Staphylococcus aureus photodynamic inactivation mechanisms by rose bengal: use of antioxidants and spectroscopic study

Staphylococcus aureus photodynamic inactivation mechanisms by rose bengal: use of antioxidants... This study aims to follow the photodynamic and spectroscopic properties of dianionic rose Bengal disodium salt (RB) on Staphylococus aureus (S. aureus) in phosphate buffer solution (PBS) at pH 7.3. It focused on: (1) the effect of several reac - tive oxygen species (ROS) antioxidants used [such as sodium azide (N aN ), l -tryptophan (l -Trp) and d -mannitol] on the RB photodynamic efficiency as a mean to identify the main ROS attributed, and (2) the possible interactions of the RB with the important singlet oxygen quencher used namely tryptophan and/or between the dye and the bacteria S. aureus thanks to a spectroscopic study. The results showed that 20 µM of RB and 10 min of visible light (50 mW/cm ) with a light fluence dose of 30 J/cm are crucial for a good photodynamic action, achieving a reduction of 79.4% in the viability. Rose Bengal photodynamic action was in part inhibited by D-mannitol and l -Trp, indicating the mediation by.OH and O , respectively. The high inhibition of the RB activity against S. aureus by l -Trp is not due only to its singlet oxygen quencher ability but it is mainly due to the interaction between RB and l -Trp as shown spectrophotometrically. Keywords Absorption spectra · Quenchers · Tryptophan · Sodium azide · Mannitol · Visible light Introduction ground state of molecular oxygen, generating singlet oxygen ( O ), the first excited state of oxygen. Both mechanisms Light induces excitation of the photosensitizer (PS) mol- may occur simultaneously (Taniellian et al. 2000; Calin and ecules to the singlet excited state which can be converted Parasca 2009) and generate a variety of oxygen species, into the triplet excited compound. This triplet state may which can lead to cell apoptosis (Moan and Peng 2003). react with surrounding molecules in two types of photooxi- In fact, singlet oxygen, due to its high electrophilicity, is dative reaction. Type I involves electron or hydrogen atom capable of oxidizing phenols, sulphides and amines (DeRosa transfer, producing radical forms of the substrate which may and Crutchley 2002) possibly through a charge transfer inter- react with oxygen to form peroxides, superoxide ions, and mediate (Gollnick 1978) and can interact with numerous hydroxyl radicals. The Type II mechanism involves energy enzymes, leading to the inhibition of protein synthesis and transfer between the excited triplet state of the PS and the to the molecular alteration of DNA strands, which alters the transcription of the genetic material during its replication and in this way, leading to microbial death (Calin and Par- * S. Sabbahi asca 2009; Agnez-Lima et al. 2012). Like nucleic acids and sabbahisonia@yahoo.fr proteins, unsaturated lipids are also prominent targets of O Laboratoire de Microbiologie des Eaux Usées et des and free radical attack. The hydroxyl radical is identified as Boues Résiduaires, Institut National de Recherche en initiating lipid peroxidation by abstracting hydrogen atoms Génie Rural Eaux et Forêts, Rue Hédi Karray, B.P. 10, from fatty-acid side chains (Shevchuk et al. 2002). Among 2080  Ariana, Tunis, Tunisie xanthenes dyes which are potent photosensitizers (PSs) gen- Laboratoire Sciences et Techniques de l’Eau, Département du erating reactive oxygen species such as singlet oxygen (Kato Génie Rural, Eaux et Forêts, Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082 Tunis, Tunisie et al. 2012), RB is one of the most efficient and widely used sources of O (80%) in polar solvents such as water (Neck- Laboratoire des Bioprocédés Environnementaux, Pôle d’Excellence Régional (PER, AUF), Centre de ers 1989; Alarcόn et al. 2009), with a high quantum yield of Biotechnologie de Sfax, Université de Sfax, Route de Sidi 0.75–0.76 (Gandin et al. 1983; Redmon and Gamlin 1999). Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisie Vol.:(0123456789) 1 3 56 Page 2 of 9 Applied Water Science (2018) 8:56 The RB, is a cyclic compound that contains three aromatic rings in a linear arrangement and an oxygen atom in the cen- tre of the ring, which absorbs light in the visible spectrum (Costa et al. 2010), exhibiting intense absorption bands in the green area of the visible spectrum (480–550 nm) (DeR- osa and Crutchley 2002) with maximum absorption around 549 nm in diluted aqueous solutions (Xu and Neckers 1987). Therefore, according to these properties, we investigated in this current study the mechanisms of photodynamic action of this PS, against a Gram-positive bacterium (S. aureus), used as a pathogenic microorganism model. The simplest approach for determining whether O and/ or free radicals are involved in a photodynamic inactiva- tion process is to study the PS efficiency in the presence of various quenchers and free radicals scavengers (Nitzan et al. Fig. 1 Chemical structure of rose bengal (RB) 1989; Tavares et al. 2011). Singlet oxygen reacts primarily with five amino acids, which are tryptophan, histidine, tyros- ine, methionine and cysteine to form peroxides (Michaeli experiments of concentration equal to 20 µM for S. aureus. and Feitelson 1995 and Michaeli and Feitelson 1997) and in d -mannitol, l -tryptophan (RPE Analyticals Farmitalia Carlo high yields especially for tryptophan, tyrosine and histidine Erba, Milano, Italy) and sodium azide (Sigma-Aldrich, Ger- residues, both free and on proteins (Gracanin et al. 2009). many) were used as antioxidants. In previous studies (Jemli et  al. 2002; Sabbahi et  al. 2008a, 2013), RB has been exploited, at laboratory scale, as Illuminations experiments a promising PS in wastewater treatment against faecal bacte- ria indicators, pathogenic bacteria and helminth ova destruc- All illumination experiments were carried out with a 500 W tion. But the inactivation mechanisms by this PS have not halogen lamp light source (OSRAM, Italy) which emits in been yet well elucidated. Consequently, it is quite difficult the range of 500–750 nm with a peak at 650 nm, as previ- to predict the PS photo disinfection behaviour against any ously described in Sabbahi et al. (2008b). To avoid heating microorganism such as bacteria. By the way, in this study, of the samples, the photocontainers (with a final volume of the ultimate objective was to identify the main mechanisms 2.5 mL) were run at 28 °C covered by ice to maintain the action involved in S. aureus strain photoinactivation by dian- temperature constant. The irradiance at the level of microor- ionic rose bengal PS, which could effectively be carried out ganism sample was 50 mW/cm as measured with solarme- in buffer (PBS). In this context, three antioxidants including ter–pyranometer (Instruments HEANNS messger.A.T.E. two singlet oxygen quenchers (sodium azide and l -trypto- Solar 118). phan) and one free radical scavenger (d -mannitol) were used to search the main oxygen species causing damage to bacte- Bacterial strain and growth conditions ria by the photoactivated RB, to design improved PS and to elucidate the best conditions for S. aureus photoinactivation. The selection of S. aureus as bacterium model tested here In addition, it is of interest to define the relationship between is based first, on its pathogenicity (Oliveira et al. 2014) and the spectroscopic, photophysical and photochemical proper- second, on its frequency in wastewater samples and resist- ties of this PS and its photosensitization effect and damage ance to conventional disinfection treatments (Thompson mechanisms for the selected microbial strain. et al. 2013). Staphylococcus aureus strain ATCC 25923 isolated derive from surgical samples collected from the micro- Materials and methods biological department of the Children’s hospital in Tunis, Tunisia. Staphylococcus aureus strain was grown aerobically Chemicals overnight at 37 °C for 18 h in liquid culture (nutrient broth, 1% tryptone, 0.5% yeast extract, 0.5% NaCl w/v) from Bio- Rose bengal, 2,4,5,7-tetraiodo-3′ ,4′ ,5′ ,6′ - Rad (Marnes-La-Coquette, France). The culture was pelleted tetrachlorofluoresein disodium salt used is from Prolabo by centrifugation (10 min at 4 °C, 1050×g), the supernatant (Fontenay-Sous-Bois, France). The chemical structure discarded and the cells were washed twice with phosphate- (Fig. 1) was used as received, as photosensitizer. The dye buffered saline (PBS, 2.7 mM KCl, 137 mM NaCl, pH 7.3) was added to the buffer solution (pH 7.3) for photodynamic (Lab Online, France) and resuspended in a total volume of 1 3 Applied Water Science (2018) 8:56 Page 3 of 9 56 2.5 mL PBS to an optical density of 0.4 at 660 nm, cor- Statistics responding to approximately 5 × 10 colony-forming units per mL (CFUs/mL). This absorbance was measured using All experiments were performed in triplicate. Survival a Spectronic 20 Genesys™ spectrophotometer (Thermo values are expressed as means ± standard deviation. The Electron Corporation, France). differences among treatments were compared by analysis of variance using one-way ANOVA test, performed with the statistical package for Social Science 20.0 for windows Experimental setup: in vitro photodynamic (SPSS IBN Corp., 2011) software and the Duncan test: p inactivation value < 0.05 was considered to be statistically significant. Cells precultured in nutrient broth were washed twice with phosphate-buffered saline (PBS, pH 7.3 containing 2.7 mM KCl and 137 mM NaCl). The cells were diluted in PBS to Results and discussion a final density of 5.1 × 10 cells per mL, corresponding to an absorbance of 0.4 at 660 nm. This bacterial suspen- This section will include two components: (1) the evaluation sion was equally distributed into a 96-well-flat-bottomed of the RB photodynamic effect against S. aureus bacterium microliter plate (Sterilin, Stafford, UK) and incubated for and its mechanisms involved by quenching the main ROS 10 min with 100 µL of PS which were added to achieve produced and (2) the spectrophotometric study to reveal the final concentration of 20 µM corresponding to a total vol- possible interactions between the tryptophan and the dye ume of 2.5 mL per beaker. The samples were protected from and/or the dye and the bacterium. light and were incubated for 10 min in the dark, at 25–30 °C with RB (20 µM), as used for the PDI experiment. Control Eec ff t of RB photosensitization on S. aureus treatments were as follows: (1) without PS but illuminated; inactivation (2) with PS, but no light and (3) only suspension (no PS, no light). In the dark control, the PS at concentration used The RB sensitization procedure on S. aureus inactivation (20 µM) was added to the beaker and they were covered with was performed in phosphate buffer instead of the culture aluminum foil. medium because this latter is providing additional protec- After irradiation, 0.1  mL samples were taken off and tion as the antioxidants themselves. Samples with RB were serially diluted (tenfold) with PBS. Aliquots (0.1 mL) were kept in the dark (dark control) for 10 and 30 min and illu- spread over selective culture medium: Chapman–Man- minated samples without RB (light control) served as con- nitol Salt Agar (Bio-Rad, Diagnostic Pasteur, Marnes-La- trols. After 10 min of dark incubation, RB did not induce a Coquette, France); the number of colony-forming units significant death neither in the presence nor in the absence per milliliter (CFUs/mL) on each plate was then counted of antioxidants (ANOVA, p > 0.05). Thus, RB showed dark following 36 h of incubation at 37 °C and likewise, plated toxicity of only 0.1 CFU log reduction after 10 min exposure on nutrient agar (Bio-Rad, Diagnostic Pasteur, Marnes-La- showing cell survival of about 76.5 ± 1.4%. After 30 min, Coquette, France) after incubation at the same temperature RB showed a much higher level of dark killing of S. aureus for 24–48 h. The survival fraction (CFU per mL), is given bacteria (0.25 CFU log reduction; 54.9 ± 1.0% survivals) by the equation N /N as previously described by Sabbahi i 0 than it did after 10 min (76.5 ± 1.4% survival) (ANOVA, et al. (2008b). Each experiment was carried out three times. p < 0.05) (Table 1). Therefore, the cytotoxic effect of RB and the bacteria inactivation were increased by rising dark incubation time (Table 1). Spectrophotometric study By the way, the dark toxicity of RB is somewhat unclear, as the dye has been described by certain groups as cyto- Spectrophotometric study was carried out with a Spec- toxic (Paulino et al. 2005; Shrestha et al. 2012) and by other tronic® 20 Genesys™ spectrophotometer (Thermo Electron groups as noncytotoxic (Chilvers et al. 1999), depending on Corporation, France). Samples for spectrophotometric meas- the PS concentrations tested, the microorganisms studied urements were prepared by dilutions of RB stock solution to and their initial cell concentration, the dark incubation time, the desired concentration (20 µM). Shortly before the meas- etc. In fact, according to Kato et al. (2012), the photoinac- urements were taken, the appropriate fresh bacterial suspen- tivation of the membrane functions of S. aureus induced by sion was added to RB solution. The absorption spectra of the xanthenes dyes such as rose bengal, phloxine B, erythrosine dye were obtained in the visible region ranging from 400 to B and eosin B showed that when these specific dyes were 700 nm where the monomer and dimer absorption band of applied with 1 µM in the dark, none of them had an effect. this dye were distinct. 1 3 56 Page 4 of 9 Applied Water Science (2018) 8:56 Table 1 Photoinactivation of S. aureus cultures in PBS (pH 7.3) in Table 2 Photoinactivation of S. aureus cultures in PBS (pH 7,3) −2 the dark with RB (20  µM) after 10 and 30  min in the presence of treated with RB (20  µM) and artificial visible light (50  mW cm ) three antioxidants (10 mM): N aN , Trp and mannitol in the presence of three antioxidants (10  mM) after 10 and 30  min −2 of phototreatment at light fluence doses of 30 and 90 J cm , respec- Medium Viability after 10 min of phototreatment tively −1 CFU ml Survival fraction (%) Inactivated Medium Viability after 10 min of phototreatment bacteria (%) −1 CFU ml Survival fraction Inactivated bac- Without RB 4.9 × 10 96.1 ± 2.0 3.9 ± 2.0 (%) teria (%) RB alone 3.9 × 10 76.5 ± 1.4 23.5 ± 1.4 Without RB 4.2 × 10 82.3 ± 5.7 17.6 ± 5.7 −a 7 RB + N 3.9 × 10 76.5 ± 1.0 23.5 ± 1.0 RB alone 10.5 × 10 20.6 ± 1.4 79.4 ± 1.4 b 7 RB + Trp 3.1 × 10 60.8 ± 0.5 39.2 ± 0.5 −a 6 RB + N 10.2 × 10 20.0 ± 0.7 80.0 ± 0.7 RB + mannitol 4.5 × 10 88.2 ± 1.3 11.8 ± 1.3 b 7 RB + Trp 5.1 × 10 > 99 ± 0.1 < 0.1 ± 0.1 Viability after 30 min of phototreatment RB + mannitol 5.1 × 10 > 99 ± 0.1 < 0.1 ± 0.1  Without RB 4.8 × 10 94.1 ± 1.9 5.9 ± 1.9 Viability after 30 min of phototreatment  RB alone 2.8 × 10 54.9 ± 1.0 45.1 ± 1.0  Without RB 2.6 × 10 51.0 ± 4.2 49.0 ± 4.2 − 7  RB + N 2.6 × 10 51.0 ± 0.6 49.0 ± 0.6  RB alone 4.1 × 10 8.0 ± 0.4 91.9 ± 0.4  RB + Trp 2.6 × 10 51.0 ± 0.5 49.0 ± 0.5 − 6  RB + N 1.5 × 10 2.9 ± 0.4 97.0 ± 0.4  RB + mannitol 4.2 × 10 82.3 ± 14.1 17.6 ± 14.1  RB + Trp 2.0 × 10 3.9 ± 1.0 96.1 ± 1.0 S. aureus Staphylococcus aureus, PBS phosphate buffer solution, RB  RB + mannitol 5.1 × 10 10.0 ± 0.2 90.0 ± 0.2 rose bengal, NaN sodium azide a − b S. aureus: Staphylococcus aureus, PBS Phosphate buffer solution, RB N anion azide reactive, Trp tryptophan rose bengal, NaN sodium azide a − b N anion azide reactive, Trp tryptophan By Paulino et al. (2005), when Streptococcus mutans was treated with different concentrations (0–10 µM) of RB, it agent (Street et al. 2009), protected significantly S. aureus was observed that in the dark, this dye was toxic only to against photoactivated RB by 78% (its survival is more than the cells tested at concentrations above 5.0 µM. Indeed, the 99%; 0.69 log units reduction on bacteria photoinactivation results presented here confirmed a dark incubation time- rate) after 10 min of phototreatment (ANOVA, p < 0.05). dependent increase in the death rate of S. aureus with RB The same extent of S. aureus protection as obtained by l - for 30 min. Trp, occurred in the presence of d -mannitol, as an alcoholic Under the same conditions and in a previous work, the sugar which has an antioxidant effect; it reacts especially effect caused only by the light with or without antioxidants with hydroxyl free radical (Liang et  al. 2008; Al-Omari used (light control) on S. aureus, showed that after 10 and and Ali 2009). Accordingly, this substance is considered 30 min of irradiation time, fractions of about 83.3 ± 5.7 and to be a strong free radical scavenger (Sagone et al. 1983; 50.7 ± 4.2% in cell survival were noted, respectively (Sab- Kamat et al. 2000). Some experiments have shown that it can bahi et al. 2008b). prevent enzymes inactivation such as glucose-6-phosphate After being exposed to visible light in the presence of dehydrogenase (G6PD), nitrate-reductase and sulphite oxi- RB (20 µM), an additional photodamaging effect was noted dase by ionizing radiations (Eichler et al. 1987). d -mannitol (ANOVA, p < 0.05). The S. aureus survival fraction had can protect supercoiled DNA from hydroxyl radical dam- decreased to 20.6 ± 1.4% (with a recorded reduction of ~ 0.6 age due to ionizing radiation (Peak et al. 1995). The rate log units on S. aureus viability) after 10 min of irradiation of photoprotection powered by l -Trp and d -mannitol was 2 2 time (50 mW/cm ; light fluence of 30 J/cm ) (Table 2). significant (ANOVA, p < 0.05) during the first 10 min of To investigate whether photo-oxidation of Type I or II are illumination (Table 2). These results prove that the RB pho- the predominant action mechanisms; d -mannitol, a quencher tosensitization reaction, was especially proceeded by the of hydroxyl radical (Type I) and tryptophan or sodium azide, Type I pathway generating predominantly hydroxyl radicals quenchers of singlet oxygen (Type II) were used (Street (.OH) and Type II ( O production). For sodium azide as et  al. 2009). The results, resumed in Table  2, show that singlet oxygen quencher, there is no bacterial protection than under these conditions, sodium azide (10 mM), which is that obtained in the presence of l -Trp which was compara- known to react especially with singlet oxygen (Costa et al. ble to that obtained with d -mannitol, used as free radical 2013) and mainly as a physical quencher of O (Bancirova scavenger. The results obtained for sodium azide and l -Trp 2011), failed to show any bacteria protection as the same with the RB are unexpected since both are considered in survival fraction (20%) for the RB alone was obtained. As the literature, as singlet oxygen quenchers (Tavares et al. shown in Table  2, l -Trp, used as singlet oxygen trapping 2011). The results obtained show that the selection of l -Trp 1 3 Applied Water Science (2018) 8:56 Page 5 of 9 56 as singlet oxygen quencher can give, depending on the PS, erroneous information about the type of mechanism involved in the photodynamic inactivation process. Thirty minutes after treatment by RB and light, the sensitized S. aureus lost more than 90% of its viability (reduction of 1 log unit). The same rate of killing occurred in the presence of quench- ers which failed to show any protection (Table 2). On the other hand, the quenchers that proved their protective ability showed that the killing rate in their presence was reduced to almost the same extent as the protection provided by them. We propose two possible explanations. The first one is that RB molecules, in the absence of culture media (in saline), might be in close contact with the bacteria, ROS produced mainly HO. and O , might not be stopped efficiently by quenchers with increasing contact time. Consequently, a high percentage of the bacteria population is killed. Simi- lar explanation was reported by Nitzan et al. (1989) under their own experimental conditions. The second explanation concerns the concentration used of the quenchers. In fact, in all the protection experiments, the amount of the pro- posed quenchers was 500-fold higher than the amount of the RB added to the culture. According to authors, a plausible explanation can be the use of low quencher concentrations. The choice of the folding between PS and quenchers used was inspired from the work of Nitzan et al. (1989), in all their protection experiments, the amount of the proposed quencher was 70–700-fold higher than the amount of the overall PS added to the culture. Spectrophotometric study The spectrophotometric study has been complimented with the RB photodynamic effect on S. aureus in vitro inactiva- tion in saline solution (PBS) with the presence of the Trp antioxidant. This experiment was conducted to reveal the possibility of interactions between the antioxidant and the dye which could be associated with further understanding of the RB mechanisms photodynamic action. The visible absorption spectra of RB disodium salt with a fixed concentration of 20 µM in PBS over 400–700 nm wavelength range with or without S. aureus suspension and l -Trp (10 mM) are summarized in Fig. 2a and b. The maximum absorption (λmax) for the RB in PBS, were observed around 500 and 549 nm (Fig. 2(1a)). The loose ion pairs exhibit the same spectral shape as does the Fig. 2 Effects of S. aureus and Trp on the spectral characteristics of RB. Absorbance curves of RB at the dye concentration of 20  µM: a Without dianion in dilute aqueous solution (Linden and Neckers bacteria: (1) in PBS protected from light and (2) after 10  min of visible 1988). These peaks intensity decreased slightly after 10 min irradiation time at a light fluence dose of 30  J/cm , (3) in the presence of visible irradiation (Fig. 2(2a)). Rose bengal spectrum in of Trp (10 mM) protected from light and (4) after 10 min of visible irra- saline solution in the presence and the absence of 10 mM diation time. b In the presence of S. aureus at a concentration of 5.1 10 −1 CFU  ml : (1) absorbance curve of S. aureus protected from light in the of l -Trp is reported in Fig.  2(3a), (4a). Before illumina- absence of RB and antioxidants and (2) after 10  min of visible irradia- tion, the peak amplitude at 549 nm decreased from 2.441 tion time, (3) in the presence of RB in PBS before and (4) after 10 min of in the absence of l -Trp (Fig.  2(1a)) to 2.046 in its pres- visible irradiation time, (5) in the presence of RB and Trp before and (6) after 10 min of visible irradiation time ence (hypochromic effect) (Fig.  2(3a)). This reduction in 1 3 56 Page 6 of 9 Applied Water Science (2018) 8:56 absorbance demonstrates that some fractions of both the they are irradiated in solvent with the possibility of prod- dye and aminoacid molecules were actively involved in ucts formation, characteristics of their reaction (Fig. 2(6b)). ground state intermolecular interactions. After 10 min of This could be explained by the fact that when irradiated, visible irradiation, the peak amplitude at 549 nm diminished l -Trp could interact with RB resulting in products, which (hypochromic effect) slowly reaching 1.964 (Fig.  2(4a)). absorb at 410 nm (Fig. 2(6b)). In another way, Inoue et al. Figure 2b demonstrates the behaviour of the RB mono- (1982) in their experimental conditions reported that RB mer absorbance following bacteria addition (5.1 × 10 CFU/ sensitized photo-oxidation of l -Trp in aqueous environment mL). S. aureus growth controls are representing in the two at pH between 6 and 7 results mainly in the formation of curves, respectively of Fig. 2(1b), (2b), meaning without another product which is the N-hydroxyperoxy-hydropy- the addition of any RB or Trp antioxidant, and respectively rolle-indole carboxylic acid (HPI). Similar behaviour has in the absence of light and after 10 min of light exposure. been reported lately with hydroperoxides formed on Trp In the presence of RB, the S. aureus addition induced a oxidation (Gracanin et al. 2009). In the presence of phos- small decrease of its absorbance where the longer wave- phate buffer, the effect of l -Trp on the absorption spectra of length peak shifts 1 nm to the red region of the spectrum RB was practically similar to that shown in Fig. 2a, and to (A = 2.40); commonly called small bathochromic effect. the RB spectra obtained in aqueous solution (Linden and 550 nm Hence, the RB absorption spectrum in the presence of S. Neckers 1988), indicating that the salinity of the medium aureus suspended in saline solution showed three sharper does not appreciably affect the binding, pointing to a minor peaks at 500, 550 and 680 nm (Fig. 2(3b)). According to importance of electrostatic interactions. The factors affecting specific conditions of Neckers (1989), it can be assumed that the interactions between components and l -Trp may include the shortest wavelength of this absorption spectrum was due hydrophobic interactions. Further, this could be explained to the contact ion pair, whereas the higher one was a result by the fact that when the dye is bound to the amino acid, at of the totally dissociated dianion. Under the same condition, low oxygen concentration, trapping by oxygen of the triplet after exposure to visible light during 10 min, the shorter becomes inefficient and type I processes could contribute to wavelength peak amplitude increased and the longer one the observed photoprocess. shifted approximately to 10 nm toward the red (bathochro- Under the same conditions, Sabbahi et al. (2008b) showed mic effect) associated to an hyperchromism (A = 2.49) that thanks to the interactions between monocationique 560 nm (Fig. 2(4b)). Abuin et al. (2007) and Alarcόn et al. (2009) methylene blue (MB) and S. aureus, a metachromatic reac- assigned this behaviour to the presence of two species: RB tion took place between MB and the bacteria, inducing addi- free (band at 548–549 nm) and RB bound to protein (band tional dimerization of MB. Then, when Trp was introduced at 560–562 nm). For Chang et al. (2008), in their proper to the medium, the dye molecules would redistribute with experimental conditions, argued that the addition of lipid a decrease in the bound dye aggregate molecules as wit- results in the red shift favour the absorption of the species nessed by the dimer peak disappearance. Therefore, the MB at ≈ 563 and ≈ 523 nm probably due to the attachment of monomer species were responsible for photodestruction of the RB to the bacteria. A disappearance of the peak centred S. aureus bacteria. at 680 nm was also observed. Linden and Neckers (1988) According to author knowledge, little is known about the reported that the extent of ionisation at C-6 is function of effect of xanthenes dyes on bacterial membrane functions. solvent polarity and concentration. The absorption maxima Recently, Kato et al. (2012) showed the amounts of the dye of the C-2′ esters were shifted; however, about 10 nm to the bound to S. aureus cells estimated to be 54% at 1 µM. This red in S. aureus suspension relative to the disodium salt as amount was correlated with the potency of dye-induced that RB can be esterified at C-2′ (the carboxylate position) photoinactivation of bacterial membrane functions. Demi- selectively. dova and Hamblin (2005) showed that the anionic dye RB The spectra of the RB, in the presence of S. aureus with had no detectable binding to S. aureus but was neverthe- and without l -Trp (10 mM) are compared in Fig. 2(3b–6b). less highly efficient in mediating PDI killing. Most com- Since the amino acids do not absorb in the visible (Martínez mon, Gram-positive species lack a barrier comparable to the et al. 1993), the species absorbing in the 500 and 549 nm outer Gram-negative-bacteria cell wall. For this reason, Dahl regions should be free RB. Obviously from the comparison et al. (1988) moved forward that anionic compounds, such of the two spectra curves, there is some broadening in the as RB, which are excluded from the Gram-negative bacte- spectrum of l -Trp introduced to S. aureus suspension in the rial cell interior, may penetrate the Gram-positive exterior presence of RB (Fig. 2(5b)). This is showing a disturbance more readily. This is in agreement with our result referring in the RB spectrum in the normally sharp pair of peaks in the to the absorption spectrum change (particularly bathochro- 500–550 nm regions, indicating a possible complex forma- mic effect) when S. aureus was irradiated for 10 min in the tion of RB with the l -Trp in saline medium. Most evident presence of RB. In its presence, two distinct behaviours were spectral changes occur with the presence of l -Trp and when observed when l -Trp and N aN were used as singlet oxygen 1 3 Applied Water Science (2018) 8:56 Page 7 of 9 56 quenchers. In fact, the presence of l -Trp (10 mM) led to a the photodynamic process. Spectral studies can be a simple bacterial protection similar to the one observed with d -man- and useful screening gate to understand more of the mecha- nitol; while in the presence of sodium azide, no protection nisms action of PSs used for wastewater disinfection. was detected. The high inhibition of the PS activity by l - In addition to the spectrophotometric study, further work Trp would not only be due to its singlet oxygen quencher will be necessary to confirm some of these hypothetical ability but also to the interaction between RB and l -Trp as explanations mentioned above through our experimental witnessed by spectrophotometric study. Many available lit- observations and to identify the products found. Neverthe- erature data have shown that RB was well-known to operate less, it can be suggested to use a higher amount of the anti- via the Type II photosensitization mechanism (Redmon and oxidant’s concentration and to use more antioxidants (singlet Gamlin 1999; Gracanin et al. 2009) which were associated to oxygen quenchers and free radicals scavengers). We believe the environment of the PS, and to the absence of pre-defined that a trial as relatively simple experimental system corre- parameters for using PDI according to their experimental sponding to an evaluation in the simultaneous presence of conditions (Rossoni et al. 2010). This paper represents a antioxidants at higher concentration than the tested one in report of the mechanisms of RB photodynamic inactivation the present work to inhibit the action of both active reactive of S. aureus in phosphate buffer solution (PBS). The main oxygen species, will have the potential to better identify RB objective was to determine which ROS is more effective for reaction mechanisms. S. aureus using RB that tend to undergo Type I and/or Type II mechanisms (OH. and O , respectively), as the two highly active oxidizing agents involved in photodynamic inactiva- Conclusions tion or phototherapy (Huang et al. 2012; Wen et al. 2017). The obtained data suggested that the RB photosensitization Our results illustrate that under visible light and in the reaction, was especially proceeded by the Type I pathway presence of 20 µM RB, a higher killing efficiency for S. (hydroxyl radicals (OH.) quenched by mannitol) and Type aureus during 10 min of phototreatment was led. The S. 1 1 II ( O quenched by Trp). Surprisingly, nonquenchable O aureus survival decreases with increasing irradiation time 2 2 was detected in the presence of sodium azide (NaN ), as in the presence of PS. The hydroxyl free radicals (Type I a well-known singlet oxygen quencher. In agreement with mechanism) plays the most important role on the photoi- the results obtained and aforementioned, an explanation nactivation process of the tested bacteria by the dianionic of mechanisms of the RB photodynamic action against S. rose bengal. However, a contribution of Type II pathway aureus is that a possible quenching of the RB excited triplet cannot be discounted, in the presence of the singlet oxygen 3 * state ( RB ) by l -Trp has been proposed, as a concomitant quencher Tryptophan. Despite the presence of sodium azide 3 * consequence of interaction between RB and l -Trp and it found to be selective singlet oxygen quencher, no significant found to give rise to a large absorption change near 410 nm, protection was noted for RB after 10 min of phototreatment. and may compete with reaction between Trp and singlet oxy- From the results obtained, the selection of l -Trp as singlet gen ( O ) in aqueous solution. Although, the precise nature oxygen quencher, depending on the PS and its environment of this interaction, products generated and their contribution complimented with the spectrophotometric study can give, have not been elucidated, it has been reported by Seret and supplementary information about the type of mechanism Van de Vorst (1990) that the quenching of triplet excited involved in the photoinactivation process. 3 * state of RB ( RB ) by Trp in aqueous solution, generated Therefore, it was of great interests to characterize further semireduced RB as a product obtained through this interac- the bacterial targets, physicochemical and spectroscopic tion, but no information about rate constants was given in properties of PSs and finally the number of antioxidants their research (Criado et al. 1996). Therefore, this interac- used. tion between RB and l -Trp would be the cause of reduction Open Access This article is distributed under the terms of the Crea- of the RB microbial killing not as a reactive product. This tive Commons Attribution 4.0 International License (http://creat iveco explains the lack of correlation between RB photoinactiva- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion efficiency and its interaction capability with Trp shown tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the by our study. 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Staphylococcus aureus photodynamic inactivation mechanisms by rose bengal: use of antioxidants and spectroscopic study

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Abstract

This study aims to follow the photodynamic and spectroscopic properties of dianionic rose Bengal disodium salt (RB) on Staphylococus aureus (S. aureus) in phosphate buffer solution (PBS) at pH 7.3. It focused on: (1) the effect of several reac - tive oxygen species (ROS) antioxidants used [such as sodium azide (N aN ), l -tryptophan (l -Trp) and d -mannitol] on the RB photodynamic efficiency as a mean to identify the main ROS attributed, and (2) the possible interactions of the RB with the important singlet oxygen quencher used namely tryptophan and/or between the dye and the bacteria S. aureus thanks to a spectroscopic study. The results showed that 20 µM of RB and 10 min of visible light (50 mW/cm ) with a light fluence dose of 30 J/cm are crucial for a good photodynamic action, achieving a reduction of 79.4% in the viability. Rose Bengal photodynamic action was in part inhibited by D-mannitol and l -Trp, indicating the mediation by.OH and O , respectively. The high inhibition of the RB activity against S. aureus by l -Trp is not due only to its singlet oxygen quencher ability but it is mainly due to the interaction between RB and l -Trp as shown spectrophotometrically. Keywords Absorption spectra · Quenchers · Tryptophan · Sodium azide · Mannitol · Visible light Introduction ground state of molecular oxygen, generating singlet oxygen ( O ), the first excited state of oxygen. Both mechanisms Light induces excitation of the photosensitizer (PS) mol- may occur simultaneously (Taniellian et al. 2000; Calin and ecules to the singlet excited state which can be converted Parasca 2009) and generate a variety of oxygen species, into the triplet excited compound. This triplet state may which can lead to cell apoptosis (Moan and Peng 2003). react with surrounding molecules in two types of photooxi- In fact, singlet oxygen, due to its high electrophilicity, is dative reaction. Type I involves electron or hydrogen atom capable of oxidizing phenols, sulphides and amines (DeRosa transfer, producing radical forms of the substrate which may and Crutchley 2002) possibly through a charge transfer inter- react with oxygen to form peroxides, superoxide ions, and mediate (Gollnick 1978) and can interact with numerous hydroxyl radicals. The Type II mechanism involves energy enzymes, leading to the inhibition of protein synthesis and transfer between the excited triplet state of the PS and the to the molecular alteration of DNA strands, which alters the transcription of the genetic material during its replication and in this way, leading to microbial death (Calin and Par- * S. Sabbahi asca 2009; Agnez-Lima et al. 2012). Like nucleic acids and sabbahisonia@yahoo.fr proteins, unsaturated lipids are also prominent targets of O Laboratoire de Microbiologie des Eaux Usées et des and free radical attack. The hydroxyl radical is identified as Boues Résiduaires, Institut National de Recherche en initiating lipid peroxidation by abstracting hydrogen atoms Génie Rural Eaux et Forêts, Rue Hédi Karray, B.P. 10, from fatty-acid side chains (Shevchuk et al. 2002). Among 2080  Ariana, Tunis, Tunisie xanthenes dyes which are potent photosensitizers (PSs) gen- Laboratoire Sciences et Techniques de l’Eau, Département du erating reactive oxygen species such as singlet oxygen (Kato Génie Rural, Eaux et Forêts, Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle, 1082 Tunis, Tunisie et al. 2012), RB is one of the most efficient and widely used sources of O (80%) in polar solvents such as water (Neck- Laboratoire des Bioprocédés Environnementaux, Pôle d’Excellence Régional (PER, AUF), Centre de ers 1989; Alarcόn et al. 2009), with a high quantum yield of Biotechnologie de Sfax, Université de Sfax, Route de Sidi 0.75–0.76 (Gandin et al. 1983; Redmon and Gamlin 1999). Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisie Vol.:(0123456789) 1 3 56 Page 2 of 9 Applied Water Science (2018) 8:56 The RB, is a cyclic compound that contains three aromatic rings in a linear arrangement and an oxygen atom in the cen- tre of the ring, which absorbs light in the visible spectrum (Costa et al. 2010), exhibiting intense absorption bands in the green area of the visible spectrum (480–550 nm) (DeR- osa and Crutchley 2002) with maximum absorption around 549 nm in diluted aqueous solutions (Xu and Neckers 1987). Therefore, according to these properties, we investigated in this current study the mechanisms of photodynamic action of this PS, against a Gram-positive bacterium (S. aureus), used as a pathogenic microorganism model. The simplest approach for determining whether O and/ or free radicals are involved in a photodynamic inactiva- tion process is to study the PS efficiency in the presence of various quenchers and free radicals scavengers (Nitzan et al. Fig. 1 Chemical structure of rose bengal (RB) 1989; Tavares et al. 2011). Singlet oxygen reacts primarily with five amino acids, which are tryptophan, histidine, tyros- ine, methionine and cysteine to form peroxides (Michaeli experiments of concentration equal to 20 µM for S. aureus. and Feitelson 1995 and Michaeli and Feitelson 1997) and in d -mannitol, l -tryptophan (RPE Analyticals Farmitalia Carlo high yields especially for tryptophan, tyrosine and histidine Erba, Milano, Italy) and sodium azide (Sigma-Aldrich, Ger- residues, both free and on proteins (Gracanin et al. 2009). many) were used as antioxidants. In previous studies (Jemli et  al. 2002; Sabbahi et  al. 2008a, 2013), RB has been exploited, at laboratory scale, as Illuminations experiments a promising PS in wastewater treatment against faecal bacte- ria indicators, pathogenic bacteria and helminth ova destruc- All illumination experiments were carried out with a 500 W tion. But the inactivation mechanisms by this PS have not halogen lamp light source (OSRAM, Italy) which emits in been yet well elucidated. Consequently, it is quite difficult the range of 500–750 nm with a peak at 650 nm, as previ- to predict the PS photo disinfection behaviour against any ously described in Sabbahi et al. (2008b). To avoid heating microorganism such as bacteria. By the way, in this study, of the samples, the photocontainers (with a final volume of the ultimate objective was to identify the main mechanisms 2.5 mL) were run at 28 °C covered by ice to maintain the action involved in S. aureus strain photoinactivation by dian- temperature constant. The irradiance at the level of microor- ionic rose bengal PS, which could effectively be carried out ganism sample was 50 mW/cm as measured with solarme- in buffer (PBS). In this context, three antioxidants including ter–pyranometer (Instruments HEANNS messger.A.T.E. two singlet oxygen quenchers (sodium azide and l -trypto- Solar 118). phan) and one free radical scavenger (d -mannitol) were used to search the main oxygen species causing damage to bacte- Bacterial strain and growth conditions ria by the photoactivated RB, to design improved PS and to elucidate the best conditions for S. aureus photoinactivation. The selection of S. aureus as bacterium model tested here In addition, it is of interest to define the relationship between is based first, on its pathogenicity (Oliveira et al. 2014) and the spectroscopic, photophysical and photochemical proper- second, on its frequency in wastewater samples and resist- ties of this PS and its photosensitization effect and damage ance to conventional disinfection treatments (Thompson mechanisms for the selected microbial strain. et al. 2013). Staphylococcus aureus strain ATCC 25923 isolated derive from surgical samples collected from the micro- Materials and methods biological department of the Children’s hospital in Tunis, Tunisia. Staphylococcus aureus strain was grown aerobically Chemicals overnight at 37 °C for 18 h in liquid culture (nutrient broth, 1% tryptone, 0.5% yeast extract, 0.5% NaCl w/v) from Bio- Rose bengal, 2,4,5,7-tetraiodo-3′ ,4′ ,5′ ,6′ - Rad (Marnes-La-Coquette, France). The culture was pelleted tetrachlorofluoresein disodium salt used is from Prolabo by centrifugation (10 min at 4 °C, 1050×g), the supernatant (Fontenay-Sous-Bois, France). The chemical structure discarded and the cells were washed twice with phosphate- (Fig. 1) was used as received, as photosensitizer. The dye buffered saline (PBS, 2.7 mM KCl, 137 mM NaCl, pH 7.3) was added to the buffer solution (pH 7.3) for photodynamic (Lab Online, France) and resuspended in a total volume of 1 3 Applied Water Science (2018) 8:56 Page 3 of 9 56 2.5 mL PBS to an optical density of 0.4 at 660 nm, cor- Statistics responding to approximately 5 × 10 colony-forming units per mL (CFUs/mL). This absorbance was measured using All experiments were performed in triplicate. Survival a Spectronic 20 Genesys™ spectrophotometer (Thermo values are expressed as means ± standard deviation. The Electron Corporation, France). differences among treatments were compared by analysis of variance using one-way ANOVA test, performed with the statistical package for Social Science 20.0 for windows Experimental setup: in vitro photodynamic (SPSS IBN Corp., 2011) software and the Duncan test: p inactivation value < 0.05 was considered to be statistically significant. Cells precultured in nutrient broth were washed twice with phosphate-buffered saline (PBS, pH 7.3 containing 2.7 mM KCl and 137 mM NaCl). The cells were diluted in PBS to Results and discussion a final density of 5.1 × 10 cells per mL, corresponding to an absorbance of 0.4 at 660 nm. This bacterial suspen- This section will include two components: (1) the evaluation sion was equally distributed into a 96-well-flat-bottomed of the RB photodynamic effect against S. aureus bacterium microliter plate (Sterilin, Stafford, UK) and incubated for and its mechanisms involved by quenching the main ROS 10 min with 100 µL of PS which were added to achieve produced and (2) the spectrophotometric study to reveal the final concentration of 20 µM corresponding to a total vol- possible interactions between the tryptophan and the dye ume of 2.5 mL per beaker. The samples were protected from and/or the dye and the bacterium. light and were incubated for 10 min in the dark, at 25–30 °C with RB (20 µM), as used for the PDI experiment. Control Eec ff t of RB photosensitization on S. aureus treatments were as follows: (1) without PS but illuminated; inactivation (2) with PS, but no light and (3) only suspension (no PS, no light). In the dark control, the PS at concentration used The RB sensitization procedure on S. aureus inactivation (20 µM) was added to the beaker and they were covered with was performed in phosphate buffer instead of the culture aluminum foil. medium because this latter is providing additional protec- After irradiation, 0.1  mL samples were taken off and tion as the antioxidants themselves. Samples with RB were serially diluted (tenfold) with PBS. Aliquots (0.1 mL) were kept in the dark (dark control) for 10 and 30 min and illu- spread over selective culture medium: Chapman–Man- minated samples without RB (light control) served as con- nitol Salt Agar (Bio-Rad, Diagnostic Pasteur, Marnes-La- trols. After 10 min of dark incubation, RB did not induce a Coquette, France); the number of colony-forming units significant death neither in the presence nor in the absence per milliliter (CFUs/mL) on each plate was then counted of antioxidants (ANOVA, p > 0.05). Thus, RB showed dark following 36 h of incubation at 37 °C and likewise, plated toxicity of only 0.1 CFU log reduction after 10 min exposure on nutrient agar (Bio-Rad, Diagnostic Pasteur, Marnes-La- showing cell survival of about 76.5 ± 1.4%. After 30 min, Coquette, France) after incubation at the same temperature RB showed a much higher level of dark killing of S. aureus for 24–48 h. The survival fraction (CFU per mL), is given bacteria (0.25 CFU log reduction; 54.9 ± 1.0% survivals) by the equation N /N as previously described by Sabbahi i 0 than it did after 10 min (76.5 ± 1.4% survival) (ANOVA, et al. (2008b). Each experiment was carried out three times. p < 0.05) (Table 1). Therefore, the cytotoxic effect of RB and the bacteria inactivation were increased by rising dark incubation time (Table 1). Spectrophotometric study By the way, the dark toxicity of RB is somewhat unclear, as the dye has been described by certain groups as cyto- Spectrophotometric study was carried out with a Spec- toxic (Paulino et al. 2005; Shrestha et al. 2012) and by other tronic® 20 Genesys™ spectrophotometer (Thermo Electron groups as noncytotoxic (Chilvers et al. 1999), depending on Corporation, France). Samples for spectrophotometric meas- the PS concentrations tested, the microorganisms studied urements were prepared by dilutions of RB stock solution to and their initial cell concentration, the dark incubation time, the desired concentration (20 µM). Shortly before the meas- etc. In fact, according to Kato et al. (2012), the photoinac- urements were taken, the appropriate fresh bacterial suspen- tivation of the membrane functions of S. aureus induced by sion was added to RB solution. The absorption spectra of the xanthenes dyes such as rose bengal, phloxine B, erythrosine dye were obtained in the visible region ranging from 400 to B and eosin B showed that when these specific dyes were 700 nm where the monomer and dimer absorption band of applied with 1 µM in the dark, none of them had an effect. this dye were distinct. 1 3 56 Page 4 of 9 Applied Water Science (2018) 8:56 Table 1 Photoinactivation of S. aureus cultures in PBS (pH 7.3) in Table 2 Photoinactivation of S. aureus cultures in PBS (pH 7,3) −2 the dark with RB (20  µM) after 10 and 30  min in the presence of treated with RB (20  µM) and artificial visible light (50  mW cm ) three antioxidants (10 mM): N aN , Trp and mannitol in the presence of three antioxidants (10  mM) after 10 and 30  min −2 of phototreatment at light fluence doses of 30 and 90 J cm , respec- Medium Viability after 10 min of phototreatment tively −1 CFU ml Survival fraction (%) Inactivated Medium Viability after 10 min of phototreatment bacteria (%) −1 CFU ml Survival fraction Inactivated bac- Without RB 4.9 × 10 96.1 ± 2.0 3.9 ± 2.0 (%) teria (%) RB alone 3.9 × 10 76.5 ± 1.4 23.5 ± 1.4 Without RB 4.2 × 10 82.3 ± 5.7 17.6 ± 5.7 −a 7 RB + N 3.9 × 10 76.5 ± 1.0 23.5 ± 1.0 RB alone 10.5 × 10 20.6 ± 1.4 79.4 ± 1.4 b 7 RB + Trp 3.1 × 10 60.8 ± 0.5 39.2 ± 0.5 −a 6 RB + N 10.2 × 10 20.0 ± 0.7 80.0 ± 0.7 RB + mannitol 4.5 × 10 88.2 ± 1.3 11.8 ± 1.3 b 7 RB + Trp 5.1 × 10 > 99 ± 0.1 < 0.1 ± 0.1 Viability after 30 min of phototreatment RB + mannitol 5.1 × 10 > 99 ± 0.1 < 0.1 ± 0.1  Without RB 4.8 × 10 94.1 ± 1.9 5.9 ± 1.9 Viability after 30 min of phototreatment  RB alone 2.8 × 10 54.9 ± 1.0 45.1 ± 1.0  Without RB 2.6 × 10 51.0 ± 4.2 49.0 ± 4.2 − 7  RB + N 2.6 × 10 51.0 ± 0.6 49.0 ± 0.6  RB alone 4.1 × 10 8.0 ± 0.4 91.9 ± 0.4  RB + Trp 2.6 × 10 51.0 ± 0.5 49.0 ± 0.5 − 6  RB + N 1.5 × 10 2.9 ± 0.4 97.0 ± 0.4  RB + mannitol 4.2 × 10 82.3 ± 14.1 17.6 ± 14.1  RB + Trp 2.0 × 10 3.9 ± 1.0 96.1 ± 1.0 S. aureus Staphylococcus aureus, PBS phosphate buffer solution, RB  RB + mannitol 5.1 × 10 10.0 ± 0.2 90.0 ± 0.2 rose bengal, NaN sodium azide a − b S. aureus: Staphylococcus aureus, PBS Phosphate buffer solution, RB N anion azide reactive, Trp tryptophan rose bengal, NaN sodium azide a − b N anion azide reactive, Trp tryptophan By Paulino et al. (2005), when Streptococcus mutans was treated with different concentrations (0–10 µM) of RB, it agent (Street et al. 2009), protected significantly S. aureus was observed that in the dark, this dye was toxic only to against photoactivated RB by 78% (its survival is more than the cells tested at concentrations above 5.0 µM. Indeed, the 99%; 0.69 log units reduction on bacteria photoinactivation results presented here confirmed a dark incubation time- rate) after 10 min of phototreatment (ANOVA, p < 0.05). dependent increase in the death rate of S. aureus with RB The same extent of S. aureus protection as obtained by l - for 30 min. Trp, occurred in the presence of d -mannitol, as an alcoholic Under the same conditions and in a previous work, the sugar which has an antioxidant effect; it reacts especially effect caused only by the light with or without antioxidants with hydroxyl free radical (Liang et  al. 2008; Al-Omari used (light control) on S. aureus, showed that after 10 and and Ali 2009). Accordingly, this substance is considered 30 min of irradiation time, fractions of about 83.3 ± 5.7 and to be a strong free radical scavenger (Sagone et al. 1983; 50.7 ± 4.2% in cell survival were noted, respectively (Sab- Kamat et al. 2000). Some experiments have shown that it can bahi et al. 2008b). prevent enzymes inactivation such as glucose-6-phosphate After being exposed to visible light in the presence of dehydrogenase (G6PD), nitrate-reductase and sulphite oxi- RB (20 µM), an additional photodamaging effect was noted dase by ionizing radiations (Eichler et al. 1987). d -mannitol (ANOVA, p < 0.05). The S. aureus survival fraction had can protect supercoiled DNA from hydroxyl radical dam- decreased to 20.6 ± 1.4% (with a recorded reduction of ~ 0.6 age due to ionizing radiation (Peak et al. 1995). The rate log units on S. aureus viability) after 10 min of irradiation of photoprotection powered by l -Trp and d -mannitol was 2 2 time (50 mW/cm ; light fluence of 30 J/cm ) (Table 2). significant (ANOVA, p < 0.05) during the first 10 min of To investigate whether photo-oxidation of Type I or II are illumination (Table 2). These results prove that the RB pho- the predominant action mechanisms; d -mannitol, a quencher tosensitization reaction, was especially proceeded by the of hydroxyl radical (Type I) and tryptophan or sodium azide, Type I pathway generating predominantly hydroxyl radicals quenchers of singlet oxygen (Type II) were used (Street (.OH) and Type II ( O production). For sodium azide as et  al. 2009). The results, resumed in Table  2, show that singlet oxygen quencher, there is no bacterial protection than under these conditions, sodium azide (10 mM), which is that obtained in the presence of l -Trp which was compara- known to react especially with singlet oxygen (Costa et al. ble to that obtained with d -mannitol, used as free radical 2013) and mainly as a physical quencher of O (Bancirova scavenger. The results obtained for sodium azide and l -Trp 2011), failed to show any bacteria protection as the same with the RB are unexpected since both are considered in survival fraction (20%) for the RB alone was obtained. As the literature, as singlet oxygen quenchers (Tavares et al. shown in Table  2, l -Trp, used as singlet oxygen trapping 2011). The results obtained show that the selection of l -Trp 1 3 Applied Water Science (2018) 8:56 Page 5 of 9 56 as singlet oxygen quencher can give, depending on the PS, erroneous information about the type of mechanism involved in the photodynamic inactivation process. Thirty minutes after treatment by RB and light, the sensitized S. aureus lost more than 90% of its viability (reduction of 1 log unit). The same rate of killing occurred in the presence of quench- ers which failed to show any protection (Table 2). On the other hand, the quenchers that proved their protective ability showed that the killing rate in their presence was reduced to almost the same extent as the protection provided by them. We propose two possible explanations. The first one is that RB molecules, in the absence of culture media (in saline), might be in close contact with the bacteria, ROS produced mainly HO. and O , might not be stopped efficiently by quenchers with increasing contact time. Consequently, a high percentage of the bacteria population is killed. Simi- lar explanation was reported by Nitzan et al. (1989) under their own experimental conditions. The second explanation concerns the concentration used of the quenchers. In fact, in all the protection experiments, the amount of the pro- posed quenchers was 500-fold higher than the amount of the RB added to the culture. According to authors, a plausible explanation can be the use of low quencher concentrations. The choice of the folding between PS and quenchers used was inspired from the work of Nitzan et al. (1989), in all their protection experiments, the amount of the proposed quencher was 70–700-fold higher than the amount of the overall PS added to the culture. Spectrophotometric study The spectrophotometric study has been complimented with the RB photodynamic effect on S. aureus in vitro inactiva- tion in saline solution (PBS) with the presence of the Trp antioxidant. This experiment was conducted to reveal the possibility of interactions between the antioxidant and the dye which could be associated with further understanding of the RB mechanisms photodynamic action. The visible absorption spectra of RB disodium salt with a fixed concentration of 20 µM in PBS over 400–700 nm wavelength range with or without S. aureus suspension and l -Trp (10 mM) are summarized in Fig. 2a and b. The maximum absorption (λmax) for the RB in PBS, were observed around 500 and 549 nm (Fig. 2(1a)). The loose ion pairs exhibit the same spectral shape as does the Fig. 2 Effects of S. aureus and Trp on the spectral characteristics of RB. Absorbance curves of RB at the dye concentration of 20  µM: a Without dianion in dilute aqueous solution (Linden and Neckers bacteria: (1) in PBS protected from light and (2) after 10  min of visible 1988). These peaks intensity decreased slightly after 10 min irradiation time at a light fluence dose of 30  J/cm , (3) in the presence of visible irradiation (Fig. 2(2a)). Rose bengal spectrum in of Trp (10 mM) protected from light and (4) after 10 min of visible irra- saline solution in the presence and the absence of 10 mM diation time. b In the presence of S. aureus at a concentration of 5.1 10 −1 CFU  ml : (1) absorbance curve of S. aureus protected from light in the of l -Trp is reported in Fig.  2(3a), (4a). Before illumina- absence of RB and antioxidants and (2) after 10  min of visible irradia- tion, the peak amplitude at 549 nm decreased from 2.441 tion time, (3) in the presence of RB in PBS before and (4) after 10 min of in the absence of l -Trp (Fig.  2(1a)) to 2.046 in its pres- visible irradiation time, (5) in the presence of RB and Trp before and (6) after 10 min of visible irradiation time ence (hypochromic effect) (Fig.  2(3a)). This reduction in 1 3 56 Page 6 of 9 Applied Water Science (2018) 8:56 absorbance demonstrates that some fractions of both the they are irradiated in solvent with the possibility of prod- dye and aminoacid molecules were actively involved in ucts formation, characteristics of their reaction (Fig. 2(6b)). ground state intermolecular interactions. After 10 min of This could be explained by the fact that when irradiated, visible irradiation, the peak amplitude at 549 nm diminished l -Trp could interact with RB resulting in products, which (hypochromic effect) slowly reaching 1.964 (Fig.  2(4a)). absorb at 410 nm (Fig. 2(6b)). In another way, Inoue et al. Figure 2b demonstrates the behaviour of the RB mono- (1982) in their experimental conditions reported that RB mer absorbance following bacteria addition (5.1 × 10 CFU/ sensitized photo-oxidation of l -Trp in aqueous environment mL). S. aureus growth controls are representing in the two at pH between 6 and 7 results mainly in the formation of curves, respectively of Fig. 2(1b), (2b), meaning without another product which is the N-hydroxyperoxy-hydropy- the addition of any RB or Trp antioxidant, and respectively rolle-indole carboxylic acid (HPI). Similar behaviour has in the absence of light and after 10 min of light exposure. been reported lately with hydroperoxides formed on Trp In the presence of RB, the S. aureus addition induced a oxidation (Gracanin et al. 2009). In the presence of phos- small decrease of its absorbance where the longer wave- phate buffer, the effect of l -Trp on the absorption spectra of length peak shifts 1 nm to the red region of the spectrum RB was practically similar to that shown in Fig. 2a, and to (A = 2.40); commonly called small bathochromic effect. the RB spectra obtained in aqueous solution (Linden and 550 nm Hence, the RB absorption spectrum in the presence of S. Neckers 1988), indicating that the salinity of the medium aureus suspended in saline solution showed three sharper does not appreciably affect the binding, pointing to a minor peaks at 500, 550 and 680 nm (Fig. 2(3b)). According to importance of electrostatic interactions. The factors affecting specific conditions of Neckers (1989), it can be assumed that the interactions between components and l -Trp may include the shortest wavelength of this absorption spectrum was due hydrophobic interactions. Further, this could be explained to the contact ion pair, whereas the higher one was a result by the fact that when the dye is bound to the amino acid, at of the totally dissociated dianion. Under the same condition, low oxygen concentration, trapping by oxygen of the triplet after exposure to visible light during 10 min, the shorter becomes inefficient and type I processes could contribute to wavelength peak amplitude increased and the longer one the observed photoprocess. shifted approximately to 10 nm toward the red (bathochro- Under the same conditions, Sabbahi et al. (2008b) showed mic effect) associated to an hyperchromism (A = 2.49) that thanks to the interactions between monocationique 560 nm (Fig. 2(4b)). Abuin et al. (2007) and Alarcόn et al. (2009) methylene blue (MB) and S. aureus, a metachromatic reac- assigned this behaviour to the presence of two species: RB tion took place between MB and the bacteria, inducing addi- free (band at 548–549 nm) and RB bound to protein (band tional dimerization of MB. Then, when Trp was introduced at 560–562 nm). For Chang et al. (2008), in their proper to the medium, the dye molecules would redistribute with experimental conditions, argued that the addition of lipid a decrease in the bound dye aggregate molecules as wit- results in the red shift favour the absorption of the species nessed by the dimer peak disappearance. Therefore, the MB at ≈ 563 and ≈ 523 nm probably due to the attachment of monomer species were responsible for photodestruction of the RB to the bacteria. A disappearance of the peak centred S. aureus bacteria. at 680 nm was also observed. Linden and Neckers (1988) According to author knowledge, little is known about the reported that the extent of ionisation at C-6 is function of effect of xanthenes dyes on bacterial membrane functions. solvent polarity and concentration. The absorption maxima Recently, Kato et al. (2012) showed the amounts of the dye of the C-2′ esters were shifted; however, about 10 nm to the bound to S. aureus cells estimated to be 54% at 1 µM. This red in S. aureus suspension relative to the disodium salt as amount was correlated with the potency of dye-induced that RB can be esterified at C-2′ (the carboxylate position) photoinactivation of bacterial membrane functions. Demi- selectively. dova and Hamblin (2005) showed that the anionic dye RB The spectra of the RB, in the presence of S. aureus with had no detectable binding to S. aureus but was neverthe- and without l -Trp (10 mM) are compared in Fig. 2(3b–6b). less highly efficient in mediating PDI killing. Most com- Since the amino acids do not absorb in the visible (Martínez mon, Gram-positive species lack a barrier comparable to the et al. 1993), the species absorbing in the 500 and 549 nm outer Gram-negative-bacteria cell wall. For this reason, Dahl regions should be free RB. Obviously from the comparison et al. (1988) moved forward that anionic compounds, such of the two spectra curves, there is some broadening in the as RB, which are excluded from the Gram-negative bacte- spectrum of l -Trp introduced to S. aureus suspension in the rial cell interior, may penetrate the Gram-positive exterior presence of RB (Fig. 2(5b)). This is showing a disturbance more readily. This is in agreement with our result referring in the RB spectrum in the normally sharp pair of peaks in the to the absorption spectrum change (particularly bathochro- 500–550 nm regions, indicating a possible complex forma- mic effect) when S. aureus was irradiated for 10 min in the tion of RB with the l -Trp in saline medium. Most evident presence of RB. In its presence, two distinct behaviours were spectral changes occur with the presence of l -Trp and when observed when l -Trp and N aN were used as singlet oxygen 1 3 Applied Water Science (2018) 8:56 Page 7 of 9 56 quenchers. In fact, the presence of l -Trp (10 mM) led to a the photodynamic process. Spectral studies can be a simple bacterial protection similar to the one observed with d -man- and useful screening gate to understand more of the mecha- nitol; while in the presence of sodium azide, no protection nisms action of PSs used for wastewater disinfection. was detected. The high inhibition of the PS activity by l - In addition to the spectrophotometric study, further work Trp would not only be due to its singlet oxygen quencher will be necessary to confirm some of these hypothetical ability but also to the interaction between RB and l -Trp as explanations mentioned above through our experimental witnessed by spectrophotometric study. Many available lit- observations and to identify the products found. Neverthe- erature data have shown that RB was well-known to operate less, it can be suggested to use a higher amount of the anti- via the Type II photosensitization mechanism (Redmon and oxidant’s concentration and to use more antioxidants (singlet Gamlin 1999; Gracanin et al. 2009) which were associated to oxygen quenchers and free radicals scavengers). We believe the environment of the PS, and to the absence of pre-defined that a trial as relatively simple experimental system corre- parameters for using PDI according to their experimental sponding to an evaluation in the simultaneous presence of conditions (Rossoni et al. 2010). This paper represents a antioxidants at higher concentration than the tested one in report of the mechanisms of RB photodynamic inactivation the present work to inhibit the action of both active reactive of S. aureus in phosphate buffer solution (PBS). The main oxygen species, will have the potential to better identify RB objective was to determine which ROS is more effective for reaction mechanisms. S. aureus using RB that tend to undergo Type I and/or Type II mechanisms (OH. and O , respectively), as the two highly active oxidizing agents involved in photodynamic inactiva- Conclusions tion or phototherapy (Huang et al. 2012; Wen et al. 2017). The obtained data suggested that the RB photosensitization Our results illustrate that under visible light and in the reaction, was especially proceeded by the Type I pathway presence of 20 µM RB, a higher killing efficiency for S. (hydroxyl radicals (OH.) quenched by mannitol) and Type aureus during 10 min of phototreatment was led. The S. 1 1 II ( O quenched by Trp). Surprisingly, nonquenchable O aureus survival decreases with increasing irradiation time 2 2 was detected in the presence of sodium azide (NaN ), as in the presence of PS. The hydroxyl free radicals (Type I a well-known singlet oxygen quencher. In agreement with mechanism) plays the most important role on the photoi- the results obtained and aforementioned, an explanation nactivation process of the tested bacteria by the dianionic of mechanisms of the RB photodynamic action against S. rose bengal. However, a contribution of Type II pathway aureus is that a possible quenching of the RB excited triplet cannot be discounted, in the presence of the singlet oxygen 3 * state ( RB ) by l -Trp has been proposed, as a concomitant quencher Tryptophan. Despite the presence of sodium azide 3 * consequence of interaction between RB and l -Trp and it found to be selective singlet oxygen quencher, no significant found to give rise to a large absorption change near 410 nm, protection was noted for RB after 10 min of phototreatment. and may compete with reaction between Trp and singlet oxy- From the results obtained, the selection of l -Trp as singlet gen ( O ) in aqueous solution. Although, the precise nature oxygen quencher, depending on the PS and its environment of this interaction, products generated and their contribution complimented with the spectrophotometric study can give, have not been elucidated, it has been reported by Seret and supplementary information about the type of mechanism Van de Vorst (1990) that the quenching of triplet excited involved in the photoinactivation process. 3 * state of RB ( RB ) by Trp in aqueous solution, generated Therefore, it was of great interests to characterize further semireduced RB as a product obtained through this interac- the bacterial targets, physicochemical and spectroscopic tion, but no information about rate constants was given in properties of PSs and finally the number of antioxidants their research (Criado et al. 1996). Therefore, this interac- used. tion between RB and l -Trp would be the cause of reduction Open Access This article is distributed under the terms of the Crea- of the RB microbial killing not as a reactive product. This tive Commons Attribution 4.0 International License (http://creat iveco explains the lack of correlation between RB photoinactiva- mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion efficiency and its interaction capability with Trp shown tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the by our study. In summary, not only the use of singlet oxy- Creative Commons license, and indicate if changes were made. gen quenchers and of the free radical scavengers as simple approach to determine which pathway(s) is (are) involved in PDI (Nitzan et al. 1989; Song et al. 1999; Huang et al. 2012) but also the spectroscopic study of possible interaction between the PS and some quenchers such as Trp, used in this study, in the presence of bacteria, are important characteris- tics for determining photochemical mechanisms involved in 1 3 56 Page 8 of 9 Applied Water Science (2018) 8:56 Gandin E, Lion Y, van de Vorst A (1983) Quantum yield of singlet References oxygen production by xanthene derivatives. Photochem Photobiol 37:271–278. https://doi.or g/10.1111/j.1751-1097.1983.tb04472.x Abuin E, Aspée A, Lissi E, León L (2007) Binding of rose bengal to Gollnick K (1978) Mechanisms and kinetics of chemical reactions of bovine serum albumin. 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Applied Water ScienceSpringer Journals

Published: Apr 4, 2018

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