光动力灭活细菌中单线态氧和氧浓度的作用
The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria
鉴于细菌对抗生素的抗性增加，需要新的抗菌策略。一种有希望的技术涉及细菌的光动力学灭活。暴露在光线下时，细菌中的光敏剂会产生单线态氧，氧化蛋白质或脂质，导致细菌死亡。
为了阐明细菌杀灭过程中发生的氧化过程，将金黄色葡萄球菌与标准光敏剂一起培养，直接通过其在1,270nm处的发光检测单线态氧的产生和衰变。在低细菌浓度下，单线态氧的时间分辨发光显示出6±2μs的衰变时间，这是膜中磷脂（14±2μs）和周围水中单线态氧衰变的中间时间（3.5± 0.5μs）。显然，在低细菌浓度下，单线态氧通过扩散足以进入金黄色葡萄球菌外的水。因此，单线态氧似乎在金黄色葡萄球菌的外细胞壁区域或相邻的细胞质膜中产生。
另外，单线态氧发光的检测可以用作细胞内氧浓度的传感器。当在较高的细菌浓度下测量单线态氧发光时，由于在这些浓度下氧耗尽，衰减时间显着增加，直至≈40μs。该观察结果是氧气供应是细菌光动力灭活效果的关键因素的重要指标，并且如果该方法用于抗多种抗性细菌将具有特别重要的意义。
结论：
鉴于细菌对抗生素的抗性增加，需要新的抗菌策略。一种有希望的技术涉及细菌的光动力学灭活。暴露在光线下时，细菌中的光敏剂会产生单线态氧，氧化蛋白质或脂质，导致细菌死亡。为了阐明细菌杀灭过程中发生的氧化过程，将金黄色葡萄球菌与标准光敏剂一起培养，直接通过其在1,270nm处的发光检测单线态氧的产生和衰变。在低细菌浓度下，单线态氧的时间分辨发光显示出6±2μs的衰变时间，这是膜中磷脂（14±2μs）和周围水中单线态氧衰变的中间时间（3.5± 0.5μs）。显然，在低细菌浓度下，单线态氧通过扩散足以进入金黄色葡萄球菌外的水。因此，单线态氧似乎在金黄色葡萄球菌的外细胞壁区域或相邻的细胞质膜中产生。另外，单线态氧发光的检测可以用作细胞内氧浓度的传感器。当在较高的细菌浓度下测量单线态氧发光时，由于在这些浓度下氧耗尽，衰减时间显着增加，直至≈40μs。该观察结果是氧气供应是细菌光动力灭活效果的关键因素的重要指标，并且如果该方法用于抗多种抗性细菌将具有特别重要的意义。
Abstract
New antibacterial strategies are required in view of the increasing resistance of bacteria to antibiotics. One promising technique involves the photodynamic inactivation of bacteria. Upon exposure to light, a photosensitizer in bacteria can generate singlet oxygen, which oxidizes proteins or lipids, leading to bacteria death. To elucidate the oxidative processes that occur during killing of bacteria, Staphylococcus aureus was incubated with a standard photosensitizer, and the generation and decay of singlet oxygen was detected directly by its luminescence at 1,270 nm. At low bacterial concentrations, the time-resolved luminescence of singlet oxygen showed a decay time of 6 ± 2 μs, which is an intermediate time for singlet oxygen decay in phospholipids of membranes (14 ± 2 μs) and in the surrounding water (3.5 ± 0.5 μs). Obviously, at low bacterial concentrations, singlet oxygen had sufficient access to water outside of S. aureus by diffusion. Thus, singlet oxygen seems to be generated in the outer cell wall areas or in adjacent cytoplasmic membranes of S. aureus. In addition, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. When singlet oxygen luminescence was measured at higher bacterial concentrations, the decay time increased significantly, up to ≈40 μs, because of oxygen depletion at these concentrations. This observation is an important indicator that oxygen supply is a crucial factor in the efficacy of photodynamic inactivation of bacteria, and will be of particular significance should this approach be used against multiresistant bacteria.
The worldwide rise in bacterial resistance to antibiotics requires the development of new antibacterial strategies. Recent reports have shown that the annual rate of resistance to methicillin increased from 13% in 1986 to 28% in 2000 and is still increasing (3). In addition, the emergence of mupirocin resistance in methicillin-resistant S. aureus emphasizes the importance and urgency of developing new topical treatment alternative to the standard antibiotic treatment for skin infections (4).
Photodynamic killing of bacteria utilizes light in combination with a photosensitizer to induce a phototoxic reaction, identical to the use of photodynamic therapy for skin cancer (5–7). Various classes of chemical compounds, including phenothiazines, phthalocyanines, and porphyrines, with photoactive properties have been successfully tested as photoinactivating agents against Gram-positive and Gram-negative bacteria (8–11). Photosensitization mechanisms are initiated by the absorption of light by a given photosensitizer (12). After absorption of light, part of the energy is transferred to the triplet state in the photosensitizer molecule. Either charge (type I reaction) or energy (type II reaction) is transferred to a substrate or to molecular oxygen to generate reactive oxygen species (13). In photodynamic action, singlet oxygen (1O2) is considered to play the major role (14, 15). This highly reactive oxygen initiates further oxidative reactions in the proximate environment, such as the bacterial cell wall, lipid membranes, enzymes, or nucleic acids (16, 17). Therefore, the photodynamic inactivation of bacteria is based on the concept that a photosensitizer can accumulate to a significant extent in or at the cytoplasmic membrane, which is the critical target for irreversible damage in bacteria after irradiation (8).
To optimize photodynamic inactivation of bacteria, it is important to understand the generation and decay of singlet oxygen in bacteria. Recently, a model was developed to estimate the singlet oxygen dose by characterization of Photofrin (Axcan Pharma, Birmingham, AL) photobleaching during photodynamic therapy of eukaryotic cells in vitro (18). Moreover, singlet oxygen can be visualized by measuring its luminescence at 1,270 nm directly in solvents (19, 20), in eukaryotic cells (21, 22), and even in human skin (23). This time-resolved decay of the luminescence should provide an indication of the site of singlet oxygen in bacteria. In addition, the generation of singlet oxygen is very sensitive to oxygen concentration, which may play a role in the photodynamic inactivation of bacteria comparable to that in eukaryotic cells (24). The experiments were carried out with Photofrin, one of the most widely used and clinically approved photosensitizers in photodynamic therapy, which also demonstrates antimicrobial efficacy (25–27).
Results
Microscopic Images at Different Bacterial Densities.
Microscopic images (spatial resolution ≈1 μm) were captured of suspensions of S. aureus in water, corresponding to protein concentrations of 0.15–1.5 mg/ml. Fig. 1 A shows some isolated bacteria and small agglomerates of bacteria at a concentration of 0.15 mg/ml. Increasing the protein concentration to 0.25 mg/ml results in larger agglomerates of bacteria (Fig. 1 B). When the protein concentration is further increased (Fig. 1 C), the agglomerates become substantially larger and have a very large extension at a protein concentration of 1.5 mg/ml, filling almost all of the space (100 × 100 μm2) on the slide (Fig. 1 D).
Fig. 1.
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Fig. 1.
Microscopic images of S. aureus at various concentrations in suspension. Suspensions of S. aureus in H2O corresponding to protein concentrations between 0.15 and 1.5 mg/ml were examined by light microscopy. The images show S. aureus at 0.15 mg/ml (A), 0.25 mg/ml (B), 1 mg/ml (C), and 1.5 mg/ml (D).
Photofrin Uptake by Bacteria.
After incubation of S. aureus suspensions with Photofrin, lysed bacterial pellets showed a clear uptake of Photofrin depending on the bacterial concentration used (Fig. 2). Using the Michaelis–Menten equation, we detected a dependence of the concentration of Photofrin in bacteria on the concentration of Photofrin during incubation (90 min) of the bacterial suspension, indicating an active uptake of Photofrin. In contrast to S. aureus, no uptake of Photofrin was seen in E. coli after incubation with different concentrations of Photofrin (data not shown). Bacteria incubated at 37°C without Photofrin served as a control and showed no absorbance (data not shown).
Fig. 2.
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Fig. 2.
Uptake of Photofrin by S. aureus incubated with concentrations of 50, 100, 200, 400, or 800 μg/ml Photofrin for 90 min. Absorption spectra of Photofrin uptake were recovered after lysis of the corresponding bacterial pellets. The solid line was fitted to the experimental data points by using the Michaelis–Menten equation.
Luminescence Experiments.
A suspension with a constant concentration of S. aureus (0.15 mg/ml protein) was incubated with 0.3 mg/ml Photofrin and subsequently excited with 60 × 103 laser pulses (pulse duration 70 ns; pulse energy 75 μJ). A clear luminescence signal of singlet oxygen was detected (Fig. 3 A). The fit of the luminescence signal (Eq. 1 from Materials and Methods and solid line in Fig. 3) yielded a signal rise with a time constant of 1.0 ± 0.5 μs, which is determined by the lifetime of the triplet T1 state of Photofrin in and aqueous suspension of S. aureus. The luminescence decay time was 6 ± 2 μs. By adding 50 nM sodium azide, a specific quencher of singlet oxygen, to the S. aureus aqueous suspensions, the decay time of singlet oxygen decreased to 3 ± 1 μs (Fig. 3 B). In the experiment shown in Fig. 4, suspensions of S. aureus (0.15 mg/ml protein) incubated with 0.3 mg/ml Photofrin were excited, and the luminescence of singlet oxygen was measured at various wavelengths (1,150–1400 nm). The results show a clear luminescence maximum at 1,270 nm. No singlet oxygen signal was detected when E. coli was incubated with Photofrin and irradiated (data not shown).
Fig. 3.
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Fig. 3.
Luminescence signal at 1,270 nm for S. aureus in suspension. Shown are results for an S. aureus suspension (0.15 mg/ml protein/0.3 mg/ml Photofrin) in H2O without NaN3 (A) and with 50 mM NaN3 (B). The solid curves were fitted to the experimental data points by using Eq. 1 .
Fig. 4.
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Fig. 4.
Luminescence signal of singlet oxygen at various wavelengths, measured in the range 1,150–1,400 nm. Each signal was generated by 0.3 mg/ml Photofrin in suspensions of 1.5 mg/ml protein (S. aureus) in H2O.
Detection of Singlet Oxygen Luminescence at Various Bacterial Protein Concentrations.
Different concentrations of S. aureus were incubated with a constant Photofrin concentration (0.3 mg/ml). Fig. 5 shows the dependence of the decay rate of singlet oxygen luminescence on the concentration of proteins of S. aureus in suspension. At high protein concentrations (>1.0 mg/ml), the decay rate achieves a limiting value. Eq. 2 (see Materials and Methods) was fitted to the results in Fig. 5 to obtain a value of k im = 0.16 ± 0.05 μs−1 for the intermediate decay rate of luminescence in isolated bacteria in water. The rate saturates at k b = 0.025 ± 0.010 μs−1 for very large agglomerates of bacteria in water. The corresponding decay times are τim = 6 ± 2 μs (low bacterial concentration) and τb = 40 ± 16 μs (high bacterial concentration), respectively. For these experiments (Fig. 5, filled circles), bacteria were irradiated with 60 × 103 laser pulses, resulting in a total applied energy of 4.5 J (75 μJ per pulse).
Fig. 5.
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Fig. 5.
Relaxation rate k D of luminescence for various concentrations of proteins of S. aureus in suspension (0.3 mg/ml Photofrin) for 60,000 pulses. The dashed line was fitted to the experimental data points by using Eq. 2 . (Inset) Decay time, τD, for 8,000 (open square), 20,000 (open triangle), 60,000 (filled circle), or 80,000 (open diamond) pulses at a constant protein concentration of 1.5 mg/ml.
Additionally, different numbers of pulses were applied at a constant protein concentration of 1.5 mg/ml. The energy applied to the bacteria ranged from 0.6 to 6 J. The decay time of luminescence clearly increased with increasing number of pulses, from 18 ± 4 μs (8,000 pulses), 22 ± 5 μs (20,000 pulses), and 30 ± 6 μs (60,000 pulses) to 39 ± 8 μs (80,000 pulses) (Fig. 5 Inset).
Measurement of Oxygen Depletion in Aqueous Bacterial Suspension.
S. aureus at protein concentrations of 0.15 or 1.5 mg/ml was incubated with 0.3 mg/ml Photofrin in suspension. The respective suspensions were transferred to a cuvette, and the oxygen saturation (in percent) was measured by using a standard oxygen electrode placed in the suspension (Fig. 6). At the beginning of oxygen measurement, oxygen saturation was measured in a range of 94–99% and decreased to 90% at high protein concentrations (1.5 mg/ml protein) or remained almost unchanged (98%) at low protein concentrations (0.15 mg/ml protein) after 40 s as a result of normal oxygen consumption by the bacteria.
Fig. 6.
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Fig. 6.
Oxygen depletion of a Photofrin-incubated S. aureus suspension measured before, during, and after irradiation by using a standard oxygen electrode. Shown are values for the high (black lines) and low (gray lines) bacterial concentrations. The vertical lines represent luminescence detection. Measurements were made with the laser switched off after luminescence detection (60,000 pulses) (solid lines) and with the irradiation extended up to 8 min (dashed lines).
Subsequently, the exciting laser was switched on at 532 nm, and singlet oxygen luminescence was detected and summed for 30 s. During this period, oxygen saturation decreased from 98% to 93% for the low protein concentration (0.15 mg/ml) and dropped from 90% to 60% at the high protein concentration (1.5 mg/ml). Then, two different procedures were carried out. First, the laser was switched off after luminescence detection. The oxygen saturation remained constant at 93% at 0.15 mg/ml protein concentration, whereas at the high protein concentration (1.5 mg/ml) the oxygen saturation showed a slight increase but decreased to 46% after 8 min. Second, the laser was not switched off after luminescence detection. In this case, oxygen saturation decreased to 82% after 8 min for the low protein concentration (0.15 mg/ml), whereas for the high protein concentration (1.5 mg/ml) the detected value of oxygen reached 0%. Furthermore, the phototoxicity against Photofrin-incubated S. aureus was measured upon laser irradiation (6 min), depending on the bacterial concentration used (Table 1). After irradiation, viability was found to be reduced to 0.1 × 104 ml−1 (20-fold) for a low bacterial concentration (2.0 × 104 ml−1) and to 1.0 × 108 ml−1 (8-fold) for a high bacterial concentration (8.0 × 108 ml−1).
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Table 1.
Survival of S. aureus at low and high concentrations upon laser irradiation
Discussion
The worldwide increase in antibiotic-resistant bacteria compels researchers to develop new antibacterial strategies. One promising technique is the photodynamic inactivation of bacteria, in which the generation of singlet oxygen plays a major role (28, 29). To enhance this approach, we investigated the generation and decay of singlet oxygen in bacteria.
Singlet oxygen was detected by its time-resolved luminescence, yielding a time-dependent luminescence curve. The course of the luminescence curve is very sensitive to parameters such as the diffusion of singlet oxygen and the oxygen concentration. By fitting the luminescence curve to Eq. 1 , we determine the rise time (τR) and decay time (τD) of luminescence. Because singlet oxygen and the photosensitizer form a system coupled by energy transfer, the luminescence curve at 1,270 nm comprises both the decay rate of photosensitizer triplet state (T1) and the decay rate of singlet oxygen. For high oxygen concentrations, the rising portion of the luminescence curve (τR) represents the decaying of the photosensitizer triplet state (phosphorescence), and the decaying portion of curve (τD) represents the decay time of singlet oxygen. The meaning of these rates and decay times is reversed at low oxygen concentrations.
The generation of singlet oxygen requires a sufficient uptake of Photofrin by bacteria, which was investigated for two different types of bacteria. The uptake of Photofrin was studied quantitatively in S. aureus and E. coli as representative Gram-positive and Gram-negative bacteria species, respectively. S. aureus showed a clear uptake of Photofrin, whereas no uptake could be detected in E. coli because of the difference in the outer cell wall structures of these species. This finding is in accordance with previously published data (30, 31), which demonstrated a complete resistance of Gram-negative bacteria to the photodynamic effects of porphyrins with no membrane-disorganizing agents. The peptidoglycan layer of the bacterial cell wall of S. aureus has a much higher permeability (e.g., for antibiotics) than does the outer membrane of Gram-negative bacteria (32). Consequently, significant concentrations of Photofrin reach the cytoplasmic membrane of Gram-positive bacteria and remain in the bacteria. In contrast, Gram-negative bacteria have an additional outer membrane consisting of a lipid bilayer with an outer leaflet of lipopolysaccharide outside of the peptidoglycan layer (33). Nevertheless, Nitzan et al. (34) demonstrated that different types of porphyrin derivatives (e.g., deuteroporphyrin IX) are able to efficiently inactivate even Gram-negative bacteria (Pseuodmonas spp.) with no cell wall-disturbing molecules, depending on the binding capacities to the outer membrane of the tested porphyrins. These authors found a correlation between the extent of deuteroporphyrin binding and the observed photodynamic effect. However, the molecular weight of deuteroporphyrin is only 583.51, which may allow easier penetration of the outer membrane barrier than with Photofrin (molecular weight 1,135) (35–37).
Singlet Oxygen Luminescence for Constant and Low Bacterial Concentrations.
Corresponding to the remarkable uptake of Photofrin, S. aureus in aqueous suspension showed a clear luminescence signal at 1,270 nm when Photofrin was excited by laser light. The decay time of the luminescence was ≈6 μs (Fig. 3 A). When 50 mM sodium azide, a specific quencher of singlet oxygen, was added to the suspension, the luminescence intensity decreased and the decay time was shortened to 3 μs (Fig. 3 B). Moreover, a definite proof for singlet oxygen is the spectrally resolved detection of the luminescence signals. We measured a clear luminescence maximum at 1,270 nm, corresponding to the transition of singlet oxygen to its ground state (Fig. 4). No singlet oxygen signal was detected in E. coli because of the low uptake of Photofrin, which is in agreement with published data (25, 38).
Localization of Singlet Oxygen and Photofrin.
The subcellular localization of a photosensitizer is usually assessed by fluorescence techniques. However, in view of the small size of bacteria, such assessment is difficult because of the limited spatial resolution of fluorescence microscopy. It was shown in eukaryotic cells (HT29) that singlet oxygen can decay inside the membrane or can escape into the surrounding water after being generated by Photofrin in the plasma membrane (22, 39). The decay time was 10 μs, which was an intermediate time for singlet oxygen decay in water (3.5 μs) and in phospholipids (14 μs) of the cell membrane.
After incubation of S. aureus with Photofrin, the luminescence at 1,270 nm also decayed monoexponentially, with an intermediate decay time of 6 μs (Fig. 3 A). This time is shorter than for HT29 cells (10 μs) but still falls between the decay time in water (3.5 μs) and that in phosphatidylcholine (14 μs). However, the decay time of 6 μs is closer to that for water than to that for lipids, suggesting a strong influence of the quenching capacity of water on singlet oxygen molecules. Therefore, many singlet oxygen molecules might have escaped into the surrounding water. This assumption is reasonable for the generation of singlet oxygen within the membranes of bacteria. The membrane of S. aureus is ≈0.2 μm in thickness, which is in the range of the diffusion length of singlet oxygen in cellular membranes (0.3 μm) (22). Furthermore, at the low protein concentration of 0.15 mg/ml (equivalent to a small number of bacteria), most S. aureus can be found as single bacteria surrounded by water (Fig. 1 A). This observation supports the suggestion that singlet oxygen was generated close to, or in, the plasma membrane of S. aureus, which might then be the localization of Photofrin.
Singlet Oxygen Luminescence for Variable and High Bacterial Concentrations.
In clinical practice, the photodynamic procedure must deal with varying bacterial concentrations. Therefore, S. aureus concentrations ranging from 0.15 to 1.5 mg/ml were incubated with a constant photosensitizer concentration (0.3 mg/ml). The increase in protein concentration is proportional to the increase in the density of bacteria in the aqueous suspension (Fig. 1), which leads to large agglomerates of bacteria, in particular at the high bacterial concentration (Fig. 1 D). Surprisingly, the relaxation rate, k D, of luminescence decreased with increasing protein concentrations (Fig. 5). Specifically, the decay time of the singlet oxygen luminescence signal increased up to 40 μs at the highest protein concentration. Thus, it can be stated that (i) the generation of singlet oxygen requires sufficient oxygen concentration (19), and (ii) singlet oxygen can diffuse through different environments, such as bacteria and water.
Singlet Oxygen Diffusion.
At a low bacterial concentration (0.15 mg/ml), most of the cells are isolated and surrounded by water (Fig. 1 A). In this situation, many singlet oxygen molecules can escape into water, leading to a strong quenching effect. Therefore, we expect and observe an intermediate decay rate, k im, of luminescence, which corresponds to a decay time close to the value in water (see explanation above).
With increasing bacterial concentrations, bacteria begin to agglomerate, leading to a displacement of water between bacteria. At the highest concentration, most of the bacteria seem to be directly surrounded by other bacteria and not by water (Fig. 1 D). Thus, singlet oxygen diffuses from one bacterium to the next. Therefore, the strong quenching of singlet oxygen by water is reduced, and singlet oxygen decays predominantly inside the bacteria. This may lead to a decay rate approaching a k b (Fig. 5) of 0.025 ± 0.010 μs−1, which corresponds to a luminescence decay time of τb = 40 ± 16 μs in the bacteria.
However, such a long decay time would require an environment for singlet oxygen with a very low quenching efficacy. So far, only lipids of cellular membranes such as phosphatidylcholine offer a long decay time (14 μs) (22). Because this decay time of luminescence is longer than expected for any bacterial constituents, diffusion alone cannot be responsible for such long decay times. This leads us to assume that oxygen depletion is occurring inside the bacteria.
Oxygen Depletion.
It is well known that the generation of singlet oxygen can lead to peroxidation of cellular constituents such as proteins and lipids (40, 41). If such oxygen-consuming processes occur during photodynamic action, the oxygen concentration may decrease during irradiation. With decreasing oxygen concentration, the quantum yield of singlet oxygen is also decreasing (42). Therefore, the light excitation of photosensitizers in photodynamic therapy of cancer is fractionated by using irradiation breaks of several minutes to allow oxygen supply to tumor areas (24, 43–45). This procedure was also used with biofilms of Streptococcus mutans, which were efficiently inactivated by light dose fractionation (46).
The water in the bacterial suspension was fully aerated before irradiation (100%), which is equivalent to an oxygen concentration of ≈150 Torr. However, because of oxygen consumption by the bacteria, the starting values of oxygen saturation in our experiments were <100% and continued to decrease slowly for the first 40 s. After that, the laser was switched on and the singlet oxygen luminescence was measured for 30 s. During this period, the oxygen concentration in the suspension dropped ≈5% for the low, and ≈34% for the high, bacterial concentrations. This finding is not surprising because oxygen is consumed by oxidative reaction in the bacteria, induced by singlet oxygen. The oxygen concentration in the environment surrounding the bacteria, as measured by our oxygen sensor, is important for effective photodynamic killing of the bacteria. Much more important is the oxygen concentration at the site of singlet oxygen generation, which is the localization of a photosensitizer (e.g., Photofrin) inside the bacteria. There, the oxygen concentration should be lower than in the surrounding water. For comparison, in eukaryotic cells the oxygen concentration is ≈6 Torr (47). In the case of singlet oxygen generation, the oxygen consumed by singlet oxygen occurs inside the bacteria, where our oxygen sensor is unable to detect the oxygen concentration. To overcome this problem, we can use the luminescence signal itself because it is sensitive to the local oxygen concentration.
Our luminescence signal comprises both the triplet state deactivation and the singlet oxygen luminescence. The decay time of singlet oxygen is independent of the oxygen concentration and cannot be used in this approach; however, the triplet state of the photosensitizer (Photofrin) is oxygen-sensitive. With a high oxygen concentration, this triplet state is efficiently deactivated by oxygen, leading to the generation of singlet oxygen, and only a few Photofrin molecules decay in its ground state, emitting phosphorescent light. Thus, the decay time of the triplet state is very short (≈1 μs) and can be identified as the rise time of our luminescence signal at 1,270 nm.
With decreasing oxygen concentration, the deactivation of the triplet state by oxygen decreases and an increasing number of Photofrin molecules return to their own ground state. This leads to prolonged phosphorescence and an increase in the decay time of the triplet state. In the absence of oxygen, the luminescence signal measured is equivalent to the triplet emission of Photofrin, which is ≈200 μs (15). Thus, when we measure the time-resolved luminescence, the signal contains information about the oxygen concentration at the site of singlet oxygen generation, as either rise or decay time.
At the lowest bacterial concentration, most of the bacteria are surrounded by water and there should be only a minor problem with oxygen supply if oxygen is consumed as a result of oxidative processes inside the bacteria. Thus, the decaying portion of luminescence should be attributed to the decay time of singlet oxygen (48).
However, with increasing bacterial concentration, the bacteria form large agglomerates, which hampers the oxygen support to all bacteria inside the agglomerates. When bacteria are irradiated in these agglomerates, the oxygen diffusion may be insufficient to compensate for the oxygen depletion during irradiation. Consequently, the oxygen concentration should decrease in the bacteria. As mentioned above, this oxygen decrease should lead to an increase in triplet decay time. At low oxygen concentrations, this triplet decay time can be identified as the decay time in our luminescence signal (τD = 1/k D). In fact, with increasing bacterial concentration, k D is decreasing, which is equivalent to an increase in τD (Fig. 5). This result can explain the quite extended decay time of the measured luminescence signal (≈40 μs) (Fig. 5). Because of this long-lasting phosphorescence signal, there is presumably no strong competitive process for triplet T1 deactivation in Photofrin, such as charge transfer (type I reaction, oxygen radicals).
To confirm this perception, conditions of low or high oxygen depletion in bacteria were created by exciting the photosensitizer for different durations. Low and high numbers of excitation pulses (8,000–80,000 pulses) were applied at the highest bacterial concentration (1.5 mg/ml protein). In fact, the higher the number of pulses, the higher the oxygen depletion and, therefore, the longer the decay time of the luminescence signal (Fig. 5 Inset). These results demonstrated that strong oxygen depletion takes place in the bacteria of agglomerates during irradiation, which should affect the efficacy of photodynamic inactivation of bacteria.
When we extended the measurement of oxygen saturation up to 8 min, the value for low protein concentration was nearly constant and decreased slightly for high concentrations, which reflects the normal oxygen consumption by living bacteria (Fig. 6, solid line). In the case where the laser was not switched off after luminescence detection, slight oxygen depletion was detectable for the low bacterial concentration and reached 0% for the high concentration (Fig. 6, dashed line). Thus, remarkable oxygen consumption occurs as a result of the generation of singlet oxygen in bacteria, leading to a decrease in oxygen concentration in the entire bacterial suspension.
Overall, the crucial point in efficient photodynamic killing of bacteria seems to be the oxygen concentration inside the bacteria at a given bacterial concentration during the irradiation process. If bacteria grow in agglomerates or biofilms with an insufficient supply of oxygen during photodynamic inactivation, efficacy in bacterial killing should be considerably attenuated. Indeed, when the viability of S. aureus was measured after an irradiation period of 6 min, the cfus decreased for the low bacterial concentration by a factor 20 but for the high bacterial concentration only by a factor 8. This finding additionally confirms the better oxygen supply for the bacteria surrounded by water (Fig. 1 A) than for bacteria in agglomerates (Fig. 1 D).
Conclusions
In this study, singlet oxygen was measured directly by its luminescence inside living bacteria. When looking at small bacterial concentrations, the luminescence decay time is an intermediate time for singlet oxygen in water and in bacteria. Therefore, it seems plausible that the luminescence signal can be attributed to singlet oxygen decaying in the cell wall and membrane areas of S. aureus. Moreover, the detection of singlet oxygen luminescence can be used as a sensor of intracellular oxygen concentration. At higher bacterial concentration, the decay time of singlet oxygen luminescence significantly increased (up to ≈40 μs) as a result of oxygen depletion at high bacterial concentrations. This observation is important because oxygen supply is a crucial factor that influences the efficacy of antibacterial photodynamic therapy for the inactivation of multiresistant pathogens.
Materials and Methods
Bacteria Culture.
The bacterial strains S. aureus (ATCC 25923) or E. coli (ATCC 25922) were grown aerobically at 37°C in brain heart infusion (BHI) broth (Gibco Life Technologies, Eggenstein, Germany). A 500 μl portion of an overnight cell culture (3 ml) was transferred to 50 ml of fresh BHI medium and grown at 37°C on a shaker. When the cultures reached the stationary phase, the cells were harvested by centrifugation (200 × g, 15 min). The cells were then washed with 10 mM PBS (Biochrom, Berlin, Germany) at pH 7.4, containing 2.7 mM KCl and 0.14 M NaCl and suspended in PBS at an optical density of 0.7 (650 nm), corresponding to 108 bacteria per milliliter, for phototoxicity experiments.
Uptake of Porphyrin by Bacteria.
The uptake of cell-bound Photofrin, after three washing steps, was estimated by spectroscopic analysis (49). Suspensions of S. aureus or E. coli were incubated in the dark for 90 min with 50, 100, 200, 400, and 800 μg/ml Photofrin at 37°C, centrifuged (200 × g, 10 min), and then washed triply with PBS. The bacterial pellets were lysed by resuspending in 2% aqueous SDS and kept overnight under gentle magnetic stirring. The suspension was then centrifuged for 5 min (200 × g) to remove bacterial protein impurities, and the supernatants of each probe were collected. The absorption spectra of each probe were recorded at room temperature with a spectrophotometer (DU640; Beckman Instruments, Munich, Germany). Bacteria without Photofrin served as a control. Values were normalized to control samples of bacteria without Photofrin incubation.
Phototoxicity of Bacteria.
The survival of bacteria was determined by counting cfus, as described in detail elsewhere (28).
Oxygen Concentration in Aqueous Bacterial Suspension.
An S. aureus (1.5 mg/ml) suspension was incubated with 0.3 mg/ml Photofrin for 90 min. After incubation, the suspension was washed triply with PBS to remove unbound Photofrin. The aqueous/air-saturated bacterial suspension was transferred to the cuvette, and a needle sensor was placed in the cuvette to measure oxygen concentration (MICROX TX; PreSens, Regensburg, Germany). The cuvette was sealed, and the oxygen concentration was determined by measuring partial vapor pressure (percentage air saturation).
Luminescence Experiments.
The protein concentration of bacteria was measured by using the BCA protein assay (Pierce, Rockford, IL) in accordance with the manufacturer's protocol. Suspensions of different bacterial concentrations (protein concentration 0.15–1.5 mg/ml) were incubated with 0.3 mg/ml Photofrin for 90 min. After three washings with PBS, bacteria with only cell-bound Photofrin were resuspended in PBS in presence or absence of 50 mM sodium azide (quencher of singlet oxygen). Thereafter, the suspensions were transferred into a cuvette (QS-101; Hellma Optika, Jena, Germany). The sensitizer was excited by using a frequency-doubled Nd:YAG laser (PhotonEnergy, Ottensoos, Germany) with a repetition rate of 2.0 kHz (wavelength 532 nm; pulse duration 70 ns; pulse energy 75 μJ). The singlet oxygen luminescence was detected in near-backward direction with respect to the excitation beam, using an IR-sensitive photomultiplier (R5509–42; Hamamatsu Photonics Deutschland, Herrsching, Germany), as detailed in ref. 22. The luminescence signal was detected at wavelengths of 1,150, 1,200, 1,250, 1,270 (emission maximum of singlet oxygen luminescence), 1,300, 1,350, or 1,400 nm by using appropriate interference filters in front of the photomultiplier. All experiments were performed by summing five repeated single experiments, always using a fresh suspension. In the single experiments, the numbers of pulses were 8 × 103, 20 × 103, 60 × 103, or 80 × 103.
Determination of Singlet Oxygen Luminescence Decay Time.
The time resolution of the complete detection system (photomultiplier and detection electronics) is ≈128 ns. The error bars of the rise and decay times of the luminescence in the present experiments are on a microsecond scale because of the signal-to-noise ratio of the weak luminescence.
Because the different environments of singlet oxygen and the oxygen concentration determine the luminescence signal, multiexponential luminescence decay can occur. When the diffusion length of singlet oxygen is large enough during its lifetime, singlet oxygen distribution in different compartments is established. In this case, the signal decay is monoexponential, with an intermediate decay time (1). The main constituents of the investigated suspensions are H2O, lipids, and proteins. Proteins yield a very short decay time because of their high quenching rate (2). In proximity to lipids, contributions to singlet oxygen luminescence were expected to show a decay time of ≈14 ± 2 μs, whereas the decay time is 3.5 ± 0.5 μs in H2O (22).
In our experiments, an exponential rise and decay of luminescence was observed. The luminescence intensity is given by
Embedded Image
where the constant C was used to fit the luminescence signal, and τD and τR are the decay and rise times, respectively. To determine the rise and decay times, the least-square fit routine of Mathematica (version 4.2; Wolfram Research, Berlin, Germany) was used, with an experimental error of ≈20%.
Given the pair of time constants, τD and τR, it is sometimes difficult to decide which time is due to the deactivation of the triplet state T1 and which is due to singlet oxygen luminescence (48). Additionally, the theory must be extended when bidirectional transfer takes place, as shown for Photofrin (15). There, the rise (β1 = 1/τR) and decay (β2 = 1/τD) rates are connected with two decay rates: K T, which is responsible for the relaxation of the T1 state of the photosensitizer, and K Δ, which is responsible for the relaxation of the singlet oxygen state. For low oxygen concentrations, the decay rate β2 can be equated with K T, which represents the decay rate of the photosensitizer triplet T1 state. For high oxygen concentrations, decay rate β2 can be equated with the relaxation rate of singlet oxygen, K Δ (15, 48).
Decay Rate of Luminescence vs. Protein Concentrations.
The phenomenological expression for the decay rate k D of luminescence in a suspension of S. aureus in water is present as a function of protein concentration c pr. For protein concentrations c pr > c pr (1), where c pr (1) = 0.15 mg/ml, the decay rate is given by
Embedded Image
where k im and k b are fit parameters that represent the intermediate decay rate for isolated bacteria in water (low protein concentrations, small density of bacteria) and the decay rate in bacteria, respectively, and where k b is the limiting value for agglomerates of bacteria in water (high protein concentration, high density of bacteria).
SOURCE:
Tim Maisch, Jürgen Baier, Barbara Franz, Max Maier, Michael Landthaler, Rolf-Markus Szeimies, and Wolfgang Bäumler
PNAS April 24, 2007 104 (17) 7223-7228; https://doi.org/10.1073/pnas.0611328104
Department of Dermatology and
†Institute of Experimental and Applied Physics, University of Regensburg, 93042 Regensburg, Germany
The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria | PNAS http://www.pnas.org/content/104/17/7223