1 Department of Microbiology and 2 Cellular Microbiology Research Group, Eastman Dental Institute for Oral Health Care Sciences, University College London, 256 Grays Inn Road, London WC1X 8LD, UK; 3 Laboratory of Hospital Infection, Central Public Health Laboratory, Colindale, London
Received 6 February 2002; returned 5 July 2002; revised 10 August 2002; accepted 20 August 2002
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Abstract |
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Methods. Various strains of MRSA were exposed to light from a helium/neon laser in the presence of an IgGSnCe6 conjugate and the survivors enumerated by viable counting. Controls consisted of suspensions irradiated in the presence or absence of the conjugate and suspensions kept in the dark in the presence of the conjugate. Similar experiments were also carried out using the unconjugated photosensitizer. The experiments were repeated using a suspension consisting of both EMRSA-16 and Streptococcus sanguis.
Results. EMRSA-16 was killed by IgGSnCe6 and SnCe6 in a light-dose- and photosensitizer-dependent manner. Greater kills were achieved with the IgGSnCe6 than with the unconjugated SnCe6 using the same light energy dose and photosensitizer concentration. Furthermore, the IgGSnCe6 conjugate, but not SnCe6, was able to kill EMRSA-16 selectively in a suspension that also contained S. sanguis without any reduction in the viable count of the latter.
Conclusion. These results demonstrate that selective lethal photosensitization of MRSA can be achieved using an IgGtin (IV) chlorin e6 conjugate. The effectiveness of killing was dependent, in part, on the particular MRSA strain used, with the clinically important EMRSA-16 strain being the most susceptible.
Keywords: MRSA, tin (IV) chlorin e6, IgG-binding protein A
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Introduction |
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One possible approach is photodynamic therapy (PDT): the use of light-activated antimicrobial agents to achieve lethal photosensitization of the infecting organism.68 Basically, this involves treating the microbe with a light-activated chemical, termed a photosensitizer. Alone, a photosensitizer generally has no, or negligible, bactericidal activity. However, upon exposure to light of a wavelength absorbed by the photosensitizer, the photosensitizer undergoes a transition from a lower-energy ground state to a higher-energy triplet state. The triplet state photosensitizer can react with solvent and substrate molecules to produce free radicals and radical ions, or with molecular oxygen resulting in the generation of singlet oxygen.9 Such reactive species, in particular singlet oxygen, can damage plasma membranes and DNA, leading to cell death.10,11 The killing of bacteria in this way has been demonstrated with a wide variety of Gram-positive and Gram-negative organisms, e.g. S. aureus, including MRSA,10,1215 streptococcal species,16,17 Escherichia coli16 and Porphyromonas gingivalis.11,18
Photodynamic therapy could be a useful and simple way of treating and controlling MRSA infections and would have two major advantages over conventional antiseptics and antimicrobials. First, as neither of the two components of the system (light and sensitizer) is inherently bactericidal, the antibacterial effect would be limited to light-irradiated regions of the sensitizer-treated area, avoiding the disruption of the normal microflora at sites other than those being targeted. Secondly, and perhaps most importantly, the development of resistance to photochemically induced killing, which is mediated predominantly by singlet oxygen, would be unlikely.
Nevertheless, there are potential problems associated with PDT of infectious diseases, one of which is a lack of specificity. The photosensitizer may bind to, or be taken up by, other cells e.g. commensal microorganisms or host cells. Since subsequent irradiation of such cells could lead to their destruction, it is desirable to direct the photodynamic activity to the target cell. This could be achieved by linking the photosensitizing molecule to an antibody against the surface of the target organism. Such specific targeting is aided by the fact that the main bactericidal species, singlet oxygen, generated by the irradiation of the photosensitizer, has a very short lifetime and a limited diffusion distance of 100 nm. The antibody-targeting approach has been used successfully to target specifically different photosensitizers to a variety of bacteria: tin (IV) chlorin e6 to Pseudomonas aeruginosa,19,20 toluidine blue O (TBO) to Porphyromonas gingivalis21 and bacteriochlorophyll to S. aureus.22
In this study, we aimed to direct a suitable photosensitizer to S. aureus, including MRSA, while retaining the photodynamic activity of the molecule. Previous studies have shown that S. aureus is sensitive to lethal photosensitization using a variety of photosensitizers, e.g. porphyrins, thiazin dyes, phthalocyanines and chlorins.10,1214,17,23,24 Since the HeNe laser we used in this study emits red light with a wavelength of 632.8 nm, it was essential to choose a photosensitizer that would produce high yields of singlet oxygen when exposed to such light. In addition, it was necessary to select a photosensitizer with side chains that would enable conjugation to targeting molecules. Tin (IV) chlorin e6 (SnCe6) fits both of these criteria; it absorbs light at 632 nm and has an improved yield of singlet oxygen over related chlorins,25 while possessing reactive carboxyl groups on side chains outside the polycyclic core. The structure of SnCe6 is shown in Figure 1.
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Materials and methods |
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The organisms used in this investigation were the prototypic UK epidemic MRSA, EMRSA-1 (NCTC 11939); the three currently prevalent EMRSA, EMRSA-3 (NCTC 13130), EMRSA-15 (NCTC 13142) and EMRSA-16 (NCTC 13143); a methicillin-susceptible strain of S. aureus (NCTC 6571); and Streptococcus sanguis (NCTC 7863). Strains were maintained by weekly subculture on Columbia Agar (Oxoid Ltd, Basingstoke, UK) supplemented with 5% (v/v) horse blood. For experimental purposes, a few colonies were inoculated into tryptone soya broth (Oxoid): S. aureus strains were grown aerobically for 16 h at 37°C, S. sanguis was grown for 16 h at 37°C in a 5% CO2 cabinet. Cells were then harvested by centrifugation and resuspended in sterile phosphate-buffered saline (PBS) to an OD600 of 0.4. For growth-phase experiments, 100 mL of tryptone soya broth was inoculated with 0.25 mL of an overnight culture and growth at 37°C monitored by measuring OD600. Cells were harvested at lag (OD600 0.05), exponential (OD600 0.55) and stationary phase (OD600 2.3).
Laser and photosensitizer
The laser used in this study was a Model 127 Stabilite heliumneon laser (Spectra Physics, Mountain View, CA, USA), with a measured output of 35 mW, which emits light in a collimated beam (diameter 1.25 mm) with a wavelength of 632.8 nm. The photosensitizer, tin (IV) chlorin e6 (SnCe6) was purchased from Frontier Scientific (Lancashire, UK).
Preparation of the IgGSnCe6 conjugate
SnCe6 was conjugated to IgG exploiting the carboxyl side chains on the SnCe6 molecule (Figure 1), and amino groups in IgG, adapting the method described in Bhatti et al.21 Basically, 2 mg of SnCe6 was dissolved in 1.5 mL conjugation buffer [0.1 M 2-(n-morpholino)ethanesulphonic acid pH 6.0, 0.5 M NaCl] along with 2 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) (Sigma, Poole, UK) and 5 mM sulpho-N-hydroxysuccinimide (S-NHS) (Pierce, Cheshire, UK). EDAC reacts with the carboxyl groups of SnCe6 in the presence of S-NHS to form a reactive ester. After 4 h at room temperature, the reaction was quenched by the addition of ß-mercaptoethanol to 20 mM. The treated SnCe6 (1.5 mL) was then added to 2 mL of a 4 mg/mL solution of rabbit IgG (Sigma) in PBS and the pH adjusted to 7.6 to allow the amine-reactive intermediate to react with amine groups of the antibody. Following a 1620 h incubation at room temperature, the conjugation reaction was terminated by the addition of ethanolamine to 20 mM. The resulting IgGSnCe6 conjugate was purified from any unconjugated SnCe6 by affinity chromatography using Protein A Hi-Trap columns (Amersham Pharmacia Biotech, Buckinghamshire, UK). The purified IgGSnCe6 was dialysed in PBS. The amount of SnCe6 in the conjugate was measured by absorbance at 633 nm and IgG by absorbance at 280 nm. The molar ratio of IgG:SnCe6 varied from batch to batch, but was, on average,
2:1. The concentration of the conjugate is expressed in terms of the concentration of SnCe6 in the conjugate.
Lethal photosensitization
Aliquots (100 µL) of a suspension of S. aureus (containing between 1 x 109 and 1 x 1010 cfu/mL) in sterile PBS, were placed into a 96-well microtitre plate. To duplicate wells, 100 µL of either SnCe6 or IgGSnCe6 was added. Unless otherwise stated, the samples treated with SnCe6 were pre-incubated for 1 min at room temperature. Samples treated with IgGSnCe6 were pre-incubated for 15 min at room temperature to allow the conjugate to bind to the cells. Samples were then exposed to a measured amount of laser light as stated, where a light dose of 21 J/cm2 corresponds to a 5 min exposure, and stirred continuously during this time. Control wells were neither sensitized nor exposed to the light source. Following exposure to light, 100 µL aliquots were taken and added to 900 µL tryptone soya broth (Oxoid). Serial dilutions were made, and 50 µL aliquots (in duplicate) from each dilution were plated on to Columbia agar, and grown overnight at 37°C to enumerate survivors. For targeting experiments using S. sanguis, either 100 µL of S. sanguis or 50 µL of both EMRSA and S. sanguis were placed into the wells and the experiments repeated as before. Serial dilutions of light-treated samples were plated on to tryptone soya agar (Oxoid) and grown at 37°C in a 5% CO2 cabinet for 16 h to enumerate S. aureus survivors and for 40 h to enumerate S. sanguis survivors. The two strains were distinguishable on the basis of colony size and appearance.
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Results |
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When S. aureus NCTC 6571 was treated with 25 µg/mL SnCe6 and exposed to 8.4 J/cm2 of red light from the HeNe laser (noted as L+S+ for exposure to light and exposure to sensitizer), a 2.5 log10 reduction in the viable count was achieved when compared with a control sample that was neither sensitized nor irradiated (noted as LS) (Figure 2). This amounted to 99.7% of the cells being killed (6.75 x 109 cfu/mL). Cells treated with SnCe6 but not irradiated (LS+), or those not treated with SnCe6 but exposed to red light (L+S) did not show a significant reduction in viability (Figure 2).
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To determine the relative effectiveness of IgGSnCe6 and SnCe6 as photosensitizers in the lethal photosensitization of MRSA, EMRSA-16 was subjected to varying concentrations of photosensitizer and different light doses (Figure 3). Figure 3(a) shows that the proportion of survivors decreased as the concentration of SnCe6 or IgGSnCe6 conjugate was increased and the IgGSnCe6 conjugate killed more cells than the equivalent concentrations of free SnCe6, using the same light energy dose of 21 J/cm2 (5 min exposure). At the highest concentration of IgGSnCe6 used, 6.9 µg/mL, over 99.99% of the bacteria were killed (a 4.9 log10 reduction in the viable count). Figure 3(b) shows the proportion of EMRSA-16 surviving following treatment with SnCe6 or IgGSnCe6 at 3 µg/mL, exposed to different doses of light from the HeNe laser. The higher the light dose, the lower the percentage of EMRSA-16 surviving and, again, IgGSnCe6 produced higher kills than SnCe6 alone. From these graphs (Figure 3a and b) it can be seen that IgGSnCe6 was much more effective than unconjugated SnCe6 at killing EMRSA-16.
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Lethal photosensitization of EMRSA-16 at different growth phases
The effect of the growth phase of EMRSA-16 on lethal photosensitization was tested using the IgGSnCe6 conjugate (Figure 4). The viability of exponential and stationary phase bacteria were both dramatically reduced by the combination of IgGSnCe6 and red light, whereas bacteria in the lag phase were less susceptible. As the expression of protein A is regulated in a growth phase-dependent manner,27,28 the lag phase bacteria may have less protein A on the cell surface to bind the IgGSnCe6 conjugate. Suspensions of lag, exponential and stationary phase bacteria were also subjected to lethal photosensitization with free (unconjugated) SnCe6 at 25 µg/mL in combination with a light dose of 8.4 J/cm2. Under these conditions the bacteria in the lag phase were again the least susceptible to lethal photosensitization; 60% of lag phase, 91% of exponential phase and 97% of stationary phase bacteria were killed.
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When EMRSA strains 1, 3, 15 and 16 were subjected to lethal photosensitization using SnCe6 and laser light (Figure 5a), the kills attained using SnCe6 at 25 µg/mL and a light dose of 21 J/cm2 varied with the strain used. Figure 5(a) shows that EMRSA-1 was the least susceptible, with <70% of the cells being killed. In comparison, the viable counts of EMRSA-15 and EMRSA-16 were reduced by >99% (Figure 5a). The effectiveness of lethal photosensitization using IgGSnCe6 was also found to be strain dependent (Figure 5b): 98.7% of EMRSA-16 were killed compared with only 16.9% of EMRSA-1 using 21 J/cm2 light and 4.25 µg/mL IgGSnCe6.
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The targeting of the IgGSnCe6 conjugate towards MRSA was tested by comparing the kills of EMRSA-16 with that of another Gram-positive bacterium, S. sanguis, in single and mixed cultures (Figure 6). When S. sanguis was treated with SnCe6 and exposed to HeNe laser light under similar conditions to those used for EMRSA-16 (Figure 5a), the viable count was reduced by 99.8% (3 log10 reduction). In contrast, when S. sanguis was treated with IgGSnCe6 under comparable conditions to those used for EMRSA-16 (Figure 5b), only 16.3% kill was achieved. Therefore, although SnCe6 is an effective photosensitizer of S. sanguis, the IgGSnCe6 conjugate is much less effective against S. sanguis compared with EMRSA-16. When the IgGSnCe6 conjugate was added to a mixed culture containing approximately equal proportions of EMRSA-16 and S. sanguis, and irradiated, 99.1% of the EMRSA-16 were killed, whereas no reduction in the number of viable S. sanguis cells was observed (Figure 6).
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Discussion |
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The greater efficacy and potency of the IgGSnCe6 conjugate over free SnCe6 is likely to be due to its targeting of protein A, enabling the SnCe6 to be in closer proximity to the cells. Logically, the greater the amount of SnCe6 bound to or associated with the cells, the more singlet oxygen the cells will be exposed to. Hence, cells treated with IgGSnCe6 may have a higher concentration of the photosensitizer at the cell surface and thus a greater exposure to singlet oxygen, than those treated with SnCe6 alone. There is evidence to suggest that photosensitizers that penetrate into the bacterial cell may be more effective than those that are at the cell surface.29,30 However, this does not mean that photosensitizers located at the cell surface are not efficacious, as this and other studies11,1821 have shown.
Although both IgGSnCe6 and SnCe6 were able to kill EMRSA-16 when irradiated with red light, there were variations in the susceptibility of the other MRSA strains to this treatment. Using concentrations of SnCe6 (unconjugated) chosen to demonstrate differences in the susceptibility of the bacteria to this treatment, rather than to achieve 100% kill, we found that, in a suspension containing 109 cells, almost all of S. aureus NCTC 6571, EMRSA-15 and EMRSA-16 were killed with a combination of 25 µg/mL SnCe6 and a light dose of 8.4 J/cm2. However, <70% of EMRSA-1 were killed under these conditions. Wainwright et al.23 also reported such an effect, when using thiazin and phenothiazinium dyes as photosensitizers against pathogenic strains of S. aureus, some of which were EMRSA strains. The authors found that although the photosensitizers were effective against all strains used, they were less effective against the MRSA strains than against S. aureus NCTC 6571. This could be due to differences in the composition of the cell wall or membrane, rendering certain strains less susceptible to the effects of singlet oxygen, or due to a variation in the uptake of the photosensitizers between the strains, or possibly even both. In contrast, another such study, using aluminium disulphonated phthalocyanine (AlPcS2) against 16 different EMRSA strains, found that there were no marked differences in susceptibility among the strains.12
The MRSA strains also had varying susceptibilities to the IgGSnCe6 conjugate; however, this was not the same as with unconjugated SnCe6. When using the conjugate, the variation between the strains may be related to the amount of protein A expressed by each strain, and its attachment to the cell wall. Indeed, different strains of S. aureus, whether methicillin resistant or methicillin sensitive, can vary in the amount of protein A expressed and localized to the cell surface31 and there is some evidence to suggest that EMRSA-16 has more surface-bound protein A than EMRSA-15, EMRSA-3 and EMRSA-1.32 This would help to explain why free SnCe6 reduced the viable counts of EMRSA-16 and EMRSA-15 by 99.1%, whereas with IgGSnCe6, EMRSA-16 was much more susceptible to killing than EMRSA-15, with 98.7% and 57.8% kills, respectively. If EMRSA-16 produces larger amounts of cell wall bound protein A than EMRSA-15, it would be expected that a greater amount of the conjugate would bind to EMRSA-16 than EMRSA-15. Hence, upon exposure to laser light, more singlet oxygen would be produced in the vicinity of EMRSA-16, resulting in higher kills of this strain.
The expression of protein A can also go some way to explaining the growth phase-dependent lethal photosensitization of MRSA. Protein A is expressed during the exponential phase of growth and the spa gene is repressed by Agr and Sar in the post-exponential phase.33,34 EMRSA-16 was most susceptible during the exponential phase, coinciding with maximal protein A expression. Cells in the lag phase of growth were the least sensitive to lethal photosensitization by IgGSnCe6. However, this effect cannot be due to protein A alone, since cells treated with unconjugated SnCe6 also showed a greater resistance to killing when in the lag phase. Conversely, studies using AlPcS2 as the sensitizer demonstrated that the killing of MRSA was not dependent on growth phase.15 Similarly, no growth-phase effects have been observed with the photosensitizers toluidine blue and azure A (M. L. Embleton, unpublished observations). Thus, it seems likely that the effect of growth phase on the susceptibility of S. aureus to lethal photosensitization is a characteristic of the photosensitizer used.
A protein A-mediated targeting approach to direct the photosensitizer bacteriochlorophyll (Bchl) to the surface of S. aureus Cowan I was also used by Gross et al.22 In these experiments, an IgGBchl conjugate was tested alongside a non-targeted serineBchl conjugate for its ability to bind to and, upon exposure to light with > 550 nm, kill the bacterium. The targeted IgGBchl conjugate was found to be much more effective than the serineBchl. The higher efficacy of the IgGBchl was explained by the exclusive positioning of the conjugate on the bacterial cell wall. However, these experiments were performed on S. aureus Cowan I, a strain that is naturally abundant in protein A35,36 and, therefore, would be very able to bind the immunoconjugate. The conjugate was not tested for its effectiveness against other strains of S. aureus, for example low, or non-producers of protein A, nor was it tested for its specificity for S. aureus in a mixed culture with other bacteria.
We have not only tested our IgGSnCe6 conjugate against different strains of S. aureus, but have also shown that IgGSnCe6 was able to kill EMRSA-16 when mixed with another Gram-positive organism. Like EMRSA-16, S. sanguis is very susceptible to killing by a combination of SnCe6 and red light. However, unlike EMRSA-16, S. sanguis was not killed efficiently (16.3%) by the IgGSnCe6 conjugate. This was to be expected, since S. sanguis has not been documented as having IgG-binding proteins on its surface. When IgGSnCe6 was used with a mixed culture of EMRSA-16 and S. sanguis, >99% of the EMRSA-16 were killed with no reduction in the viable count of S. sanguis. Thus, IgGSnCe6 is able to kill EMRSA-16 selectively.
In this investigation, we have achieved our aims by demonstrating that a photosensitizer can be targeted to an EMRSA and retain its photodynamic activity. Upon exposure to light of an appropriate wavelength, the targeted bacteria are killed, whereas untargeted microorganisms in close proximity to the target MRSA are not. The effectiveness of this IgGSnCe6 conjugate against the clinically important EMRSA-16 strain may make it a good candidate for treatment of topical EMRSA-16 infections, e.g. wounds and burns, ulcers, pressure sores and prosthetic or catheter biofilm-associated infections. Furthermore, it could be used to prevent the spread of EMRSA-16 by eliminating the organism from carriage sites such as the nares, throat and skin. However, this may not be a cost-effective strategy in the longer term in view of the waxing and waning of EMRSAs in the UK.
The experiments have also demonstrated that future targeting strategies may have to use cell wall targets that are more uniformly distributed. Providing the antibodies become, or are made, available, other surface proteins could be used as targets for antibody-directed lethal photosensitization of infections caused by different MRSA strains. We are now pursuing alternative targeting strategies to improve the killing of MRSA and to reduce the difference in susceptibilities between strains. Indeed, with the selection of a suitable target and photosensitizer combination, targeted lethal photosensitization could be used to prevent and treat infections caused by a variety of bacterial species.
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Acknowledgements |
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Footnotes |
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