( Figure 7A ), inflammation was evident in the form of exudate with abundant neutrophilic infiltration confined to the epidermis, accompanied by clear evidence of epidermal regeneration, confirming ongoing re-epithelialization. In contrast, mice subjected to tape-stripping followed by colonization with S. aureus ( Figure 7B ), exhibited severe pathological alterations. The epidermis was absent, and dense bacterial aggregates were observed on the skin surface. A pronounced neutrophilic infiltrate extended beyond the dermis into subcutaneous adipose tissue and underlying musculature, indicating a robust and deep-seated inflammatory response. Exposure to green light alone ( Figure 7C ) or topical application of RB at a high concentration (50 µM, Figure 7D ) did not induce additional pathological changes or exacerbate inflammation. No blistering was observed in the group exposed only to light. However, the loss of Tegaderm dressings during the experiment led to increased mechanical irritation and drying of the wounds, which explains the different appearance of the skin in this group. Notably, skin samples from mice treated with a single ( Figure 7E ) or double ( Figure 7F ) aPDI showed no separation of the epidermis from the dermis. In both aPDI-treated groups, neutrophilic infiltration was present in the dermis, with no consistent reduction in neutrophil numbers between single and double treatments. Nevertheless, the inflammatory response remained adequate to control bacterial colonization. These findings suggest that both single and double aPDI treatments are histologically safe and do not aggravate preexisting tissue damage.

Histological images of mouse skin samples collected on the 5th day of the experiment. The samples were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to assess tissue structure. Bacterial presence was visualized using a crystal violet solution. A) Skin subjected to the tape-stripping procedure (magnification 4×). The red ellipse highlights neutrophil infiltration, indicating an inflammatory response. B) Tape-stripped skin colonized with S. aureus (magnification 4×). Clusters of S. aureus bacteria are circled in red. C) Tape-stripped, S. aureus-colonized skin irradiated with green light (magnification 4×). D) Tape-stripped, S. aureus-colonized skin treated with rose bengal (50 µM) applied directly to the skin surface (magnification 4×). E) and F) Tape-stripped, S. aureus-colonized skin treated with a single (E) or double (F) aPDI session (magnification 4×). G) SEA protein concentrations after aPDI. Each bar represents the mean SEA concentration from swab samples collected from six mice, with error bars indicating the standard error of the mean (SEM). Statistical significance is determined in comparison to the control group (tape-stripped, S. aureus-colonized mice) with p < 0.05 denoted by (*); aPDIx1 – tape-stripped, S. aureus-colonized mice subjected to a single aPDI treatment (50 µM RB, 10.6 mW/cm², 6.36 J/cm²); aPDIx2 – tape-stripped, S. aureus-colonized mice subjected to a double aPDI treatment (50 µM RB, 10.6 mW/cm², 6.36 J/cm²).

The final step of the study involved quantifying the levels of the SEA protein from mouse skin swabs. Three experimental groups were analyzed: (i) a control group consisting of tape-stripped, S. aureus-colonized mice, (ii) tape-stripped, S. aureus-colonized mice subjected to a single aPDI treatment, and (iii) tape-stripped, S. aureus-colonized mice subjected to a double aPDI treatment. In the control group, SEA protein levels were comparable to those observed in both in vitro and ex vivo studies, confirming consistent toxin production. However, aPDI treatment resulted in a statistically significant reduction in SEA levels, with a progressive trend of decrease following subsequent treatments [L (–) PS (–): 4.8 µg/mL; aPDIx1: 2.47 µg/mL; aPDIx2: 2.05 µg/mL] ( Figure 7G ). These findings demonstrate that aPDI effectively reduces bacterial virulence factors, highlighting its potential as an antimicrobial strategy against S. aureus.