Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

The impact of inducing germination of Bacillus anthracis and Bacillus thuringiensis spores on potential secondary decontamination strategies T.O. Omotade, R.C. Bernhards, C.P. Klimko, M.E. Matthews, A.J. Hill, M.S. Hunter, W.M. Webster, J.A. Bozue, S.L. Welkos and C.K. Cote Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Frederick, MD, USA

Keywords anthrax, Bacillus anthracis, decontamination, germination, spores. Correspondence Christopher K. Cote, Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Frederick, MD 217025011, USA. E-mail: [email protected] 2014/1440: received 14 July 2014, revised 3 September 2014 and accepted 3 September 2014 doi:10.1111/jam.12644

Abstract Aims: Decontamination and remediation of a site contaminated by the accidental or intentional release of fully virulent Bacillus anthracis spores are difficult, costly and potentially damaging to the environment. Development of novel decontamination strategies that have minimal environmental impacts remains a high priority. Although ungerminated spores are amongst the most resilient organisms known, once exposed to germinants, the germinating spores, in some cases, become susceptible to antimicrobial environments. We evaluated the concept that once germinated, B. anthracis spores would be less hazardous and significantly easier to remediate than ungerminated dormant spores. Methods and Results: Through in vitro germination and sensitivity assays, we demonstrated that upon germination, B. anthracis Ames spores and Bacillus thuringiensis Al Hakam spores (serving as a surrogate for B. anthracis) become susceptible to environmental stressors. The majority of these germinated B. anthracis and B. thuringiensis spores were nonviable after exposure to a defined minimal germination-inducing solution for prolonged periods of time. Additionally, we examined the impact of potential secondary disinfectant strategies including bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UV-B and UV-C radiation, employed after a 60-min germinationinduction step. Each secondary disinfectant employs a unique mechanism of killing; as a result, germination-induction strategies are better suited for some secondary disinfectants than others. Conclusions: These results provide evidence that the deployment of an optimal combination strategy of germination-induction/secondary disinfection may be a promising aspect of wide-area decontamination following a B. anthracis contamination event. Significance and Impact of the Study: By inducing spores to germinate, our data confirm that the resulting cells exhibit sensitivities that can be leveraged when paired with certain decontamination measures. This increased susceptibility could be exploited to devise more efficient and safe decontamination measures and may obviate the need for more stringent methods that are currently in place.

Introduction The Gram-positive, spore-forming bacterium Bacillus anthracis is the aetiological agent of anthrax and a

category A biothreat agent. Widespread contamination from an accidental or intentional B. anthracis spore release can result in significant mortality rates, exemplified by the release from a Soviet military laboratory in Sverdlovsk

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(now Yekaterinburg, Russia) in 1979 (Meselson et al. 1994) and the US anthrax letter attacks in 2001 (Jernigan et al. 2001). Current decontamination methods are harsh on the environment, time-consuming and extremely expensive as evidenced by the estimated $320 million spent on total decontamination efforts following the anthrax letter attacks (Schmitt and Zacchia 2012). Highly resistant spores must germinate to be metabolically active cells capable of replication or causing disease (Driks 2002, 2003, 2009). Once germinated, however, a spore becomes significantly more susceptible to environmental insults (i.e. desiccation, antibiotics and disinfectants). Spore germination is a complex, yet rapid series of events (Setlow 2003, 2014; Moir 2006). Germination, once initiated, takes place within minutes, and under adequate conditions, the germinated spore will begin to transition (outgrow) into a replicative cell (Setlow 2003, 2014; Abee et al. 2011; Paredes-Sabja et al. 2011; Giebel et al. 2009). A subpopulation of bacterial spores, termed ‘superdomant spores’, is characterized by their significantly slow germination rates compared to other spores. Superdormant spores have been identified in several species of bacteria (Gould 1970; Ghosh et al. 2009; Nerandzic and Donskey 2010). Developing a cohesive and effective remediation strategy also calls for the selection of an appropriate nonpathogenic surrogate system that can serve as a simulant in possible field trials. As a result, the Gram-positive spore-forming bacterium, B. thuringiensis, has been identified as a potential surrogate for B. anthracis in these widespread decontamination tests (Rice et al. 2005; Greenberg et al. 2010; Raber and Burklund 2010; Buhr et al. 2012; Omotade et al. 2013; Bishop and Robinson 2014; White et al. 2014). Bacillus thuringiensis has been proposed as a possible surrogate species because it possesses chromosomal sequences that are very similar to B. anthracis (Helgason et al. 2000; Read et al. 2003), responds similarly in parallel studies (Rice et al. 2005; Buhr et al. 2012, 2013; Omotade et al. 2013) and has been shown to be generally nonhazardous to humans through extensive testing as a widespread commercial insecticide (Green et al. 1990; Bernstein et al. 1999). The ungerminated B. anthracis spore is the infectious particle and germinated spores or vegetative bacilli are generally noninfectious (McKevitt et al. 2007; Cote et al. 2009). Implementing germination-induction pretreatment may reduce costs associated with a contamination event, mitigate the danger it poses to first responders and alleviate damage inflicted by more stringent remediation methods. The three types of germination media used in this study were AI (L-alanine and inosine), AAC (L-alanine, adenosine and casamino acids) and AAC + YE (AAC and yeast extract) (Hachisuka 1969; Welkos et al. 2004). We previously demonstrated that germinated spores exposed 2

to AAC produce higher concentrations of protective antigen (PA) compared to spores germinated in the more minimal AI medium, indicating that metabolic properties can vary depending on which germination-induction solution is used (Cote et al. 2005). Germinated B. anthracis spores were demonstrated to cause significantly less disease in mice compared to ungerminated spores (McKevitt et al. 2007; Cote et al. 2009). Thus, germinating/germinated spores should pose less of a threat to first responders at a contaminated site allowing for potentially safer secondary decontamination and may also mitigate risk associated with re-aerosolization events (Layshock et al. 2012). This current study aimed to characterize the vulnerability of germinated spores from B. anthracis and B. thuringiensis suspended in nutritionally limited solutions, as well as secondary decontamination methods including bleach, hydrogen peroxide, formaldehyde and UV radiation. Bleach is one of the most common and cost-effective disinfectants and is a potent sporicidal agent (Kelsey et al. 1974; Rutala and Weber 1997). Sodium hypochlorite, the active ingredient in bleach, induces oxidative protein unfolding causing thermolabile proteins to irreversibly aggregate (Winter et al. 2008). Studies have shown that sodium hypochlorite causes the formation of chloramines that interfere in cellular metabolism, and it has also been implicated in fatty acid degradation (Miller and Britigan 1997; Estrela et al. 2002). Studies conducted with spores of Bacillus subtilis reveal that sodium hypochlorite may render spores defective in germination by severely damaging the spore inner membrane (Young and Setlow 2003). The drawbacks of using this sporicidal reagent are that it is highly corrosive and can cause damage to metals and equipment over time (Berutti et al. 2006). It is also harmful to humans and potentially detrimental to the environment. Sodium hypochlorite concentrations between 2 and 15% have been shown to be effective in killing Bacillus spores (Kelsey et al. 1974; Raber and Burklund 2010). Hydrogen peroxide has been proven to be sporicidal at high concentrations with current research focusing on decontamination of Clostridium spores (Baldry 1983; Whitney et al. 2003; Boyce et al. 2008; Meyer et al. 2013). In vegetative bacterial cells, hydrogen peroxide disrupts cell integrity by oxidizing a variety of cellular components, especially DNA (Imlay et al. 1988; Juven and Pierson 1996). Spores, on the other hand, possess biochemical and structural barriers, which reduce the permeability of the spore, thus inhibiting the entrance of oxidizing agent. Other important factors, including low water content, protein-saturated spore DNA, enzymes and a protein-rich coat protect spore DNA from some levels of peroxide bombardment (Setlow and Setlow

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1993; Riesenman and Nicholson 2000; Driks 2004; Tu et al. 2012). Some studies propose that the spore’s inner membrane sustains the most damage, and this disrupts proper cell outgrowth (Shin et al. 1994; Melly et al. 2002; Cortezzo et al. 2004). Disturbing the integrity of the inner membrane may increase the permeability of the spore core and permit toxic compounds to transverse the inner membrane and damage spore DNA (Shin et al. 1994; Melly et al. 2002; Cowan et al. 2004). Formaldehyde can be used as a spore disinfectant in both its liquid and gaseous states (Manchee et al. 1994; Rogers et al. 2007). It has been used in previous B. anthracis decontamination attempts and is considered effective in inactivating spores (Young et al. 1970; Cross and Lach 1990; Manchee et al. 1994; Rogers et al. 2007). Formaldehyde inactivates bacteria by alkylating the amino and sulfhydryl groups of proteins and nitrogen atoms of purine bases (Loshon et al. 1999). While formaldehyde is known to be effective, as demonstrated by the successful decontamination of Gruinard Island (Manchee et al. 1994), the concentration needed to kill B. anthracis spores is not considered safe for human exposure (Bernstein et al. 1984). As a known carcinogen, the US Occupational Safety and Health Administration (OSHA) has deemed high concentrations of formaldehyde to be detrimental to human health (OSHA 1993). The bactericidal properties associated with the UV-C portion of the ultraviolet spectrum are widely used as a means of inactivating micro-organisms for disinfection purposes (e.g. hospital settings and food sterilization) (Keyser et al. 2008; Moeller et al. 2010). The ultraviolet spectrum is traditionally divided into three regions characterized by wavelength and associated energy. As the most highly energized region, the UV-C range (200–280 nm) exerts deleterious effects on cellular DNA but is almost entirely incapable of penetrating the ozone layer (Sinha and Hader 2002). The UV-A (315–400 nm) and UV-B (280–315 nm) ranges, respectively, are present in the environment; they play a minor role in disinfectant purposes. UV-C radiation is a commonly used disinfectant method because its range includes the 254 nm emission that is readily absorbed by DNA. In vegetative cells, UV-C exposure induces the formation of potentially lethal photoproducts. Spores demonstrate increased resistance to UV light through a variety of defences including a robust repair system (Nicholson et al. 2000; Moeller et al. 2009). However, the most vital component appears to be the association of small acid-soluble spore proteins (SASPs) with spore DNA (Nicholson et al. 1991, 2002). This complex confers unique photoreactivity characteristics, which makes the spore more resilient than vegetative cells (Setlow 1988, 1995, 2007; Moeller et al. 2009). In this study, we present data confirming that germinated

Spore germination and decontamination

spores are more susceptible to some decontamination strategies (i.e. hydrogen peroxide and formaldehyde and potentially UV irradiation). Materials and methods Bacterial strains, bacteriological media and germinationinduction solutions The fully virulent Ames strain of B. anthracis was used in most experiments (Little and Knudson 1986). B. anthracis spores were produced in Leighton and Doi medium and purified with Hypaque/Omnipaque (GE Healthcare, Silver Spring, MD) as previously described (Leighton and Doi 1971; Cote et al. 2006). Bacillus thuringiensis subsp. Al Hakam, a naturally acrystalliferous strain (Challacombe et al. 2007), was obtained from the Unified Culture Collection (UCC) at USAMRIID. B. thuringiensis spores were produced in a similar manner to B. anthracis spores using Leighton and Doi medium or were produced in NBYS medium (Lecadet et al. 1980) for 96 h under 30°C incubation conditions. The B. thuringiensis spores were not density purified, but were successfully purified by washing in sterile phosphate-buffered saline (PBS) (Lonza, Walkersville, MD) to remove bacillary debris and other particulate matter. Due to differences in hydrophobicity observed between B. anthracis Ames and B. thuringiensis Al Hakam (White et al. 2014), B. anthracis spores were manipulated in water for injection (WFI) (Corning Cellgro, Corning, NY) or germination-induction solutions prepared in WFI, while B. thuringiensis spores were manipulated in PBS or germination-induction solutions prepared in PBS. Under our purification conditions, we observed that resuspending B. thuringiensis Al Hakam spores in PBS alleviates the tendency to aggregate. Unless otherwise noted, media components were purchased from Sigma-Aldrich (St. Louis, MO). Germination-induction media used in assays described below include AI (10 mmol l 1 L-alanine and 10 mmol l 1 inosine), AI + DCS (10 mmol l 1 L-alanine, 10 mmol l 1 inosine and 10 mmol l 1 D-cycloserine), AAC (18
7 mmol l 1 L-alanine, 6
2 mmol l 1 adenosine and 0
33% casamino acids [Becton Dickinson, Franklin Lakes, NJ]), AAC + YE (AAC medium + 0
33% yeast extract [Becton Dickinson, Franklin Lakes, NJ]) and D-AI (10 mmol l 1 D-alanine and 10 mmol l 1 inosine). All colony forming unit (CFU) determinations were performed using sheep blood agar (SBA) plates (Fisher Scientific, Pittsburg, PA). Additionally, unless otherwise noted, all spores used in germination or sensitivity assays were heat-activated for 30 min at 65°C and then briefly chilled on ice. The final spore concentration for these experiments was approximately 1–2 9 107spores ml 1.

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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significantly less than mice receiving ungerminated spores, suggesting that germinated spores could not survive the immune response mounted by the mice (Cote et al. 2009). In this study, concentrations of hydrogen peroxide or formaldehyde that failed to inactivate ungerminated spores were sufficient to render the germinated spores nonviable (Figs 6 and 7). Thus, our results indicate that remediation measures that employ the use of either disinfectant may use decreased concentrations, if a germination-induction step is implemented. Using reduced chemical concentrations may mitigate decontaminationassociated costs, collateral damage to infrastructure and health risks associated with exposure to these chemicals (Campbell et al. 2012). Furthermore, we chose to leave the germinationinduction solutions in the presence of spores as secondary 12

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Figure 7 Representative data demonstrating the impact of germination of B. anthracis (a) spores and B. thuringiensis (b) spores on sensitivity to formaldehyde. The X-axis labels indicate the germination-induction solutions used as well as the final concentration of formaldehyde used (0
2 or 2
0%). CFU ml 1 is depicted + SD. These are representative of data collected from three experiments.

decontaminants were added. We considered this to be an important variable to investigate because these solutions (or at least components of these solutions) would likely remain in place in real-world situations and may impact the disinfectant strategies employed. The presence of germinants had little effect on the impact of hydrogen peroxide and formaldehyde as both disinfectants were significantly more effective against germinated spores compared to ungerminated spores (Figs 6 and 7, respectively). However, the presence of these germinants reduced the efficacy of disinfection with bleach and UV radiation (Figs 4, 8–10). It is well documented that organic debris can interfere with the efficacy of bleach (Fukayama et al. 1986; Hawkins and Davies 1999; Hilgren et al. 2007). Once the germinants were removed, the cells were sensitive to the effects of bleach; however, it would not be feasible to induce germination and then remove the nutritive

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T.O. Omotade et al.

light source was monitored and measured with a UV-A/B meter (Sper Scientific, Scottsdale, AZ) and a UV-C meter (Sper Scientific), respectively. The recorded average intensities were 990–994lW cm 2 during UV-A exposure, 2900–3000lW cm 2 during UV-B exposure and 2700– 3200lW cm 2 during UV-C exposure. The samples were suspended in each respective medium in borosilicate glass tubes (VWR, Bridgeport, NJ) and subsequently irradiated at a distance of 15
2 cm from each light source for 5, 15 and 60 min, in solution. Additional experiments were performed to investigate to what extent the germinationinduction media would interfere with UV-C inactivation assays (e.g. shielding and scattering). The first method involved removing the germination-induction media following the 60-min incubation period (as described in the bleach sensitivity assays) and resuspending the samples in PBS. The samples were then irradiated for 60 min and cell survival was determined. In the second method, the germination-induction medium was not removed prior to UV-C irradiation. Rather, spores were incubated in PBS, AI or D-AI for 60 min, and each sample was subsequently irradiated. D-alanine is a potent germination inhibitor; thus, the D-AI medium would allow us to assess the impact of UV-C irradiation on ungerminated spores suspended in similar concentrations of nutrients as the germinated spores suspended in AI medium. Following each UV exposure, the samples were diluted and plated on SBA plates and incubated at 37°C. Colonies were then enumerated to determine CFU ml 1. Results Germination-induction media of varying complexities impact spore germination Significant debate remains as to the best methodology that would accomplish germination-induction so as to better kill spores subsequently in a real-world scenario. One important criterion to evaluate is the extent to which the potential germination-induction media would support germination in spores and whether this would correlate with an increased susceptibility to various decontamination methods (Campbell et al. 2012; Omotade et al. 2013). Accordingly, we characterized the effects of several germination media of various nutritional complexities to identify advantages or disadvantages associated with each medium. The media included the most defined minimal medium AI, the more nutritive and less defined AAC, and then the most nutritive AAC + YE. Germinated spores were routinely compared to ungerminated spores suspended in either water or PBS. We initially examined the extent of germination as measured by loss of heat resistance (exposure to 65°C for 30 min). As shown in

Spore germination and decontamination

Fig. 1, the extent of germination within 1 h was fairly comparable regardless of the germination-induction medium used. This was true for both B. anthracis (Fig. 1a) and B. thuringiensis (Fig. 1b) spores. In our hands, B. thuringiensis spores generally germinated to a greater extent following the 60-min incubation period in each germination-induction medium (approximately 99
96– 99
99%), compared to B. anthracis spores (approximately 97–99%). After determining the germination potential supported by each medium, we sought to better characterize the impacts of the germination media on the spores. As shown in Fig. 2, after 1 h of exposure to the germinationinduction solutions, the B. anthracis cells were appreciably different as compared to ungerminated B. anthracis spores in water (Fig. 2a). During this early time point, the morphological difference between spores germinated in AI compared to AAC or AAC + YE was apparent. The AIexposed cells appeared to resemble the ungerminated spore, but substantial rehydration and potential coat alterations were appreciable (Fig. 2b), and as indicated in Fig. 1a, these cells were shown to be mainly heat sensitive. In contrast, the characteristics of spores exposed to the more complex media (Fig. 2c,d) exhibited more extensive physical changes. We also examined the samples following a 24-h incubation period in each germination-induction medium and compared them to samples retained in water (Fig. 2e). As expected, the AI-exposed cells retained the exosporium and showed no indication of vegetative replication (Fig. 2f), but prolonged exposure to either AAC (Fig. 2g) or AAC + YE (Fig. 2h) demonstrated vegetative cell outgrowth and probable vegetative replication. The micrographs indicated apparent vegetative cell division, and this appeared to be more prominent within AAC + YE compared to the AAC-germinated spores. B. thuringiensis samples were also examined by phase contrast microscopy, and we observed similar effects of these germination-induction solutions (data not shown). These data suggest that our most minimal medium will likely result in germinated spores, which would be susceptible to environmental stressors, but the use of the minimal medium will not result in an increased risk of a secondary sporulation event. Suspending spores in a germination-induction solution that does not support outgrowth The samples of B. anthracis and B. thuringiensis that were germinated in our most minimal medium (AI) sustained a significant reduction in cell viability during the course of the prolonged incubation (162h). The B. anthracis unheated viable counts declined within the first 66 h, after which there was little additional germination (and

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Hilgren, J., Swanson, K.M., Diez-Gonzalez, F. and Cords, B. (2007) Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue. Appl Environ Microbiol 73, 6370–6377. Imlay, J.A., Chin, S.M. and Linn, S. (1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240, 640–642. Indest, K.J., Buchholz, W.G., Faeder, J.R. and Setlow, P. (2009) Workshop report: modeling the molecular mechanism of bacterial spore germination and elucidating reasons for germination heterogeneity. J Food Sci 74, R73–R78. Jernigan, J.A., Stephens, D.S., Ashford, D.A., Omenaca, C., Topiel, M.S., Galbraith, M., Tapper, M., Fisk, T.L. et al. and Anthrax Bioterrorism Investigation Team. (2001) Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 7, 933–944. Juven, B.J. and Pierson, M.D. (1996) Antibacterial effects of hydrogen peroxide and methods for its detection and quantitation. J Food Prot 59, 1233–1241. Kang, T.J., Fenton, M.J., Weiner, M.A., Hibbs, S., Basu, S., Baillie, L. and Cross, A.S. (2005) Murine macrophages kill the vegetative form of Bacillus anthracis. Infect Immun 73, 7495–7501. Kelsey, J.C., Mackinnon, I.H. and Maurer, I.M. (1974) Sporicidal activity of hospital disinfectants. J Clin Pathol 27, 632–638. Keyser, M., Muller, I.A., Cilliers, F.P., Nel, W. and Gouws, P.A. (2008) Ultraviolet radiation as a non-thermal treatment for the inactivation of microorganisms in fruit juice. Innov Food Sci Emerg Technol 9, 348–354. Koutchma, T. (2008) UV Light for Processing Foods. In IUVA NEWS. pp. 24–29. Layshock, J.A., Pearson, B., Crockett, K., Brown, M.J., Van Cuyk, S., Daniel, W.B. and Omberg, K.M. (2012) Reaerosolization of Bacillus spp. in outdoor environments: a review of the experimental literature. Biosecur Bioterror 10, 299–303. Lecadet, M.M., Blondel, M.O. and Ribier, J. (1980) Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber. J Gen Microbiol 121, 203–212. Leighton, T.J. and Doi, R.H. (1971) The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J Biol Chem 246, 3189–3195. Little, S.F. and Knudson, G.B. (1986) Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect Immun 52, 509–512. Loshon, C.A., Genest, P.C., Setlow, B. and Setlow, P. (1999) Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the alpha/beta-type protect spores against this DNA damage. J Appl Microbiol 87, 8–14. Manchee, R.J., Broster, M.G., Stagg, A.J. and Hibbs, S.E. (1994) Formaldehyde solution effectively inactivates spores

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(b)

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200 nm

200 nm (c)

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2 µm (g)

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Figure 2 Transmission electron micrographs demonstrating the resulting germination of B. anthracis Ames cells after 1-h (a–d) or 24-h exposure (e–h) to various germination-induction solutions. Spores were incubated in water for injection (a and e), AI germination-induction medium (b and f), AAC germination-induction medium (c and g) or AAC + YE germination-induction medium (d and h). The exosporium fibres are identified by a black arrow head, and the spore coat is identified with a white arrow head.

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(a) 1·E+08 1·E+07

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Figure 3 Representative data demonstrating the impact of suspending spores in a germination-induction solution, which does not support outgrowth on long-term viability of germinated B. anthracis spores (a) or B. thuringiensis (b) spores in the presence or the absence of D-cycloserine. The X-axis labels indicate the germination-induction solutions used and whether the sample was heated (H) and length of time each sample was permitted to incubate in media, CFU ml 1 is depicted + SD. These are representative of data collected from at least three experiments.

germination-induction solutions (AI, AAC, or AAC + YE) at room temperature for 60 min prior to the introduction of secondary disinfection strategies. We tested bleach, hydrogen peroxide, formaldehyde and artificial UV-A, UVB and UV-C light as secondary decontamination strategies. Bleach When B. anthracis Ames and B. thuringiensis Al Hakam spores incubated in germination-induction solutions were 8

subsequently treated with bleach, the germinated spores were nearly unaffected by the selected concentrations of bleach when compared to ungerminated spores treated with either sterile water or PBS (Fig. 4). These data suggested that germinated spores are not more susceptible to the effects of bleach compared to ungerminated spores; however, it is well documented that organic matter can interfere with bleach efficacy (Hilgren et al. 2007; Baert et al. 2009). Our results demonstrated that potentially even a mixture of 10 mmol l 1 L-alanine and

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

T.O. Omotade et al.

Spore germination and decontamination

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Hydrogen peroxide and formaldehyde Germinated spores of B. anthracis and B. thuringiensis were significantly more susceptible to the antimicrobial effects of hydrogen peroxide and formaldehyde as compared to ungerminated spores suspended in WFI or PBS (Figs 6 and 7). This was observed for B. anthracis (Figs 6a and 7a) and B. thuringiensis (Figs 6b and 7b). The ungerminated spores were not significantly killed under the disinfectant concentrations or contact times employed in our experiments. In contrast, the preliminary germination-induction step with AI, AAC and AAC + YE elicited substantial loss in viability of both species. In some cases, the more nutritive medium AAC + YE resulted in a greater susceptibility to secondary disinfection compared to AI or AAC, but the extent and significance of this benefit varied. Importantly, the presence of the germinants in the media did not negate the killing efficacy of either hydrogen peroxide or formaldehyde, as was seen with bleach (Fig. 4).

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10 mmol l 1 inosine (AI) is sufficient to completely negate the effects of bleach. For confirmation, experiments were performed with germinated spores that were resuspended in water or PBS after the AI germination medium was removed. After removing the germinationinduction medium, germinated spores of B. anthracis Ames (Fig. 5a) and B. thuringiensis Al Hakam (Fig. 5b) were susceptible to bleach in a concentration-dependent manner, which clearly demonstrates that the germination media neutralized bleach activity in the initial assays. Interestingly, after removing the germination media, the germinated spores were similarly susceptible as compared to ungerminated spores, and under our conditions, there was no significant advantage to pregerminating the spores in the context of bleach disinfection.

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Figure 4 Representative data demonstrating the impact of germination state of B. anthracis spores on sensitivity to bleach. The X-axis labels indicate the germinationinduction solutions used as well as the final concentration of bleach used (0
01% or 0
1%). CFU ml 1 is depicted + SD. The germination-induction solutions were not removed from the germinated spores prior to the addition of the bleach. These are representative of data collected from at least two experiments.

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Artificial UV-A, UV-B and UV-C radiation Neither ungerminated nor germinated B. anthracis (nor B. thuringiensis) spores exhibited a reduction in viability under artificial UV-A exposure conditions (Figs 8a and 9a). In comparison, UV-B irradiated samples were significantly reduced in a dose-dependent manner (Figs 8b and 9b), and this was further augmented in the presence of AI and AAC germination-induction media. The AAC germinated samples exhibited the greatest loss in viability for both B. anthracis and B. thuringiensis samples (greater than a 4-log decrease in viability in either species after 60 min of exposure). Although the AAC + YE germinated samples were killed during prolonged UV-B exposure, they proved to be the least susceptible to UV-B radiation. Under our UV-C exposure conditions, ungerminated spores (PBS or WFI) suffered the greatest reduction in viability; prolonged exposure resulted in approximately 1
5–2 log decreases in spore recovery for both B. anthracis and B. thuringiensis (Figs 8c and 9c). Our findings suggested that more nutritive germination conditions did not enhance the UV-C-associated killing response. Rather, the presence of even our most minimal germination medium (AI) seemed to largely prevent the UV-C-associated killing of germinated B. anthracis and B. thuringiensis spores. To explore this further, we removed the germinants after the 60-min germination period and suspended the cells in PBS. These samples were then irradiated under our standard exposure conditions. The germinated spores, once the AI was removed and they were resuspended in PBS, were more susceptible to UV-C disinfection (Fig. 10a) than ungerminated spores (P < 0
0001). Our results support previous findings which suggest that similar to what we observed in our bleach sensitivity assays, UV-absorbing compounds (i.e. purines) inhibit the killing normally associated with germicidal UV-C light (Nicholson and

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Galeano 2003; Caron et al. 2007; Coohill and Sagripanti 2008). As shown in Fig. 10b, we also incubated ungerminated spores in D-alanine, a potent transient germination inhibitor (Fey et al. 1964; McKevitt et al. 2007), and inosine. We confirmed that B. thuringiensis spores suspended in D-AI medium remained largely ungerminated (based upon heat resistance properties) throughout the course of the experiment (data not shown). Spores suspended in PBS (ungerminated), AI (germinated) and D-AI (ungerminated) were irradiated under our UV-C inactivation conditions. As expected, the ungerminated spores suspended in D-AI appear more resistant to UV-C radiation as compared to ungerminated spores suspended in PBS. This loss of sensitivity indicated that even the components of our most minimal germination-induction medium were sufficient to hinder the efficacy of UV-C irradiation (Fig. 10b). 10

1

Figure 5 Impact of bleach on AI-germinated B. anthracis (a) and B. thuringiensis (b) spores. The germination-induction solutions were removed prior to the addition of bleach. Germinated spores were exposed to bleach concentrations of 0%, 0
01%, 0
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Our UV exposure experiments were performed with samples contained in borosilicate glass tubes, a material which is known to block some UV irradiation and which can thus partially shield spores from its deleterious effects. Nonetheless, our proof of concept results clearly showed the sporicidal effects of UV-B and UV-C and demonstrated the relative susceptibilities of the cell types (and treatments) under the conditions tested. Our data also underscored potential pitfalls associated with choosing UV irradiation as a means of secondary decontamination following a germination-induction period. Discussion We explored whether more nutritionally complex germination-induction media (AAC and AAC + YE) would further sensitize the newly germinated spore to subsequent

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

T.O. Omotade et al.

Spore germination and decontamination

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Figure 6 Representative data demonstrating the impact of germination of B. anthracis spores (a) and B. thuringiensis (b) spores on sensitivity to hydrogen peroxide. The X-axis labels indicates the germination-induction solutions used as well as the final concentration of hydrogen peroxide used (0
1% or 1%). CFU ml 1 is depicted + SD. These are representative of data collected from three individual experiments.

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decontamination measures as compared to the more minimal AI medium. Although each medium employed in this study supported comparable levels of germination in both Bacillus species (Fig. 1a,b), they also induced phenotypically distinct levels of outgrowth depending upon the ‘richness’ of the medium (Fig. 2). Our findings also indicate that, in general, our most minimal germinationinduction solution (AI) sensitized spores to subsequent treatment as compared to our most nutritionally complex solution (AAC + YE). Thus, the use of a more nutritive germination-induction medium that could induce bacterial replication possibly causing a subsequent sporulation event or promoting the growth of other undesirable environmental organisms (i.e. algal blooms) may not be the most appropriate strategy. It has been demonstrated that germinated spores are more susceptible to antimicrobial-associated killing in both in vivo and in vitro models. Gut et al. (2011) illustrated that subjecting B. anthracis spores to a diverse

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panel of germinating conditions effectively sensitized them to phagocytic killing. This study also demonstrated that intracellular B. anthracis viability was significantly reduced when spores were pregerminated in minimal (10 mmol l 1 AI) and nutritive (10% FBS) media. Kang et al. (2005) and Basu et al. (2007) emphasized the heightened vulnerability to killing of newly germinated spores compared to ungerminated spores. Using spores with defined genetic mutations, Giorno et al. (2007) demonstrated that spores that germinated rapidly were more susceptible to the killing effect of macrophages, whereas spores with a slower germination rate were resistant to killing for an extended period of time. Correspondingly, mice receiving germinated spores via intranasal instillation were significantly more likely to survive infection as compared to mice receiving ungerminated spores (McKevitt et al. 2007; Cote et al. 2009). Importantly, it was also demonstrated that the bacterial burden of mice challenged with the germinated spores was

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Spore germination and decontamination

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significantly less than mice receiving ungerminated spores, suggesting that germinated spores could not survive the immune response mounted by the mice (Cote et al. 2009). In this study, concentrations of hydrogen peroxide or formaldehyde that failed to inactivate ungerminated spores were sufficient to render the germinated spores nonviable (Figs 6 and 7). Thus, our results indicate that remediation measures that employ the use of either disinfectant may use decreased concentrations, if a germination-induction step is implemented. Using reduced chemical concentrations may mitigate decontaminationassociated costs, collateral damage to infrastructure and health risks associated with exposure to these chemicals (Campbell et al. 2012). Furthermore, we chose to leave the germinationinduction solutions in the presence of spores as secondary 12

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Figure 7 Representative data demonstrating the impact of germination of B. anthracis (a) spores and B. thuringiensis (b) spores on sensitivity to formaldehyde. The X-axis labels indicate the germination-induction solutions used as well as the final concentration of formaldehyde used (0
2 or 2
0%). CFU ml 1 is depicted + SD. These are representative of data collected from three experiments.

decontaminants were added. We considered this to be an important variable to investigate because these solutions (or at least components of these solutions) would likely remain in place in real-world situations and may impact the disinfectant strategies employed. The presence of germinants had little effect on the impact of hydrogen peroxide and formaldehyde as both disinfectants were significantly more effective against germinated spores compared to ungerminated spores (Figs 6 and 7, respectively). However, the presence of these germinants reduced the efficacy of disinfection with bleach and UV radiation (Figs 4, 8–10). It is well documented that organic debris can interfere with the efficacy of bleach (Fukayama et al. 1986; Hawkins and Davies 1999; Hilgren et al. 2007). Once the germinants were removed, the cells were sensitive to the effects of bleach; however, it would not be feasible to induce germination and then remove the nutritive

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

T.O. Omotade et al.

Spore germination and decontamination

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Figure 8 UV-A, UV-B and UV-C irradiation of ungerminated and germinated B. anthracis spores. Ungerminated (water for injection) and germinated (AI, AAC or AAC + YE) B. anthracis spores were irradiated under artificial UV-A (a), UV-B (b) and UV-C (c) conditions. Under each respective exposure condition, the samples were irradiated for 0, 5, 15 and 60 min. CFU ml 1 is depicted + SD. These data are the average of five experiments.

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Figure 9 UV-A, UV-B and UV-C irradiation of ungerminated and germinated B. thuringiensis spores. Ungerminated (PBS) and germinated (AI, AAC or AAC + YE) B. thuringiensis spores were irradiated under artificial UV-A (a), UV-B (b) and UV-C (c) conditions. Under each respective exposure condition, the samples were irradiated for 0, 5, 15 and 60 min. CFU ml 1 is depicted + SD. These data are the average of five experiments.

14

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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Spore germination and decontamination

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Figure 10 Data demonstrating the impact of germination-induction medium on UV-C inactivation efficiency with Bacillus thuringiensis spores irradiated in borosilicate glass tubes. (a) Following the 60-min germination period, the germinationinduction medium was removed prior to UV-C exposure. Each sample was then irradiated under artificial UV-C conditions for 60 min, while control samples were not exposed, 0 min. (b) Experiments were also performed on ungerminated B. thuringiensis spores suspended in D-AI (germinationinhibition solution). All samples were exposed under our standard UV-C conditions for 0 min or 60 min. CFU ml 1 is depicted + SD, and the data are an average of three experiments.

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germinants prior to secondary decontamination in an actual remediation event. We also examined UV irradiation as a secondary decontamination strategy. Each region of the UV spectrum (UV-A, UV-B and UV-C) may induce varying detrimental effects on spores (Xue and Nicholson 1996; Slieman and Nicholson 2001; Moeller et al. 2009). Interestingly, some studies have identified a highly resistant transient stage in the germinating spore that is short-lived, yet appears to be photobiologically distinct from the dormant spore and the

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vegetative bacilli (Stafford and Donnellan 1968; Munakata 1974). Under our experimental UV-C inactivation conditions (using samples contained in glass tubes), the results indicated that germinated spores were more resistant than the ungerminated samples (Figs 8c and 9c). However, it is known that attenuation of the UV-C intensity (and associated germicidal activity) may be due to a number of factors including organic debris (Keyser et al. 2008), the efficiency of the light source (Skowron et al. 2014), dry or wet conditions (Slieman and Nicholson 2001), the presence of

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

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UV-absorbing compounds (Koutchma 2008) and other physical variables. Once we removed the AI medium from the germinated samples, the newly germinated spores were more susceptible to UV-C inactivation than ungerminated spores (Fig. 10a). Collectively, these findings suggest the germinated spores are relatively more vulnerable to UV-C radiation, but the presence of certain compounds may strongly inhibit or dampen UV-C inactivation. Our experiments evaluated samples contained in borosilicate glass tubes. Accordingly, these experimental conditions also resulted in a certain amount of shielding associated with the glass tubes. While not ideal for establishing actual kill curves associated with UV irradiation, this experimental design allowed us to test many samples and replicates including fully virulent B. anthracis spores under BSL-3 conditions. Even with the associated shielding effect of the tubes, we achieved ≥95% reduction in spore viability after UV-C irradiation. This experimental model allowed us to gather proof of concept data demonstrating that UV radiation, as a potential secondary decontamination strategy, would likely be significantly hindered by the presence of UV-absorbing germinants (i.e. purines) (Owens et al. 2005; Fredericks et al. 2011). Most importantly, as we also observed in our bleach disinfection assays, obstructions that undermine the inactivation of spores by a disinfectant further highlight the importance of designing germinationinduction and decontamination strategies that account for these variables. In this report, we provide a series of data demonstrating the increased susceptibility of germinated B. anthracis and B. thuringiensis spores to certain disinfection strategies. These data also add to evidence suggesting that B. thuringiensis may serve as a reasonable surrogate for B. anthracis in proposed field trials. However, all of our experiments were performed in solution. To more completely investigate the potential of these proposed strategies (including completely unshielded UV exposure experiments), experiments need to be conducted on materials that mimic natural surfaces that may be contaminated (e.g. concrete, asphalt and soil). There are several complex aspects to consider in such studies. When sampling surfaces for ungerminated spores, a great amount of variation exists depending upon the type of surface and the sampling/collection methods used (Estill et al. 2009; Piepel et al. 2012; Da Silva et al. 2013). This variation may be further exacerbated when germinated spores are used. To reliably measure the impact of secondary decontamination methods, it is important to accurately calculate the amount of germinated spores recovered and to ensure that sampling procedures do not inadvertently inactivate them. Each secondary disinfectant tested possesses a unique inactivation mechanism that might not be suitable 16

under specific remediation events. A comprehensive decontamination plan will most likely be comprised of different combined disinfectant strategies. Therefore, it is also important to consider how these strategies will work in tandem and if they will work antagonistically or in concert. It has been reported that certain disinfectants (i.e. UV-C and hydrogen peroxide) could be combined to maximize the advantages of both treatments (Rutherford et al. 2000; Reidmiller et al. 2003; Zhang et al. 2014). When considering a germination-induction pretreatment plan, it is critical to account for the diversity that is intrinsic to germinating spore populations. Specifically, the ‘superdormant’ spores possess different germination kinetics and germination requirements, which may also correlate with different physiological traits that should be taken into account (Ghosh and Setlow 2010; Indest et al. 2009). These factors, amongst others, will greatly influence the efficacy and feasibility of a proposed germination-induction measure. Although there are many variables involved in wide-area remediation, data presented here serve as a proof of principle for germination-induction and its potential utility in wide-area spore decontamination. Acknowledgements The authors wish to thank Ms. Andrea Keefer and Ms. Avery Quirk for their excellent technical assistance. The research described herein was sponsored by the Defense Threat Reduction Agency JSTO-CBD plan CBTPHM-12-CB2-CB3831 to SLW and CKC. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army. The authors have no conflict of interest to declare. Conflict of interest No conflict of interest declared. References Abee, T., Groot, M.N., Tempelaars, M., Zwietering, M., Moezelaar, R. and van der Voort, M. (2011) Germination and outgrowth of spores of Bacillus cereus group members: diversity and role of germinant receptors. Food Microbiol 28, 199–208. Baert, L., Vandekinderen, I., Devlieghere, F., Van Coillie, E., Debevere, J. and Uyttendaele, M. (2009) Efficacy of sodium hypochlorite and peroxyacetic acid to reduce murine norovirus 1, B40-8, Listeria monocytogenes, and Escherichia coli O157:H7 on shredded iceberg lettuce and in residual wash water. J Food Prot 72, 1047–1054.

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