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Article

With Blue Light against Biofilms: Berberine as Natural Photosensitizer for Photodynamic Inactivation of Human Pathogens

by
Annette Wimmer
1,
Michael Glueck
1,
Jun Liu
2,
Michael Fefer
3 and
Kristjan Plaetzer
1,*
1
Laboratory of Photodynamic Inactivation of Microorganisms, Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
2
Nutrien Ag Solutions, 13131 Lake Fraser Drive SE, Calgary, AB T2J 7E8, Canada
3
Whitby Ag Consulting, 23 Clovelly Drive, Whitby, ON L1N 7A7, Canada
*
Author to whom correspondence should be addressed.
Photonics 2024, 11(7), 647; https://doi.org/10.3390/photonics11070647
Submission received: 7 June 2024 / Revised: 27 June 2024 / Accepted: 2 July 2024 / Published: 8 July 2024
(This article belongs to the Section Biophotonics and Biomedical Optics)
Browse Figures
Versions Notes

Abstract

:
Evolving antibiotic resistance of bacteria is a prevailing global challenge in health care and requires the development of safe and efficient alternatives to classic antibiotics. Photodynamic Inactivation (PDI) has proven to be a promising alternative for treatment of a broad range of microorganisms. Photodynamic Inactivation uses photoactive molecules that generate reactive oxygen species (ROS) upon illumination and in the presence of oxygen, which immediately kill pathogenic target organisms. Relevant photoactive properties are provided by berberine. Originally extracted from Barberry (Berberis vulgaris), it is a natural compound widely used in Traditional Chinese Medicine for its antimicrobial and anti-inflammatory effects. With this study, we demonstrated the potential of berberine chloride hydrate (Ber) as a photosensitizer for PDI of important human pathogens, Gram(+) Staphylococcus capitis subsp. capitis, Gram(+) Staphylococcus aureus, and Gram(−) Escherichia coli. In vitro experiments on planktonic and biofilm cultures were conducted focusing on Ber activated with visible light in the blue wavelength range. The number of planktonic S. capitis cells was reduced by 7 log10 steps using 100 µM Ber (5 min incubation, illumination with 435 nm LED array, radiant exposure 25 J/cm2). For an antibacterial effect of 4 log10 steps, static S. capitis biofilms required 1 mM Ber, a drug-to-light interval of 60 min, and illumination with 100 J/cm2. Almost all planktonic cells of Staphylococcus aureus could be photokilled using 100 µM Ber (drug-to-light interval of 30 min, radiant exposure 25 J/cm2). Biofilms of S. aureus could be phototreated (3 log10 steps inactivation) when using 1 mM Ber incubated for 5 min and photoactivated with 100 J/cm2. The study is highlighted by the proof that PDI treatment using Ber showed an antibacterial effect on Gram(−) E. coli. Planktonic cells could be reduced by 3 log10 steps with 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). With 5 mM ethylenediamine tetraacetic acid disodium salt dihydrate (Na2EDTA) or 1.2% polyaspartic acid (PASA) in addition, a relative inactivation of 4 log10 steps and 7 log10 steps, respectively, was detectable. Furthermore, we showed that an antibacterial effect of 3.4 log10 towards E. coli biofilms was given when using 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). These results underscore the significance of PDI-treatment with Ber as a natural compound in combination with blue light as valuable antimicrobial application.

1. Introduction

One of the major challenges in global health care is the rapidly evolving resistance of human pathogens to various antimicrobials [1] causing prolonged illness, higher mortality rates, and increased healthcare costs in hospitals after surgeries and prosthetic device infections [2,3,4]. Rapidly increasing antibiotic resistance, on one hand, and the lack of new antibiotics, on the other hand, complicate the treatment of wound infections [5]. Bacteria that pose a severe problem to human health are, for example, Gram(+) Staphylococcus capitis subsp. capitis, Staphylococcus aureus, and the Gram(−) Escherichia coli. These nosocomial pathogens are able to form protective biofilms with much higher tolerance towards antibiotic therapies [6].
Staphylococcus capitis is part of the normal skin microbiome colonizing the human scalp [7]. However, these bacteria can also act as inflammatory-induced pathogens and cause skin infections like cysts and folliculitis [8]. Standard therapy for S. capitis infections is a combination antibiotic treatment of vancomycin, gentamicin, and rifampin [9]. In addition to rapidly increasing numbers of resistant strains, treatment of S. capitis infections also remains challenging due to its capability to form biofilms [10]. S. capitis can become problematic especially for elderly or immunosuppressed patients. Infections of joint prostheses bear the risk of S. capitis entry into soft tissues, which can lead to serious infections and in line-associated bacteremias [4]. Staphylococcus aureus is responsible for a wide range of conditions ranging from superficial skin infections to severe, invasive diseases [11]. S. aureus shows a strong capacity to produce biofilms and is thus able to persist therapeutic treatments. This is even more critical when it comes to indwelling medical devices, such as catheters and prosthetic joints [12,13].
Gram(−) bacteria cause life-threatening infections in humans. They are of particular concern because their outer membrane provides a significant penetration barrier to antimicrobials. Particularly, uncharged or anionic agents are unable to overcome this barrier, thus making Gram(−) bacteria more resistant to such interventions [14]. Escherichia coli serves as an etiological agent for nosocomial infections, causes a broad spectrum of diseases, and manifests an increasing resistance to conventional therapeutic modalities [15].
The development of alternative treatment strategies captures the attention of scientists around the world. Photodynamic Inactivation (PDI) represents an innovative method for killing microorganisms [16]. Mode of action is the synergistic interaction of a photoactive substance, light, and molecular oxygen. Subsequently produced reactive oxygen species (ROS) damage different compartments of bacterial cells in an unspecific manner and thus elicits toxicity [17]. The photosensitizer (PS) plays a key role in PDI, and photoactive molecules derived from herbs, such as chlorophyllin, hypericin, and curcumin, have already demonstrated their potential for PDI treatment [18]. The PDI process is illustrated in Figure 1.
Berberine is an isoquinoline alkaloid with photoactive properties [19]. Extracted from Barberry (Berberis vulgaris) and many other plants, it is a natural compound widely used in Traditional Chinese Medicine [20]. As a medicinal herb, it possesses antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, and anti-cancer as well as antimicrobial effects [21,22,23,24]. The chloride or sulfate salt of berberine is commonly employed for clinical applications due to its enhanced water solubility compared to the natural form [25]. Berberine carries a positive charge. That positive charge could be of key relevance in PDI when it comes to treatment of Gram(−) E. coli overcoming its complex barrier system [26].
The aim of our study was to investigate in vitro the photodynamic efficacy of berberine chloride hydrate (Ber) as a natural PS photoactivated with blue light towards S. capitis, S. aureus and E. coli in planktonic and biofilm cultures.

2. Materials and Methods

2.1. Photosensitizer Stock Solutions

Aqueous stock solutions of 10 mM Ber (CAS No. 141433-60-5, Carl Roth GmbH + Co.KG, Karlsruhe, Germany) were prepared in sterile ddH2O and stored at −20 °C in the dark until usage.

2.2. Microorganisms

Gram(+) Staphylococcus capitis subsp. capitis (ATCC 27840), Gram(+) Staphylococcus aureus (ATCC 25923), and Gram(−) Escherichia coli (ATCC 25955) were obtained from the Leibnitz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany and stored as cryo-cultures supplemented with 10% glycerol at −196 °C.
S. aureus and E. coli were grown overnight (o/n) in medium containing 30 g/L Caso-Bouillon (Roth), and S. capitis was grown in media containing 30 g/L Caso-Bouillon (Roth) and 3 g/L yeast extract (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 37 °C under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA). For static biofilm formation, the respective media were enriched with 1% glucose (Roth). Petri dishes were prepared with the respective media without glucose but supplemented with 15 g/L Agar-Agar (Roth).

2.3. Photoactivity Testing of Berberine Chloride Hydrate

Photoactivity of Ber was assessed by absorbance measurement using an UV-VIS spectrophotometer (Implen, Munich, Germany). For this method, 50 µM Ber were prepared in 1 mL Dulbecco’s phosphate-buffered saline (DPBS, Sigma-Aldrich Chemie GmbH). The absorbance was measured against a blank containing DPBS.

2.4. Photodynamic Inactivation of Bacteria in Planktonic Culture

Bacterial strains were cultivated o/n in the respective broth as described under 2.2. Before each experiment, the optical density (OD) at 600 nm of the bacterial cultures was measured using an Infinite M200 microplate reader (Tecan, Grödig, Austria), and cultures were diluted to an OD600nm of 0.05, which corresponded to a concentration of approximately 107 cells/mL. After two hours incubation at 37 °C, bacteria were centrifuged with 833 RCF for 3 min at 4 °C (Centrifuge 5417R, Eppendorf, Hamburg, Germany). Bacterial pellets were resuspended in DPBS containing either 0 µM, 1 µM, 10 µM, or 100 µM Ber. If required, cell-wall-permeabilization was achieved by further supplementation with 5 mM ethylenediamine tetraacetic acid disodium salt dihydrate (Na2EDTA; VWR International, Leuven, Belgium) or 1.2% Polyaspartic acid (PASA; Baypure®DS100; Kurt Obermeier GmbH & Co. KG, Bad Berleburg, Germany). Triplets of 500 µL of each sample were transferred into 24-well plates and incubated for 5 or 30 min in the dark followed by illumination from below under constant agitation (Flow Laboratories, DSG Titertek, microplate shaker). As a light source, an in-house manufactured LED array (480 LEDs, 435 nm dominant wavelength, LED435-12-30, Roithner Lasertechnik, Vienna, Austria) was used, providing a radiant exposure of 25 J/cm2. Dark controls were treated in the same way but excluded from light with aluminum foil. Aliquots of treated and control samples were serially diluted up to 10 −7, plated on agar plates with the respective medium, and incubated for 24 h at 37 °C in the dark to count the colony-forming units (CFU).

2.5. Photodynamic Inactivation of Bacteria Grown in Static Biofilm Formations

To induce the formation of static biofilms of S. capitis and S. aureus, bacterial strains were precultured o/n in the respective medium described under 2.2. Then, 250 µL of this preculture were added to 13 mL fresh medium supplemented with 1% glucose. Samples of 150 µL were transferred into 96-well plates and incubated for 24 h at 37 °C. For static biofilms of E. coli bacteria were cultured o/n in Caso-Bouillon. After adjusting the OD600nm to 0.1, suspensions were mixed in a ratio of 1:9 with liquid Caso-Bouillon containing 1% glucose. A 150 µL of bacterial suspensions were transferred to 96-well plates, and biofilms were obtained within 48 h at 37 °C. The medium was renewed after 24 h.
For PDI of biofilm cultures, supernatants were removed from biofilms and 150 µL of Ber were added and incubated for 5 or 60 min. After incubation, Ber was removed by careful aspiration and replaced with 150 µL DPBS. Illumination was performed from below with a 435 nm LED-array resulting in a radiant exposure of 100 J/cm2. For quantification of CFUs, PDI-treated suspensions were serially diluted, plated on agar dishes, and incubated for 24 h.

2.6. Data Evaluation

The PDI efficacy was expressed as relative inactivation, calculated as
log [CFUCo−/−/CFUsample]
for each biological replicate [27]. If no CFUs were visible (equal to the detection limit), the CFU of the Co−/− was divided by 1. Results are depicted as mean values (mean) of all biological replicates with standard deviations (SD). All sets of experiments have been replicated at least three times. Data visualization was performed using OriginPro, Version 2021b (OriginLab Corporation, Northampton, MA, USA). Based on the standards of the American Society for Microbiology, an antibacterial effect is considered to be present if the relative inactivation of bacteria exceeds three orders of magnitude, which is equivalent to the removal of 99.9% of bacterial cells [28]. A red dashed line in the graphs depicts this antibacterial effect. All data of the experiments presented in this study can be found in the “Supplementary Materials” section.

3. Results

3.1. Photoactivity of Berberine Chloride Hydrate

The absorption spectrum of Ber is shown in Figure 2. In the UV spectral range, Ber showed absorption at 229 nm, 263 nm, and 340 nm. For PDI experiments, the peak at 420 nm was used.

3.2. Phototoxicity of Berberine Chloride Hydrate towards Staphylococcus capitis and Staphylococcus aureus

For planktonic cells of S. capitis (Figure 3A), a relative inactivation of 1.2 × 103 was determined at a concentration of 10 µM Ber after 5 min incubation, based on 1.5 × 108 CFU/100 µL in the double negative control (Co−/−). Prolonged incubation of 30 min led to an amplification of Ber-induced relative inactivation to 1.3 × 106. At a concentration of 100 µM, Ber caused relative inactivation of 2.6 × 107 and 2.0 × 107 after 5 or 30 min incubation, respectively.
For static biofilms of S. capitis (Figure 3B), an inactivation of 3.8 × 104 CFU/100 µL was observed at a concentration of 1 mM Ber incubated for 60 min, relative to 5.9 × 107 CFU/100 µL in the Co−/−. This protocol induced a slight dark toxicity of approximately one log10 step.
Planktonic cells of S. aureus (Figure 3C) showed an average of 8.5 × 106 CFU/100 µL in the untreated control. An antibacterial effect of 1.4 × 105 relative inactivation was measured at a concentration of 100 µM Ber and 5 min incubation. The effect of 100 µM Ber could be amplified to 3.2 × 106 when incubation was prolonged to 30 min.
Berberine was phototoxic towards static biofilms of S. aureus (Figure 3D). After 5 min incubation in the dark and subsequent illumination with 100 J/cm2 1 mM, Ber induced a relative inactivation of 1.9 × 103. The CFU count of untreated controls was 1.0 × 107 CFU/100 µL. Prolonged incubation up to 60 min did not show any additional effect (relative inactivation of 5.4 × 102).

3.3. Phototoxicity of Berberine Chloride Hydrate towards Gram(−) Escherichia coli

Figure 4 shows a comparison of PDI experiments using Ber without additives (Figure 4A,D) and with 5 mM Na2EDTA (Figure 4B) or 1.2% PASA (Figure 4C) against E. coli (5 or 30 min incubation, 435 nm, 25 J/cm2). At a concentration of 100 µM, Ber presented an antibacterial effect of 1.2 × 103 after 5 min incubation and 2.3 × 103 after 30 min towards planktonic E. coli cells, based on 7.6 × 107 CFU/100 µL in the untreated controls. The photoeffect could be enhanced when 100 µM Ber was supplemented with 5 mM Na2EDTA, resulting in a photokilling effect of 1.1 × 104 and 1.3 × 104, respectively, after 5 or 30 min incubation (based on 1.1 × 108 CFU/100 µL). Addition of 1.2% PASA further enhanced the photoantimicrobial effect of Ber. Moreover, 100 µM Ber supplemented with 1.2% PASA resulted in a relative inactivation of 9.2 × 106 after 5 min incubation. Extending the incubation period to 30 min did not enhance the photoeffect, with a relative inactivation of 1.0 × 106 (based on 7.2 × 107 CFU/100 µL). However, increasing the radiant exposure from 25 J/cm2 up to 100 J/cm2 enhanced the photoeffect of Ber without the use of additives (Figure 4D). A 100 µM concentration of Ber presented an antibacterial effect of 8.7 × 105 after 5 min incubation. A concentration of 10 µM Ber fulfilled the three-log criterion for an antibacterial effect with 1.2 × 103 relative inactivation (based on 1.4 × 108 CFU/100 µL in the Co−/−).
The PDI treatment using Ber against E. coli biofilms required a higher concentration of Ber in combination with an increased radiant exposure. A concentration of 1 mM Ber and a radiant exposure of 100 J/cm2 resulted in an antibacterial effect of 4.1 × 103 (based on 6.4 × 107 CFU/100 µL in the Co−/−) after 5 min incubation (Figure 5). Compared to 5 min incubation, prolonging the incubation of 1 mM Ber up to 60 min showed a reduced photoeffect of 5.1 × 101.

4. Discussion

For PDI experiments in this study, the absorption of Ber in the blue light range at 420 nm was used, although the absorption maximum of Ber is at 340 nm in the UVA range. UVA elicits skin damage and is suspected of being mutagenic [29,30]. To avoid these negative effects, in this study, Ber was photoactivated at its minor absorption band at 420 nm in the visible blue light range. Blue light with its high energy and therefore low tissue penetration is attractive to employ for treatment of superficial infections in a future clinical application [31]. The lower absorptivity of Ber at 420 nm and thus lower PS excitation was compensated by a higher light intensity in order to achieve the same high quantum efficiency.
The abundance of antibiotic-resistant S. capitis infections is strikingly increasing and indicates the need for alternative therapies. Photodynamic treatment of S. capitis with Ber as a PS has shown a clear effect on planktonic cultures (Figure 3A). An antibacterial effect of 3 log10 steps was already visible at a concentration of 10 µM Ber after 5 min incubation before illumination with a 435 nm LED array, resulting in a radiant exposure of 25 J/cm2. The effect of 10 µM Ber could be amplified to 6 log10 steps when the incubation period was prolonged to 30 min. A 7 log10 steps relative inactivation could be reached at a concentration of 100 µM (after 5 and 30 min incubation, respectively). In previous studies, Methylene Blue (MB) was used for the PDI treatment of S. capitis. Miyabe et al. [32] treated planktonic cultures of S. capitis with 3 mM MB illuminated with a 660 nm gallium-aluminum-arsenide laser (radiant exposure of 26.3 J/cm2) and observed a relative inactivation of 6 log10 steps. A similar result was shown by the study of Karner et al. [33], who achieved a relative inactivation of 5 log10 units of S. capitis with 20 µM MB photoactivated by a 632 nm pulsed LED light with 30 J/cm2. Thus, our results show that PDI with Ber needs far lower concentrations or lower radiant exposure to achieve a comparable photokilling effect.
Precedent findings from PDI studies of S. capitis indicate that biofilms generally require a higher concentration of PS, a longer incubation period as well as a higher radiant exposure to penetrate the polysaccharide matrix. As shown in Figure 3B, an antibacterial effect of 4 log10 steps was achieved by PDI with 1 mM Ber incubated for 60 min in the dark succeeded by illumination with a radiant exposure of 100 J/cm2. As a result of the high concentration required, a dark toxicity (reduction of CFUs in the presence of PS but without illumination) of approximately one log10 step was observed. Minor toxic effects for the dark controls were observed but remained below the respective SD and were most likely caused by the in general high PS concentrations required for biofilm treatments.
We demonstrated that Ber is a potent PS for PDI of planktonic and biofilm cultures of S. capitis, indicating its potential to treat pathologic skin infections caused by S. capitis, which are becoming increasingly challenging to control by classic antibiotics due to increasing numbers of resistant strains.
This study also investigated the PDI effect of Ber on S. aureus. At 100 µM Ber, the CFU of planktonic S. aureus was reduced by 5 log10 steps and 6 log10 steps (after 5 or 30 min incubation, respectively). In the PDI-related literature, MB and toluidine blue O-based PS (TBO) have been widely used as PS. Muehler et al. [34] reported that for PDI with MB (3.9 µM) and TMPyP (3.9 µM), CFU reductions of up to 6.3 log10 and 6.7 log10 steps, respectively, were observed for planktonic cultures of S. aureus. Bacteria were incubated with PS for 10 min and illuminated for 10 min, resulting in radiant exposures of 12 J/cm2 (PDT 1200L Waldmann lamp) for MB and 10.8 J/cm2 (UV 236 Waldmann lamp) for TMPyP. The photoantibacterial efficiency of these PS is comparable to Ber. However, the advantage of Ber is that it is a natural PS and, to date, no side effects have been reported. On the contrary, MB is toxic per se and proofed to be harmful to human health [35]. And TBO can lead to adverse effects because of its harmfulness to mitochondrial energy metabolism [36].
Hasenleitner et al. [37] reported that 5 µM Chlorophyllin (Chl), another natural PS, photoactivated by a 433 nm Repuls7PDI-blue light source (radiant exposure 6.6 J/cm2) killed 7 log10 steps of S. aureus.
In the S. aureus static biofilm assay, a higher concentration of Ber, as well as an increased radiant exposure, was needed. One mM of Ber incubated for 5 min and illuminated with 100 J/cm2 resulted in a relative inactivation of 3 log10 steps with only a slight dark toxicity lower than 1 log10 step. Safai et al. reported that with 150 µg/mL and 500 µg/mL (~400 µM/1.3 mM) of Ber, S. aureus biofilms were totally eradicated in the PDI samples (60 min incubation, 465 nm LED irradiation for 15 min) and also in the dark controls, whereby 50 µg/mL Ber (~134 µM) did not affect biofilm formation [38].
Due to the more permeable cell wall structure of Gram(+) bacteria, PS can penetrate more readily and induce bacterial cell death without additional permeabilization. In contrast, phototreatment of Gram(−) bacteria, with their complex outer membrane, necessitates the application of cell-wall-permeabilizing agents, such as Na2EDTA or PASA, to achieve substantial bacterial inactivation. These agents are essential because they disrupt the outer membrane, allowing PS like Ber to penetrate and effectively exert its photodynamic efficacy [39,40,41].
Therefore, the PDI of E. coli could be enhanced up to 4 log10 steps when 100 µM Ber was supplemented with 5 mM Na2EDTA. Addition of 1.2% PASA further enhanced the photoeffect of 100 µM Ber up to almost 7 log10 steps. Photoactivation of 3.9 µM MB or 3.9 µM TMPyP reduced the CFU of planktonic E. coli up to 6.4 log10 and 6.5 log10 steps [34]. Two other natural PS, 500 µM Aloe emodin or 500 µM Curcumin (irradiance 0.06 mW/cm2, illumination 750 s, 208 s respectively), were tested and showed no significant inhibition of planktonic E. coli [42]. In contrast, the positively charged Ber was able to attach to the negatively charged outer membrane of E. coli. Beyond that, the outer membrane of Gram(−) bacteria possesses a highly organized structure forming a physical and functional barrier [43]. Hence, one of the approaches for a successful PDI treatment of Gram(−) E. coli could be the proven combination of positively charged Ber and PASA as cell-wall-permeabilizing agent.
In this study, PDI treatment using Ber against E. coli biofilms was proven to be successful for the first time, to the best of our knowledge. An antibacterial effect of 3.4 log10 steps could be realized with 1 mM Ber after short incubation of 5 min. However, it required a radiant exposure of 100 J/cm2. Comparable results were reported by Vilela et al. [44] using 3 mM Malachite green (MG) incubated for 5 min and illuminated by a 660 nm laser light, realizing an antibacterial effect of 4.1 log10 steps towards E. coli biofilms.
A 300 µM MB and 150 µM TBO were also tested, but only showed a negligible effect. As a result, a threefold higher concentration of MG is required to achieve the same antibacterial effect as Ber, whereby, to date, there is no evidence on the safety of MG as PS for medical application.

5. Conclusions

In this basic research study, the natural compound Ber was employed as ecofriendly and economical PS for PDI treatment towards human pathogens. The results of the conducted in vitro experiments showed that Ber had an antibacterial effect towards the tested Gram(+) and Gram(−) bacteria, in planktonic form as well as in biofilm formation. In Table 1, the most important results are summarized.
To our knowledge, this study is the first to show that Ber photoactivated with blue light has an antibacterial effect on Gram(−) E. coli, even in biofilm formation. The photoeffect towards E. coli was lower than that towards S. capitis or S. aureus; however, it could be enhanced by adding Na2EDTA or PASA as cell-wall-permeabilizing agents or by increasing the radiant exposure. For future investigations, an approach to be pursued in the fight against human pathogens especially in their biofilm formations, is the combination of PDI treatment with Ber and biofilm destructing agents, advantageous to support healing processes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/photonics11070647/s1, Table S1: PDI towards planktonic S. capitis using Ber (at 1 µM, 10 µM, 100 µM), 5 or 30 min drug-to-light interval, illumination at 435 nm, 25 J/cm2, n = 3; Table S2: PDI towards S. capitis biofilms using Ber (at 100 µM, 1 mM), 5 or 60 min drug-to-light interval, illumination at 435 nm, 100 J/cm2, n = 4; Table S3: PDI towards planktonic S. aureus using Ber (at 1 µM, 10 µM, 100 µM), 5 or 30 min drug-to-light interval, illumination at 435 nm, 25 J/cm2, n = 3; Table S4: PDI towards S. aureus biofilms using Ber (at 100 µM, 1 mM), 5 or 60 min drug-to-light interval, illumination at 435 nm, 100 J/cm2, n = 3; Table S5: PDI towards planktonic E. coli using Ber (at 1 µM, 10 µM, 100 µM), 5 or 30 min drug-to-light interval, illumination at 435 nm, 25 J/cm2, n = 3; Table S6: PDI towards planktonic E. coli using Ber (at 1 µM, 10 µM, 100 µM) and 5 mM Na2EDTA, 5 or 30 min drug-to-light interval, illumination at 435 nm, 25 J/cm2, n = 3; Table S7: PDI towards planktonic E. coli using Ber (at 1 µM, 10 µM, 100 µM) and 1.2% PASA, 5 or 30 min drug-to-light interval, illumination at 435 nm, 25 J/cm2, n = 3; Table S8: PDI towards planktonic E. coli using Ber (at 1 µM, 10 µM, 100 µM), 5 or 30 min drug-to-light interval, illumination at 435 nm, 100 J/cm2, n = 5; Table S9: PDI towards E. coli biofilms using Ber (at 100 µM, 1 mM), 5 or 60 min drug-to-light interval, illumination at 435 nm, 100 J/cm2, n = 3.

Author Contributions

Conceptualization, A.W. and K.P.; methodology, A.W., M.G. and K.P.; validation, A.W. and K.P.; investigation, A.W.; resources, J.L., M.F. and K.P.; data curation, A.W.; writing—original draft preparation, A.W.; writing—review and editing, A.W., J.L., M.F. and K.P.; visualization, A.W.; supervision, K.P.; project administration, K.P.; funding acquisition, K.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

The authors are grateful to Aleksandar Paunovic and Anabel Santiago Cenit for technical and experimental support, as well as Linda Jernej and Anna Müller (all from the Department of Biosciences and Medical Biology, Paris Lodron University Salzburg) for proofreading the manuscript.

Conflicts of Interest

Authors Jun Liu and Michael Fefer are employed by Nutrien Ag Solutions. Author Kristjan Plaetzer has received research funding from Nutrien Ag Solution. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This study was conducted at the Paris Lodron University Salzburg. The selection of study topics, experimental design, collection and analysis of data were all conducted for academic research purposes. Jun Liu and Michael Fefer collaborated in the scientific discussions, manuscript review and editing. There are no potential perceived conflicts. The authors declare no conflicts of interest.

Abbreviations

BerBerberine chloride hydrate
CFUColony-forming unit(s)
Co−/−Double-negative control (no light, no PS)
DPBSDulbecco’s phosphate-buffered saline
E. coliEscherichia coli
meanMean value
MGMalachite green
MBMethylene Blue
Na2EDTAEthylenediamine tetraacetic acid disodium salt dihydrate
PASAPolyaspartic acid
PDIPhotodynamic Inactivation
PSPhotosensitizer
PS controlPhotosensitizer control (highest PS concentration, longest incubation period, no light)
ROSReactive oxygen species
SDStandard deviation
S. aureusStaphylococcus aureus
S. capitisStaphylococcus capitis subsp. capitis
TBOToluidine blue O-based PS

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Photonics 11 00647 g001
Figure 1. Simplified illustration of the photodynamic principle based on photophysical und photochemical reactions between a PS and light in the presence of oxygen.
Figure 1. Simplified illustration of the photodynamic principle based on photophysical und photochemical reactions between a PS and light in the presence of oxygen.
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Photonics 11 00647 g002
Figure 2. Absorption spectrum of 50 µM Ber dissolved in DPBS in the range of 200–800 nm wavelength and the chemical structure of the photoactive molecule Ber.
Figure 2. Absorption spectrum of 50 µM Ber dissolved in DPBS in the range of 200–800 nm wavelength and the chemical structure of the photoactive molecule Ber.
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Photonics 11 00647 g003
Figure 3. Relative inactivation of S. capitis (upper panel) and S. aureus (lower panel) after PDI with Ber at different concentrations and incubation periods. (A) Planktonic and (B) biofilm cultures of S. capitis; (C) planktonic and (D) biofilm culture of S aureus. Graph shows the mean and standard deviation of relative inactivation. The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. Abbreviations used in the graphs: PS (photosensitizer), Co−/− (no light, no PS), Light control (light but no PS), PS control (highest PS concentration, longest incubation period, no light). The number of planktonic S. capitis cells was reduced by 7 log10 steps using 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). For an antibacterial effect of 4 log10 steps, static S. capitis biofilm formations required 1 mM Ber (60 min, 435 nm, 100 J/cm2). Planktonic S. aureus was photokilled by 5 log10 steps using 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). Static biofilms of S. aureus were phototreated resulting in a 3 log10 steps reduction by using 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). Biological replicates n ≥ 3.
Figure 3. Relative inactivation of S. capitis (upper panel) and S. aureus (lower panel) after PDI with Ber at different concentrations and incubation periods. (A) Planktonic and (B) biofilm cultures of S. capitis; (C) planktonic and (D) biofilm culture of S aureus. Graph shows the mean and standard deviation of relative inactivation. The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. Abbreviations used in the graphs: PS (photosensitizer), Co−/− (no light, no PS), Light control (light but no PS), PS control (highest PS concentration, longest incubation period, no light). The number of planktonic S. capitis cells was reduced by 7 log10 steps using 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). For an antibacterial effect of 4 log10 steps, static S. capitis biofilm formations required 1 mM Ber (60 min, 435 nm, 100 J/cm2). Planktonic S. aureus was photokilled by 5 log10 steps using 100 µM Ber (5 min incubation, 435 nm, 25 J/cm2). Static biofilms of S. aureus were phototreated resulting in a 3 log10 steps reduction by using 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). Biological replicates n ≥ 3.
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Figure 4. Relative inactivation of E. coli in planktonic culture using Ber at different concentrations without additives (A) and with Na2EDTA (B) supplemented with PASA (C), or without additives but an increased radiant exposure of 100 J/cm2 (D). The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. The PDI treatment of planktonic cells of Gram(−) E. coli showed an antibacterial effect with 100 µM Ber (5 and 30 min incubation, 435 nm, 25 J/cm2). In presence of 5 mM Na2EDTA a relative inactivation of 4 log10 steps was measured with 100 µM Ber. After photoactivation of 100 µM Ber and 1.2% PASA (5 min incubation, 435 nm, 25 J/cm2), the photokilling effect could enhanced up to 7 log10 steps. Increased radiant exposure up to 100 J/cm2 also enhanced the photoeffect of 100 µM Ber up to almost 6 log10 steps and even 10 µM showed an antibacterial effect of 3 log10 steps. n ≥ 3.
Figure 4. Relative inactivation of E. coli in planktonic culture using Ber at different concentrations without additives (A) and with Na2EDTA (B) supplemented with PASA (C), or without additives but an increased radiant exposure of 100 J/cm2 (D). The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. The PDI treatment of planktonic cells of Gram(−) E. coli showed an antibacterial effect with 100 µM Ber (5 and 30 min incubation, 435 nm, 25 J/cm2). In presence of 5 mM Na2EDTA a relative inactivation of 4 log10 steps was measured with 100 µM Ber. After photoactivation of 100 µM Ber and 1.2% PASA (5 min incubation, 435 nm, 25 J/cm2), the photokilling effect could enhanced up to 7 log10 steps. Increased radiant exposure up to 100 J/cm2 also enhanced the photoeffect of 100 µM Ber up to almost 6 log10 steps and even 10 µM showed an antibacterial effect of 3 log10 steps. n ≥ 3.
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Photonics 11 00647 g005
Figure 5. Relative inactivation of static biofilms of E. coli using Ber at different concentrations with a radiant exposure of 100 J/cm2. The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. The PDI treatment Gram(−) E. coli static biofilms showed an antibacterial effect (3 log10 steps) with 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). Prolonging the incubation up to 60 min did not enhance the photokilling effect. n = 3.
Figure 5. Relative inactivation of static biofilms of E. coli using Ber at different concentrations with a radiant exposure of 100 J/cm2. The red dashed line marks a reduction of 3 log10 steps indicating an antibacterial effect. The PDI treatment Gram(−) E. coli static biofilms showed an antibacterial effect (3 log10 steps) with 1 mM Ber (5 min incubation, 435 nm, 100 J/cm2). Prolonging the incubation up to 60 min did not enhance the photokilling effect. n = 3.
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Table 1. Key results for each bacterial strain tested, including conditions and outcome.
Table 1. Key results for each bacterial strain tested, including conditions and outcome.
BacteriaLife FormConcentration of BerAdditiveDrug-to-Light Interval [min]Radiant Exposure [J/cm2]Relative Inactivation
S. capitisplanktonic100 µM-5257 log10
biofilm1 mM-601004 log10
S. aureusplanktonic100 µM-30256 log10
biofilm1 mM-51003 log10
E. coliplanktonic100 µM1.2% PASA5257 log10
biofilm1 mM-51003 log10
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Wimmer, A.; Glueck, M.; Liu, J.; Fefer, M.; Plaetzer, K. With Blue Light against Biofilms: Berberine as Natural Photosensitizer for Photodynamic Inactivation of Human Pathogens. Photonics 2024, 11, 647. https://doi.org/10.3390/photonics11070647

AMA Style

Wimmer A, Glueck M, Liu J, Fefer M, Plaetzer K. With Blue Light against Biofilms: Berberine as Natural Photosensitizer for Photodynamic Inactivation of Human Pathogens. Photonics. 2024; 11(7):647. https://doi.org/10.3390/photonics11070647

Chicago/Turabian Style

Wimmer, Annette, Michael Glueck, Jun Liu, Michael Fefer, and Kristjan Plaetzer. 2024. "With Blue Light against Biofilms: Berberine as Natural Photosensitizer for Photodynamic Inactivation of Human Pathogens" Photonics 11, no. 7: 647. https://doi.org/10.3390/photonics11070647

APA Style

Wimmer, A., Glueck, M., Liu, J., Fefer, M., & Plaetzer, K. (2024). With Blue Light against Biofilms: Berberine as Natural Photosensitizer for Photodynamic Inactivation of Human Pathogens. Photonics, 11(7), 647. https://doi.org/10.3390/photonics11070647

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