Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria

Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria Abstract Photodynamic treatment combining light and a photosensitizer molecule can be an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 as an efficient photosensitizer for ultraviolet light A (UVA)-induced bacterial inactivation. Six bacterial species, Bacillus cereus (vegetative form), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa* and Staphylococcus aureus*, were suspended in aqueous solutions with or without vitamin K5 and exposed to UVA irradiation. UVA irradiation (5.8 J cm−2) with vitamin K5 (1600 μmol l−1) reduced the colony forming units (CFU) of these bacteria by three to seven logs. Antibiotic resistant bacteria were also susceptible to the bactericidal effects of UVA and vitamin K5 combination treatment. Inactivation of bacteria in human plasma required higher doses of UVA light and vitamin K5. UVA irradiation (30 J cm−2) with vitamin K5 (2000 μmol l−1) reduced E. coli and S. aureus spiked into human plasma by seven logs CFU/ml. Reactive oxygen species, such as superoxide anion radicals and hydroxyl radicals, were found to be generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria. ultraviolet light, photodynamic inactivation, vitamin K5, reactive oxygen species INTRODUCTION Photosensitization is a chemical reaction that takes place when light interacts with a photosensitizer and excites it to a higher energy state. This energy can be transferred to other molecules to generate reactive oxygen species (ROS), such as hydroxyl radicals and superoxides (Sibata et al.2001). ROS have destructive effects on living organisms through oxidation of biological substrates such as amino acids in proteins, the nucleic acids of DNA and RNA and a variety of unsaturated lipids (Ridley et al.2009). The ability to induce damage in biological systems by photosensitization has been utilized in clinical applications where unintended damage can be minimized by control of the light dose and photosensitizer concentrations. Several photosensitizer-based drugs have been approved for photodynamic therapy for human cancer or skin disorders. As an example, psoralen (8-methoxypsoralen) is used to treat psoriasis with the irradiation of ultraviolet A light. (Gupta and Anderson 1987; Honigsmann 1990; Pathak and Fitzpatrick 1992; Bethea et al.1999; Archier et al.2012). Photosensitization has also been proven to be an effective method to inactivate microbial contamination in water, food and natural and clinical environments (Hamamoto et al.2007; Matafonova and Batoev 2012; Carrascosa, Plana and Ferrandiz 2013; Maisch et al.2014; Ballatore et al.2015; Omarova et al.2015; Sobotta et al.2015; Tim 2015). In a blood bank setting, bacterial contamination of blood components can lead to transfusion transmitted septic reactions (Goodnough, Shander and Brecher 2003). To improve safety of transfusions, platelet and plasma products can be treated with a psoralen derivative photosensitizer, amotosalen (S-59) along with ultraviolet light A (UVA) light (Irsch and Lin 2011). Similarly, combination of vitamin B2 with a broad UV light spectrum (265–375 nm) was developed to inactivate viruses, bacteria and parasites capable of contaminating blood products (Marschner and Goodrich 2011). In addition, vitamin B6 has also been shown as an effective photosensitizer of UVA to inactivate bacteria (Maeda et al.2000). Vitamin K5 (VK5) is a synthetic derivative of vitamin K, a naturally occurring molecule present in normal human metabolism. VK5 has been found to have limited antimicrobial activity against many bacteria, molds and yeasts and has been proposed as a food preservative due to its low order of toxicity (Merrifield and Yang 1965a,b). It is also found to have some antitumor effects (Hitomi et al.2005; Kuriyama et al.2005; Ogawa et al.2007; Chen et al.2012). Recently, we reported that vitamin K3 (VK3) can function as a photosensitizer to UVA light in phosphate-buffered saline (PBS) solution and this combination can effectively inhibit bacterial growth (Xu and Vostal 2014). However, VK3 is not water soluble that could have limitation for its use. VK5 is water soluble and has a similar structure to VK3, it was thus reasonable for us to evaluate its potential as an UVA photosensitizer to inactivate bacteria in PBS and in human plasma. MATERIALS AND METHODS Materials Acetylacetone, ammonium iron (ii) sulfate hexahydrate, ammonium acetate, D-mannitol l-ascorbic acid bioxtra, dimethyl sulfoxide, nitro-blue tetrazolium (NBT), superoxide dismutase (SOD), trichloroacetic acid (TCA), carbonate and phosphate buffers and VK5 were purchased from Sigma-Aldrich (St. Louis, MO). VK5 solution was made fresh in sterilized water in a sterilized tube before each experiment. Platelet Additive Solution (PAS) was from Fenwal (Lake Zurich, IL). Human plasma collected by Fenwal Amicus Cell Separator was obtained from the National Institutes of Health blood bank. TC-6 well plates were from Corning (Corning, NY). GyroTwister 3-D Laboratory Shaker was from Labnet (Edison, NJ). 2,2-Bis(hydroxymethyl)-2,2΄,2″-nitrilotriethanol (1 mol l−1, pH 6.5) was purchased from Hampton Research (Aliso Viejo, CA). D-PBS was purchased from Life Technologies (Walkersville, MD). Gelatin veronal buffer (GVB; 5 m mol l−1 Veronal, 150 m mol l−1 NaCl, 0.05% gelatin, pH 7.4) was purchased from Boston BioProducts (Boston, MA). Triton X-100, Nunc 96-well polypropylene microplates (0.5 ml, white) and Nunc 96-well clear flat bottom microplates were obtained from Fisher Scientific (Pittsburgh, PA). Lyophilized DV-1, a commercially manufactured intravenous immunoglobulin (IVIG) lot was provided by the National Institute of Biological Standards and Control (NIBSC code 14/160, High titer anti-A in IVIG; Hertfordshire, UK). Bacteria and UVA light irradiation All bacteria were purchased from ATCC (Manassas, VA). Bacteria strain and corresponding ATCC catalog number are listed below. Bacillus cereus 11778, Escherichia coli 25922, Klebsiella pneumoniae 9997, Pseudomonas aeruginosa 27853, Staphylococcus aureus 35548, Staphylococcus epidermidis 49134 and two antibiotic resistant bacteria strains, Pseudomonas aeruginosa* BAA-2108 and Staphylococcus aureus* BAA-1747. UVA lamp with a wavelength of 350 nm (Sylvania 21623 - F15 - T8 - 350BL - ECO) was used for UVA irradiation. The dose of UVA light was measured with a radiometer (Model UVX-36, UVP, Upland, CA). All UVA irradiations were conducted at room temperature. The bacteria grown in log phase were centrifuged at 2000 g for 10 min and resuspended in PBS. Approximately 104 ml−1 colony forming unit (CFU) bacteria were used for the UVA dose and VK5 concentration studies (Fig. 1). Approximately 108 ml−1 CFU bacteria were used for the log reduction studies (Table 1) with the exception of S. aureus and S. aureus* that was used at 104 ml−1 CFU. A total of 2 ml of bacteria mixed with 0 to 1600 μmol l−1 of VK5 were placed in 6-well plates and exposed to dark or UVA irradiation for 10–20 min corresponding to 2.9 and 5.8 J cm−2 following our previous procedures (Xu and Vostal 2014). For bacterial inactivation in plasma, bacteria were mixed with 500–2000 μmol l−1 of VK5 in 35% or 50% plasma diluted with PAS in a total volume of 2 ml. Plasma inoculated with bacteria in 6-well plates was placed on a shaker at the speed of 10 rpm while exposed to UVA irradiation at 6 and 30 J cm−2. After UVA irradiation, PBS and plasma bacterial suspensions were plated on culture plates and grown at 37°C overnight prior to counting the colony forming units. Relative survival in Fig. 1 was calculated with the CFU count of different treatment divided by the CFU count after the irradiation of 2.9 J cm−2 without VK5 added. Figure 1. View largeDownload slide Escherichia coli survival was significantly reduced by VK5 plus UVA treatments in PBS solution. Escherichia coli suspended in PBS with 0–1600 μmol l−1 VK5 were irradiated with 2.9 J cm−2 or 5.8 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance ***P < 0.001 and **P < 0.01. Figure 1. View largeDownload slide Escherichia coli survival was significantly reduced by VK5 plus UVA treatments in PBS solution. Escherichia coli suspended in PBS with 0–1600 μmol l−1 VK5 were irradiated with 2.9 J cm−2 or 5.8 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance ***P < 0.001 and **P < 0.01. Table 1. Inactivation of bacteria with VK5 irradiated with UVA.   Bacterial log reduction factor  Bacteria  VK5-UVA  UVA-VK5  VK5 + UVA  B. cereus  0.14 ± 0.05  1.01 ± 0.06  6.76 ± 1.39**  E. coli  0.29 ± 0.11  0.16 ± 0.06  6.83 ± 1.14**  K. pneumoniae  0.07 ± 0.11  0.31 ± 0.08  7.12 ± 1.33**  P. aeruginosa  1.68 ± 0.13  1.00 ± 0.07  6.07 ± 0.59**  P. aeruginosa*  1.09 ± 0.23  1.18 ± 0.05  5.88 ± 0.84**  S. aureus  0.13 ± 0.14  0.41 ± 0.09  3.03 ± 0.10**  S. aureus*  0.23 ± 0.24  0.29 ± 0.08  3.09 ± 0.19**  S. epidermidis  0.33 ± 0.14  1.07 ± 0.14  6.55 ± 1.61*    Bacterial log reduction factor  Bacteria  VK5-UVA  UVA-VK5  VK5 + UVA  B. cereus  0.14 ± 0.05  1.01 ± 0.06  6.76 ± 1.39**  E. coli  0.29 ± 0.11  0.16 ± 0.06  6.83 ± 1.14**  K. pneumoniae  0.07 ± 0.11  0.31 ± 0.08  7.12 ± 1.33**  P. aeruginosa  1.68 ± 0.13  1.00 ± 0.07  6.07 ± 0.59**  P. aeruginosa*  1.09 ± 0.23  1.18 ± 0.05  5.88 ± 0.84**  S. aureus  0.13 ± 0.14  0.41 ± 0.09  3.03 ± 0.10**  S. aureus*  0.23 ± 0.24  0.29 ± 0.08  3.09 ± 0.19**  S. epidermidis  0.33 ± 0.14  1.07 ± 0.14  6.55 ± 1.61*  Note: All bacteria were resuspended in VK5 at 1600 μmol l−1 or PBS and irradiated with or without UVA at 5.8 J cm−2. Data are represented as mean ± SD (n = 3). **P < 0.01 and *P < 0.05. View Large Determination of superoxide anion radical (O2⋅−) The generation of O2.− was determined by recording the photosensitized reduction of NBT to nitroblue diformazan resulting in an increase in absorbance at 560 nm. NBT solution (1.67 × 10−4 mol l−1) prepared in 0.01 mol l−1 sodium carbonate buffer (pH = 10) was added into 6-well plate that has VK5 solution at 0–1000 μmol l−1 and irradiated with UVA dose at 2.9 J cm−2 following previous study (Dwivedi et al.2012; Xu and Vostal 2014). To confirm the generation of O2.−, SOD was used as a specific quencher in the NBT reduction assay. Quenching (%) in Fig. 4b was calculated based on the reduction of absorbance at 560 nm with the SOD concentration increased from 0 to 640 units ml−1 with UVA irradiation at 2.9 J cm−2. Determination of hydroxyl radical (⋅OH) Hydroxyl radical generation was determined by the formaldehyde production that has absorbance at 412 nm (Dwivedi et al.2012). This assay was performed in an ascorbic acid-iron-ethylenediaminetetraacetic acid system where ascorbic acid in the study was replaced with 0–1000 μmol l−1 VK5 following previous studies (Tullius and Dombroski 1986; Dwivedi et al.2012; Xu and Vostal 2014). Mannitol was used as a specific .OH quencher in the assay (Dwivedi et al.2012). Quenching (%) in Fig. 5b was calculated based on the reduction of absorbance at 412 nm with the mannitol concentration increased from 0 to 1 mol l−1 with UVA irradiation at 2.9 J cm−2. Measurement of complement activities The assay was performed in quadruplicate in a total volume of 150 μl in 96-well white round bottom microplates using a hemolysis assay procedure (Wang and Scott 2017). Briefly, 40 μl of DV-1 (anti-A IgG) was added to wells, followed by addition of 110 μl of GVB. After brief mixing, 35 μl of a 3% solution of red blood cell (RBC; ABO type A) in GVB buffer was added to each well. In separate wells, RBC was prepared and lysed with 2% Triton X-100 to provide a 100% cell lysis control. Plates were covered and incubated at 37°C for 20–30 min while shaking (400 rpm). After incubation plates were spun at 314 g (1200 rpm) in Sorvall Legend XTR centrifuge [Thermo Fisher Scientific (Waltham, MA)] for 2 min to pellet RBC. The supernatant was aspirated and 150 μl of test plasma was added to the wells. VK5 treated plasmas (test plasma) of blood type A were prepared by mixing with 0, 250, 500 and 1000 μ mol l−1 VK5 and irradiated under UVA (2.9 J cm−2). RBCs were incubated at 37°C with agitation at 400 rpm with a microplate shaker for 90 min. One hundred microliter of supernatant from each well was transferred to a flat-bottom enzyme-linked immunosorbent assay microplate. Hemoglobin levels were quantitated by measuring OD 414/OD 690, using a 96-well microplate reader (Molecular Devices, Sunnyvale, CA). The degree of cell lysis was calculated as a percentage of a positive lysis control [samples (OD 414 – OD 690]/[100% cell lysis (OD 414 – OD 690)]. Statistical analysis Microsoft Excel software Student's t test was used for statistical analysis with the significance set at a P value for *<0.05, **<0.01 and ***<0.001. All experiments were performed in triplicate. RESULTS AND DISCUSSION Inactivation of bacteria with vitamin K5 under UVA irradiation VK5 (0–1600 μmol l−1) was added to bacterium Escherichia coli suspended in PBS and the bacterial suspension was exposed to 2.9 or 5.8 J cm−2 of UVA. A representative survival of VK5 and UVA treated E. coli, as determined by a CFU count, is shown in Fig. 1. In the presence of VK5, E. coli became much more sensitive to UVA treatment. A dose effect of UV light was evident when the UVA dose was increased from 2.9 to 5.8 J cm−2 with a fixed VK5 concentration. Similarly, a concentration effect of VK5 was evident when the UVA dose was fixed. As shown in Fig. 1, the reduced survivals of E. coli at VK5 concentrations of 100 to 1600 μmol l−1 with a constant UVA dose of 2.9 or 5.8 J cm−2 were significantly lower than control groups without VK5. Specifically, the survival of E. coli dropped from 100% to 5% as the VK5 concentration was increased from 0 to 1600 μmol l−1 with a constant UVA dose of 2.9 J cm−2. The higher dose of UVA had substantial bactericidal effect on its own that reduced the survival of E. coli bacteria to 20% and this was further potentiated by increasing concentrations of VK5. Similar bactericidal effect of UVA doses and VK5 concentrations was found in all other bacteria, including Bacillus cereus (vegetative form), Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae. The reduction factor for all bacteria resuspended in VK5 at 1600 μmol l−1 and irradiated under UVA at 5.8 J cm−2 was between six to seven logs with the exception of S. aureus that was only reduced by three logs (Table 1). In contrast, VK5 alone at 1600 μmol l−1 or UVA alone at 5.8 J cm−2 treatment had limited bactericidal effects of approximately one log reduction. There was no substantial difference in log reduction between antibiotic-resistant and non-resistant bacteria P. aeruginosa or S. aureus after VK5 and UVA combined treatment (Table 1). These results indicate that antibiotic-resistant bacteria are equally susceptible to the photodynamic treatment and are consistent with the UVA-riboflavin bactericidal effect in antibiotic resistant bacteria (Makdoumi and Backman 2016). Together, it suggests the efficacy of photodynamic UVA photosensitizer treatment for pathogen inactivation regardless of bacteria sensitivity to antibiotics treatment. As such, this photodynamic UVA treatment may provide an alternative for bacterial contamination control in clinical practice. We previously tested VK3 (1600 μM) under UVA (5.8 J cm−2) irradiation for inactivation of bacteria in aqueous solution (Xu and Vostal 2014). The reduction factors for VK3 and UVA light were over 8 logs for the same group of bacteria with the exception of P. aeruginosa that only showed inactivation by 4 logs. The results with VK5 and UVA at equal concentration and UVA dose were different in that the same strain of S. aureus that was less susceptible to VK5 (3 logs vs 8 logs reduction for VK5 and VK3, respectively). The two vitamins also had different efficacy for P. aeruginosa that was reduced by six logs of CFU with vitamin K5 and only four logs of CFU with VK3. These differences may reflect variability in uptake of the vitamins by the bacteria or a variable susceptibility of S. aureus and other bacteria to the types of free radicals generated by VK5 and VK3. Similar mechanisms have been proposed to contribute to variable antibiotic resistance of bacteria (Nikaido 1989; Li, Livermore and Nikaido 1994; Fernandez and Hancock 2012; Olivares et al.2013). We extended the application of VK5/UVA to pathogen reduction of plasma products intentionally contaminated with gram positive and negative bacteria, E. coli and S. aureus. Human plasma, diluted with PAS to a final concentration of 35% or 50% plasma, was spiked with E. coli or S. aureus bacteria. Spiked plasmas were mixed with 500, 1000 or 2000 μmol l−1 VK5 and irradiated with 6 or 30 J/cm2 UVA. As shown in Fig. 2a, there is a clear VK5 concentration and UVA dose response in E. coli log reduction. When VK5 was mixed with bacterial solution to a final concentration of 500, 1000 and 2000 μmol l−1 and irradiated with 6 J/cm2 UVA, Escherichia coli reduction was 2.11, 2.39 and 4.2 logs in 35% plasma, and 1.94, 2.27 and 3.53 logs in 50% plasma, respectively. After 30 J/cm2 UVA irradiation with 500, 1000 and 2000 μmol l−1 VK5, E. coli log reduction was 2.46, 7.12 and 7.51 in 35% plasma and 2.99, 4.65 and 7.51 in 50% plasma, respectively. At concentrations of 1000 and 2000 μmol l−1 VK5 irradiated with 30 J/cm2 UVA, the E. coli log reduction was increased to statistically significant levels compared to 6 J/cm2 UVA in both 35% and 50% plasma. These results demonstrated the potential for a seven logs reduction of E. coli in 35% or 50% plasma after treatment with 2000 μmol l−1 VK5 and 30 J/cm2 UVA irradiation. Similarly in Fig. 2b at concentrations of 500, 1000 and 2000 μmol l−1 VK5 and irradiation with 30 J/cm2 UVA, S. aureus log reduction was significantly increased compared to 6 J/cm2 UVA in both 35% and 50% plasma. Staphylococcus aureus, spiked into 35% or 50% plasma mixed with 500, 1000 and 2000 μmol l−1 VK5, was reduced approximately seven logs after 30 J/cm2 UVA irradiation. Figure 2. View largeDownload slide Escherichia coli (a) and S. aureus (b) were significantly reduced by VK5 plus UVA treatments in plasma. Bacteria suspended in plasma with VK5 at 500–2000 μmol l−1 were irradiated with 6 J cm−2 or 30 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Figure 2. View largeDownload slide Escherichia coli (a) and S. aureus (b) were significantly reduced by VK5 plus UVA treatments in plasma. Bacteria suspended in plasma with VK5 at 500–2000 μmol l−1 were irradiated with 6 J cm−2 or 30 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Vitamin K5 structure alteration after UVA irradiation To study if the structure of VK5 was altered by UVA irradiation, 1600 μmol l−1 of VK5 PBS solution was irradiated with 5.8 J cm−2 of UVA. After irradiation, the VK5 solution absorption spectrum was measured by a spectrophotometer. As shown in Fig. 3, the absorption of spectra of VK5 was altered after UVA irradiation, suggesting VK5 structure was modified by the UVA irradiation, similar chemical structure alteration has been reported with the vitamin B6 solution irradiated with UVA (Maeda et al.2000). It will be of interests in the future to study what changes in the chemical structure and any toxicity of this new structure modified molecule. Figure 3. View largeDownload slide The changes of absorption spectrum of vitamin K5 before and after UVA irradiation. PBS solution with 1600 μmol l−1 of VK5 was irradiated with 5.8 J cm−2 of UVA. Figure 3. View largeDownload slide The changes of absorption spectrum of vitamin K5 before and after UVA irradiation. PBS solution with 1600 μmol l−1 of VK5 was irradiated with 5.8 J cm−2 of UVA. Generation of superoxide anion radical (O2.−) The generation of superoxide anion radical (O2.−) in UVA-irradiated-VK5 PBS solution was dependent on the concentration of VK5. At a fixed UVA dose of 2.9 J cm−2, the generation of O2.− was significantly increased when VK5 concentration was increased from 250 to 1000 μmol l−1 (Fig. 4a). To further confirm that O2.− was generated, we used a specific O2.− quencher, SOD. The production of O2.− was significantly reduced as the SOD concentration was increased from 160 to 640 units ml−1 in a 500 μmol l−1 VK5 solution irradiated with a UVA (2.9 J cm−2), as shown in Fig. 4b. Figure 4. View largeDownload slide Photochemical generation of superoxide (a) in PBS measured by the OD at 560 nm was induced by VK5 (0–1000 μmol l−1) under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of superoxide (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of super oxide dismutase (SOD) from 0–640 units ml−1. Data are represented as mean ± SD (n = 3); significance ***P < 0.001, **P < 0.01 and *P < 0.05. Figure 4. View largeDownload slide Photochemical generation of superoxide (a) in PBS measured by the OD at 560 nm was induced by VK5 (0–1000 μmol l−1) under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of superoxide (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of super oxide dismutase (SOD) from 0–640 units ml−1. Data are represented as mean ± SD (n = 3); significance ***P < 0.001, **P < 0.01 and *P < 0.05. Generation of hydroxyl radical (⋅OH) VK5 and UVA light combination also caused a statistically significant release of ⋅OH as the VK5 concentrations increased from 250 to 1000 μmol l−1 with a fixed UVA dose of 2.9 J cm−2 (Fig. 5a). Generation of ⋅OH was further confirmed by inclusion of mannitol, a specific quencher for ⋅OH. Significantly, less ⋅OH was produced when mannitol concentration was increased from 0.25 to 1 mol l−1 in the presence of a fixed VK5 concentration of 500 μmol l−1 and a UVA dose of 2.9 J cm−2 (Fig. 5b). Figure 5. View largeDownload slide Photochemical generation of hydroxyl radical in PBS (a) measured by the OD at 412 nm was induced by VK5 (0–1000 μmol l−1) Under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of hydroxyl radical (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of mannitol from 0 to 1 mol l−1. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Figure 5. View largeDownload slide Photochemical generation of hydroxyl radical in PBS (a) measured by the OD at 412 nm was induced by VK5 (0–1000 μmol l−1) Under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of hydroxyl radical (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of mannitol from 0 to 1 mol l−1. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. In our previous study, we found that irradiation of VK3 solutions with UVA light resulted in the release of ROS, such as singlet oxygen, hydroxyl radicals and superoxide anion radicals. In this study, we found that hydroxyl radicals and superoxide anion radicals, but not singlet oxygen (data not shown), were generated in VK5 solutions under UVA light irradiation, while under the same conditions singlet oxygen was detected in VK3 solutions (Xu and Vostal 2014). These results suggest that there is a different type of photosensitization reactions between VK3 and VK5 when irradiated with UVA and this altered profile of ROS species may cause the variable bactericidal effects on the same species of bacteria, such as S. aureus. Living organisms are susceptible to UVA irradiation due to the cytotoxic effect of ROS generated during the UVA exposure. ROS can cause lipid peroxidation, protein oxidation and DNA breakage, all of which are likely to contribute to the cytotoxic effects (Carraro and Pathak 1988; Cadet et al.1999; Maverakis et al.2010; Bracchitta et al.2013; Cadet and Wagner 2013). A previously described alternate method of light-based bacterial decontamination that utilizes UV light with vitamin B2 have been proposed to act through modification of nucleic acids, primarily on guanine residues to mediate the pathogen inactivation (Marschner and Goodrich 2011). It is likely that the ROS produced in UVA irradiated VK5 solution also mediates the bacterial inactivation. Measurement of complement activity Our experiments indicate that VK5 and UVA irradiation can reduce bacterial load in plasma diluted to 50% or 35% with PAS and thus this treatment could be potentially used for decontamination of plasma for transfusion or plasma for manufacture of plasma derivatives. To measure if this process can reduce activity of proteins found in plasma, we evaluated the effect VK5 and UVA treatment has on the plasma complement system. This system consists of a highly complex cascade of proteins present in plasma that assemble in response to an antibody binding a cell (Rogers, Veeramani and Weiner 2014; Wouters and Zeerleder 2015). The classical pathway initiates the assembly of a C3 convertase complex (proteins C2aC4b) that converts C3 into C3b on the surface of the cell. C3b then mediates conversion of protein C5 to C5b and mediates the formation of a membrane attack complex. The attack complex is an agglomerate of at least five separate proteins that assemble on the surface of the cell and introduce pores into the membrane that lead to cell lysis. We chose this system to test the impact of the VK5 and UVA treatment because it has multiple proteins and alteration to any one of the proteins could interfere with the cascade response. As shown in Fig. 6, there was a slight but not significant decrease of complement-mediated cell lysis after the UVA (2.9 J cm−2) irradiation in plasma mixed with 0, 250, 500 and 1000 μmol l−1 VK5. These results indicate that complement functions were slightly damaged after treatment with VK5 under UVA irradiation, although these detrimental effects to the complement system were not statistically significant. Thus, the impact of the VK5 and UVA treatment on plasma proteins appears to be relatively modest. However, depending on the application of the plasma after this treatment, the impact on other proteins relevant to the application should be studied further in the future. Figure 6. View largeDownload slide Measurement of complement activities in plasma with 0–1000 μmol l−1 VK5 irradiated under UVA (2.9 J cm−2). Data are represented as mean ± SD (n = 3). Figure 6. View largeDownload slide Measurement of complement activities in plasma with 0–1000 μmol l−1 VK5 irradiated under UVA (2.9 J cm−2). Data are represented as mean ± SD (n = 3). Our results show that VK5 and UVA photodynamic treatment can reduce bacteria in aqueous solutions and has efficacy against antibiotic-resistant and non-resistant bacteria. We also demonstrated that the photodynamic treatment could effectively reduce bacterial contamination in human plasma, although the concentration of VK5 and the dose of UVA needed to be increased and/or the plasma diluted. These results indicate that the conditions of this method could be optimized for different applications that require reduction of bacteria. FUNDING Food and Drug Administration, Center for Biologics Evaluation and Research. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. 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Google Scholar CrossRef Search ADS PubMed  Xu F, Vostal JG. Inactivation of bacteria via photosensitization of vitamin K3 by UV-A light. FEMS Microbiol Lett  2014; 358: 98– 105. Google Scholar CrossRef Search ADS PubMed  Published by Oxford University Press on behalf of FEMS 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Letters Oxford University Press

Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria

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Abstract

Abstract Photodynamic treatment combining light and a photosensitizer molecule can be an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 as an efficient photosensitizer for ultraviolet light A (UVA)-induced bacterial inactivation. Six bacterial species, Bacillus cereus (vegetative form), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa* and Staphylococcus aureus*, were suspended in aqueous solutions with or without vitamin K5 and exposed to UVA irradiation. UVA irradiation (5.8 J cm−2) with vitamin K5 (1600 μmol l−1) reduced the colony forming units (CFU) of these bacteria by three to seven logs. Antibiotic resistant bacteria were also susceptible to the bactericidal effects of UVA and vitamin K5 combination treatment. Inactivation of bacteria in human plasma required higher doses of UVA light and vitamin K5. UVA irradiation (30 J cm−2) with vitamin K5 (2000 μmol l−1) reduced E. coli and S. aureus spiked into human plasma by seven logs CFU/ml. Reactive oxygen species, such as superoxide anion radicals and hydroxyl radicals, were found to be generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria. ultraviolet light, photodynamic inactivation, vitamin K5, reactive oxygen species INTRODUCTION Photosensitization is a chemical reaction that takes place when light interacts with a photosensitizer and excites it to a higher energy state. This energy can be transferred to other molecules to generate reactive oxygen species (ROS), such as hydroxyl radicals and superoxides (Sibata et al.2001). ROS have destructive effects on living organisms through oxidation of biological substrates such as amino acids in proteins, the nucleic acids of DNA and RNA and a variety of unsaturated lipids (Ridley et al.2009). The ability to induce damage in biological systems by photosensitization has been utilized in clinical applications where unintended damage can be minimized by control of the light dose and photosensitizer concentrations. Several photosensitizer-based drugs have been approved for photodynamic therapy for human cancer or skin disorders. As an example, psoralen (8-methoxypsoralen) is used to treat psoriasis with the irradiation of ultraviolet A light. (Gupta and Anderson 1987; Honigsmann 1990; Pathak and Fitzpatrick 1992; Bethea et al.1999; Archier et al.2012). Photosensitization has also been proven to be an effective method to inactivate microbial contamination in water, food and natural and clinical environments (Hamamoto et al.2007; Matafonova and Batoev 2012; Carrascosa, Plana and Ferrandiz 2013; Maisch et al.2014; Ballatore et al.2015; Omarova et al.2015; Sobotta et al.2015; Tim 2015). In a blood bank setting, bacterial contamination of blood components can lead to transfusion transmitted septic reactions (Goodnough, Shander and Brecher 2003). To improve safety of transfusions, platelet and plasma products can be treated with a psoralen derivative photosensitizer, amotosalen (S-59) along with ultraviolet light A (UVA) light (Irsch and Lin 2011). Similarly, combination of vitamin B2 with a broad UV light spectrum (265–375 nm) was developed to inactivate viruses, bacteria and parasites capable of contaminating blood products (Marschner and Goodrich 2011). In addition, vitamin B6 has also been shown as an effective photosensitizer of UVA to inactivate bacteria (Maeda et al.2000). Vitamin K5 (VK5) is a synthetic derivative of vitamin K, a naturally occurring molecule present in normal human metabolism. VK5 has been found to have limited antimicrobial activity against many bacteria, molds and yeasts and has been proposed as a food preservative due to its low order of toxicity (Merrifield and Yang 1965a,b). It is also found to have some antitumor effects (Hitomi et al.2005; Kuriyama et al.2005; Ogawa et al.2007; Chen et al.2012). Recently, we reported that vitamin K3 (VK3) can function as a photosensitizer to UVA light in phosphate-buffered saline (PBS) solution and this combination can effectively inhibit bacterial growth (Xu and Vostal 2014). However, VK3 is not water soluble that could have limitation for its use. VK5 is water soluble and has a similar structure to VK3, it was thus reasonable for us to evaluate its potential as an UVA photosensitizer to inactivate bacteria in PBS and in human plasma. MATERIALS AND METHODS Materials Acetylacetone, ammonium iron (ii) sulfate hexahydrate, ammonium acetate, D-mannitol l-ascorbic acid bioxtra, dimethyl sulfoxide, nitro-blue tetrazolium (NBT), superoxide dismutase (SOD), trichloroacetic acid (TCA), carbonate and phosphate buffers and VK5 were purchased from Sigma-Aldrich (St. Louis, MO). VK5 solution was made fresh in sterilized water in a sterilized tube before each experiment. Platelet Additive Solution (PAS) was from Fenwal (Lake Zurich, IL). Human plasma collected by Fenwal Amicus Cell Separator was obtained from the National Institutes of Health blood bank. TC-6 well plates were from Corning (Corning, NY). GyroTwister 3-D Laboratory Shaker was from Labnet (Edison, NJ). 2,2-Bis(hydroxymethyl)-2,2΄,2″-nitrilotriethanol (1 mol l−1, pH 6.5) was purchased from Hampton Research (Aliso Viejo, CA). D-PBS was purchased from Life Technologies (Walkersville, MD). Gelatin veronal buffer (GVB; 5 m mol l−1 Veronal, 150 m mol l−1 NaCl, 0.05% gelatin, pH 7.4) was purchased from Boston BioProducts (Boston, MA). Triton X-100, Nunc 96-well polypropylene microplates (0.5 ml, white) and Nunc 96-well clear flat bottom microplates were obtained from Fisher Scientific (Pittsburgh, PA). Lyophilized DV-1, a commercially manufactured intravenous immunoglobulin (IVIG) lot was provided by the National Institute of Biological Standards and Control (NIBSC code 14/160, High titer anti-A in IVIG; Hertfordshire, UK). Bacteria and UVA light irradiation All bacteria were purchased from ATCC (Manassas, VA). Bacteria strain and corresponding ATCC catalog number are listed below. Bacillus cereus 11778, Escherichia coli 25922, Klebsiella pneumoniae 9997, Pseudomonas aeruginosa 27853, Staphylococcus aureus 35548, Staphylococcus epidermidis 49134 and two antibiotic resistant bacteria strains, Pseudomonas aeruginosa* BAA-2108 and Staphylococcus aureus* BAA-1747. UVA lamp with a wavelength of 350 nm (Sylvania 21623 - F15 - T8 - 350BL - ECO) was used for UVA irradiation. The dose of UVA light was measured with a radiometer (Model UVX-36, UVP, Upland, CA). All UVA irradiations were conducted at room temperature. The bacteria grown in log phase were centrifuged at 2000 g for 10 min and resuspended in PBS. Approximately 104 ml−1 colony forming unit (CFU) bacteria were used for the UVA dose and VK5 concentration studies (Fig. 1). Approximately 108 ml−1 CFU bacteria were used for the log reduction studies (Table 1) with the exception of S. aureus and S. aureus* that was used at 104 ml−1 CFU. A total of 2 ml of bacteria mixed with 0 to 1600 μmol l−1 of VK5 were placed in 6-well plates and exposed to dark or UVA irradiation for 10–20 min corresponding to 2.9 and 5.8 J cm−2 following our previous procedures (Xu and Vostal 2014). For bacterial inactivation in plasma, bacteria were mixed with 500–2000 μmol l−1 of VK5 in 35% or 50% plasma diluted with PAS in a total volume of 2 ml. Plasma inoculated with bacteria in 6-well plates was placed on a shaker at the speed of 10 rpm while exposed to UVA irradiation at 6 and 30 J cm−2. After UVA irradiation, PBS and plasma bacterial suspensions were plated on culture plates and grown at 37°C overnight prior to counting the colony forming units. Relative survival in Fig. 1 was calculated with the CFU count of different treatment divided by the CFU count after the irradiation of 2.9 J cm−2 without VK5 added. Figure 1. View largeDownload slide Escherichia coli survival was significantly reduced by VK5 plus UVA treatments in PBS solution. Escherichia coli suspended in PBS with 0–1600 μmol l−1 VK5 were irradiated with 2.9 J cm−2 or 5.8 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance ***P < 0.001 and **P < 0.01. Figure 1. View largeDownload slide Escherichia coli survival was significantly reduced by VK5 plus UVA treatments in PBS solution. Escherichia coli suspended in PBS with 0–1600 μmol l−1 VK5 were irradiated with 2.9 J cm−2 or 5.8 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance ***P < 0.001 and **P < 0.01. Table 1. Inactivation of bacteria with VK5 irradiated with UVA.   Bacterial log reduction factor  Bacteria  VK5-UVA  UVA-VK5  VK5 + UVA  B. cereus  0.14 ± 0.05  1.01 ± 0.06  6.76 ± 1.39**  E. coli  0.29 ± 0.11  0.16 ± 0.06  6.83 ± 1.14**  K. pneumoniae  0.07 ± 0.11  0.31 ± 0.08  7.12 ± 1.33**  P. aeruginosa  1.68 ± 0.13  1.00 ± 0.07  6.07 ± 0.59**  P. aeruginosa*  1.09 ± 0.23  1.18 ± 0.05  5.88 ± 0.84**  S. aureus  0.13 ± 0.14  0.41 ± 0.09  3.03 ± 0.10**  S. aureus*  0.23 ± 0.24  0.29 ± 0.08  3.09 ± 0.19**  S. epidermidis  0.33 ± 0.14  1.07 ± 0.14  6.55 ± 1.61*    Bacterial log reduction factor  Bacteria  VK5-UVA  UVA-VK5  VK5 + UVA  B. cereus  0.14 ± 0.05  1.01 ± 0.06  6.76 ± 1.39**  E. coli  0.29 ± 0.11  0.16 ± 0.06  6.83 ± 1.14**  K. pneumoniae  0.07 ± 0.11  0.31 ± 0.08  7.12 ± 1.33**  P. aeruginosa  1.68 ± 0.13  1.00 ± 0.07  6.07 ± 0.59**  P. aeruginosa*  1.09 ± 0.23  1.18 ± 0.05  5.88 ± 0.84**  S. aureus  0.13 ± 0.14  0.41 ± 0.09  3.03 ± 0.10**  S. aureus*  0.23 ± 0.24  0.29 ± 0.08  3.09 ± 0.19**  S. epidermidis  0.33 ± 0.14  1.07 ± 0.14  6.55 ± 1.61*  Note: All bacteria were resuspended in VK5 at 1600 μmol l−1 or PBS and irradiated with or without UVA at 5.8 J cm−2. Data are represented as mean ± SD (n = 3). **P < 0.01 and *P < 0.05. View Large Determination of superoxide anion radical (O2⋅−) The generation of O2.− was determined by recording the photosensitized reduction of NBT to nitroblue diformazan resulting in an increase in absorbance at 560 nm. NBT solution (1.67 × 10−4 mol l−1) prepared in 0.01 mol l−1 sodium carbonate buffer (pH = 10) was added into 6-well plate that has VK5 solution at 0–1000 μmol l−1 and irradiated with UVA dose at 2.9 J cm−2 following previous study (Dwivedi et al.2012; Xu and Vostal 2014). To confirm the generation of O2.−, SOD was used as a specific quencher in the NBT reduction assay. Quenching (%) in Fig. 4b was calculated based on the reduction of absorbance at 560 nm with the SOD concentration increased from 0 to 640 units ml−1 with UVA irradiation at 2.9 J cm−2. Determination of hydroxyl radical (⋅OH) Hydroxyl radical generation was determined by the formaldehyde production that has absorbance at 412 nm (Dwivedi et al.2012). This assay was performed in an ascorbic acid-iron-ethylenediaminetetraacetic acid system where ascorbic acid in the study was replaced with 0–1000 μmol l−1 VK5 following previous studies (Tullius and Dombroski 1986; Dwivedi et al.2012; Xu and Vostal 2014). Mannitol was used as a specific .OH quencher in the assay (Dwivedi et al.2012). Quenching (%) in Fig. 5b was calculated based on the reduction of absorbance at 412 nm with the mannitol concentration increased from 0 to 1 mol l−1 with UVA irradiation at 2.9 J cm−2. Measurement of complement activities The assay was performed in quadruplicate in a total volume of 150 μl in 96-well white round bottom microplates using a hemolysis assay procedure (Wang and Scott 2017). Briefly, 40 μl of DV-1 (anti-A IgG) was added to wells, followed by addition of 110 μl of GVB. After brief mixing, 35 μl of a 3% solution of red blood cell (RBC; ABO type A) in GVB buffer was added to each well. In separate wells, RBC was prepared and lysed with 2% Triton X-100 to provide a 100% cell lysis control. Plates were covered and incubated at 37°C for 20–30 min while shaking (400 rpm). After incubation plates were spun at 314 g (1200 rpm) in Sorvall Legend XTR centrifuge [Thermo Fisher Scientific (Waltham, MA)] for 2 min to pellet RBC. The supernatant was aspirated and 150 μl of test plasma was added to the wells. VK5 treated plasmas (test plasma) of blood type A were prepared by mixing with 0, 250, 500 and 1000 μ mol l−1 VK5 and irradiated under UVA (2.9 J cm−2). RBCs were incubated at 37°C with agitation at 400 rpm with a microplate shaker for 90 min. One hundred microliter of supernatant from each well was transferred to a flat-bottom enzyme-linked immunosorbent assay microplate. Hemoglobin levels were quantitated by measuring OD 414/OD 690, using a 96-well microplate reader (Molecular Devices, Sunnyvale, CA). The degree of cell lysis was calculated as a percentage of a positive lysis control [samples (OD 414 – OD 690]/[100% cell lysis (OD 414 – OD 690)]. Statistical analysis Microsoft Excel software Student's t test was used for statistical analysis with the significance set at a P value for *<0.05, **<0.01 and ***<0.001. All experiments were performed in triplicate. RESULTS AND DISCUSSION Inactivation of bacteria with vitamin K5 under UVA irradiation VK5 (0–1600 μmol l−1) was added to bacterium Escherichia coli suspended in PBS and the bacterial suspension was exposed to 2.9 or 5.8 J cm−2 of UVA. A representative survival of VK5 and UVA treated E. coli, as determined by a CFU count, is shown in Fig. 1. In the presence of VK5, E. coli became much more sensitive to UVA treatment. A dose effect of UV light was evident when the UVA dose was increased from 2.9 to 5.8 J cm−2 with a fixed VK5 concentration. Similarly, a concentration effect of VK5 was evident when the UVA dose was fixed. As shown in Fig. 1, the reduced survivals of E. coli at VK5 concentrations of 100 to 1600 μmol l−1 with a constant UVA dose of 2.9 or 5.8 J cm−2 were significantly lower than control groups without VK5. Specifically, the survival of E. coli dropped from 100% to 5% as the VK5 concentration was increased from 0 to 1600 μmol l−1 with a constant UVA dose of 2.9 J cm−2. The higher dose of UVA had substantial bactericidal effect on its own that reduced the survival of E. coli bacteria to 20% and this was further potentiated by increasing concentrations of VK5. Similar bactericidal effect of UVA doses and VK5 concentrations was found in all other bacteria, including Bacillus cereus (vegetative form), Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae. The reduction factor for all bacteria resuspended in VK5 at 1600 μmol l−1 and irradiated under UVA at 5.8 J cm−2 was between six to seven logs with the exception of S. aureus that was only reduced by three logs (Table 1). In contrast, VK5 alone at 1600 μmol l−1 or UVA alone at 5.8 J cm−2 treatment had limited bactericidal effects of approximately one log reduction. There was no substantial difference in log reduction between antibiotic-resistant and non-resistant bacteria P. aeruginosa or S. aureus after VK5 and UVA combined treatment (Table 1). These results indicate that antibiotic-resistant bacteria are equally susceptible to the photodynamic treatment and are consistent with the UVA-riboflavin bactericidal effect in antibiotic resistant bacteria (Makdoumi and Backman 2016). Together, it suggests the efficacy of photodynamic UVA photosensitizer treatment for pathogen inactivation regardless of bacteria sensitivity to antibiotics treatment. As such, this photodynamic UVA treatment may provide an alternative for bacterial contamination control in clinical practice. We previously tested VK3 (1600 μM) under UVA (5.8 J cm−2) irradiation for inactivation of bacteria in aqueous solution (Xu and Vostal 2014). The reduction factors for VK3 and UVA light were over 8 logs for the same group of bacteria with the exception of P. aeruginosa that only showed inactivation by 4 logs. The results with VK5 and UVA at equal concentration and UVA dose were different in that the same strain of S. aureus that was less susceptible to VK5 (3 logs vs 8 logs reduction for VK5 and VK3, respectively). The two vitamins also had different efficacy for P. aeruginosa that was reduced by six logs of CFU with vitamin K5 and only four logs of CFU with VK3. These differences may reflect variability in uptake of the vitamins by the bacteria or a variable susceptibility of S. aureus and other bacteria to the types of free radicals generated by VK5 and VK3. Similar mechanisms have been proposed to contribute to variable antibiotic resistance of bacteria (Nikaido 1989; Li, Livermore and Nikaido 1994; Fernandez and Hancock 2012; Olivares et al.2013). We extended the application of VK5/UVA to pathogen reduction of plasma products intentionally contaminated with gram positive and negative bacteria, E. coli and S. aureus. Human plasma, diluted with PAS to a final concentration of 35% or 50% plasma, was spiked with E. coli or S. aureus bacteria. Spiked plasmas were mixed with 500, 1000 or 2000 μmol l−1 VK5 and irradiated with 6 or 30 J/cm2 UVA. As shown in Fig. 2a, there is a clear VK5 concentration and UVA dose response in E. coli log reduction. When VK5 was mixed with bacterial solution to a final concentration of 500, 1000 and 2000 μmol l−1 and irradiated with 6 J/cm2 UVA, Escherichia coli reduction was 2.11, 2.39 and 4.2 logs in 35% plasma, and 1.94, 2.27 and 3.53 logs in 50% plasma, respectively. After 30 J/cm2 UVA irradiation with 500, 1000 and 2000 μmol l−1 VK5, E. coli log reduction was 2.46, 7.12 and 7.51 in 35% plasma and 2.99, 4.65 and 7.51 in 50% plasma, respectively. At concentrations of 1000 and 2000 μmol l−1 VK5 irradiated with 30 J/cm2 UVA, the E. coli log reduction was increased to statistically significant levels compared to 6 J/cm2 UVA in both 35% and 50% plasma. These results demonstrated the potential for a seven logs reduction of E. coli in 35% or 50% plasma after treatment with 2000 μmol l−1 VK5 and 30 J/cm2 UVA irradiation. Similarly in Fig. 2b at concentrations of 500, 1000 and 2000 μmol l−1 VK5 and irradiation with 30 J/cm2 UVA, S. aureus log reduction was significantly increased compared to 6 J/cm2 UVA in both 35% and 50% plasma. Staphylococcus aureus, spiked into 35% or 50% plasma mixed with 500, 1000 and 2000 μmol l−1 VK5, was reduced approximately seven logs after 30 J/cm2 UVA irradiation. Figure 2. View largeDownload slide Escherichia coli (a) and S. aureus (b) were significantly reduced by VK5 plus UVA treatments in plasma. Bacteria suspended in plasma with VK5 at 500–2000 μmol l−1 were irradiated with 6 J cm−2 or 30 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Figure 2. View largeDownload slide Escherichia coli (a) and S. aureus (b) were significantly reduced by VK5 plus UVA treatments in plasma. Bacteria suspended in plasma with VK5 at 500–2000 μmol l−1 were irradiated with 6 J cm−2 or 30 J cm−2 of UVA. The bacteria colony forming units were quantitated after treatment. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Vitamin K5 structure alteration after UVA irradiation To study if the structure of VK5 was altered by UVA irradiation, 1600 μmol l−1 of VK5 PBS solution was irradiated with 5.8 J cm−2 of UVA. After irradiation, the VK5 solution absorption spectrum was measured by a spectrophotometer. As shown in Fig. 3, the absorption of spectra of VK5 was altered after UVA irradiation, suggesting VK5 structure was modified by the UVA irradiation, similar chemical structure alteration has been reported with the vitamin B6 solution irradiated with UVA (Maeda et al.2000). It will be of interests in the future to study what changes in the chemical structure and any toxicity of this new structure modified molecule. Figure 3. View largeDownload slide The changes of absorption spectrum of vitamin K5 before and after UVA irradiation. PBS solution with 1600 μmol l−1 of VK5 was irradiated with 5.8 J cm−2 of UVA. Figure 3. View largeDownload slide The changes of absorption spectrum of vitamin K5 before and after UVA irradiation. PBS solution with 1600 μmol l−1 of VK5 was irradiated with 5.8 J cm−2 of UVA. Generation of superoxide anion radical (O2.−) The generation of superoxide anion radical (O2.−) in UVA-irradiated-VK5 PBS solution was dependent on the concentration of VK5. At a fixed UVA dose of 2.9 J cm−2, the generation of O2.− was significantly increased when VK5 concentration was increased from 250 to 1000 μmol l−1 (Fig. 4a). To further confirm that O2.− was generated, we used a specific O2.− quencher, SOD. The production of O2.− was significantly reduced as the SOD concentration was increased from 160 to 640 units ml−1 in a 500 μmol l−1 VK5 solution irradiated with a UVA (2.9 J cm−2), as shown in Fig. 4b. Figure 4. View largeDownload slide Photochemical generation of superoxide (a) in PBS measured by the OD at 560 nm was induced by VK5 (0–1000 μmol l−1) under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of superoxide (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of super oxide dismutase (SOD) from 0–640 units ml−1. Data are represented as mean ± SD (n = 3); significance ***P < 0.001, **P < 0.01 and *P < 0.05. Figure 4. View largeDownload slide Photochemical generation of superoxide (a) in PBS measured by the OD at 560 nm was induced by VK5 (0–1000 μmol l−1) under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of superoxide (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of super oxide dismutase (SOD) from 0–640 units ml−1. Data are represented as mean ± SD (n = 3); significance ***P < 0.001, **P < 0.01 and *P < 0.05. Generation of hydroxyl radical (⋅OH) VK5 and UVA light combination also caused a statistically significant release of ⋅OH as the VK5 concentrations increased from 250 to 1000 μmol l−1 with a fixed UVA dose of 2.9 J cm−2 (Fig. 5a). Generation of ⋅OH was further confirmed by inclusion of mannitol, a specific quencher for ⋅OH. Significantly, less ⋅OH was produced when mannitol concentration was increased from 0.25 to 1 mol l−1 in the presence of a fixed VK5 concentration of 500 μmol l−1 and a UVA dose of 2.9 J cm−2 (Fig. 5b). Figure 5. View largeDownload slide Photochemical generation of hydroxyl radical in PBS (a) measured by the OD at 412 nm was induced by VK5 (0–1000 μmol l−1) Under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of hydroxyl radical (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of mannitol from 0 to 1 mol l−1. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. Figure 5. View largeDownload slide Photochemical generation of hydroxyl radical in PBS (a) measured by the OD at 412 nm was induced by VK5 (0–1000 μmol l−1) Under UVA irradiation (2.9 J cm−2). Quenching of photochemical generation of hydroxyl radical (b) in PBS with 500 μmol l−1 VK5 and UVA irradiation (2.9 J cm−2) by increasing concentrations of mannitol from 0 to 1 mol l−1. Data are represented as mean ± SD (n = 3); significance **P < 0.01 and *P < 0.05. In our previous study, we found that irradiation of VK3 solutions with UVA light resulted in the release of ROS, such as singlet oxygen, hydroxyl radicals and superoxide anion radicals. In this study, we found that hydroxyl radicals and superoxide anion radicals, but not singlet oxygen (data not shown), were generated in VK5 solutions under UVA light irradiation, while under the same conditions singlet oxygen was detected in VK3 solutions (Xu and Vostal 2014). These results suggest that there is a different type of photosensitization reactions between VK3 and VK5 when irradiated with UVA and this altered profile of ROS species may cause the variable bactericidal effects on the same species of bacteria, such as S. aureus. Living organisms are susceptible to UVA irradiation due to the cytotoxic effect of ROS generated during the UVA exposure. ROS can cause lipid peroxidation, protein oxidation and DNA breakage, all of which are likely to contribute to the cytotoxic effects (Carraro and Pathak 1988; Cadet et al.1999; Maverakis et al.2010; Bracchitta et al.2013; Cadet and Wagner 2013). A previously described alternate method of light-based bacterial decontamination that utilizes UV light with vitamin B2 have been proposed to act through modification of nucleic acids, primarily on guanine residues to mediate the pathogen inactivation (Marschner and Goodrich 2011). It is likely that the ROS produced in UVA irradiated VK5 solution also mediates the bacterial inactivation. Measurement of complement activity Our experiments indicate that VK5 and UVA irradiation can reduce bacterial load in plasma diluted to 50% or 35% with PAS and thus this treatment could be potentially used for decontamination of plasma for transfusion or plasma for manufacture of plasma derivatives. To measure if this process can reduce activity of proteins found in plasma, we evaluated the effect VK5 and UVA treatment has on the plasma complement system. This system consists of a highly complex cascade of proteins present in plasma that assemble in response to an antibody binding a cell (Rogers, Veeramani and Weiner 2014; Wouters and Zeerleder 2015). The classical pathway initiates the assembly of a C3 convertase complex (proteins C2aC4b) that converts C3 into C3b on the surface of the cell. C3b then mediates conversion of protein C5 to C5b and mediates the formation of a membrane attack complex. The attack complex is an agglomerate of at least five separate proteins that assemble on the surface of the cell and introduce pores into the membrane that lead to cell lysis. We chose this system to test the impact of the VK5 and UVA treatment because it has multiple proteins and alteration to any one of the proteins could interfere with the cascade response. As shown in Fig. 6, there was a slight but not significant decrease of complement-mediated cell lysis after the UVA (2.9 J cm−2) irradiation in plasma mixed with 0, 250, 500 and 1000 μmol l−1 VK5. These results indicate that complement functions were slightly damaged after treatment with VK5 under UVA irradiation, although these detrimental effects to the complement system were not statistically significant. Thus, the impact of the VK5 and UVA treatment on plasma proteins appears to be relatively modest. However, depending on the application of the plasma after this treatment, the impact on other proteins relevant to the application should be studied further in the future. Figure 6. View largeDownload slide Measurement of complement activities in plasma with 0–1000 μmol l−1 VK5 irradiated under UVA (2.9 J cm−2). Data are represented as mean ± SD (n = 3). Figure 6. View largeDownload slide Measurement of complement activities in plasma with 0–1000 μmol l−1 VK5 irradiated under UVA (2.9 J cm−2). Data are represented as mean ± SD (n = 3). Our results show that VK5 and UVA photodynamic treatment can reduce bacteria in aqueous solutions and has efficacy against antibiotic-resistant and non-resistant bacteria. We also demonstrated that the photodynamic treatment could effectively reduce bacterial contamination in human plasma, although the concentration of VK5 and the dose of UVA needed to be increased and/or the plasma diluted. These results indicate that the conditions of this method could be optimized for different applications that require reduction of bacteria. FUNDING Food and Drug Administration, Center for Biologics Evaluation and Research. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. 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FEMS Microbiology LettersOxford University Press

Published: Feb 1, 2018

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