Bacteria
A total of 21 clinical isolates including P. aeruginosa (n = 3), E. coli (n = 2), A. baumannii (n = 14), K. pneumoniae (n = 2) were used. These clinical isolates with MDR were obtained from İzmir Katip Çelebi University Faculty of Medicine, Microbiology Laboratory. Antibiotic susceptibilities of bacteria using routine tests according to Clinical Laboratory Standards Institute-2018 (CLSI-2018) recommendations were evaluated (Institute 2018), and the results was as shown in supplementary 1.
Photosensitizer
CPDs named PM, PE, PN and, PL were synthesized by researchers at Ege University Faculty of Pharmacy Pharmaceutical Chemistry Department. The chemical structure and absorbance values of the CPDs which previously evaluated for aPDT efficacy on MRSA by researchers (Taslı et al. 2018) were shown in Fig. 1a. CPDs can absorb a wavelength in the broad spectrum varying from 250 to 800 nm and, the maximum light absorption was at 422 ± 3 nm (Fig. 1b).
Chemistry: The infrared (IR) spectra of the compounds were monitored by attenuated total reflectance (ATR) (PerkinElmer Spectrum 100 FT-IR, Waltham, MA, USA). 1H NMR spectra were recorded on an Agilent 600 MHz Premium COMPACT NMR spectrometer (Santa Clara, CA, USA) by using tetramethylsilane (TMS) as an internal standard and DMSO-d6 as a solvent. Chemical shifts were measured in parts per million (δ). The J values were given in Hz. Abbreviations for data quoted are: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sxt, sextet. All reagents and solvents were of reagent-grade quality and obtained from commercial suppliers (Sigma, Acros and Merck). Elemental analyses (C, H, N) were performed by Leco TruSpec Micro (Leco, St. Joseph, MI, USA).
General procedure for the synthesis of the final compounds: CPDs were prepared according to the method of Gomes et al., 2011 (M. Gomes et al. 2011). Excess of the corresponding alkyl halide (iodomethane, ethyl bromide, propyl bromide, 3-Bromo-1-propanol) (65 mmol) was added to a suspension of 5,10,15,20-tetrakis(4-pyridyl)porphyrin (120 mg, 193.9 µmol) in DMF (30 mL). After refluxing for 1–8 h, the mixture was cooled to room temperature and the obtained precipitate was filtered and washed with diethyl ether or ethanol. The crude product was taken in acetone-water (1:1) and then filtered, washed with acetone. The structures of the final compounds were confirmed by FT-IR, 1H NMR, and elemental analysis. The purity levels of compounds were determined by elemental analysis (C, H, N), and the results were within ± 0.4% of the calculated values. Spectral and elemental analysis data of the compounds were reported in the supporting information (Supplementary 2).
Minimal inhibitory concentrations (MIC) of the CPDs for E.coli-1, P. aeruginosa-1, K. pneumoniae-1, and A. baumannii-1 were evaluated by the micro-dilution method according to CLSI-2018 to determine antimicrobial activity and predict maximal PS concentration levels that could be used in photoinactivation experiments (Institute 2018). The MIC experiments evaluated at Ege University Pharmaceutical Microbiology Laboratory was done as described in the study (Taslı et al. 2018). 1 mL of Tryptic Soy Broth (TSB) containing bacteria was centrifuged (3000 rpm at 4 ° C for 10 minutes) and the supernatant was discharged without touching the pellet. 1 ml of phosphate buffer saline (PBS) was added onto the pellet remaining at the bottom and suspended. Bacterial suspensions were adjusted to standard McFarland 0.5 (1.5×108 CFU/mL) turbidity and further diluted to give a final inoculum size of 5×105 CFU/mL per well. 50 µL of Mueller Hinton Broth (MHB) was transferred to each well of 96 well plates. The two-fold serial dilutions were made by adding 50 µL PS dissolved in PBS/antibiotic to the first wells of the plates. In the evaluation, ciprofloxacin was adjusted from an initial concentration of 16 µg/mL to a final concentration of 0.016 µg/mL and, PSs were adjusted from an initial concentration of 5.000 µg/mL and final concentrations of 2.441 µg/mL. 50 µL of bacteria suspension (5×105 CFU/mL) was added to each well of the plates and, the plates were allowed to incubate at 37°C for 16–18 h. After the incubation, the MIC values of the CPDs were determined by calculating the lowest compound concentrations that prevent the growth of bacteria. All experiments were performed in triplicate, each being repeated at least three times.
Laser device and optical set up
A diode laser with a wavelength of 655 nm was used as a light source (PSU III.LED; Changchun New Industries Optoelectronics Co. Ltd., Changchun, China). The distance between the optical plate and the fiber tip was 8.7 cm, and the light illuminated an area of 3.14 cm₂ on the optical plate from this distance. The output power of light was measured as 41.5 milliwatts (mW) at the plate surface.
Photoinactivation experiments
Photoinactivation experiments were carried out in two stages at Izmir Katip Çelebi University Central Research Laboratories Biomedical Optics and Laser Applications Laboratory. Firstly, bacterial photoinactivation evaluation was carried out by using combinations of different energy density (J/cm²) and PS concentrations (\({\mu }\text{M}\)) on a single clinical isolate representing each species. Because clinical isolates of each species had a similar antibiotic resistance profile (Supplementary 1), randomization was used to select representative isolates. In these pioneering experiments, the photoinactivation efficiency of aPDT combinations on representative clinical isolates was determined. The combinations for each PS were determined as follows: aPDT experiments were started with low energy density and PS concentration. The energy density was kept constant and the PS concentration gradually increased until a strong antimicrobial activity was achieved. After this stage, the energy density was increased and the PS concentrations were gradually reduced until limited antimicrobial efficacy was developed. In determining the maximum PS concentration that can be used in the combinations, the MIC values of PSs were taken into account. In the second step, the photoinactivation effect on the other strains of the bacteria species was evaluated for the combination that had a strong photoinactivation on the selected bacterial strain. The following groups were formed in each of the aPDT experiments:
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Laser group (L) where light was applied alone,
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PS group where PS was applied alone,
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aPDT group where both the light and PS were applied together,
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Control group (C) where PBS was applied alone.
50 µL of the bacterial suspension that adjusted to standard McFarland 0.5 (1.5×108 CFU/mL) turbidity was transferred to specific wells of each of 96 well plates identified as PS, aPDT, L, and C. 50 µL PS from the stock suspensions at specific concentrations was added to wells of the PS and aPDT group plates containing bacteria. 50 µL of PBS was added to the wells of the L and C group plates with bacteria. All four groups were incubated for 15 min at room temperature. The aPDT and L group plates were exposed to light. After light exposure, bacterial suspensions in all groups were diluted by serial dilution using PBS. 100 µL of aliquot was taken from the dilutions and plated on tryptic soy agar and left for an overnight incubation in the dark area at 37 ºC. After incubation, bacterial survival was calculated at CFU/mL. Each experiment was repeated three times in triplicate.
Toxicity on the fibroblast cells
These experiments were carried out as described in study (Topaloğlu et al. 2020) at Izmir Katip Çelebi University Central Research Laboratories Biomedical Optics and Laser Applications Laboratory. A healthy mouse skin fibroblast cell line (L929) was used in the study. The cells were cultured in 75 cm₂ tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, Germany) solution, containing 1% L Glutamine (Gibco, USA), 10 % Fetal Bovine Serum (FBS) (Gibco, USA) solution, and 1% Penicillin/Streptomycin (Gibco, USA). These cells were incubated in a humidified environment containing 95% air and 5% CO₂ until they form a confluent culture in a single layer. The cells reaching 80% confluence were washed with PBS and trypsinized using 0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) (Biological Industries, Israel). 2×104 fibroblast cells were seeded into each well of 96-well plates and allowed to incubate at 37°C for 24 h so that the cells adhere to the wells of the plate. Then the cell culture medium was discarded and the experimental process continued as described in the groups below.
The dark toxicity of the concentrations ranging from 25 to 600 µM for PM, 3.125 to 400 µM for PE, PN, and PL on the fibroblast was performed using 15 min and 24 h incubation. In the experiment, 100 µL PS suspension from stock suspension dissolved at specific concentrations in cell culture medium for dark toxicity groups was transferred to plate wells containing fibroblasts. 100 µL cell culture medium without PS was transferred to control group plate wells. The plates wrapped with aluminum foil to create a dark environment and allowed to incubate at 37°C incubate for 15 min or 24 h.
The toxicity of aPDT on the fibroblast cells was performed using 15 min and 24 h incubation. 100 µL from PS suspension dissolved at specific concentrations in cell culture medium was transferred to plate wells containing fibroblasts cells (Combinations were as in Fig. 7b). 100 ml cell culture medium without PS was placed in the control and light group plate wells containing fibroblast cells. The plates wrapped with aluminum foil to create a dark environment and allowed to incubate at 37°C incubate for 15 min. Phototoxicity or L (50 J/cm², 100 J/cm², and 150 J/cm²) and aPDT groups were irradiated at an appropriate energy density with a diode laser. Control and PS groups were taken from the incubator but not exposed to ambient light or laser. After the light application to aPDT or light groups was finished, all the plates wrapped with aluminum foil again and allowed to incubate at 37°C incubate for 15 min or 24 h.
Following, cell culture medium or PSs added to the plate wells were removed. Cells in the wells were washed with PBS. 100 µL MTT (4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (5 mg/mL) (Sigma, St. Louis, MO, USA) was added to each well. After 2 h of incubation, the formazan crystals were dissolved with 100 µL of DMSO, and the absorbance was measured at 570 nm with a microplate reader (iMark, Bio-Rad Lab., USA.). The absorbance values were used to determine the change in survival in fibroblast cells. Control groups were used for each experiment. Each experiment was repeated three times in triplicate.
Data analysis and Evaluation
In the photoinactivation experiments, the calculations were done as described below: First, bacterial survival in CFU/mL for each plate was calculated according to formula 1.
Formula 1
The control group was taken as a reference for determining survival reduction of the aPDT, PS, or L groups. Survival reductions were calculated as logarithmic as shown in formula 2.
Formula 2
$$Reduction={log}_{10 }\left(\frac{Number of colonies per mL in the control group}{Number of colonies per mL in the application group}\right)$$
For the toxicity on the fibroblast cells, the calculations were done as follows. The control group was taken as a reference for determining the toxic effect of dark toxicity, phototoxicity, or aPDT applications on fibroblast cells. Percentage changes of the fibroblast cell survival based on the absorbance values of the groups were calculated according to formula 3.
Formula 3
$$Cell viablility \left(\%\right) =\frac{\left(Absorbance value of control group-Absorbance value of the application group\right)x100}{Absorbance value of the control group}$$
SPSS 16.0 was used for data analysis. "Paired sample t-test" was used to determine the differences of laser or aPDT groups compared to the control. In the comparisons, differences with p < 0.05 were accepted as statistically significant.