Staphylococcus aureus remains a leading cause of skin and soft-tissue infections [1]. In recent years, antibiotic resistance in strains of this microorganism has continued to increase, making treatment choices more difficult for superficial bacterial infections.

Dermatologists and skincare specialists often need to control the bacterial burden on the skin locally—for example, after contamination, when caring for patients with impaired skin-barrier function, or before invasive interventions. Approaches that do not depend on the classical targets of antibiotics are therefore of practical interest. They may reduce selective pressure on the microbiota and potentially limit further resistance development.

Principles of photodynamic and sonodynamic therapy

Photodynamic therapy (PDT) relies on photosensitizers absorbing light at a specific wavelength. The absorbed energy transfers to oxygen molecules, forming reactive oxygen species. These highly reactive compounds damage bacterial cell structures and may ultimately lead to bacterial death.

Sonodynamic therapy (SDT) uses ultrasound. It is thought to facilitate sensitizer penetration into tissue and further enhance reactive oxygen species formation. For this reason, researchers have explored combining SDT with PDT to increase local antimicrobial activity without increasing photosensitizer concentration.

Methylene blue is a long-established medicinal and diagnostic agent that is already used in clinical practice for a range of indications. In PDT, however, it serves a different function: it acts as a photosensitizer. Because of its physicochemical properties, it may interact with bacterial cell surfaces and penetrate them. Once activated by red light, it initiates the formation of reactive oxygen species. These compounds can damage the bacterial cell wall and membrane, as well as other cellular structures, potentially resulting in bacterial lysis and death.

The selectivity of this approach should not be understood as an absolute effect limited exclusively to bacteria. It relates to the photosensitizer's interaction primarily with microbial cells, while the generated reactive oxygen species act locally and persist only briefly. As a result, the damaging effect develops mainly where methylene blue is present and where light exposure is subsequently delivered. In this study, however, the authors assessed the approach's safety on the skin only in a small pilot study involving healthy volunteers; no dermatologic adverse events were reported in any of the 20 participants.

Thus, the authors did not investigate methylene blue as a standalone antiseptic. They aimed to evaluate a new approach to its local use—in combination with red light and ultrasound—to reduce the skin burden of Staphylococcus aureus.

Study design

The authors conducted a two-stage sequential study.

During the first stage, they examined the effect of methylene blue on an S. aureus culture under different red-light exposure protocols and in combination with ultrasound. The study used the S. aureus CCM 4223 strain. Bacterial growth was assessed by optical density, the minimum inhibitory and bactericidal concentrations of methylene blue were determined, and cellular damage was visualized using scanning electron microscopy.

During the second stage, an open-label pilot clinical study was conducted with 20 healthy volunteers. A suspension of S. aureus (ATCC 6538) was artificially applied to areas of intact skin on the upper extremities. We then administered different treatment protocols and assessed the reduction in bacterial burden.

Laboratory findings

Without methylene blue, neither red light nor ultrasound had a noticeable effect on the growth of S. aureus. After adding methylene blue, the effect depended on its concentration and the treatment protocol.

• At a concentration of 0.5 μM, the greatest suppression of bacterial growth was observed after repeated light exposure—two sessions of 30 J/cm² separated by a 2-hour interval—particularly when ultrasound was added.
• At 1 μM, a single light exposure completely suppressed culture growth under the experimental conditions.

Scanning electron microscopy showed marked cell-wall damage, bacterial lysis, and loss of the characteristic coccal shape after combined treatment with repeated exposure. In the control samples and in the samples treated with ultrasound alone, the bacteria retained their structural integrity.

Clinical study

The pilot study included volunteers with intact forearm skin. Methylene blue (25 μM) was applied to limited areas of the skin. PDT was then performed: the site received either a single exposure to red light at 625–630 nm with a dose of 30 J/cm² or two such exposures separated by a 2-hour interval.

The SDT protocols also included ultrasound at 1 MHz and 0.7 W/cm² for 5 minutes. The primary endpoint was the reduction in viable Staphylococcus aureus compared with baseline. A dermatologist monitored participants’ skin, and an ethics committee approved the study.

Clinical results

All treatment protocols produced a marked reduction in the skin burden of Staphylococcus aureus:

• PDT with a single red-light exposure reduced the number of viable bacteria by 99.16%;
• PDT with two red-light exposures separated by a 2-hour interval produced a 99.20% reduction;
• PDT combined with SDT and a single red-light exposure reduced the bacterial burden by 99.39%;
• PDT combined with SDT and two red-light exposures separated by a 2-hour interval produced a 99.62% reduction, the strongest result among all protocols studied.

The greatest effect was therefore observed when methylene blue was combined with ultrasound and two sequential light exposures. Only this protocol exceeded the investigators' 99% reduction threshold with statistical significance, corresponding to a 100-fold decrease in viable microorganisms (p = 0.009). At the same time, each methylene-blue protocol produced a statistically significant reduction in bacterial burden of more than 90% from baseline (p < 0.0001).

Tolerability

None of the 20 healthy volunteers experienced dermatologic adverse events, visible skin changes, or other procedure-related complications.

Study limitations

The study had an open-label design and included a small sample. The authors investigated artificial contamination of intact skin in healthy volunteers rather than treating a clinically apparent infection. The authors evaluated only one type of microorganism. The study did not examine biofilms, chronic wounds, deep infections, or resistant strains under real-world clinical conditions. The authors note that using this approach in more complex situations would require consideration of light penetration depth, oxygen availability, and the structural complexity of biofilms.

Practical interpretation

The findings demonstrate that combining methylene blue, red light, and ultrasound can reduce the skin burden of S. aureus. The approach acts independently of the traditional mechanisms underlying antibiotic resistance and may be considered a promising local technology. However, this study does not support conclusions about replacing antibiotics or the method's readiness for routine clinical use in skin infections. Further studies involving patients, standardized treatment parameters, and evaluation in real-world clinical scenarios are needed before the approach can be introduced into practice.

Conclusion

In a pilot “bench-to-clinic” study, the combination of photodynamic and sonodynamic therapy with methylene blue demonstrated the ability to reduce the number of viable S. aureus bacteria on intact skin. The most effective protocol involved repeated light exposure combined with ultrasound.

The findings suggest that this approach may be a useful additional local tool for controlling bacterial contamination. Further studies are needed to confirm its clinical relevance and determine its place in practice.

Source

Kolarova H., Valkova L., Kolarikova M. et al. Methylene blue-enhanced photodynamic and sonodynamic therapy against Staphylococcus aureus: From laboratory research to clinical evaluation. Photodiagnosis Photodyn Ther 2026; 58: 105382.