Photodynamic Inactivation of Bacillus Spores, Mediated by ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2005, p. 6918–6925 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.11.6918–6925.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 11

Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes Tatiana N. Demidova1,2 and Michael R. Hamblin1,3,4* Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, Massachusetts1; Graduate Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts2; Department of Dermatology, Harvard Medical School, Boston, Massachusetts3; and Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts4 Received 18 October 2004/Accepted 28 June 2005

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.

The genus Bacillus comprises a group of gram-positive endospore-forming rod-shaped aerobic bacteria and is widely distributed in soil environments. Sporulation is a protective mechanism whereby bacteria can survive in the environment for long time periods, even at physical extremes for life forms on earth. Bacillus anthracis is a pathogen, and in 1887, was the first bacterium shown to be the cause of a disease by Robert Koch, who demonstrated its ability to form endospores and produced experimental anthrax by injecting it into animals (31). B. anthracis has been proposed to be an important organism in biological warfare and bioterrorism due to its ability to be weaponized by producing a finely ground highly infective spore powder that could be widely dispersed (12). B. anthracis is a member of a closely related family of six species (22), including B. cereus, which is less pathogenic to humans (1, 5), and B. thuringiensis, which is pathogenic to insects (45). Spores are relatively resistant to many of the disinfectant and antiseptic agents that routinely destroy vegetative bacteria, such as alcohols, phenols, chlorhexidine, and benzalkonium compounds (37, 39). Agents commonly cited as being sporicidal include heat greater than 121°C, formaldehyde or glutaraldehyde, strong hypochlorite solutions, chlorine dioxide, and ionizing or UV radiation (38, 55). Photodynamic therapy (PDT) is a therapy for cancer and other diseases that has received regulatory approvals for sev-

eral indications in many countries (10). It employs nontoxic dyes known as photosensitizers (PS) and visible light of the appropriate wavelength to excite the PS molecule to the excited singlet state. This excited state undergoes intersystem crossing to the long-lived triplet state, which can react with molecular oxygen to produce cytotoxic species such as singlet oxygen, superoxide, and radicals (9, 11). These reactive oxygen species can then oxidize many biological molecules, such as proteins, nucleic acids, and lipids, leading to cell death. PDT has the advantage over other therapies of dual selectivity: not only is the PS targeted to the affected tissue or cell type, but the light can also be accurately delivered to the appropriate area. Although originally developed as a cancer treatment, PDT has been successful as a treatment for age-related macular degeneration in ophthalmology (2) and is under investigation as a treatment for infectious disease (16). The rapidly increasing emergence of antibiotic resistance among pathogenic bacteria may be bringing to an end a period extending over the past 50 years termed “the antibiotic era” (59) and has led to a major research effort to find alternative antibacterial therapeutics (6), such as PDT, to which we have hypothesized bacteria will not be easily able to develop resistance (8). Indeed, Lauro et al. failed to demonstrate the development of resistance in Actinobacillus actinomycetemcomitans and Peptostreptococcus micros after 10 cycles of subtotal photodynamic inactivation (PDI) (28). Because the delivery of visible light is almost by definition a localized process, PDT for infections is likely to be applied exclusively to localized disease by local delivery of the PS into the infected area by methods

* Corresponding author. Mailing address: Wellman Center for Photomedicine, Massachusetts General Hospital, BAR414, 40 Blossom Street, Boston, MA 02114. Phone: (617) 726-6182. Fax: (617) 7268566. E-mail: [email protected]. 6918

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such as topical application, instillation, interstitial injection, or aerosol delivery (16). It has been known since the first days of PDT early in the 1900s that certain microorganisms can be killed by the appropriate combination of dyes and light in vitro, but in the last 15 years progress has been made in defining the precise molecular features of the PS necessary for binding to and subsequently killing by photodynamic action microbes such as bacteria, fungi, and viruses (51). Bacillus subtilis spores, however, have been reported to be resistant to PDT using the dye Rose Bengal under conditions which easily led to the killing of vegetative cells (40). We decided to carry out a study examining candidate antimicrobial PS for their ability to mediate the light-induced destruction of bacterial spores. We describe here for the first time the efficient photoinactivation (PDI) of Bacillus spores, including those closely related to anthrax, mediated by members of the family of phenothiazinium dyes. MATERIALS AND METHODS Bacterial strains and culture conditions. The following Bacillus strains were used: B. atrophaeus (ATCC 9372), B. cereus (ATCC14579), B. megaterium (ATCC14581), B. thuringiensis (ATCC 33740), and B. subtilis (ATCC 6051). Vegetative cells were routinely cultured in brain heart infusion (BHI) broth with shaking and on BHI agar, both at 37°C. For initial experiments, spores of B. atrophaeus and B. cereus were purchased from SGM Biotech, Inc. (Bozeman, MT). Subsequently, spores of all species were prepared in our laboratory, using sporulation broth for B. atrophaeus, B. megaterium, and B. subtilis and sporulation agar (4, 32) for B. cereus and B. thuringiensis. The sporulation medium consisted of 16.0 g nutrient broth (Difco), 2.0 g KCl, and 0.5 g MgSO4 per liter; 17 g of agar was added if needed. The pH of the medium was adjusted to 7, and then the medium was autoclaved and cooled. After the medium was cooled, 1 ml of 1 M Ca2(NO3)2, 1 ml of 0.1 M MnCl2 · 4H2O, 1 ml of 1 mM FeSO4, and 2 ml of 50% glucose were added. Bacteria were grown in sporulation medium for 3 days. For spore purification, the mixture of spores and cells was centrifuged at 1,300 ⫻ g for 20 min, washed with 5 volumes 1 M KCl–0.5 M NaCl, rinsed with sterile deionized water, washed with 1 M NaCl, and rinsed with sterile deionized water again. Lysozyme (50 ␮g/ml) was added in the presence of buffer (5 volumes 0.05 M Tris-Cl, pH 7.2) and incubated with constant stirring at 4°C overnight. Lysozyme was removed by centrifugation eight times (at 1,300 ⫻ g) and washing with sterile deionized water. In a control experiment, B. cereus spores were prepared without the use of lysozyme. The purity of spore suspensions was checked using Wirtz-Conklin stain (5% aqueous malachite green and safranine O) (20) and transmission electron microscopy. Spores were ⬎95% pure, with the remaining material being fragments of vegetative sporangium (data not shown). Spores were then frozen at ⫺80°C in phosphate-buffered saline without Ca2⫹ or Mg2⫹ (PBS) with 10% glycerol and stored until use. To avoid germination, spores were used immediately after defrosting. Photosensitizers and light sources. Eight PS were employed (Fig. 1 shows their structures). Rose Bengal (RB), toluidine blue O (TBO), methylene blue (MB), new methylene blue N (zinc chloride double salt; NMBN), and 1,9dimethylmethylene blue chloride (DMMB) were purchased from Sigma (St. Louis, MO). These PS were dissolved in distilled water to give stock solutions with a dye concentration of 2 mM. 5-Phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin chloride [TriP(4)] was generous gift of G. M. T. Smijs and R. van der Steen (Leiden University Medical Center, The Netherlands), and a stock solution of 1 mM was prepared in PBS. The poly-L-lysine chlorin(e6) conjugate had an average of two ce6 molecules attached per pl chain, with an average degree of polymerization of 167 lysine residues, and was prepared as described previously (17) and stored as a 1.6 mM stock solution in water. A benzoporphyrin derivative (BPD) was a gift of QLT Inc. (Vancouver, Canada) and was dissolved in dimethyl sulfoxide at a concentration of 300 ␮M. All PS stock solutions were stored at 4°C in the dark for no more than 2 weeks, and immediately before experiments, were diluted in PBS without Ca2⫹ or Mg2⫹. Spectra of stock solutions diluted 140- to 280-fold in methanol were recorded on a UV-visible spectroscopy system (Waldbronn, Germany). A noncoherent light source with interchangeable fiber bundles (LumaCare, London,

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United Kingdom) was employed. Thirty-nanometer-band-pass filters at ranges of 540 ⫾ 15 nm for RB, 635 ⫾ 15 nm for TBO, DMMB, and TriP(4), 660 ⫾ 15 nm for MB, NMBN, and pLce6, and 690 ⫾ 15 nm for BPD were used. The total power output provided out of the fiber bundle ranged from 300 to 700 mW. Uptake studies. Uptake studies were performed with all spore species and TBO. Preliminary studies had shown that inactive PS such as RB gave no detectable uptake of dye by the spores (data not shown). Spores at a density of 107/ml were incubated for 3 h with PS in the dark at 37°C. Unbound PS was washed out by centrifugation of the mixture of dye and microorganisms for 6 min at 1,550 ⫻ g, followed by the resuspension of washed pellets in 10 ml PBS without Ca2⫹ or Mg2⫹. Aliquots (200 ␮l) of these suspensions were used for PDI experiments, the remaining suspensions were centrifuged again, and the pellets were dissolved in 6 ml 0.1 M NaOH–1% sodium dodecyl sulfate (SDS) for at least 24 h to give a clear solution. These solutions were used for PS uptake measurements. The fluorescence of dissolved pellets was measured on a spectrofluorimeter (FluoroMax3; SPEX Industries, Edison, NJ). The excitation wavelength was 620 nm, and the range for emission was 627 to 720 nm. A calibration curve was constructed from pure TBO at different concentrations dissolved in NaOH-SDS and used for determination of the PS concentration in dissolved pellets. Uptake values were obtained by dividing the number of nmol of PS in the dissolved pellet by the number of CFU obtained by serial dilutions and the number of PS molecules/cell, calculated by using Avogadro’s number. PDI studies. Suspensions of spores or bacteria at 107/ml were incubated with PS in the dark at 37°C. The incubation time was 3 h, and the PS concentration was varied from 5 to 1,600 ␮M. Illumination was performed either after or before excess dye was washed out. Fluence levels ranged from 0 to 200 J/cm2 at an irradiance of 200 to 400 mW/cm2. During illumination after defined fluences had been delivered, aliquots of 20 ␮l were taken to determine the CFU. The contents of the wells were mixed before sampling. The aliquots were serially diluted 10-fold in PBS without Ca2⫹ or Mg2⫹ to give dilutions of 10⫺1 to 10⫺6 times the original concentrations and were streaked horizontally on square BHI agar plates as described by Jett et al. (24). Plates were incubated at 37°C overnight. Colonies were counted, and the survival fraction was determined relative to an untreated control. No additional treatment to enhance spore germination was used. Two types of control conditions were employed, namely, illumination in the absence of PS and incubation with PS in the dark. PS were generally not toxic for spores or vegetative cells in the dark, and light alone did not cause cell destruction. All experiments were performed in triplicate.

RESULTS Spores are resistant to photoinactivation, except that by TBO. We initially screened a panel of PS to test PDI of B. cereus spores (see chemical structures in Fig. 1). BPD is a clinically used PS that has found wide applicability in ophthalmology and has undergone testing against cancer. We previously reported that it was active in mediating PDI against gram-positive bacteria (44). TriP(4) is a cationic porphyrin that has been reported to be active in mediating PDI against both gram-positive and gram-negative bacteria (27) as well as fungi (43) and viruses (47). The pL-ce6 conjugate is a member of a class of macromolecular conjugates between polycationic polymers and PS that show a high degree of efficiency in mediating PDI of both classes of bacteria in vitro and in vivo (16, 18, 19). RB was previously reported to be ineffective at killing B. subtilis spores but is known to kill vegetative bacterial cells efficiently (40). TBO is the most frequently used member of the class of phenothiazinium dyes for mediating PDI of bacteria and fungi (52, 57, 58). Because we initially did not know whether the dyes would bind to the spores, we did not wash the spores after incubation with the dye. As shown in Fig. 2, only TBO (50 ␮M) demonstrated a light-dose-dependent loss of viability of B. cereus spores, with 40 J/cm2 of 630-nm light leading to 99.999% killing. All of the other PS, even when used at a 100 ␮M concentration and with 100 J/cm2 of the appropriate light, led to no loss of viability

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DEMIDOVA AND HAMBLIN

APPL. ENVIRON. MICROBIOL.

FIG. 1. Chemical structures of the PS used.

whatsoever. We repeated the TBO PDI experiment with B. cereus spores that had been prepared without the use of lysozyme. There was no difference in killing between spores that had been treated with lysozyme and those that had not (data not shown). This shows that lysozyme does not remove an outer coat layer or part of the cortex from the spores, thus increasing the penetration of the dye into the spore interior. We checked to be sure that the incubation of B. cereus spores for 3 h with 100 ␮M TBO solution at 37°C did not cause germination, using phase-contrast microscopy, and that incubated spores were as resistant to 70% ethanol as control spores (data not shown). Phenothiazinium dyes as a class photoinactivate spores. Since TBO had shown significant and surprising activity as an

antimicrobial PS against B. cereus spores, we examined other related members of the class of phenothiazinium dyes (see structures in Fig. 1). MB has been used almost as often as TBO for antimicrobial PDT (30, 46, 48, 61). DMMB was found by O’Neill et al. to be the most effective PS compared to other phenothiazinium dyes tested against the gram-positive bacterium Streptococcus sanguis (34). NMBN was found to be the most active PS compared to other phenothiazinium dyes tested against the gram-negative bacterium Yersinia enterocolitica (54). Because our first experiments had shown that TBO appeared to bind to B. cereus spores, we washed the spores after phenothiazinium dye incubation and resuspended them in PBS before PDI. Figure 3 shows that there are three broad levels of effective-

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FIG. 2. PDI of B. cereus spores incubated with different PS at a concentration of 100 ␮M (50 ␮M for TBO) for 3 h at 37°C. The spores were illuminated without a wash. The appropriate wavelength of light was used for each dye (see Materials and Methods), and the fluence rate was 200 mW/cm2.

ness of the different phenothiazinium dyes tested (50 ␮M) in killing B. cereus spores. MB is the least powerful, killing only about 99% of spores after treatment at 40 J/cm2; DMMB had an almost identical efficiency to that found with TBO (99.999%) after treatment with 40 J/cm2. NMBN was the most powerful of the compounds we tested, leading to ⬎99.999% killing with only half the amount of light (20 J/cm2). Differential susceptibility of Bacillus spores. We next asked whether spores from other Bacillus species were also suscep-

FIG. 3. PDI of B. cereus spores incubated with different phenothiazinium dyes at 50 ␮M for 3 h at 37°C. The spores were illuminated after a wash. The appropriate wavelength of light was used for each dye (see Materials and Methods), and the fluence rate was 200 mW/ cm2.


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FIG. 4. PDI of different Bacillus spores incubated with different concentrations of TBO for 3 h at 37°C. The spores were illuminated after a wash. The light used was 635 ⫾ 15 nm, and 40 J/cm2 was delivered at a fluence rate of 200 mW/cm2. *, B. subtilis and B. atrophaeus had significantly different killing curves (P ⬍ 0.05 for comparison of slopes by nonlinear regression analysis).

tible to PDI with TBO. B. thuringiensis is a species that is closely related to B. cereus (and B. anthracis) (22). B. subtilis and B. atrophaeus (formerly B. subtilis var. niger) are a pair of closely related species (3). B. megaterium has large cells both in the vegetative state and as spores (33, 35). Again, these experiments were carried out with a wash after incubation. There were large differences in susceptibility to TBO-mediated PDI between spores of different Bacillus species. In order to display these in Fig. 4, we used a series of TBO concentrations in the incubation mixture and a constant fluence of 40 J/cm2 of 630-nm light. B. cereus and B. thuringiensis were both highly susceptible to PDI, with a TBO concentration of 50 ␮M leading to an almost complete loss of viability. In contrast, B. subtilis and B. atrophaeus were much less sensitive and needed concentrations as high as 1.6 mM to achieve killing of ⬎99.9% of cells. B. atrophaeus was significantly more sensitive than B. subtilis (P ⬍ 0.05 for comparison of slopes by nonlinear regression analysis). B. megaterium spores were completely resistant to PDI under these conditions. Photoinactivation is better after a wash. Most of the experiments described above were carried out after the spores were incubated with the dye and excess dye in solution was removed by pelleting the spores by centrifugation and resuspending them in PBS before illumination. This demonstrated that the dye actually bound to the spores, rather than generating reactive oxygen species in solution that subsequently attacked the spores. We compared the effectiveness of this technique with the alternative method of leaving the dye in the incubation mixture. Figure 5 (top panel) shows that in the case of B. cereus spores, where we only needed to use TBO concentrations of up to 50 ␮M, the washing out of excess dye gave a somewhat better rate of killing than leaving the dye in solution. In contrast, for B. atrophaeus (Fig. 5, bottom panel), where much

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FIG. 6. Comparison of PDI of spores and vegetative cells. B. cereus and B. subtilis spores and vegetative cells were incubated with TBO at different concentrations for 3 h at 37°C. Cells were washed and illuminated with 40 J/cm2 of 635- ⫾ 15-nm light at a fluence rate of 200 mW/cm2.

FIG. 5. Comparison of PDI of spores with and without a wash. (Top) B. cereus spores were incubated with TBO at 50 ␮M for 3 h at 37°C. Suspensions were either washed or not and were illuminated with 635- ⫾ 15-nm light at a fluence rate of 200 mW/cm2. (Bottom) B. subtilis spores were incubated with TBO at different concentrations for 3 h at 37°C. Suspensions were either washed or not and were illuminated with 40 J/cm2 of 635- ⫾ 15-nm light at a fluence rate of 200 mW/cm2.

higher TBO concentrations were required, we found that when the dye was left in solution, killing reached a plateau with a value of only 80% at 200 ␮M TBO, while it continued to increase with increasing TBO concentrations when the spores were washed free from excess dye. We interpreted these results by postulating a major absorbance of the light by the dye in solution, thus significantly reducing the amount of light that

can penetrate to the dye-containing spores in suspension. The absorbance of the B. atrophaeus dye mixture was much higher than that of the B. cereus dye mixture due to the higher concentrations of TBO required to kill B. atrophaeus, and therefore the optical screening effect of the excess dye was more pronounced, leading to almost no additional killing of B. atrophaeus spores at concentrations of TBO above 100 ␮M. Comparison of photosensitivity between spores and vegetative cells. We compared the relative sensitivities to PDI of spores and vegetative cells from two Bacillus species (B. cereus and B. subtilis). Because there is such a large difference in the concentrations of TBO needed to kill these four entities, we plotted the TBO concentrations on a log scale (Fig. 6). The sensitivities of vegetative cells from the two species were fairly similar, except that B. cereus was killed more than B. subtilis at low TBO concentrations (up to 8 ␮M). There was a very large difference in susceptibility between spores of B. cereus and spores of B. subtilis, which was similar to that found previously (Fig. 4) and meant that there was a much smaller difference in sensitivity between spores and vegetative cells of B. cereus (the TBO concentration needed to kill spores was 3 to 4 times higher than that needed to kill vegetative cells) than between spores and vegetative cells of B. subtilis (⬎100 times the TBO concentration was needed to kill spores compared to vegetative cells). Dye uptake by Bacillus spores. The fact that the spores could be washed free of dye in the incubation mixture by centrifugation allowed us to quantify the amount of dye taken up by each spore by previously established methods of chemical extraction and fluorescence spectrophotometry. Figure 7 shows the relationship between the uptake of TBO by spores after incubation and increasing concentrations of dye. No detectable

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FIG. 7. Uptake of TBO by spores. Spores were incubated with TBO at different concentrations for 3 h at 37°C, washed, and dissolved in NaOH-SDS, and dye uptake was measured by spectrofluorimetry. Spore numbers were measured by plating, and a calibration curve gave moles of dye. Points are means of three determinations, and bars are standard errors of the means. The dotted line indicates the approximate level of dye uptake necessary for efficient PDI. *, significant differences (P ⬍ 0.01 by unpaired two-tailed Student’s t test) between B. cereus and B. thuringiensis and between both of these and B. subtilis/B. atrophaeus.

uptake could be measured with spores of B. megaterium. Although there was a significant difference between the uptake values of B. cereus and B. thuringiensis at high TBO concentrations (⬎100 ␮M), at lower concentrations where PDI is performed (50 ␮M) the uptakes were similar, which explains why B. cereus and B. thuringiensis showed similar susceptibilities to PDI (Fig. 4). Both B. subtilis and B. atrophaeus had much lower uptakes, and in fact it is possible to estimate an uptake value in the region of 3 ⫻ 107 molecules per spore being necessary for PDI to occur (see the dotted line in Fig. 7). However, although there is clearly a correlation between the uptake of dye by spores and susceptibility to killing, we cannot exclude that there are also intrinsic differences in susceptibility between different Bacillus spores independent of dye uptake. DISCUSSION This study has demonstrated PDI of Bacillus spores for the first time. We discovered that dyes from the phenothiazinium structural group were the only compounds that were active among several PS we tested that had previously been shown to be efficient against vegetative bacterial cells. Previously, Schafer et al. had shown that spores of B. subtilis were resistant to PDI by using 2 ␮M of RB and 90 J/cm2 visible light (40). The phenothiazinium dyes bound to the spores and in fact were more active after excess dye was washed from the spore suspensions. This was likely due to the dye remaining in solution acting as an optical screen, thus preventing the light from reaching the spores containing active bound dye. It was necessary to incubate the spores for 3 h with dyes at a temperature of 37°C. Shorter incubation times and lower incubation tem-

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peratures were less effective (data not shown), suggesting that the dyes penetrated into the spores by a process of passive diffusion. Diffusion is significantly time dependent and is enhanced at higher temperatures. A future study will systematically examine the effects of various incubation times and temperatures on the process of spore PDI. The difficulty that certain exogenous molecules face in diffusing into spores is due to the impermeable nature of the spore coat and is thought to be responsible for much of the resistance of spores to biocidal chemicals (41). Phenothiazinium dyes are small molecules that have an intrinsic molecular cationic charge that allows them to bind to the generally anionic outer membranes of bacteria and fungi (36, 53, 54). We investigated the possibility that small cationic PS as a broad class may therefore be effective against spores. However, we failed to demonstrate any PDI of spores using a tricationic porphyrin (another small molecule) that has been effectively used to mediate PDI of various bacterial (27) and fungal (43) cells. There is therefore clearly another molecular feature involved besides the cationic charge and small molecular size. This may be related to the ability of phenothiazinium dyes to form dimers and oligomers. Usacheva et al. (48–50) showed that TBO was a better antimicrobial PS than MB partly because it was able to self-associate into dimers that were supposed to preferentially interact with lipopolysaccharide and other components of bacterial membranes. The differential sensitivity of spores from different Bacillus species is probably related to differences in spore structure. B. cereus and B. thuringiensis are closely related species from a family that also contains B. anthracis (23, 26) and three other species (21). They are characterized by the presence of an outer structure beyond the spore coat termed an exosporium. The exosporium of B. cereus contains two main layers: the outer layer is made up of a nap of hairlike projections, while the inner layer has a hexagonally perforated surface pattern of holes, and the intact basal membrane is about 19 nm thick (15). It is possible that the presence of the exosporium is involved in the high dye accumulation found with B. cereus and B. thuringiensis spores and not found with nonexosporium-forming spores. If the exosporium traps more dye than the “naked” spore, then it may allow more dye diffusion into the interior of the spore, where fatal photodamage could occur. The nonsusceptibility of B. megaterium spores may simply be related to their much larger size. We have found that Candida albicans cells are less sensitive to PDI than both gram-positive and gram-negative bacterial cells, partly because the fungal cells are 20 to 30 times bigger than the bacterial cells (7). However, the complete lack of detectable TBO uptake by B. megaterium spores suggests that the dye either does not bind to the spore or cannot penetrate to a sufficient extent to carry out photodamage of essential components. It is possible that differences in spore coat permeability are primarily responsible for the marked differences in PDI between species. In particular, B. megaterium spores are thought to have a lower permeability to small molecules, as described by Gerhardt and coworkers (14, 25). Another distinguishing factor that might explain the nonsusceptibility of B. megaterium spores is the much more hydrophilic nature of their surfaces than those of spores of B. subtilis and B. cereus (56). Since phenothiazinium dyes (in common with most PS) have a planar aromatic molecular structure, they

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will have amphiphilic regions that can bind to hydrophobic surfaces despite the polar cationic charges that are also on the molecule. If the dyes failed to bind to the spores, then the process of diffusion into the spore would be much reduced. Setlow and coworkers have demonstrated that B. subtilis spores are susceptible to agents that produce reactive oxygen species. These include aqueous ozone (60), hydrogen peroxide (29), peroxynitrite (13), and the Fenton reagent, consisting of cuprous chloride and ascorbic acid (42). The fact that PDI produces reactive oxygen species such as singlet oxygen, superoxide, and hydroxyl radicals is in agreement with the observed sporicidal effects of oxidizing agents. Future work will investigate the mechanisms of PDI of Bacillus spores and will also study spores of Clostridium species. It is fairly well established that PDI of vegetative bacterial cells involves damage to the cytoplasmic membrane and leakage of essential cellular constituents into the medium. It may be possible to identify which proteins in the spore coat or the spore core are damaged by the reactive oxygen species produced by PDI. Spores produced by mutant bacteria are known that have deficiencies in the permeability barrier, and these may give some useful insights into the mechanisms of phenothiazinium dye uptake by spores. ACKNOWLEDGMENTS This work was funded by the Department of Defense’s Program to Develop Biomedical Applications of the Free Electron Laser (contract number N00014-94-1-0927) and by the National Institute of Allergy and Infectious Diseases (grant number R01 AI050875). We are grateful to Maleha Khan for technical assistance and to Tayyaba Hasan for support. We thank QLT Inc. for the gift of BPD and Saskia Lambrechts, Threes Smijs, and Ruud van der Steen for the gift of TriP(4). We thank George P. Tegos for a critical reading of the manuscript.


13. 14. 15. 16. 17.

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