Ravi S. Kane, Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, CBIS 4105, 110 8th Street, Troy, NY 12180, USA. E-mail: email@example.com
The objective of this study was to develop porphyrin-based formulations to inactivate Bacillus spores. We probed the effect of porphyrins alone and in combination with germinants against both Bacillus cereus and Bacillus anthracis spores in the presence of light.
Methods and Results
We tested the effect of two different porphyrins, amine-modified protoporphyrin IX (PPIX) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). Treatment with the porphyrins alone did not significantly influence spore viability. However, when spores were pretreated with a solution containing the germinants, l-alanine and inosine, the spore viability dropped by as much as 4·5 logs in the presence of light. The extent of inactivation depended on the germination conditions and the type of porphyrin used, with TMP being more effective.
Porphyrins can be used effectively in combination with germinants to inactivate Bacillus spores.
Significance and Impact of the Study
The results of this study provide evidence that porphyrins can be used to inactivate Bacillus spores in the presence of germinants and light irradiation. This finding may be general and may be extended to spores of other pathogens.
Herein, we report a strategy for light-based inactivation of bacterial spores by using a combination of germinating agents and porphyrins. Some bacteria produce endospores when they sense unfavourable environmental conditions. These spores can survive in the environment for long periods of time resisting both high temperatures and a lack of nutrients. When the spores find a suitable host, such as the human body, they germinate and can become pathogenic. Moreover, antibiotic-resistant strains of spore-forming bacteria can be genetically engineered to form deadly bioweapons. An example of a biowarfare agent is Bacillus anthracis, which is one of the most dangerous and widely known pathogens from the Bacillus group and the causative agent for the disease anthrax. Outside the host environment, B. anthracis exists as spores, which can infect our body and germinate to form vegetative cells that can replicate and spread throughout the body causing life-threatening conditions (Bryskier 2002; Athamna et al. 2004; Koehler 2009). Another spore-forming bacterium, B. cereus, is found in the soil and is known to cause severe foodborne illness (Luksiene et al. 2009).
Endospores are highly resistant to mild bactericidal agents, such as alcohols, phenols, chlorhexidine and benzalkonium compounds (Oliveira et al. 2009). Treatments commonly used to inactivate spores include strong chemicals (e.g. formaldehyde or glutaraldehyde, concentrated hypochlorite solutions, chlorine dioxide), high heat or ionizing radiation (Russell 2003). The strong chemicals mentioned above may also be harmful to the environment. The use of UV radiation for decontamination is also associated with health hazards.
The inactivation of bacterial endospores has been regarded as a challenge for human health, environmental quality and food safety. On the other hand, inactivation of vegetative bacterial cells is relatively easy. Fischetti and coworkers have shown that a mixture of a germinating agent (l-alanine) and a bacteriolytic enzyme (PlyG) can effectively inactivate Bacillus spores (Schuch et al. 2002). The germinant first converted the spores into vegetative cells, which were easily inactivated by PlyG. Nerandzic et al. reported that Clostridium spores become more susceptible to UV radiation when treated with a germinating solution (Nerandzic and Donskey 2010). However, enzymes may only target specific strains and the use of UV radiation may pose health hazards as noted above.
Photodynamic therapy using porphyrins has been shown to be highly effective against a number of bacterial strains (Malik et al. 1990; Stojiljkovic et al. 2001; Banfi et al. 2006; Yu et al. 2009; Banerjee et al. 2010) and viruses (Malik et al. 1994; Casteel et al. 2004; Wen et al. 2009; Takehara et al. 2010; Banerjee et al. 2012). Porphyrins act by producing reactive oxygen species (ROS) that act on multiple targets in the pathogen; consequently, it is less likely that pathogens will develop resistance. Furthermore, such a treatment could be effective against a wide range of pathogens (Malik et al. 1990, 1994; Stojiljkovic et al. 2001; Banfi et al. 2006; Yu et al. 2009; Banerjee et al. 2010; Casteel et al. 2004; Wen et al. 2009; Takehara et al. 2010; Banerjee et al. 2012), because the damaging effects of ROS are not specific towards any given strain. Therefore, we hypothesized that porphyrins alone or in combination with germinants (e.g. l-alanine and inosine) could be used to inactivate spores. Such a mixture may be applied to decontaminate various surfaces. Another advantage of this approach is that this mixture is stable for several months when stored in dark and at 4°C. We treated both B. cereus and B. anthracis spores with two different porphyrins – amine-modified protoporphyrin IX (PPIX) and meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP). The porphyrins were strongly sporicidal in the presence of l-alanine and inosine upon irradiation, but were not significantly active against the spores in the absence of the germinants. In addition, TMP was more active against the spores than PPIX in the presence of the germinants. We envision that a mixture of germinants and a porphyrin will serve as an effective strategy in the decontamination of Bacillus spores, and may also be extended to target spores of other pathogens.
Materials and methods
Protoporphyrin IX (Sigma Chemical Co., St Louis, MO, USA) was modified with N-Boc-ethylenediamine by 1-(3-(dimethylamino) propyl)-3-ethylcarbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling. The Boc-protecting groups in the product were removed by the treatment with trifluoroacetic acid (TFA) to give the amine-functionalized product, PPIX-NH2. The details of the synthetic procedure have been reported by us previously (Banerjee et al. 2010). This PPIX-NH2 thus obtained (from now on referred to as PPIX in this manuscript) was used in this study. TMP (Frontier Scientific, Logan, UT) was used as obtained from the supplier.
Purification of spores
To obtain Bacillus spores (Nicholson and Setlow 1990), Difco sporulation media (DSM) were prepared in Millipore water by dissolving Bacto nutrient broth (Difco) (8 g l−1), 0·10% (w/v) KCl, and 0·012% (w/v) MgSO4·7H2O, and 1 mol l−1 NaOH was added to set pH to 7·6. The media were autoclaved and cooled to 50°C. Prior to use, 1 ml sterile solutions of 1 mol l−1 Ca(NO3)2, 0·01 mol l−1 MnCl2 and 1 mmol l−1 FeSO4 were added to 1 l of the autoclaved media. B. cereus (ATCC 4342) or B. anthracis Sterne (34F2; Colorado Serum Company, Denver, CO, USA) was first grown in 25 ml DSM at 37°C and 150 rev min−1 until mid-log phase (0·45 < A600 < 0·6) was reached (usually 2 h). This culture was then transferred to 225 ml of prewarmed (37°C) DSM in a 1-l flask. The mixture was further incubated for 72 h at 37°C in a shaker at 150 rev min−1, and the culture was periodically observed during growth until spore purity of >90% was achieved. The spore-containing 250-ml culture was washed several times in sterile Millipore water (SMW) by centrifuging at 4000 g for 10 min. Finally, the pellet was resuspended in 200 ml cold SMW. The spores were examined for purity using an optical microscope; under phase-contrast optics, the spores appeared phase-bright. The washing steps were repeated until >99% spore purity was obtained. The spore suspension was then stored at 4°C and protected from light.
Photoirradiation of spores
The suspension of Bacillus spores stored at 4°C was first heated at 80°C on a hot plate for 20 min to eliminate any bacteria that may be present because of germination over time. The heat-treated spores were first cooled in an ice bath and diluted in SMW such that the final concentration of the spores was 2 × 108 colony forming units ml−1 (CFU ml−1). To test the effect of light and porphyrin on spores, 100 μl of the spore suspension was mixed with an equal volume of PPIX solution such that the final concentration of PPIX was 200 μg ml−1. This mixture was irradiated in a bovine serum albumin (BSA)-coated 96-well plate for 2 h under visible light from a sunlite compact fluorescent lamp (350 W and 4200 Lumens; BulbAmerica, Brooklyn, NY, USA). The 96-well plate was placed in an ice bath during the irradiation, and a NIR filter (short pass 800 nm; Andover Corp., Salem, NH, USA) was used to prevent heating of the mixture. As controls, the spores were irradiated under light without PPIX or incubated in dark with or without PPIX. After these treatments, the spores were serially diluted, plated onto NB agar plates and incubated at 37°C overnight. The number of colonies that formed on the plates was counted the following day to determine the CFU ml−1.
Photoirradiation of spores pretreated with germinants
As indicated above, the suspension of Bacillus spores in SMW (stored at 4°C) was first heated at 80°C on a hot plate for 20 min to eliminate any bacteria that may be present because of germination over time. The heat-treated spores were then cooled in an ice bath for c. 5 min. For the germination step, the spores were diluted in either SMW or tryptic soy broth (TSB) containing 10 mmol l−1 of l-alanine and 5 mmol l−1 of inosine, such that the final concentration of the spores was c. 2 × 107 CFU ml−1. This suspension was incubated at room temperature for 30 min to trigger germination. Spores germinated with l-alanine and inosine in TSB were centrifuged at 3300 g for 15 min and resuspended in SMW.
In order to estimate the extent of germination after the 30-min incubation, part of the spore suspensions were heated at 80°C for 20 min. After heat treatment, the suspensions were serially diluted and plated on NB agar plates. The plates were incubated overnight at 37°C. As vegetative cells cannot survive at 80°C, the colonies that grew on these plates the following day corresponded to ungerminated spores (Shah et al. 2008).
For the photoirradiation experiments, 100 μl of the germinant-treated spore suspensions was mixed with equal volumes of the porphyrin (PPIX or TMP) solutions in SMW. The experiments were carried out using different concentrations of the porphyrins. These mixtures were irradiated in a BSA-coated 96-well plate for different periods of time ranging from 0 to 30 min under visible light from a compact fluorescent lamp in the same manner as described above. To establish the synergistic effect of light and porphyrin, control experiments were carried out in which the spores were mixed with only water and incubated under light or were stored with porphyrin in the dark for 30 min. After these different treatments, the spore suspensions were diluted serially and plated on NB agar plates in duplicate and incubated overnight at 37°C. The number of colonies in each of the plates was counted the following day, and the CFU ml−1 corresponding to each treatment was compared.
Effect of light irradiation with PPIX on B. cereus spores
Amine-modified protoporphyrin IX alone was ineffective against B. cereus spores in the presence of light (Fig. 1a). The spore viability did not decrease even after treatment with 200 μg ml−1 of PPIX in SMW under light for 2 h. We next tested the effect of PPIX on B. cereus spores, which were pretreated with 10 mmol l−1 of l-alanine and 5 mmol l−1 of inosine in SMW for 30 min. A mixture of l-alanine and inosine has been widely used to germinate Bacillus spores effectively (Johnstone 1994). Upon treatment with 20 μg ml−1 of PPIX, the spore viability dropped by nearly 2 logs within 30 min of light irradiation (Fig. 1b). Control experiments were performed in which pregerminated spores were incubated with the same amount of PPIX in the dark or without PPIX in light/dark. In these cases, the spore viability did not change significantly, confirming the synergistic effect of PPIX and light on survival. The duration of light irradiation influenced germinated spore viability in the presence of 20 μg ml−1 of PPIX (Fig. 1c). The spore viability decreased sharply by about a log within 5 min of light irradiation and continued to decrease further as the time of irradiation was increased.
Effect of light irradiation with TMP on B. cereus spores in SMW
While we obtained nearly two log reduction in spore viability using PPIX, we wanted to check whether we could achieve a greater reduction using TMP, which is a tetracationic porphyrin. Cationic porphyrins are known to be highly active against bacteria because the presence of positive charge allows them to bind more strongly to the negatively charged bacterial membrane and hence target the bacteria more effectively (Donnelly et al. 2009). Exposure to light for 30 min in the presence of 20 μg ml−1 of TMP resulted in a 2·8 log reduction in the B. cereus spore viability following pretreatment with 10 mmol l−1 of l-alanine and 5 mmol l−1 of inosine in SMW (Fig. 2a). Controls performed with TMP in the dark also gave a decrease in spore viability by as much as 0·7 log. Polycations are known to disrupt the cell membrane of bacteria, thereby causing damage (Tiller et al. 2001). Thus, it is possible that the tetracationic TMP caused damage to the germinated spores even in the dark by a similar mechanism. However, this decrease was not significant when compared to the reduction in spore viability for the light-irradiated samples. Controls performed without TMP under light showed no significant decrease in spore viability, when normalized with respect to spores stored in the dark without TMP. A dose–response experiment was performed (Fig. 2b) to test the effect of TMP concentration on the viability of germinated B. cereus spores. Spore viability decreased sharply when the TMP concentration was increased from 0 to 2 μg ml−1, yet no additional sporicidal activity was achieved above this concentration up to 20 μg ml−1. The kinetics of pregerminated B. cereus spore inactivation was determined in the presence of 2 μg ml−1 TMP and with varying duration of light irradiation up to 30 min (Fig. 2c). Spore viability dropped sharply within the first 5 min of light exposure and then decreased slowly thereafter up to 30 min.
Effect of light irradiation with TMP or PPIX on B. anthracis spores in SMW
Following these initial experiments with B. cereus, we endeavoured to determine whether the aforementioned strategy could be used to inactivate B. anthracis. We used the relatively nonpathogenic B. anthracis Sterne strain, which has lost its ability to produce the outer protective capsule that prevents B. anthracis from getting phagocytosed. The effects of both PPIX and TMP on B. anthracis spores, which had been pretreated with 10 mmol l−1 of l-alanine and 5 mmol l−1 of inosine in SMW for 30 min, were tested in an identical manner to those with B. cereus spores. A reduction in B. anthracis viability was obtained when the germinated B. anthracis spores were treated with 20 μg ml−1 of PPIX or TMP under light for 30 min. Fig. 3 shows that the spore viability was reduced much more in the presence of TMP than with PPIX. It was seen that spore viability was reduced to 64% and 2% (as compared to the spores stored in dark) when treated with PPIX and TMP, respectively. The decrease in spore viability because of the treatment with TMP was nearly identical to that of the germinated spores after heating at 80°C for 20 min. Unlike vegetative cells, spores can survive at 80°C; thus, the decrease in the spore viability on heat treatment indicates the extent of germination (Shah et al. 2008). Hence, this result suggests that the decrease in spore viability upon treatment with light-activated TMP was likely limited by the extent of germination.
Effect of light irradiation with TMP on B. anthracis spores in TSB
We next tested whether the use of TSB as a medium would enable greater germination efficiency. The results described above suggested that spore inactivation was limited by the extent of germination. We reasoned that the use of TSB, a bacterial growth medium, would cause better germination and allow us to achieve a greater reduction in spore viability. In a similar experiment with light-activated TMP as depicted in Fig. 3, B. anthracis spore viability decreased by more than 4 logs (Fig. 4). This reduction in spore viability was clearly much greater than that obtained when the germination was carried out in SMW (Fig. 3). Figure 4 also reveals that reduction in spore viability upon treatment with the TMP for 30 min had nearly the same effect as that obtained on heating the germinated spores at 80°C for 20 min. This result again indicates that the decrease in spore viability was limited by the extent of germination in case of experiments performed in both SMW and TSB.
Decontamination of spores is regarded as one of the toughest challenges. Porphyrins have been widely used for targeting pathogens and are quite attractive as there are little or no chances for bacteria to develop resistance. We therefore tested two different porphyrins (PPIX and TMP) against Bacillus spores under different conditions.
Our results indicate that PPIX did not have significant sporicidal activity, even in the presence of light. Spores have multiple protective layers surrounding the inner core that probably prevents ROS from damaging the inner spore core. ROS have a very short lifetime of less than a few nanoseconds in water (Wilkinson et al. 1995), as a result of which they are unable to diffuse through the multiple protective layers and damage the inner core. However, when molecules like l-alanine and inosine were used to germinate B. cereus spores, subsequent treatment with PPIX and TMP caused as much as 2 and 2·8 log reductions in spore viability, respectively. The early events in germination trigger the removal of the outer protective coat and the hydrolysis of the spore cortex, thus making the inner core more susceptible to ROS (Setlow 2003; Moir 2006).
We found that TMP was more effective against Bacillus spores than PPIX. This is because TMP is tetracationic, which possibly allows it to bind more effectively to the negatively charged bacterial cell wall, and show greater bactericidal activity than PPIX in the presence of light. However, it was observed that the activity of both TMP and PPIX was much less in the case of B. anthracis spores. Interestingly, we observed that the viability of the germinated spores after heat inactivation (80°C for 20 min) was the same as that obtained after treatment with TMP and light for 30 min. As the germinated spores cannot survive at 80°C, this result indicated that spore inactivation was limited by the extent of germination. When the spores were germinated in TSB, which is a bacterial growth media, it was seen that the viability of B. anthracis spores dropped dramatically in the presence of light and TMP. Our future research will explore more effective germinants to further increase the extent of decontamination.
We believe that this study may help in designing decontaminant formulations composed of a germinant (l-alanine, inosine) and a photosensitizer which may be used to inactivate Bacillus spores. The strategy can also be extended to decontaminate other types of spores, such as spores of Clostridium. The advantage of using a porphyrin is that it is effective in small quantities and acts very fast by producing ROS that acts on multiple targets on pathogens and hence the probability of development of resistance is unlikely. However, one potential pitfall of this approach would be the lack of microbicidal activity in the dark and the possible photobleaching of the photosensitizer. In this study, we have used porphyrins but the strategy is not limited to photosensitizers. Thus, this approach may open up the use of a wide range of antibacterial materials for the decontamination of spores of different pathogens.
We acknowledge the support from a NYSTAR faculty development award to R.S.K. Any opinions, findings, conclusions or recommendations expressed are those of the authors and do not necessarily reflect the views of NYSTAR. We thank Dr Elena Paskaleva for helpful discussions.