Evaluation of natural and synthetic photosensitizers in visible light-mediated antimicrobial photodynamic therapy against pathogenic bacteria

INTRODUCTION

Antimicrobial resistance, particularly multi-drug resistance, has become a global threat in recent years due to the ongoing misuse of antibiotics.^[1] An April 2014 report from the World Health Organization stated that the “post-antibiotic” period was nearing and that people would die even from minor diseases or injuries due to increasing ineffectiveness of common antibiotics.^[2] Bangladesh faces a high mortality burden attributable to AMR: An Institute for Health Metrics and Evaluation analysis estimated around 23,500 deaths directly attributable to AMR and nearly 97,000 deaths associated with AMR in 2021, underscoring the scale of the crisis.^[3] The rapidly deteriorating scenario of antibiotic resistance in bacteria has necessitated the critical search for alternate therapeutic approaches and has revealed antimicrobial photodynamic therapy (APDT) as a unique strategy to minimize bacterial biofilms and plankton.^[4,5]

Antimicrobial photodynamic therapy (APDT), also known as photodynamic inactivation, is a potential approach for eradicating harmful microorganisms^[6] including Gram-negative bacteria and Gram-positive bacteria found in a variety of contexts such as medical, dental, industrial, environmental, food safety, and cosmetics.^[1] Furthermore, not only bacterial but also fungal infections, as well as cancer, have been treated with APDT.^[7] Although the clinical implementation of APDT in Bangladesh is still a nascent field, it holds potential as a strong tool in response to widespread antimicrobial resistance and ineffectiveness of common antibiotics.

APDT relies on a photosensitizer (PS), light of a suitable wavelength (typically in the visible spectrum between 400 and 700 nm), and molecular oxygen in the target environment. The PS is typically absorbed by the bacterial shell, and an excited singlet state (IPS) is created when bacteria are exposed to light of a particular wavelength, releasing a reactive oxygen species (ROS).^[8] Nowadays, several natural compounds are recognized as PS, such as hypericin (Hyp). Hyp is a naturally occurring highly lipophilic anthraquinone that is extracted from Hypericum perforatum L. It is commonly known as St. John’s Wort. Several studies have revealed HYP as a potent PS, exhibiting anti-carcinogenic, antibacterial, antifungal, antiviral, and anti-biofilm actions.^[8,9] There are also chemically derived PS that works effectively for inhibiting microorganisms. Methylthioninium chloride, or methylene blue (MB), is a hydrophilic phenothiazine byproduct. MB has been found to be an efficient PS that absorbs 660 nm wavelength light and can produce ROS.^[10,11] Gram-positive bacteria are more susceptible to APDT compared to Gram-negative bacteria due to differences in their cell wall structure and differences in the uptake of PSs, which result in lower activity during APDT.^[12] Damage to non-specific targets by the ROS leads to the destruction of both planktonic cells and biofilms^[1] [Figure 1].

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Figure 1: Mechanism of antimicrobial photodynamic treatment (APDT) against pathogenic bacteria. Antimicrobial photodynamic therapy (APDT) uses a photosensitizer (PS), visible light, and molecular oxygen to generate reactive oxygen species (ROS). Hypericin or methylene blue, activated by light, produces ROS through Type I and Type II reactions. These ROS damage bacterial membranes, proteins, enzymes, and nucleic acids, leading to membrane disruption, loss of viability, and bacterial cell death. This mechanism may reduce the possibility of bacterial resistance development. Figure created with the assistance of OpenAI ChatGPT and finalized by the authors.

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This study aimed to test a simple and cheap in vitro setup for exposing bacteria to chemical PS and light to inhibit Staphylococcus aureus and Escherichia coli. The findings suggest that APDT could be implemented using readily available components in Bangladesh, thus paving the way for developing more sophisticated clinical setups and rapidly expanding the application of this technique to counter the critical issue of multidrug resistance in clinical isolates in Bangladesh.

MATERIALS AND METHODS

PDT

Bacteria were grown in liquid Luria-Bertani broth at 37°C overnight for 14–16 h to reach the stationary phase. The cultures were then diluted with normal saline and adjusted to appropriate optical density values to give initial cell numbers of approximately 10⁷–10⁸ colony-forming units (CFU/mL).

S. aureus or E. coli broth culture was incubated with PS (1 mM/mL MB or 10 μg/mL Hyp) for 30 min at room temperature under shaking conditions at 120 rpm. Azithromycin (400 μg/mL) was used as a positive control for S. aureus whereas tetracycline was used as a positive control for E. coli. Following incubation, 4 mL of each culture was transferred into sterile small Petri dish (5 cm) and exposed to orange LED light positioned 1 cm below the culture surface for a duration of 15 min.

Post-treatment, bacterial viability was assessed by enumeration of CFU/mL using the standard spread plate technique [Figure 2]. Treated samples were plated onto Nutrient Agar. Triplicate agar plates were used for each CFU determination, and three independent experiments were performed. All plates were incubated at 37°C for 24 h before colony counting.

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Figure 2: Schematic for antimicrobial photodynamic treatment for bacterial inhibition. Bacteria and Photosensitizer were treated for 30 min and then light exposure was given for 15 min. Next, samples were diluted and spread plate was done in Nutrient agar (NA). The plates were incubated at 37°C for 24h. Figure created in Canva and Adobe Illustrator.

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RESULTS

APDT is effectively against the Gram-positive bacterium S. aureus. First, APDT was evaluated on the Gram-positive bacterium S. aureus to test the suitability of the APDT setup in 3 independent experiments. Azithromycin (400 μg/mL) was used as a positive control. The optimum APDT setup was Hype (10 μg/mL), incubation for 30 min followed by orange light exposure for 15 min. The data show that the combined treatment of Hyp with orange light significantly decreased the growth of S. aureus compared to that of the control. As shown in Figure 3, the log reduction of bacterial growth was up to 3.1 for APDT of S. aureus with Hyp and light, and with MB (1 mM), the APDT effect was 2.8 log reduction. Moreover, the effect of APDT was significantly higher than that of the conventional azithromycin treatment against S. aureus.

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Figure 3: Antimicrobial photodynamic treatment effects on Staphylococcus aureus with hypericin and light or methylene blue and light (compared to controls), APDT: Antimicrobial photodynamic treatment. ***Significant differences is expressed with *** for p < 0.001 relative to the vehicle control.

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In Figure 4, the effect of APDT was also measured with E. coli with Hyp or MB co-treatment with light. There was no significant reduction in bacterial counts observed with MB and light, but with Hyp, it was found that only 1.5 log reduction had occurred. All the experiments were performed in duplicate.

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Figure 4: Antimicrobial photodynamic treatment effects on Escherichia coli with hypericin and light or methylene blue and light (compared to controls), ns: not significant, APDT: Antimicrobial photodynamic treatment. *Significant differences is expressed with * for p < 0.05, relative to the vehicle control. NS: Not significant.

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The antibiogram showed that among 10 and 13 commercial antibiotic discs which are regularly used to treat infections involving S. aureus and E. coli, respectively, the bacteria were found to be resistant to 4 antibiotics. In addition, it was evident that vancomycin resistance was on the rise and was going to be an issue soon. Bacteria showed less sensitivity to vancomycin compared to ciprofloxacin, which is a common broad-spectrum antibiotic used to treat infection for wide range of bacteria. The study showed that E. coli was less sensitive to cefuroxime compared to ciprofloxacin [Figure 5 and 6]. These antibiogram results showed that antibiotic effectiveness varied and had big differences (sensitive, intermediate, and resistant). The structure and the mechanism of action were summarized in supplementary Table 1 (see the supplementary data).

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Figure 5: Antibiogram profiling of Staphylococcus aureus by zone of inhibition (mm) by Kirby-Baur Disk Diffusion method.

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Figure 6: Antibiogram profiling of Escherichia coli by zone of inhibition (mm) by Kirby-Baur Disk Diffusion method.

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DISCUSSION

Worldwide, drug-resistant microorganisms are causing major public health issues and it is a major concern to find alternative treatments. Multidrug-resistant bacteria are a critical concern in Bangladesh and a source of mortality. APDT using various naturally and chemically derived PSs has shown significant bacterial inhibition^[10] and there is growing academic interest in Bangladesh; however, the technique has not yet been clinically deployed. This study focused on in vitro inhibition of S. aureus and E. coli by APDT with Hyp and MB with orange light, using a setup with easily and cheaply acquired components. 3-fold reduction of S. aureus confirmed previous findings of efficient APDT against Gram-positive organisms [Figure 3].^[1,8] Lower reduction of E. coli also mirrored previous results [Figure 4].^[8] This in vitro confirmation of APDT in a Dhaka-based laboratory, along with previous studies,^[1,6,8] suggests a pipeline of practical expertise that can be translated to local pathogens and clinical applications in Bangladesh. The next steps would involve translating the findings of this paper to resource-limited hospitals, as well as standardizing PS formulations, light conditions, and treatment parameters under in vivo setups for regulatory approval and clinical guideline development.

Hyp was chosen as a natural PS due to its higher oxidative power and its ability to produce singlet oxygen effectively. The low concentration of Hyp was found effective in APDT.^[6] It is commercially available and is non-toxic in the dark, with minimal side effects at low concentrations.^[4] MB, which is cationic, is also a known chemical PS that can effectively kill microorganisms in low concentration (300 ng/mL) while used in APDT against S. aureus and E. coli with several combination treatments.^[14] The phototoxic effects of these PS trigger ROS production and thus lead to cell death. ROS diffusion potential is influenced by three factors: (i) the maximal time-limited diffusion length, particularly for singlet oxygen (¹O₂), (ii) photostability in the environment, and (iii) the chemical properties of PSs (e.g., molecular size, charge, lipophilicity, stability), which impact their interactions with target microorganisms. Photoinactivation affects photochemical processes in bacterial cells based on the PS and its charge.^[8]

For the human body, chlorins and phenothiazinium dyes are better used in APDT due to less toxicity. MB is one of the PSs among the phenothiazinium compounds and has been found to be effective for photodynamic antimicrobial activity by producing high quantum yields of singlet oxygen.^[15-18] The tendency of MB to aggregate is governed by several factors, including the properties of the medium, its concentration, and the pH. These factors influence both the nature and effectiveness of the photochemical reactions that take place. Specifically, monomeric MB is associated with the production of singlet oxygen (¹O₂), whereas its dimeric form tends to generate free radicals. Consequently, the formation of MB aggregates reduces the yield of ¹O₂, thereby compromising the efficiency of photosensitization.^[19] MB APDT is also useful for treating simple cutaneous superficial fungal infections and diabetic feet.^[20] Due to its safety profile and efficacy, MB is a promising candidate for use as a PS in antimicrobial photodynamic therapy (APDT) for skin infections caused by Methicillin sensitive Staphylococcus aureus (MSSA) and Methicillin resistant Staphylococcus aureus (MRSA).^[21]

Antibiogram profiling conducted with a number of commercial antibiotic drugs for both S. aureus and E. coli revealed that S. aureus was resistant to doxycycline, gentamicin, and chloramphenicol while E. coli was resistant to ciprofloxacin, nalidixic acid, and cefixime [Figures 5 and 6]. Many conventional antibiotics depend on specific bacterial targets or pathways, such as cell-wall synthesis, ribosomal protein synthesis, nucleic-acid synthesis, or defined enzymes.

A single target alteration, drug-inactivating enzyme, reduced uptake, or increased efflux may therefore reduce antibiotic activity. In contrast, APDT combines a PS, visible light, and molecular oxygen to generate ROS. These oxidants can attack the cell envelope, membrane-associated proteins, cytosolic enzymes, and other intracellular components simultaneously.^[22]

It is alarming that soon bacteria will become resistant to many more antibiotics, and the treatment of various infections caused by pathogenic microorganisms will become a life-threatening problem. Antimicrobial photodynamic therapy offers a powerful alternative approach by generating ROS that can effectively combat antibiotic-resistant bacteria, whether in their planktonic (free-swimming) or biofilm state.^[17]

In APDT, Type I reactions involve electron or hydrogen transfer, producing radical ions and free radicals, whereas Type II reactions involve energy transfer to molecular oxygen, resulting in singlet oxygen. These highly reactive species induce irreversible oxidative damage to multiple cellular targets, particularly the cytoplasmic membrane, membrane-associated enzymes, transport systems, and, to a lesser extent, DNA. Although some DNA damage may be repaired, membrane disruption and leakage of intracellular components are considered major contributors to microbial death. The multi-target and non-specific mode of action of APDT offers several advantages, including limited side effects, reduced microbial regrowth, and a low probability of resistance development. Because PSs can act on cell walls and membranes without requiring intracellular uptake, microorganisms are less able to develop resistance through reduced drug entry, increased efflux, or metabolic detoxification. Repeated-exposure studies using MB and other PSs have shown no significant reduction in susceptibility in antibiotic-sensitive and antibiotic-resistant strains of S. aureus, including methicillin-resistant S. aureus (MRSA), or in E. coli. These findings indicate that resistance to APDT is unlikely to develop even after multiple treatment cycles. Nevertheless, the potential for resistance should continue to be evaluated whenever new PSs or treatment protocols are introduced.^[22]

Piska et al., (2023) described that from 2000 to 2022 year MB APDT was found 84% studies were done within bacteria 56%, fungi 29%, viruses 8%, parasites 4.5%, plants 1.5% and animals 1%.^[23] Among bacteria, the most studied strain was S. aureus (23%) including MRSA and E. coli (12.5%), followed by Enterococcus faecalis (10.5%), Pseudomonas aeruginosa (9%), and Streptococcus mutans (8%). S. aureus is the most studied bacterial target in antimicrobial photodynamic therapy (APDT) as it is a common skin pathogen of clinical importance and is often associated with antibiotic resistance. In APDT studies, Gram-positive bacteria represent around 55% of the reported targets among the bacterial species examined, while Gram-negative bacteria represent approximately 45%. In the Gram-positive group, S. aureus represented about 41% of the species studied, underscoring its relevance as a model organism for assessing APDT efficacy. In contrast, E. coli, the most studied Gram-negative bacterium, accounts for about 27% of APDT studies with Gram-negative pathogens. In addition to bacteria, MB-mediated APDT has demonstrated antiviral activity against several medically important viruses, including vesicular stomatitis virus, bovine viral diarrhea virus, dengue virus, West Nile virus, hepatitis C virus, herpes simplex virus type 1, and severe acute respiratory syndrome coronavirus 2, indicating its broad-spectrum antimicrobial potential.^[24] APDT has gained attention for a long time due to their nonresistance properties among microorganisms. The mechanism antibiotic versus PSs in APDT is quite different. Antibiotics are usually target-specific, for example, ribosomes, penicillin-binding proteins/cell wall synthesis, DNA gyrase, RNA polymerase, folate synthesis, or cell-wall synthesis. APDT causes oxidative injury at multiple sites such as lipids, proteins, enzymes, DNA, and membranes simultaneously, so a single resistance mechanism is less likely to protect the bacterial cell. ROS from APDT do not behave like one antibiotic molecule binding to one target. After light activation, the PS generates ROS such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. These reactive molecules oxidize several bacterial components at once: Membrane lipids, proteins, enzymes, transport systems, and nucleic acids. Tavares et al. describe APDT as a multi-target process and report no bacterial viability recovery after one treatment and no resistance after repeated treatment cycles in their experimental model. Therefore, resistance development against APDT is considered less likely than resistance development against conventional antibiotics, although bacterial tolerance mechanisms such as biofilm formation, efflux pumps, catalase, superoxide dismutase, and oxidative-stress responses should still be evaluated in future studies.^[21] The role of inactivation of microorganisms by PS (Hyp and MB) and antibiotics differs in many ways including structures. Hyp differs fundamentally from conventional antibiotics in both chemical structure and antimicrobial mechanism. Unlike antibiotics, which inhibit specific cellular targets such as cell wall synthesis, protein synthesis, or nucleic acid replication, Hyp functions as a photoactivatable PS. Its highly conjugated polycyclic aromatic quinone structure, containing multiple hydroxyl and carbonyl groups, enables efficient absorption of visible light and a high singlet oxygen quantum yield. The resulting ROS induce simultaneous oxidative damage to bacterial membranes, proteins, nucleic acids, and other essential biomolecules, producing a multi-target antimicrobial effect.^[23,24] Although MB also acts through ROS generation, its cationic phenothiazinium structure provides greater aqueous solubility and facilitates electrostatic interactions with negatively charged bacterial surfaces, particularly in Gram-negative organisms.^[25] In contrast, Hyp exhibits superior photophysical properties and greater singlet oxygen production, making it one of the most potent naturally occurring PSs for antimicrobial photodynamic therapy. This multi-target oxidative mechanism substantially reduces the likelihood of resistance development compared with conventional antibiotics, which typically act on a single molecular target.^[21]

Here, a safety issue for applying APDT for treating infection is noticeable. As previous studies have demonstrated, the favorable safety profile and multifunctionality of natural PSs – combined with the inherent adaptability of APDT – position this approach as both a valuable complement and in many cases, a potent alternative to conventional antimicrobial treatments. Caenorhabditis elegans is a known animal model, and in previous study, it was found that APDT is safe because after APDT, the gut permeability was significantly improved and it was also evident that the growth rate and reproduction were not affected. In addition, only light treatment for a certain short period of time did not have any toxicity in C. elegans.^[6,7] APDT has already made many significant discoveries for many diseases including antimicrobial, antifungal, antiviral, and anti-tumor. Mouse and rat models were also used to check the efficacy of APDT for treating bacteria and tumor which showed sufficient improvement comparative to control.^[6]

CONCLUSION

The findings underscore the efficacy of APDT in inhibiting bacterial growth and suggest further exploration into its mechanism of action and broader application in clinical settings. Continued research in this area could pave the way for novel therapeutic strategies to combat antibiotic-resistant infections. To combat the antibiotic resistance problem, alternative treatment is a must needed. APDT can be the next applicable treatment by adding more research fields such as cell biology, biochemistry, and pharmacology. A remarkable parameter is the light sources for APDT. The light exposure, different wavelengths of light, and intensity may differ in the efficiency of the treatment. Low radiant lights maybe less effective compared to high radiant light. Moreover, this study was limited by the range of photosensitizer concentrations and treatment durations tested. Multiple combinations with PS may add more therapeutic options in the treatment of patients who suffer from antibiotic resistance.