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Antimicrobial photodynamic therapy with Ligularia fischeri against methicillin-resistant Staphylococcus aureus infection in Caenorhabditis elegans model

Antimicrobial photodynamic therapy with Ligularia fischeri against methicillin-resistant... The high prevalence of methicillin‑resistant Staphylococcus aureus (MRSA) infection threatens the effectiveness of current clinical settings. Antimicrobial photodynamic therapy (APDT ) is a promising alternative to antibiotics for treating infections due to its low resistance. This study aimed to evaluate the antibacterial properties of APDT with L. fischeri extract (LFE) against MRSA and various skin and oral pathogens in vitro and its photopharmaceutical actions in Caenorhabditis elegans. The antimicrobial activities of APDT with LFE against pathogens were evaluated using plate counting method. The chemical profile was characterized using high‑performance liquid chromatography and spec‑ trophotometry. The growth rate assay, lifespan assay, and bacterial attachment on worms were performed to assess the therapeutics effects in C. elegans. The swab method was used for the detection of pathogens on the micropig skin surface. The APDT treatment with L. fischeri extract (LFE, 20 µg/mL) and red light (intensity of 120 W/m ) reduced 4.3–4.9 log (colony forming unit/mL) of Staphylococcus aureus, MRSA, Cutibacterium acnes, Streptococcus mutans; and 2.4 log (CFU/mL) of Candida albicans. Chemical analysis revealed that LFE enriched three active photosensitizers. APDT reduced bacterial populations on worms, recovered growth retardation, and improved lifespan in MRSA‑infected C. elegans without causing severe side effects. The surface eradication of MRSA after exposure to LFE with red light was demonstrated on micropig skin. These findings highlight the significance of L. fischeri as a natural resource for the safe phototreatment of MRSA infection in the biomedical and cosmeceutical industries. Keywords Antimicrobial photodynamic treatment, C. elegans, Ligularia fischeri, Pheophorbide, MRSA, Staphylococcus aureus † 2 Ngoc Minh Ha and Hoseong Hwang are contributed equally to this work Division of Bio‑Medical Science & Technology, KIST School, University of Science and Technology (UST ), Gangneung, Gangwon‑do 25451, *Correspondence: Republic of Korea Jaeyoung Kwon Natural Product Research Center, Gangneung Institute of Natural kjy1207@kist.re.kr Products, Korea Institute of Science and Technology, Gangneung, Kyungsu Kang Gangwon‑do 25451, Republic of Korea kskang@kist.re.kr Natural Product Informatics Research Center, Gangneung Institute of Natural Products, Korea Institute of Science and Technology, 679 Saimdang‑ro, Gangnneung, Gangwon‑do 25451, Republic of Korea © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Ha et al. Applied Biological Chemistry (2023) 66:19 Page 2 of 13 antimicrobial material and evaluated its therapeutic Introduction impact on C. elegans. Infectious diseases caused by methicillin-resistant In this study, Caenorhabditis elegans was used as a Staphylococcus aureus (MRSA) continue to be a sig- model to evaluate the impact of ADPT with LFE. Thanks nificant global concern. Although antibiotics are quick to the short lifespan and high percentage of genes that are and effective, drug resistance in pathogenic bacteria homologous to human genes; this invertebrate nematode increases the likelihood of treatment failure and eco- has proven to be a useful model for the screening of bio- nomic burden [1]. Therefore, more sustainable treat - active compounds in therapeutic fields involving antiag - ment with lower resistance risk is desirable. ing, antineurodegeneration, gut health improvement, and One of the promising options for treating micro- metabolic disorders [9–18]. Regarding pathogen infec- bial infection is antimicrobial photodynamic therapy tions, live-C. elegans infection model was established to (APDT). In APDT, a photoactive substance in the pres- evaluate the in vivo efficacy against bacteria Enterococcus ence of oxygen undergoes a photochemical activation faecalis, Streptococcus pyogenes, Staphylococcus aureus, by light that absorbs and transfers energy to the oxygen and fungi Candida albicans [19–23]. Here, it is the first molecule to generate reactive oxygen species (ROS) like time APDT with LFE was evaluated and its therapeutic singlet oxygen and superoxides. ROS has potent oxida- impacts was illustrated on C. elegans. tive properties that can permanently modify cellular The present investigation aimed to evaluate the anti- constituents like fatty acids, amino acids, and nucle- infective effects of LFE in combination with red light obases that cause the elimination of harmful bacteria against different pathogenic microorganisms, particularly [2, 3]. It has several advantages, including high kill- MRSA in  vitro. The therapeutic effects of APDT treat - ing selectivity, minimal invasiveness, and a lower risk ment against MRSA infection were further assessed in of toxicity induction in nearby host tissues due to the C. elegans model. Figure  1 illustrates the experimental selective accumulation of target pathogens around the scheme of the study. photosensitizers and the restriction of light application to a specific local area [4 –6]. APDT treats infectious Methods diseases caused by bacteria and fungi in  vitro and in Sample collection animal models, such as skin infections, peri-implanti- The LFE for rapid screening was provided from the tis, periodontitis, and diabetic foot ulcers [4, 5, 7]. natural product library, Korea Institute of Science and As plants are a rich source of photoactive chemicals Technology, Gangneung Institute of Natural Products with diverse secondary metabolite chemical groups (Accession number KHG2016-01-0007; Gangwon-do, and distinct structures that have advantages in terms South Korea). Dried L. fischeri was purchased from the of minimal side effects and economics, attempts are herbal market in Jeongseon (Gangwon-do, South Korea). therefore needed for the screening of plant-derived material for APDT. Ligularia fischeri is a wild vegeta - Extraction and compound isolation ble plant with yellow composite flowers often used in The L. fischeri samples were dried (900  g) and ground, folk medicine to treat jaundice, scarlet fever, rheuma- and 101.1  g of dark green extract was collected under toid arthritis, and liver diseases [7]. It is known for its 2-hour-reflux with ethanol (9 × 2  L), followed by filtra - anti-oxidation, anti-cancer, anti-obesity, anti-hepa- tion and evaporation at low pressure. Bioassay-guided totoxic, and anti-inflammation properties [8 ]. How- fractionation scheme was applied for the isolation of ever, no study has used this plant as a photodynamic Fig. 1 Experimental scheme of the present study, antimicrobial photodynamic therapy (APDT ) with Ligularia fischeri extract (LFE) Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 3 of 13 bioactive substances. The screening method was used to production was recorded at 440  nm. A DMSO-water detect the singlet oxygen generation ( O ). LFE (14.5  g) mixture was served as a negative control. was adsorbed on Celite, separated by a column filled with Diaion HP-20 resin (130  g, 2.5 × 18  cm) and eluted with Bacterial strains and bacterial suspension preparations ethanol/water/acetone (3:2:0, 4:1:0, 1:0:0, and 0:1:1). The Cutibacterium acnes KCTC3314, Staphylococcus aureus four fractions ranged from F1 to F4. F4 (215.1 mg) were KCTC3881, Candida albicans KCTC7965, and Strepto- separated by a Gilson semipreparative high-pressure liq- coccus mutans KCTC3065 strains were purchased from uid chromatography (HPLC, Phenomenex Luna 10  μm the Korean Collections for Type Cultures (Jeongeup, C (2) column [10  μm, 250 × 21.2  mm]) and eluted Korea). Pseudomonas aeruginosa PAO1 was purchased using gradient conditions (acetonitrile/water, 77:23 to from American Type Culture Collection, and MRSA 3:97 in 120  min, flow rate 8 mL/min, 420  nm) to collect 2659 was acquired as described previously [20]. Luria- compounds 1 (1.6  mg, t = 36.1  min), 2 (2.7  mg, t = Bertani (LB) broth was used to culture MRSA, S. aureus, R R 57.8 min), and 3 (1.6 mg, t = 65.4 min). S. mutans, P. aeruginosa, and Sabouraud broth was used to culture C. albicans. C. acnes was grown in brain heart Structural identification of active compounds infusion (BHI) broth. The bacterial suspensions were pre - The chemical structures of compounds 1–3 were identi - pared by diluting fresh cultures with the corresponding fied using a combination of spectroscopy and spectrome - liquid broth and incubating at 37 °C overnight. Bacterial try analysis (Nuclear magnetic resonance [NMR]: Varian suspensions were adjusted to an optical density of 0.01 500  MHz NMR spectrometer; HPLC-MS: Agilent 1200 at 600 nm for APDT treatments. The initial bacterial cell HPLC and a 6120 quadrupole mass spectrometry [MS] numbers were determined as described earlier [19]. with a Phenomenex Luna C (2) column [5 μm, 250 mm × 10  mm]). The NMR and MS data of compounds 1–3 APDT against various pathogenic microorgansims in vitro are appended in the Supplementary material. In vitro APDT assays were conducted with LED red light (660 nm wavelength). The light intensity was determined Ultra‑performance liquid chromatography (UPLC) analysis by a radiometer (LI-250  A LICOR, Bioscience). Extract Waters Acquity H-Class system (Waters, Milford, MA, (20  µg/mL or 50  µg/mL) or fractions (20  µg/mL) or iso- USA) was used to analyze the sample. Column separation lated compounds (10  µg/mL) were added to bacterial (Phenomenex Kinetex XB-C18 column, 2.6 μm, 100 mm suspensions and incubated at 25 °C for 30 min (S. aureus, × 2.1  mm) was conducted at a detection wavelength of MRSA, S. mutans, and C. acnes) or 1 h (P. aeruginosa and 420 nm. Two µL of sample was injected and the column C. albicans). After incubation, 100 µL bacterial suspen- temperature was remained stable at 30  °C. After elution sion was aliquoted in a transparent 96-well plate (SPL from the column, the extract and the isolated compounds Life Science, Pocheon, Korea) and subjected to LED red were dissolved in 1 mL of internal standard 10  µg/mL. light at 120 W/m from the top for 15 min. APDT against The calibration curves were made from at least five dif - P. aeruginosa and C. albicans was performed at 600  W/ ferent concentrations of the compounds. The intra- and m . After 24-48  h incubation, CFU was determined and inter-day precision and accuracy were estimated by ana- expressed in log (CFU/mL). Ampicillin, vancomycin, or lyzing at least three replicates within a single day and on gentamycin (100  µg/mL) was used as a positive control five subsequent days, respectively. Quantification and for bacteria, and nystatin (20  µg/mL) was used as the validation experiments of compounds 2 and 3 were per- anti-fungal positive control. At least three independences formed using only compound 2 because both compounds were performed in all experiments. are stereoisomers. C. elegans maintenance Singlet oxygen generation assay Wild-type C. elegans N were purchased from the Cae- The singlet oxygen ( O ) generation was measured norhabditis Genetics Center (Minneapolis, MN, USA). C. through the imidazole-RNO (N,N-dimethyl-4-ni- elegans was maintained on a nematode growth medium trosoaniline) method [19]. In brief, mixtures of RNO (NGM) agar plate at 20  °C supplied with E. coli OP50. − 4 − 1 0.25 × 10   M and l-histidine 0.125 × 10   M was pre- Egg synchronization was prepared as described previ- − 1 pared in sodium phosphate buffer 0.25 × 10   M (pH ously [18]. 7.0) and then aliquoted into a 96-well plate. Plates were prepared by dissolving samples in water–dimethyl sul- Growth rate assay in C. elegans after APDT foxide (DMSO) mixtures (1:1) and then subjected to red The pathogen preparation and toxicity test under two light (655  nm wavelength, S-Tech light emitting diode conditions (pathogen-prefed and infected C. elegans) 2 1 (LED)) at the intensity of 200 W/m for 30 min. The O were conducted as described previously [19]. Briefly, in 2 Ha et al. Applied Biological Chemistry (2023) 66:19 Page 4 of 13 the prefeeding model, MRSA or S. aureus KCTC3881 performed in duplicates or triplicates with at least three suspension was treated with LFE-APDT under red light independences. (120  W/m , 15  min) and then was coated on an NGM plate and fed to C. elegans eggs. The body length was Results measured after 96  h using a stereoscopic microscope. LFE as a novel active photosensitizer based on the singlet In the infection condition, the eggs were fed MRSA or oxygen generation assay S. aureus and LFE until reaching the L1 stage. Infected Fifty different ethanol extracts of plants (Natural prod - worms were subjected to red light (600  W/m , 10  min) uct library, KIST) were rapidly screened for their ability and recorded the body length on the fourth day from the to generate O (red light was used since long-wavelength egg stage. is sufficient for penetration and safety of APDT [19, 24, 25]), among which LFE showed the best activity (Addi- tional file  1: Figure S1), and then the fractionation (F1– In vivo APDT efficacy test in the MRSA‑infected C. elegans F4) from LFE was performed for further identification of model the photoactive compounds. Adult worms were incubated in MRSA suspension (400 µL, OD = 0.1) for 24  h at 20  °C in a 24-well plate. After In vitro APDT effect with LFE the infection, the wells were incubated with LFE (20 µg/ APDT with LFE inhibited the growth of various pathogens mL, 30  min) in a shaker. Control was incubated with in vitro DMSO. Then, 50 worms were allowed to crawl in blank LFE or red light alone did not suppress bacterial growth, plates twice to remove the excess MRSA in the platinum showing that the current light dose or LFE was not haz- wire. The final NGM plate was irradiated at 600 W/m for ardous to bacterial cells (Fig. 2). APDT treatment, on the 5 min. Five worms from each treatment plate were moved other hand, considerably reduced the growth of all gram- to 0.5 mL LB broth and samples were spread on LB agar positive bacteria tested, with log reductions of 4.5, 4.7, plates. The reliability of this assay was tested as described 4.9, and 4.3 of viable cells of S. aureus, MRSA, S. mutans, previously. It was illustrated that the number of bacteria and C. acnes, respectively. The log decrease achieved on the platinum wire or NGM plate were insignificant by the APDT with LFE against S. aureus, MRSA, and S. compared to that on the C. elegans body [19]. Survivabil- mutans was 2.2-, 2.3-, and 7.0-fold greater than the anti- ity was also evaluated after APDT treatment by transfer- biotic therapy. Pathogenic fungi C. albicans reduced via- ring to blank NGM plates and monitoring the number of ble cells by 2.4 log (CFU/mL), which was 0.5 log lower living, dead, and censored worms every day [14]. than the nystatin therapy. With a 0.8 log decrease, APDT had a weak effect against gram-negative P. aeruginosa. Removal of the MRSA from the micropig skin surface Micropig skin was purchased from APURES Co., Ltd. APDT with LFE under different light conditions in vitro (Gyeonggi-do, South Korea). Prior to the experiment, the The APDT effect with LFE against MRSA was investi - skin was sterilized with 70% ethanol, followed by UVA gated to validate the applicable range of APDT condi- exposure for 15  min. Fresh MRSA or C. acnes suspen- tions with different doses of red light. At the milder sion (OD 0.01) mixed with or without LFE (20  µg/mL) 2 light intensity 20  W/m , APDT LFE showed sufficient was spread on the micropig skin at 5 µL/cm . Ampicillin antimicrobial activities (Fig.  3). Light exposure of photo (100 µg/mL) was served as a positive control. The coated materials for 30 min reduced 3.7 log (CFU/mL). LFE was skin was irradiated under red light (20  W/m , 30  min). effective after 10 min of exposure, inhibiting cell growth The skin in each treatment group was stamped on LB 2 by 3.0 log (CFU/mL) reduction. APDT with 1 W/m red or BHI agar and incubated for 48  h. After treatment, light for 10  min had a weak antibacterial action against the bacterial load on the skin was determined using the MRSA, with the log decrease of 0.3 (data not shown). swabbing method as described previously [18]. Identification of photoactive compounds Statistical analysis for the standardization of LFE Data were analyzed using GraphPad Prism 7.0 (La Jolla, Compound identification of LFE CA, USA). Statistical analysis of lifespan assay was Although F3 fraction generated slightly higher singlet conducted with JMP software (version 10, SAS Insti- oxygen than those by LFE or other fractions (Additional tute, Cary, NC, USA) using the log-rank test. A value of file  1: Figure S2), only LFE and F4 were analyzed using p < 0.05 was considered statistically significant. The data UPLC–PDA to identify photoactive compounds after are presented as the means ± SD, and experiments were carefully considering the yield and activity of LFE and Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 5 of 13 Fig. 2 In vitro antimicrobial activities of APDT with LFE against various pathogens. A APDT against S. aureus KCTC3881 (30 min incubation; red 2 2 light intensity at 120 W/m for 15 min), B APDT against MRSA (30 min incubation; red light intensity at 120 W/m for 15 min). C APDT against P. aeruginosa (60 min incubation; red light intensity at 600 W/m for 1 h). D APDT against C. albicans (60 min incubation; red light intensity at 600 W/ 2 2 m for 15 min). E APDT against S. mutans (30 min incubation; red light intensity at 120 W/m for 15 min). F APDT against C. acnes (30 min incubation; red light intensity at 120 W/m for 15 min). Suspensions of S. mutans, MRSA, S. aureus, and C. acnes were treated with LFE (20 µg/mL); P. aeruginosa and C. albicans were treated with LFE (50 µg/mL). Nystatin (20 µg/mL) and vancomycin or ampicillin (100 µg/mL) were used as positive controls. The data are expressed as mean ± standard error (n = 2), and representative of at least two independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column fractions in vitro (Additional file  1: Figure S3). Three main characteristic signal pattern of the cyclic tetrapyrrole core peaks were detected in the chromatogram at 420  nm structure (δ 9.52, 9.40, and 8.56) with additional olefinic (Fig.  4A), showing characteristic UV absorption around proton signals (δ 7.99, 6.29, and 6.18). Furthermore, it 420 and 660 nm due to highly conjugated π-electron sys- had five different methyl signals (δ 3.68, 3.40, 3.24, 1.81, tems. The chemical database metabolite annotation using and 1.69). This data was similar to that of pheophorbide m/z values showed that these compounds might contain a. The   H NMR data and the m/z value of compound 3 a porphyrin skeleton. were superimposable to that of compound 2. Previous Three compounds (1 − 3) were isolated using vari- literature have shown that compound 3 was considered ous chromatographic techniques (Fig.  4B). Among 21-epi-pheophorbide a [19]. According to mass data, the them, compound 2, which had the molecular formula of molecular formula of compound 1 was C H N O . Data 35 34 4 6 C H N O according to mass spectrum, was the most also showed a characteristic signal pattern of the cyclic 35 36 4 5 abundant. The   H NMR data of compound 2 showed a tetrapyrrole core structure (δ 10.44, 9.72, and 8.55). H Ha et al. Applied Biological Chemistry (2023) 66:19 Page 6 of 13 However, an aldehyde proton signal (δ 11.20) and four methyl signals were only displayed. Therefore, compound 1 was deduced as pheophorbide b [19]. Pheophorbides as active compounds for the APDT effects All three pheophorbide-related compounds isolated from LFE significantly suppressed the growth of S. aureus, with the log reduction of 3.9, 3.8, and 3.6 for compounds C1-C3, respectively (Fig. 4C). Quantitative analysis of LFE The bioactivity analysis of LFE showed that the three active compounds potentially contribute to its APDT activity. Next, we performed quantitative chemical analy- sis for the standardization of LFE and its APDT quality control. For the chemical analysis of LFE, the UPLC– PDA method was developed, where dimethyl curcumin was used as the internal standard. The established method was then validated to check the specificity, lin - earity, accuracy, and precision, suggesting this method is reliable (Table  1). The quantitative analysis (w/w) Fig. 3 In vitro antimicrobial activities of LFE under different light suggested the amounts of compounds 1 − 3 as 0.22% ± conditions. APDT with LFE (20 µg/mL; incubation time, 30 min) 0.01% (1), 0.47% ± 0.01% (2), and 0.07% ± 0.01% (3), against MRSA under a red light intensity of 20 W/m for 10 or 30 min. respectively. The experiments were carried out in tripli - Ampicillin (100 µg/mL) was used as the positive control. The data cates. From these results, we concluded that LFE could are expressed as mean ± standard error (n = 2) and representative of three independences. ns indicates no statistical significance relative be a valuable photosensitizer candidate due to the abun- to the control group. Significant differences are expressed with ** dance of photoactive compounds, potentially leading to for p < 0.01 and *** for p < 0.001 relative to the vehicle control. The effective APDT. log reductions compared with vehicle control are presented in each column Fig. 4 Chemical profiling of LFE. A UPLC–PDA chromatogram of LFE. B The chemical structures of isolated compounds 1 − 3. C Antimicrobial activities against S. aureus KCTC3881 of isolated compounds C1–C3. APDT with isolated compounds (10 µg/mL; incubation time, 30 min) against S. aureus under red light (20 W/m ; 30 min). Ampicillin (100 µg/mL) and APDT with pheophorbide were used as positive controls. The data are expressed as mean ± standard error (n = 2) and representative of three independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 7 of 13 Table 1 Linearity, accuracy, and precision of results (n = 3) No Linear range Regression equation r Precision (%RSD) Accuracy (µg/mL) Intra‑ day Inter‑ day Intra‑ day Inter‑ day 1 6.3–100.0 y = 0.0023x–0.0036 0.999 1.1 ± 1.0 1.2 ± 1.0 101.0 ± 3.2 100.9 ± 3.3 2 6.3–100.0 y = 0.0152x–0.0148 0.999 0.8 ± 0.3 0.6 ± 0.3 97.5 ± 0.8 96.5 ± 0.4 APDT with LFE in the C. elegans model MRSA, indicating its promising therapeutic effects in Effects of APDT on the growth rate of the S. aureus antibiotic-resistant bacterial infection. and MRSA‑infected C. elegans Based on the in  vitro results, the therapeutic effects Effects of APDT on survivability of C. elegans under MRSA of considerable pathogen eradication were examined infection using key physiological parameters in the C. elegans. Next, we evaluated the effects of APDT on the sur - The efficacy and side effects of APDT were first evalu - vivability of the MRSA-infected worms, because ated in vivo using the standardized LFE in two different survivability is the most reliable indicator of the ther- pathogen-infected C. elegans models. apeutic effect and side effects of the antimicrobial In the prefeeding model, worms were supplied with treatment [19]. MRSA killed worms (n = 25) within 8 APDT or ampicillin-treated pathogens in NGM plate. days (Fig.  7A), and the single treatment of red light or During S. aureus infection, worms treated with APDT LFE did not affect the lifespan of the MRSA-infected with LFE could decrease growth retardation effects, worms. However, after the APDT treatment of LFE and with the body length of 1,057 mm, which was similar to red light, the survival rate of worms was extended, with the vehicle control and ampicillin treatment (1,083 mm a mean lifespan of 4.72 ± 0.25 days, significantly differ - and 1,121  mm, respectively) (Fig.  5A, B). After 4 days, ent from the MRSA-infected worms (3.88 ± 0.20 days , the body length of worms fed with untreated MRSA p = 0.0078). The number of worms-associated patho - decreased significantly (0.53-fold of the vehicle control, genic bacteria was also assessed (Fig.  7B). During the p < 0.001). The MRSA-induced decrease in the worm infection, number of MRSA associated with worm size was recovered using ampicillin by only 13.11%, was 9700 ± 300 (CFU/worm); in contrast, after APDT whereas APDT treatment using LFE could significantly treatment, the number of pathogen on worms reduced increase the growth by 71.98% compared with the remarkably to 10 ± 2 (CFU/worm). Similar significant MRSA-infected worms (Fig. 5C, D). antimicrobial effects were also observed in C. elegans In addition, we determined the APDT efficacy in the infected with normal S. aureus KCTC3881 following infection model, which is a more reliable experimen- APDT treatment (Additional file  1: Figure S4). These tal model [19]. APDT treatment at the L1 stage in the results indicate that the killing of MRSA by APDT infection model with MRSA or S. aureus showed a det- helped to cure C. elegans against infection. rimental effect on the growth rate of worms. Worm treated with APDT using LFE against S. aureus recov- ered to 1,027  mm of body length, which is similar to Eradication of MRSA and C. acnes load on micropig skin that of control healthy worms (1,074 mm) (Fig. 6A , B). by LFE‑APDT Regarding MRSA, the infection caused considerable APDT efficacy was tested to eradicate MRSA or C. acnes decrease in the body length of infected worms com- from micropig skin and to evaluate its biomedical poten- pared with that of healthy worms in the vehicle control tial for infective skin diseases. The inoculation of MRSA (693  mm and 1,019  mm, respectively). Notably, ampi- on micropig kin was about 5.8 log (CFU/cm ) (Fig.  8A, cillin treatment could not rescue the growth retarda- B). This bacterial load slightly reduced under the sole tion caused by MRSA at all, whereas APDT treatment treatment of ampicillin or LFE, with the log reduction significantly increased the growth, with the mean worm of 0.5 and 0.1 respectively. When a combination of red length of 930 mm, which was 237 mm longer than that light and LFE was used, the MRSA concentration on the of the MRSA-infected worms (Fig. 6C, D). skin was significantly reduced by 3.4 log (CFU/cm ) com- In the prefeeding and infection C. elegans mod- pared to the control. Similarly, the APDT LFE inhibited els, ampicillin treatment was only effective against S. the C. acnes growth on skin, with the log reduction of 2.5, aureus, whereas APDT with LFE could alleviate the which was 2.1 log lower than the ampicillin treatment toxic effects on body length caused by S. aureus and (Fig. 8C, D). Ha et al. Applied Biological Chemistry (2023) 66:19 Page 8 of 13 Fig. 5 Eec ff t of APDT ‑LFE on the growth of C. elegans in the prefeeding model. S. aureus KCTC3881 or MRSA was pretreated with APDT using LFE (20 µg/mL; 30 min incubation) and red light 120 W/m for 15 min. The treatment with ampicillin (100 µg/mL) served as the positive control. Synchronized eggs were fed with E. coli OP50 as vehicle control, or treated MRSA or S. aureus with APDT or ampicillin. The body length A, C and the microscopic images of the worms B, D were evaluated four days after the egg state. White scale bar = 1 mm. The data are representative of three independent experiments. Analysis of variance test (n = 20). ns indicates no statistical significance relative to the control group; significant ## ### differences are expressed with *** for p < 0.001 relative to the vehicle control group; for p < 0.01, for p < 0.001 relative to the pathogen single treatment group Discussion the combination of LFE and red light with a wide range Human infectious disorders are strongly linked to tem- of intensity exhibited strong anti-infective effects due poral dysbiosis caused by specific pathogenic bacteria. to the strong ROS production, causing the reduction of The bacteria tested in this study have been reported to bacterial load higher than 3 log (CFU/mL), which is more be opportunistic pathogens of humans and the primary effective than antibiotics treatments. drivers of skin and dental diseases [26, 27]. Among them, Our chemical study confirmed the high amount of MRSA is one of the most common antibiotic-resistant photosensitizers, which aided in chemical profiling and bacterial strains, posing threat in the treatment of soft standardization for future clinical approval. The photo - tissue and skin infections in systemic diseases, includ- active ingredient in LFE was discovered to be pheophor- ing toxic shock syndrome in the community and health- bide, a metabolic breakdown product of chlorophyll. care settings [28]. The in vitro results demonstrated that This is a promising photosensitizer with gold standard Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 9 of 13 Fig. 6 Eec ff t of APDT ‑LFE on the growth rate of C. elegans in MRSA and S. aureus infection model. Eggs were fed with LFE‑incubated MRSA or S. aureus (LFE concentration:20 µg/mL; incubation time: 30 min) or ampicillin as the positive control. NGM plate coated with E. coli was used as vehicle control. After reaching the L1 stage, the plates were irradiated with a red light at 600 W/m for 10 min. The body length A, C and the microscopic images of the worms B, D were evaluated on the fourth day after the egg state. The results are representative of three independent experiments. White scale bar = 1 mm. Analysis of variance test (n = 10). ns indicates no statistical significance relative to the control group; significant differences ## ### are expressed with *** for p < 0.001 relative to the vehicle control group; for p < 0.01, for p < 0.001 relative to the pathogen single treatment group photophysical and photobiological properties such as derivatives, can be used to treat skin, oral, and surface near infrared light absorption, high singlet oxygen yield disinfection. S. aureus, C. albicans, and Artemia salina (through both the type I and type II photoprocesses were killed by pheophorbide concentrations ranging from with the extended π-π conjugated system), better selec- 20 to 100  µg/mL combined with 6  J/cm of light [32]. tivity (in microorganism cells compared to mammalian The viable cells of C. albicans were deemed 3.67 ± 0.18 cells), and photostability, which maintains a long life at log(CFU/mL) under the APDT treatment of porphy- excited state during irradiation [29–32]. Furthermore, rin derivatives and LED at 440–460  nm [33]. A clinical owing to the biosynthetic relation to the protoporphyrin investigation conducted by Song and colleagues showed IX present in higher organisms, biocompatibility such that the phototherapy of chlorophyll-a with blue light as pharmacokinetic clearance is anticipated [32]. Many (λmax = 430  nm) and red light (λmax = 660  nm) under pieces of research have shown that APDT of tetrapyrrole- radiance influence of 1800 and 1170 J/cm improved the based chemicals, such as porphyrins, chlorins, and their inflammatory states in Acnes vulgaris [34]. Unlike prior Ha et al. Applied Biological Chemistry (2023) 66:19 Page 10 of 13 Fig. 7 Eec ff ts of APDT efficacy on lifespan A and bacterial attachment B in MRSA‑infected C. elegans. Adult worms were incubated with fresh MRSA suspension for 24 h. Then, LFE (20 µg/mL) was treated for 30 min. DMSO was used as the control group. Next, the worms were allowed to crawl to blank NGM agar twice before the irradiation to red light at 600 W/m for 5 min. The worms were then assessed for survival rate every day (A). The p‑ value according to the MRSA single treatment is presented. The data are representative of three independent experiments. Five worms from each treatment plate were also moved to fresh LB broth and spread onto LB agar for CFU determination of viable bacteria associated with C. elegans (B). The data are reported as mean ± standard error of duplicates and representative of three independent experiments. ns indicates no statistical significance relative to MRSA; significant differences are expressed with *** for p < 0.001 relative to MRSA. The log reductions compared with MRSA are presented in each column studies, we used the plant L. fischeri as a new photosen - on gram-negative membranes contribute to this limita- sitizer source instead of single compounds as photosensi- tion [31, 32]. The unfavorable result has been observed in tizers. Our study first demonstrated the APDT efficacy of previous studies, which encourages the development of this food plant, especially against life-threatening MRSA. new strategies to improve efficacy against gram-negative The synthesis of high-purity compounds is usually labori - bacteria, such as combining different photosensitizers, ous and more expensive, while the usage of natural plant conjugating inorganic salts and antibiotics, or the devel- extract is simple, ecofriendly, and cost effective [35, 36]. opment of nanodelivery systems [20, 40]. The bioactive constituents in extracts may contribute to As light dosimetry is a critical aspect of the success of the synergistic therapeutic effects [20]. Phytochemical APDT, our study emphasizes the range of photodynamic investigation of this plant revealed that some constitu- conditions for sustained light-inactivation effect from ents like caffeoylquinic acids and quercetin derivatives high to mild red light intensity. Our research demon- exerted the antioxidant, anti-inflammatory activities and strated that the initial light energy of 120  W/m against inhibited elastase, tyrosinase, which offered the anti- bacteria and 600  W/m against fungi is the safe dose wrinkle effects [8, 37]. in  vivo testing without harming the growth rate and In our in vitro investigation, we discovered that APDT reproduction of C. elegans [19]. APDT with light inten- therapy for effectively controlling fungi C. albicans sity of 120 and 20 W/m showed antimicrobial activities required a longer incubation time and a higher light against MRSA. Milder intensity 20  W/m may be more intensity than gram-positive bacteria. Glycoproteins, suitable for commercial use, particularly in cosmeceutical soluble and insoluble polymers embedded in the mem- therapy such as LED masks. It is worth noting that the brane, large cell size, and the presence of the nuclear LED red light utilized as the source for photodynamic membrane are some factors that cause photosensitizers action is not only coherent with maximum light absorp- to take longer pre-irradiation time to penetrate the cells tion at 660  nm [41] but also minimizes the toxicity of [38]. Previous investigations have shown that higher light light to local tissue and penetrating deeply into tissues energy and photosensitizer doses of methylene blue, [24, 25]. While blue light only reaches the stratum cor- rose bengal, and a chlorin(e6) conjugate are required to neum, the red light can penetrate the stratum corneum kill C. albicans more effectively than bacteria [38, 39]. and hair follicles, enabling the killing effects on the exter - However, LFE showed weak effectiveness in the inacti - nal surface and inactivating the virulent factors such as vation of gram-negative P. aeruginosa. The hydropho - extracellular enzymes by dermatophytes [42]. bicity of the pheophorbides, the repulsion between the In C. elegans, APDT treatment improved illness con- carboxylate group, and the additional negative charge ditions by reducing the number of bacteria adhered to Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 11 of 13 Fig. 8 APDT effects against MRSA and C. acnes with LFE on micropig skin. Overnight culture of MRSA or C. acnes was incubated with or without LFE (20 µg/mL; 30 min incubation) and spread on the micropig skin at 5 µL/cm . Ampicillin (100 µg/mL) was used as a positive control. The coated skin was then illuminated under red light at 20 W/m for 30 min. The treated skin was imprinted on BHI agar to illustrate the inhibition of C. acnes or MRSA on the surface of the skin by APDT. Representative images of MRSA growth on LB plates (A) and C. acnes growth on BHI plates (C) were recorded after 48‑hour incubation. The differently treated skins were also washed with phosphate ‑buffered saline using a swab. The total content of bacteria on the swab of each treatment was plated on LB or BHI agar plates. B, D) CFU formation after 48 h was quantified and represented in the graph as a log (CFU/cm ). The data are reported as mean ± standard error (n = 2) and representative of at least two independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column worms. C. elegans, an invertebrate model with ease in APDT methods using in  vitro, C. elegans, and micropig experimental manipulation, short lifespan, and no ethical skin promotes a screening platform without live mam- concerns, was successfully used as a long-term and quick malian animal experiments. screening tool for APDT efficacy [16]. C. elegans can be Despite the satisfactory photodynamic antimicro- used to learn more about the molecular mechanisms of bial effects in  vitro and in  vivo nematode model, our pathogen-host interaction. The assay using micropig skin study carries some limitations. In our linked study, attempts to simulate the actual cellular infections, given wound healing effect of LFE phototreatment in a rodent that the binding of photosensitizer at proper doses is model would highlight the utility of the existing APDT critical for effective APDT. Our approach to discover new approach in treating various infectious diseases in Ha et al. Applied Biological Chemistry (2023) 66:19 Page 12 of 13 mammalian systems. However, bioassays for the under- relative to the vehicle control. The log reductions compared with vehicle lying effect of ROS on pathogen virulence factors are control are presented in each column. Fig. S4 Eec ff ts of APDT efficacy on lifespan (A) and bacterial attachment (B) in S. aureus KCTC3881 and lacking. The tested bacterial collection is well known infected C. elegans. Adult C. elegans were treated with overnight S. aureus for its ability to produce biofilms, which mediate patho - culture for a day. Then, LFE (20 μg/mL) was treated for 30 min. DMSO was gen adhesion and dissemination to host cells, allowing used as the control group. Next, the worms were allowed to crawl to blank NGM agar twice before the irradiation to red light (600 W/m for 5 min). pathogens to spread broadly and prolong the infection. The worms were then assessed for survivability every day (A). The p‑ value Biofilms are less responsive to antimicrobial therapy according to the S. aureus single treatment is presented. The results are due to extracellular matrix components and quorum representative of three independent experiments. Five worms from each treatment plate were also moved to fresh LB broth and spread onto LB sensing [43]. Therefore, further research into the bio - agar for CFU determination of viable bacteria associated with C. elegans film inhibitory effects of APDT with LFE is required. (B). The data are reported as mean ± standard error of duplicates and rep‑ Moreover, infectious conditions may be caused by a resentative of three independent experiments. ns indicates no statistical significance relative to S. aureus; significant differences are expressed with diverse community of pathogens. Therefore, a multi - ** for p < 0.01 and *** for p < 0.001 relative to S. aureus. The log reductions species infections simulation model should be estab- compared with S. aureus are presented in each column. lished to offer insight into the action of APDT. The present study revealed that the treatment of LFE Acknowledgements with red light showed a substantial inhibitory effect The Caenorhabditis elegans strains were provided by the Caenorhabditis Genet‑ against MRSA and common pathogenic bacteria involved ics Center (Minneapolis, MN, USA). in infectious diseases. Taken together with the therapeu- Author contributions tic effects of increasing survival and reversing growth Conceptualization: HK, JK, and KK; Methodology: NMH, HH, STA, UTTN, SL, JK, retardation under infection in C. elegans model, we and KK; Investigation: NMH, STA, HH, SL, UTTN, JK; Resources: HH, SL, JP, JK, and HK; Supervision: JK and KK; Writing: NMH, JK, and KK. All authors read and foresee that LFE holds excellent promise as a new pho- approved the final manuscript. tosensitizer source for the antimicrobial photodynamic treatment of MRSA infectious diseases in this post-anti- Funding This work was supported by an intramural research grant from KIST (2E32611, biotic era. However, in-depth studies on the underlying 2E31881) and a grant from the Ministry of Trade, Industry and Energy (MOTIE, mechanism of bacterial cell death caused by APDT and Republic of Korea, 20008861). the interaction of pathogen-host-APDT in mammalian Availability of data and materials models are required to guarantee its efficacy and safety in All data generated or analysed during this study are included in this published clinical settings. article and its Additional files. Declarations Abbreviations APDT Antimicrobial photodynamic therapy Competing interests BHI Brain heart infusion The authors declare that they have no competing interests. CFU Colony‑forming unit HPLC High‑pressure liquid chromatography LB Luria‑Bertani LED Light emitting diode Received: 10 December 2022 Accepted: 26 February 2023 LFE Ligulariafischeri extract MRSA Methicillin‑resistant Staphylococcus aureus MS Mass spectrometry NGM Nematode growth medium NMR Nuclear magnetic resonance References ROS Reactive oxygen species 1. Pendleton JN, Gorman SP, Gilmore BF (2013) Clinical relevance of the UPLC Ultra‑performance liquid chromatography ESKAPE pathogens. Expert Rev Anti Infect Ther 11(3):297–308 2. Ochsner M (1997) Photophysical and photobiological processes in the Supplementary Information photodynamic therapy of tumours. J Photochem Photobiol B 39(1):1–18 3. Hamblin MR (2016) Antimicrobial photodynamic inactivation: a bright The online version contains supplementary material available at https:// doi. new technique to kill resistant microbes. Curr Opin Microbiol 33:67–73 org/ 10. 1186/ s13765‑ 023‑ 00778‑2. 4. 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Antimicrobial photodynamic therapy with Ligularia fischeri against methicillin-resistant Staphylococcus aureus infection in Caenorhabditis elegans model

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Springer Journals
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Copyright © The Author(s) 2023
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10.1186/s13765-023-00778-2
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Abstract

The high prevalence of methicillin‑resistant Staphylococcus aureus (MRSA) infection threatens the effectiveness of current clinical settings. Antimicrobial photodynamic therapy (APDT ) is a promising alternative to antibiotics for treating infections due to its low resistance. This study aimed to evaluate the antibacterial properties of APDT with L. fischeri extract (LFE) against MRSA and various skin and oral pathogens in vitro and its photopharmaceutical actions in Caenorhabditis elegans. The antimicrobial activities of APDT with LFE against pathogens were evaluated using plate counting method. The chemical profile was characterized using high‑performance liquid chromatography and spec‑ trophotometry. The growth rate assay, lifespan assay, and bacterial attachment on worms were performed to assess the therapeutics effects in C. elegans. The swab method was used for the detection of pathogens on the micropig skin surface. The APDT treatment with L. fischeri extract (LFE, 20 µg/mL) and red light (intensity of 120 W/m ) reduced 4.3–4.9 log (colony forming unit/mL) of Staphylococcus aureus, MRSA, Cutibacterium acnes, Streptococcus mutans; and 2.4 log (CFU/mL) of Candida albicans. Chemical analysis revealed that LFE enriched three active photosensitizers. APDT reduced bacterial populations on worms, recovered growth retardation, and improved lifespan in MRSA‑infected C. elegans without causing severe side effects. The surface eradication of MRSA after exposure to LFE with red light was demonstrated on micropig skin. These findings highlight the significance of L. fischeri as a natural resource for the safe phototreatment of MRSA infection in the biomedical and cosmeceutical industries. Keywords Antimicrobial photodynamic treatment, C. elegans, Ligularia fischeri, Pheophorbide, MRSA, Staphylococcus aureus † 2 Ngoc Minh Ha and Hoseong Hwang are contributed equally to this work Division of Bio‑Medical Science & Technology, KIST School, University of Science and Technology (UST ), Gangneung, Gangwon‑do 25451, *Correspondence: Republic of Korea Jaeyoung Kwon Natural Product Research Center, Gangneung Institute of Natural kjy1207@kist.re.kr Products, Korea Institute of Science and Technology, Gangneung, Kyungsu Kang Gangwon‑do 25451, Republic of Korea kskang@kist.re.kr Natural Product Informatics Research Center, Gangneung Institute of Natural Products, Korea Institute of Science and Technology, 679 Saimdang‑ro, Gangnneung, Gangwon‑do 25451, Republic of Korea © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Ha et al. Applied Biological Chemistry (2023) 66:19 Page 2 of 13 antimicrobial material and evaluated its therapeutic Introduction impact on C. elegans. Infectious diseases caused by methicillin-resistant In this study, Caenorhabditis elegans was used as a Staphylococcus aureus (MRSA) continue to be a sig- model to evaluate the impact of ADPT with LFE. Thanks nificant global concern. Although antibiotics are quick to the short lifespan and high percentage of genes that are and effective, drug resistance in pathogenic bacteria homologous to human genes; this invertebrate nematode increases the likelihood of treatment failure and eco- has proven to be a useful model for the screening of bio- nomic burden [1]. Therefore, more sustainable treat - active compounds in therapeutic fields involving antiag - ment with lower resistance risk is desirable. ing, antineurodegeneration, gut health improvement, and One of the promising options for treating micro- metabolic disorders [9–18]. Regarding pathogen infec- bial infection is antimicrobial photodynamic therapy tions, live-C. elegans infection model was established to (APDT). In APDT, a photoactive substance in the pres- evaluate the in vivo efficacy against bacteria Enterococcus ence of oxygen undergoes a photochemical activation faecalis, Streptococcus pyogenes, Staphylococcus aureus, by light that absorbs and transfers energy to the oxygen and fungi Candida albicans [19–23]. Here, it is the first molecule to generate reactive oxygen species (ROS) like time APDT with LFE was evaluated and its therapeutic singlet oxygen and superoxides. ROS has potent oxida- impacts was illustrated on C. elegans. tive properties that can permanently modify cellular The present investigation aimed to evaluate the anti- constituents like fatty acids, amino acids, and nucle- infective effects of LFE in combination with red light obases that cause the elimination of harmful bacteria against different pathogenic microorganisms, particularly [2, 3]. It has several advantages, including high kill- MRSA in  vitro. The therapeutic effects of APDT treat - ing selectivity, minimal invasiveness, and a lower risk ment against MRSA infection were further assessed in of toxicity induction in nearby host tissues due to the C. elegans model. Figure  1 illustrates the experimental selective accumulation of target pathogens around the scheme of the study. photosensitizers and the restriction of light application to a specific local area [4 –6]. APDT treats infectious Methods diseases caused by bacteria and fungi in  vitro and in Sample collection animal models, such as skin infections, peri-implanti- The LFE for rapid screening was provided from the tis, periodontitis, and diabetic foot ulcers [4, 5, 7]. natural product library, Korea Institute of Science and As plants are a rich source of photoactive chemicals Technology, Gangneung Institute of Natural Products with diverse secondary metabolite chemical groups (Accession number KHG2016-01-0007; Gangwon-do, and distinct structures that have advantages in terms South Korea). Dried L. fischeri was purchased from the of minimal side effects and economics, attempts are herbal market in Jeongseon (Gangwon-do, South Korea). therefore needed for the screening of plant-derived material for APDT. Ligularia fischeri is a wild vegeta - Extraction and compound isolation ble plant with yellow composite flowers often used in The L. fischeri samples were dried (900  g) and ground, folk medicine to treat jaundice, scarlet fever, rheuma- and 101.1  g of dark green extract was collected under toid arthritis, and liver diseases [7]. It is known for its 2-hour-reflux with ethanol (9 × 2  L), followed by filtra - anti-oxidation, anti-cancer, anti-obesity, anti-hepa- tion and evaporation at low pressure. Bioassay-guided totoxic, and anti-inflammation properties [8 ]. How- fractionation scheme was applied for the isolation of ever, no study has used this plant as a photodynamic Fig. 1 Experimental scheme of the present study, antimicrobial photodynamic therapy (APDT ) with Ligularia fischeri extract (LFE) Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 3 of 13 bioactive substances. The screening method was used to production was recorded at 440  nm. A DMSO-water detect the singlet oxygen generation ( O ). LFE (14.5  g) mixture was served as a negative control. was adsorbed on Celite, separated by a column filled with Diaion HP-20 resin (130  g, 2.5 × 18  cm) and eluted with Bacterial strains and bacterial suspension preparations ethanol/water/acetone (3:2:0, 4:1:0, 1:0:0, and 0:1:1). The Cutibacterium acnes KCTC3314, Staphylococcus aureus four fractions ranged from F1 to F4. F4 (215.1 mg) were KCTC3881, Candida albicans KCTC7965, and Strepto- separated by a Gilson semipreparative high-pressure liq- coccus mutans KCTC3065 strains were purchased from uid chromatography (HPLC, Phenomenex Luna 10  μm the Korean Collections for Type Cultures (Jeongeup, C (2) column [10  μm, 250 × 21.2  mm]) and eluted Korea). Pseudomonas aeruginosa PAO1 was purchased using gradient conditions (acetonitrile/water, 77:23 to from American Type Culture Collection, and MRSA 3:97 in 120  min, flow rate 8 mL/min, 420  nm) to collect 2659 was acquired as described previously [20]. Luria- compounds 1 (1.6  mg, t = 36.1  min), 2 (2.7  mg, t = Bertani (LB) broth was used to culture MRSA, S. aureus, R R 57.8 min), and 3 (1.6 mg, t = 65.4 min). S. mutans, P. aeruginosa, and Sabouraud broth was used to culture C. albicans. C. acnes was grown in brain heart Structural identification of active compounds infusion (BHI) broth. The bacterial suspensions were pre - The chemical structures of compounds 1–3 were identi - pared by diluting fresh cultures with the corresponding fied using a combination of spectroscopy and spectrome - liquid broth and incubating at 37 °C overnight. Bacterial try analysis (Nuclear magnetic resonance [NMR]: Varian suspensions were adjusted to an optical density of 0.01 500  MHz NMR spectrometer; HPLC-MS: Agilent 1200 at 600 nm for APDT treatments. The initial bacterial cell HPLC and a 6120 quadrupole mass spectrometry [MS] numbers were determined as described earlier [19]. with a Phenomenex Luna C (2) column [5 μm, 250 mm × 10  mm]). The NMR and MS data of compounds 1–3 APDT against various pathogenic microorgansims in vitro are appended in the Supplementary material. In vitro APDT assays were conducted with LED red light (660 nm wavelength). The light intensity was determined Ultra‑performance liquid chromatography (UPLC) analysis by a radiometer (LI-250  A LICOR, Bioscience). Extract Waters Acquity H-Class system (Waters, Milford, MA, (20  µg/mL or 50  µg/mL) or fractions (20  µg/mL) or iso- USA) was used to analyze the sample. Column separation lated compounds (10  µg/mL) were added to bacterial (Phenomenex Kinetex XB-C18 column, 2.6 μm, 100 mm suspensions and incubated at 25 °C for 30 min (S. aureus, × 2.1  mm) was conducted at a detection wavelength of MRSA, S. mutans, and C. acnes) or 1 h (P. aeruginosa and 420 nm. Two µL of sample was injected and the column C. albicans). After incubation, 100 µL bacterial suspen- temperature was remained stable at 30  °C. After elution sion was aliquoted in a transparent 96-well plate (SPL from the column, the extract and the isolated compounds Life Science, Pocheon, Korea) and subjected to LED red were dissolved in 1 mL of internal standard 10  µg/mL. light at 120 W/m from the top for 15 min. APDT against The calibration curves were made from at least five dif - P. aeruginosa and C. albicans was performed at 600  W/ ferent concentrations of the compounds. The intra- and m . After 24-48  h incubation, CFU was determined and inter-day precision and accuracy were estimated by ana- expressed in log (CFU/mL). Ampicillin, vancomycin, or lyzing at least three replicates within a single day and on gentamycin (100  µg/mL) was used as a positive control five subsequent days, respectively. Quantification and for bacteria, and nystatin (20  µg/mL) was used as the validation experiments of compounds 2 and 3 were per- anti-fungal positive control. At least three independences formed using only compound 2 because both compounds were performed in all experiments. are stereoisomers. C. elegans maintenance Singlet oxygen generation assay Wild-type C. elegans N were purchased from the Cae- The singlet oxygen ( O ) generation was measured norhabditis Genetics Center (Minneapolis, MN, USA). C. through the imidazole-RNO (N,N-dimethyl-4-ni- elegans was maintained on a nematode growth medium trosoaniline) method [19]. In brief, mixtures of RNO (NGM) agar plate at 20  °C supplied with E. coli OP50. − 4 − 1 0.25 × 10   M and l-histidine 0.125 × 10   M was pre- Egg synchronization was prepared as described previ- − 1 pared in sodium phosphate buffer 0.25 × 10   M (pH ously [18]. 7.0) and then aliquoted into a 96-well plate. Plates were prepared by dissolving samples in water–dimethyl sul- Growth rate assay in C. elegans after APDT foxide (DMSO) mixtures (1:1) and then subjected to red The pathogen preparation and toxicity test under two light (655  nm wavelength, S-Tech light emitting diode conditions (pathogen-prefed and infected C. elegans) 2 1 (LED)) at the intensity of 200 W/m for 30 min. The O were conducted as described previously [19]. Briefly, in 2 Ha et al. Applied Biological Chemistry (2023) 66:19 Page 4 of 13 the prefeeding model, MRSA or S. aureus KCTC3881 performed in duplicates or triplicates with at least three suspension was treated with LFE-APDT under red light independences. (120  W/m , 15  min) and then was coated on an NGM plate and fed to C. elegans eggs. The body length was Results measured after 96  h using a stereoscopic microscope. LFE as a novel active photosensitizer based on the singlet In the infection condition, the eggs were fed MRSA or oxygen generation assay S. aureus and LFE until reaching the L1 stage. Infected Fifty different ethanol extracts of plants (Natural prod - worms were subjected to red light (600  W/m , 10  min) uct library, KIST) were rapidly screened for their ability and recorded the body length on the fourth day from the to generate O (red light was used since long-wavelength egg stage. is sufficient for penetration and safety of APDT [19, 24, 25]), among which LFE showed the best activity (Addi- tional file  1: Figure S1), and then the fractionation (F1– In vivo APDT efficacy test in the MRSA‑infected C. elegans F4) from LFE was performed for further identification of model the photoactive compounds. Adult worms were incubated in MRSA suspension (400 µL, OD = 0.1) for 24  h at 20  °C in a 24-well plate. After In vitro APDT effect with LFE the infection, the wells were incubated with LFE (20 µg/ APDT with LFE inhibited the growth of various pathogens mL, 30  min) in a shaker. Control was incubated with in vitro DMSO. Then, 50 worms were allowed to crawl in blank LFE or red light alone did not suppress bacterial growth, plates twice to remove the excess MRSA in the platinum showing that the current light dose or LFE was not haz- wire. The final NGM plate was irradiated at 600 W/m for ardous to bacterial cells (Fig. 2). APDT treatment, on the 5 min. Five worms from each treatment plate were moved other hand, considerably reduced the growth of all gram- to 0.5 mL LB broth and samples were spread on LB agar positive bacteria tested, with log reductions of 4.5, 4.7, plates. The reliability of this assay was tested as described 4.9, and 4.3 of viable cells of S. aureus, MRSA, S. mutans, previously. It was illustrated that the number of bacteria and C. acnes, respectively. The log decrease achieved on the platinum wire or NGM plate were insignificant by the APDT with LFE against S. aureus, MRSA, and S. compared to that on the C. elegans body [19]. Survivabil- mutans was 2.2-, 2.3-, and 7.0-fold greater than the anti- ity was also evaluated after APDT treatment by transfer- biotic therapy. Pathogenic fungi C. albicans reduced via- ring to blank NGM plates and monitoring the number of ble cells by 2.4 log (CFU/mL), which was 0.5 log lower living, dead, and censored worms every day [14]. than the nystatin therapy. With a 0.8 log decrease, APDT had a weak effect against gram-negative P. aeruginosa. Removal of the MRSA from the micropig skin surface Micropig skin was purchased from APURES Co., Ltd. APDT with LFE under different light conditions in vitro (Gyeonggi-do, South Korea). Prior to the experiment, the The APDT effect with LFE against MRSA was investi - skin was sterilized with 70% ethanol, followed by UVA gated to validate the applicable range of APDT condi- exposure for 15  min. Fresh MRSA or C. acnes suspen- tions with different doses of red light. At the milder sion (OD 0.01) mixed with or without LFE (20  µg/mL) 2 light intensity 20  W/m , APDT LFE showed sufficient was spread on the micropig skin at 5 µL/cm . Ampicillin antimicrobial activities (Fig.  3). Light exposure of photo (100 µg/mL) was served as a positive control. The coated materials for 30 min reduced 3.7 log (CFU/mL). LFE was skin was irradiated under red light (20  W/m , 30  min). effective after 10 min of exposure, inhibiting cell growth The skin in each treatment group was stamped on LB 2 by 3.0 log (CFU/mL) reduction. APDT with 1 W/m red or BHI agar and incubated for 48  h. After treatment, light for 10  min had a weak antibacterial action against the bacterial load on the skin was determined using the MRSA, with the log decrease of 0.3 (data not shown). swabbing method as described previously [18]. Identification of photoactive compounds Statistical analysis for the standardization of LFE Data were analyzed using GraphPad Prism 7.0 (La Jolla, Compound identification of LFE CA, USA). Statistical analysis of lifespan assay was Although F3 fraction generated slightly higher singlet conducted with JMP software (version 10, SAS Insti- oxygen than those by LFE or other fractions (Additional tute, Cary, NC, USA) using the log-rank test. A value of file  1: Figure S2), only LFE and F4 were analyzed using p < 0.05 was considered statistically significant. The data UPLC–PDA to identify photoactive compounds after are presented as the means ± SD, and experiments were carefully considering the yield and activity of LFE and Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 5 of 13 Fig. 2 In vitro antimicrobial activities of APDT with LFE against various pathogens. A APDT against S. aureus KCTC3881 (30 min incubation; red 2 2 light intensity at 120 W/m for 15 min), B APDT against MRSA (30 min incubation; red light intensity at 120 W/m for 15 min). C APDT against P. aeruginosa (60 min incubation; red light intensity at 600 W/m for 1 h). D APDT against C. albicans (60 min incubation; red light intensity at 600 W/ 2 2 m for 15 min). E APDT against S. mutans (30 min incubation; red light intensity at 120 W/m for 15 min). F APDT against C. acnes (30 min incubation; red light intensity at 120 W/m for 15 min). Suspensions of S. mutans, MRSA, S. aureus, and C. acnes were treated with LFE (20 µg/mL); P. aeruginosa and C. albicans were treated with LFE (50 µg/mL). Nystatin (20 µg/mL) and vancomycin or ampicillin (100 µg/mL) were used as positive controls. The data are expressed as mean ± standard error (n = 2), and representative of at least two independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column fractions in vitro (Additional file  1: Figure S3). Three main characteristic signal pattern of the cyclic tetrapyrrole core peaks were detected in the chromatogram at 420  nm structure (δ 9.52, 9.40, and 8.56) with additional olefinic (Fig.  4A), showing characteristic UV absorption around proton signals (δ 7.99, 6.29, and 6.18). Furthermore, it 420 and 660 nm due to highly conjugated π-electron sys- had five different methyl signals (δ 3.68, 3.40, 3.24, 1.81, tems. The chemical database metabolite annotation using and 1.69). This data was similar to that of pheophorbide m/z values showed that these compounds might contain a. The   H NMR data and the m/z value of compound 3 a porphyrin skeleton. were superimposable to that of compound 2. Previous Three compounds (1 − 3) were isolated using vari- literature have shown that compound 3 was considered ous chromatographic techniques (Fig.  4B). Among 21-epi-pheophorbide a [19]. According to mass data, the them, compound 2, which had the molecular formula of molecular formula of compound 1 was C H N O . Data 35 34 4 6 C H N O according to mass spectrum, was the most also showed a characteristic signal pattern of the cyclic 35 36 4 5 abundant. The   H NMR data of compound 2 showed a tetrapyrrole core structure (δ 10.44, 9.72, and 8.55). H Ha et al. Applied Biological Chemistry (2023) 66:19 Page 6 of 13 However, an aldehyde proton signal (δ 11.20) and four methyl signals were only displayed. Therefore, compound 1 was deduced as pheophorbide b [19]. Pheophorbides as active compounds for the APDT effects All three pheophorbide-related compounds isolated from LFE significantly suppressed the growth of S. aureus, with the log reduction of 3.9, 3.8, and 3.6 for compounds C1-C3, respectively (Fig. 4C). Quantitative analysis of LFE The bioactivity analysis of LFE showed that the three active compounds potentially contribute to its APDT activity. Next, we performed quantitative chemical analy- sis for the standardization of LFE and its APDT quality control. For the chemical analysis of LFE, the UPLC– PDA method was developed, where dimethyl curcumin was used as the internal standard. The established method was then validated to check the specificity, lin - earity, accuracy, and precision, suggesting this method is reliable (Table  1). The quantitative analysis (w/w) Fig. 3 In vitro antimicrobial activities of LFE under different light suggested the amounts of compounds 1 − 3 as 0.22% ± conditions. APDT with LFE (20 µg/mL; incubation time, 30 min) 0.01% (1), 0.47% ± 0.01% (2), and 0.07% ± 0.01% (3), against MRSA under a red light intensity of 20 W/m for 10 or 30 min. respectively. The experiments were carried out in tripli - Ampicillin (100 µg/mL) was used as the positive control. The data cates. From these results, we concluded that LFE could are expressed as mean ± standard error (n = 2) and representative of three independences. ns indicates no statistical significance relative be a valuable photosensitizer candidate due to the abun- to the control group. Significant differences are expressed with ** dance of photoactive compounds, potentially leading to for p < 0.01 and *** for p < 0.001 relative to the vehicle control. The effective APDT. log reductions compared with vehicle control are presented in each column Fig. 4 Chemical profiling of LFE. A UPLC–PDA chromatogram of LFE. B The chemical structures of isolated compounds 1 − 3. C Antimicrobial activities against S. aureus KCTC3881 of isolated compounds C1–C3. APDT with isolated compounds (10 µg/mL; incubation time, 30 min) against S. aureus under red light (20 W/m ; 30 min). Ampicillin (100 µg/mL) and APDT with pheophorbide were used as positive controls. The data are expressed as mean ± standard error (n = 2) and representative of three independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 7 of 13 Table 1 Linearity, accuracy, and precision of results (n = 3) No Linear range Regression equation r Precision (%RSD) Accuracy (µg/mL) Intra‑ day Inter‑ day Intra‑ day Inter‑ day 1 6.3–100.0 y = 0.0023x–0.0036 0.999 1.1 ± 1.0 1.2 ± 1.0 101.0 ± 3.2 100.9 ± 3.3 2 6.3–100.0 y = 0.0152x–0.0148 0.999 0.8 ± 0.3 0.6 ± 0.3 97.5 ± 0.8 96.5 ± 0.4 APDT with LFE in the C. elegans model MRSA, indicating its promising therapeutic effects in Effects of APDT on the growth rate of the S. aureus antibiotic-resistant bacterial infection. and MRSA‑infected C. elegans Based on the in  vitro results, the therapeutic effects Effects of APDT on survivability of C. elegans under MRSA of considerable pathogen eradication were examined infection using key physiological parameters in the C. elegans. Next, we evaluated the effects of APDT on the sur - The efficacy and side effects of APDT were first evalu - vivability of the MRSA-infected worms, because ated in vivo using the standardized LFE in two different survivability is the most reliable indicator of the ther- pathogen-infected C. elegans models. apeutic effect and side effects of the antimicrobial In the prefeeding model, worms were supplied with treatment [19]. MRSA killed worms (n = 25) within 8 APDT or ampicillin-treated pathogens in NGM plate. days (Fig.  7A), and the single treatment of red light or During S. aureus infection, worms treated with APDT LFE did not affect the lifespan of the MRSA-infected with LFE could decrease growth retardation effects, worms. However, after the APDT treatment of LFE and with the body length of 1,057 mm, which was similar to red light, the survival rate of worms was extended, with the vehicle control and ampicillin treatment (1,083 mm a mean lifespan of 4.72 ± 0.25 days, significantly differ - and 1,121  mm, respectively) (Fig.  5A, B). After 4 days, ent from the MRSA-infected worms (3.88 ± 0.20 days , the body length of worms fed with untreated MRSA p = 0.0078). The number of worms-associated patho - decreased significantly (0.53-fold of the vehicle control, genic bacteria was also assessed (Fig.  7B). During the p < 0.001). The MRSA-induced decrease in the worm infection, number of MRSA associated with worm size was recovered using ampicillin by only 13.11%, was 9700 ± 300 (CFU/worm); in contrast, after APDT whereas APDT treatment using LFE could significantly treatment, the number of pathogen on worms reduced increase the growth by 71.98% compared with the remarkably to 10 ± 2 (CFU/worm). Similar significant MRSA-infected worms (Fig. 5C, D). antimicrobial effects were also observed in C. elegans In addition, we determined the APDT efficacy in the infected with normal S. aureus KCTC3881 following infection model, which is a more reliable experimen- APDT treatment (Additional file  1: Figure S4). These tal model [19]. APDT treatment at the L1 stage in the results indicate that the killing of MRSA by APDT infection model with MRSA or S. aureus showed a det- helped to cure C. elegans against infection. rimental effect on the growth rate of worms. Worm treated with APDT using LFE against S. aureus recov- ered to 1,027  mm of body length, which is similar to Eradication of MRSA and C. acnes load on micropig skin that of control healthy worms (1,074 mm) (Fig. 6A , B). by LFE‑APDT Regarding MRSA, the infection caused considerable APDT efficacy was tested to eradicate MRSA or C. acnes decrease in the body length of infected worms com- from micropig skin and to evaluate its biomedical poten- pared with that of healthy worms in the vehicle control tial for infective skin diseases. The inoculation of MRSA (693  mm and 1,019  mm, respectively). Notably, ampi- on micropig kin was about 5.8 log (CFU/cm ) (Fig.  8A, cillin treatment could not rescue the growth retarda- B). This bacterial load slightly reduced under the sole tion caused by MRSA at all, whereas APDT treatment treatment of ampicillin or LFE, with the log reduction significantly increased the growth, with the mean worm of 0.5 and 0.1 respectively. When a combination of red length of 930 mm, which was 237 mm longer than that light and LFE was used, the MRSA concentration on the of the MRSA-infected worms (Fig. 6C, D). skin was significantly reduced by 3.4 log (CFU/cm ) com- In the prefeeding and infection C. elegans mod- pared to the control. Similarly, the APDT LFE inhibited els, ampicillin treatment was only effective against S. the C. acnes growth on skin, with the log reduction of 2.5, aureus, whereas APDT with LFE could alleviate the which was 2.1 log lower than the ampicillin treatment toxic effects on body length caused by S. aureus and (Fig. 8C, D). Ha et al. Applied Biological Chemistry (2023) 66:19 Page 8 of 13 Fig. 5 Eec ff t of APDT ‑LFE on the growth of C. elegans in the prefeeding model. S. aureus KCTC3881 or MRSA was pretreated with APDT using LFE (20 µg/mL; 30 min incubation) and red light 120 W/m for 15 min. The treatment with ampicillin (100 µg/mL) served as the positive control. Synchronized eggs were fed with E. coli OP50 as vehicle control, or treated MRSA or S. aureus with APDT or ampicillin. The body length A, C and the microscopic images of the worms B, D were evaluated four days after the egg state. White scale bar = 1 mm. The data are representative of three independent experiments. Analysis of variance test (n = 20). ns indicates no statistical significance relative to the control group; significant ## ### differences are expressed with *** for p < 0.001 relative to the vehicle control group; for p < 0.01, for p < 0.001 relative to the pathogen single treatment group Discussion the combination of LFE and red light with a wide range Human infectious disorders are strongly linked to tem- of intensity exhibited strong anti-infective effects due poral dysbiosis caused by specific pathogenic bacteria. to the strong ROS production, causing the reduction of The bacteria tested in this study have been reported to bacterial load higher than 3 log (CFU/mL), which is more be opportunistic pathogens of humans and the primary effective than antibiotics treatments. drivers of skin and dental diseases [26, 27]. Among them, Our chemical study confirmed the high amount of MRSA is one of the most common antibiotic-resistant photosensitizers, which aided in chemical profiling and bacterial strains, posing threat in the treatment of soft standardization for future clinical approval. The photo - tissue and skin infections in systemic diseases, includ- active ingredient in LFE was discovered to be pheophor- ing toxic shock syndrome in the community and health- bide, a metabolic breakdown product of chlorophyll. care settings [28]. The in vitro results demonstrated that This is a promising photosensitizer with gold standard Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 9 of 13 Fig. 6 Eec ff t of APDT ‑LFE on the growth rate of C. elegans in MRSA and S. aureus infection model. Eggs were fed with LFE‑incubated MRSA or S. aureus (LFE concentration:20 µg/mL; incubation time: 30 min) or ampicillin as the positive control. NGM plate coated with E. coli was used as vehicle control. After reaching the L1 stage, the plates were irradiated with a red light at 600 W/m for 10 min. The body length A, C and the microscopic images of the worms B, D were evaluated on the fourth day after the egg state. The results are representative of three independent experiments. White scale bar = 1 mm. Analysis of variance test (n = 10). ns indicates no statistical significance relative to the control group; significant differences ## ### are expressed with *** for p < 0.001 relative to the vehicle control group; for p < 0.01, for p < 0.001 relative to the pathogen single treatment group photophysical and photobiological properties such as derivatives, can be used to treat skin, oral, and surface near infrared light absorption, high singlet oxygen yield disinfection. S. aureus, C. albicans, and Artemia salina (through both the type I and type II photoprocesses were killed by pheophorbide concentrations ranging from with the extended π-π conjugated system), better selec- 20 to 100  µg/mL combined with 6  J/cm of light [32]. tivity (in microorganism cells compared to mammalian The viable cells of C. albicans were deemed 3.67 ± 0.18 cells), and photostability, which maintains a long life at log(CFU/mL) under the APDT treatment of porphy- excited state during irradiation [29–32]. Furthermore, rin derivatives and LED at 440–460  nm [33]. A clinical owing to the biosynthetic relation to the protoporphyrin investigation conducted by Song and colleagues showed IX present in higher organisms, biocompatibility such that the phototherapy of chlorophyll-a with blue light as pharmacokinetic clearance is anticipated [32]. Many (λmax = 430  nm) and red light (λmax = 660  nm) under pieces of research have shown that APDT of tetrapyrrole- radiance influence of 1800 and 1170 J/cm improved the based chemicals, such as porphyrins, chlorins, and their inflammatory states in Acnes vulgaris [34]. Unlike prior Ha et al. Applied Biological Chemistry (2023) 66:19 Page 10 of 13 Fig. 7 Eec ff ts of APDT efficacy on lifespan A and bacterial attachment B in MRSA‑infected C. elegans. Adult worms were incubated with fresh MRSA suspension for 24 h. Then, LFE (20 µg/mL) was treated for 30 min. DMSO was used as the control group. Next, the worms were allowed to crawl to blank NGM agar twice before the irradiation to red light at 600 W/m for 5 min. The worms were then assessed for survival rate every day (A). The p‑ value according to the MRSA single treatment is presented. The data are representative of three independent experiments. Five worms from each treatment plate were also moved to fresh LB broth and spread onto LB agar for CFU determination of viable bacteria associated with C. elegans (B). The data are reported as mean ± standard error of duplicates and representative of three independent experiments. ns indicates no statistical significance relative to MRSA; significant differences are expressed with *** for p < 0.001 relative to MRSA. The log reductions compared with MRSA are presented in each column studies, we used the plant L. fischeri as a new photosen - on gram-negative membranes contribute to this limita- sitizer source instead of single compounds as photosensi- tion [31, 32]. The unfavorable result has been observed in tizers. Our study first demonstrated the APDT efficacy of previous studies, which encourages the development of this food plant, especially against life-threatening MRSA. new strategies to improve efficacy against gram-negative The synthesis of high-purity compounds is usually labori - bacteria, such as combining different photosensitizers, ous and more expensive, while the usage of natural plant conjugating inorganic salts and antibiotics, or the devel- extract is simple, ecofriendly, and cost effective [35, 36]. opment of nanodelivery systems [20, 40]. The bioactive constituents in extracts may contribute to As light dosimetry is a critical aspect of the success of the synergistic therapeutic effects [20]. Phytochemical APDT, our study emphasizes the range of photodynamic investigation of this plant revealed that some constitu- conditions for sustained light-inactivation effect from ents like caffeoylquinic acids and quercetin derivatives high to mild red light intensity. Our research demon- exerted the antioxidant, anti-inflammatory activities and strated that the initial light energy of 120  W/m against inhibited elastase, tyrosinase, which offered the anti- bacteria and 600  W/m against fungi is the safe dose wrinkle effects [8, 37]. in  vivo testing without harming the growth rate and In our in vitro investigation, we discovered that APDT reproduction of C. elegans [19]. APDT with light inten- therapy for effectively controlling fungi C. albicans sity of 120 and 20 W/m showed antimicrobial activities required a longer incubation time and a higher light against MRSA. Milder intensity 20  W/m may be more intensity than gram-positive bacteria. Glycoproteins, suitable for commercial use, particularly in cosmeceutical soluble and insoluble polymers embedded in the mem- therapy such as LED masks. It is worth noting that the brane, large cell size, and the presence of the nuclear LED red light utilized as the source for photodynamic membrane are some factors that cause photosensitizers action is not only coherent with maximum light absorp- to take longer pre-irradiation time to penetrate the cells tion at 660  nm [41] but also minimizes the toxicity of [38]. Previous investigations have shown that higher light light to local tissue and penetrating deeply into tissues energy and photosensitizer doses of methylene blue, [24, 25]. While blue light only reaches the stratum cor- rose bengal, and a chlorin(e6) conjugate are required to neum, the red light can penetrate the stratum corneum kill C. albicans more effectively than bacteria [38, 39]. and hair follicles, enabling the killing effects on the exter - However, LFE showed weak effectiveness in the inacti - nal surface and inactivating the virulent factors such as vation of gram-negative P. aeruginosa. The hydropho - extracellular enzymes by dermatophytes [42]. bicity of the pheophorbides, the repulsion between the In C. elegans, APDT treatment improved illness con- carboxylate group, and the additional negative charge ditions by reducing the number of bacteria adhered to Ha  et al. Applied Biological Chemistry (2023) 66:19 Page 11 of 13 Fig. 8 APDT effects against MRSA and C. acnes with LFE on micropig skin. Overnight culture of MRSA or C. acnes was incubated with or without LFE (20 µg/mL; 30 min incubation) and spread on the micropig skin at 5 µL/cm . Ampicillin (100 µg/mL) was used as a positive control. The coated skin was then illuminated under red light at 20 W/m for 30 min. The treated skin was imprinted on BHI agar to illustrate the inhibition of C. acnes or MRSA on the surface of the skin by APDT. Representative images of MRSA growth on LB plates (A) and C. acnes growth on BHI plates (C) were recorded after 48‑hour incubation. The differently treated skins were also washed with phosphate ‑buffered saline using a swab. The total content of bacteria on the swab of each treatment was plated on LB or BHI agar plates. B, D) CFU formation after 48 h was quantified and represented in the graph as a log (CFU/cm ). The data are reported as mean ± standard error (n = 2) and representative of at least two independences. ns indicates no statistical significance relative to the control group. Significant differences are expressed with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 relative to the vehicle control. The log reductions compared with vehicle control are presented in each column worms. C. elegans, an invertebrate model with ease in APDT methods using in  vitro, C. elegans, and micropig experimental manipulation, short lifespan, and no ethical skin promotes a screening platform without live mam- concerns, was successfully used as a long-term and quick malian animal experiments. screening tool for APDT efficacy [16]. C. elegans can be Despite the satisfactory photodynamic antimicro- used to learn more about the molecular mechanisms of bial effects in  vitro and in  vivo nematode model, our pathogen-host interaction. The assay using micropig skin study carries some limitations. In our linked study, attempts to simulate the actual cellular infections, given wound healing effect of LFE phototreatment in a rodent that the binding of photosensitizer at proper doses is model would highlight the utility of the existing APDT critical for effective APDT. Our approach to discover new approach in treating various infectious diseases in Ha et al. Applied Biological Chemistry (2023) 66:19 Page 12 of 13 mammalian systems. However, bioassays for the under- relative to the vehicle control. The log reductions compared with vehicle lying effect of ROS on pathogen virulence factors are control are presented in each column. Fig. S4 Eec ff ts of APDT efficacy on lifespan (A) and bacterial attachment (B) in S. aureus KCTC3881 and lacking. The tested bacterial collection is well known infected C. elegans. Adult C. elegans were treated with overnight S. aureus for its ability to produce biofilms, which mediate patho - culture for a day. Then, LFE (20 μg/mL) was treated for 30 min. DMSO was gen adhesion and dissemination to host cells, allowing used as the control group. Next, the worms were allowed to crawl to blank NGM agar twice before the irradiation to red light (600 W/m for 5 min). pathogens to spread broadly and prolong the infection. The worms were then assessed for survivability every day (A). The p‑ value Biofilms are less responsive to antimicrobial therapy according to the S. aureus single treatment is presented. The results are due to extracellular matrix components and quorum representative of three independent experiments. Five worms from each treatment plate were also moved to fresh LB broth and spread onto LB sensing [43]. Therefore, further research into the bio - agar for CFU determination of viable bacteria associated with C. elegans film inhibitory effects of APDT with LFE is required. (B). The data are reported as mean ± standard error of duplicates and rep‑ Moreover, infectious conditions may be caused by a resentative of three independent experiments. ns indicates no statistical significance relative to S. aureus; significant differences are expressed with diverse community of pathogens. Therefore, a multi - ** for p < 0.01 and *** for p < 0.001 relative to S. aureus. The log reductions species infections simulation model should be estab- compared with S. aureus are presented in each column. lished to offer insight into the action of APDT. The present study revealed that the treatment of LFE Acknowledgements with red light showed a substantial inhibitory effect The Caenorhabditis elegans strains were provided by the Caenorhabditis Genet‑ against MRSA and common pathogenic bacteria involved ics Center (Minneapolis, MN, USA). in infectious diseases. Taken together with the therapeu- Author contributions tic effects of increasing survival and reversing growth Conceptualization: HK, JK, and KK; Methodology: NMH, HH, STA, UTTN, SL, JK, retardation under infection in C. elegans model, we and KK; Investigation: NMH, STA, HH, SL, UTTN, JK; Resources: HH, SL, JP, JK, and HK; Supervision: JK and KK; Writing: NMH, JK, and KK. All authors read and foresee that LFE holds excellent promise as a new pho- approved the final manuscript. tosensitizer source for the antimicrobial photodynamic treatment of MRSA infectious diseases in this post-anti- Funding This work was supported by an intramural research grant from KIST (2E32611, biotic era. However, in-depth studies on the underlying 2E31881) and a grant from the Ministry of Trade, Industry and Energy (MOTIE, mechanism of bacterial cell death caused by APDT and Republic of Korea, 20008861). the interaction of pathogen-host-APDT in mammalian Availability of data and materials models are required to guarantee its efficacy and safety in All data generated or analysed during this study are included in this published clinical settings. article and its Additional files. Declarations Abbreviations APDT Antimicrobial photodynamic therapy Competing interests BHI Brain heart infusion The authors declare that they have no competing interests. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Mar 13, 2023

Keywords: Antimicrobial photodynamic treatment; C. elegans; Ligularia fischeri; Pheophorbide; MRSA; Staphylococcus aureus

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