Editor s Choice. Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Analysis of the germination kinetics of individual Bacillus subtilis spores treated with hydrogen peroxide or sodium hypochlorite B. Setlow 1,J.Yu 2, Y.-Q. Li 2 and P. Setlow 1 1 Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA 2 Department of Physics, East Carolina University, Greeneville, NC, USA Editor s Choice Significance and Impact of the Study: This work shows that with Bacillus subtilis spore populations in which approximately 95% of individual spores were killed by several oxidizing agents, >95% of the spores in these populations germinated with nutrients, albeit slowly. This is important, as assay of an early germination event, release of dipicolinic acid, has been suggested as a rapid assay for spore viability and would give false-positive readings for the level of the killing of oxidizing agent-treated spore populations. Analysis of the germination kinetics of multiple individual untreated or oxidizing agenttreated spores also provides new information on proteins damaged by oxidizing agent treatment, and at least some of which are in spores inner membrane. Keywords Bacillus, hydrogen peroxide, hypochlorite, spores, spore germination, sterilization. Correspondence Peter Setlow, Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT , USA. setlow@nso2.uchc.edu 213/639: received 2 April 213, revised 4 June 213 and accepted 4 June 213 doi:1.1111/lam Abstract More than 95% of individuals in populations of Bacillus subtilis spores killed approximately 95% by hydrogen peroxide or hypochlorite germinated with a nutrient, although the germination of the treated spores was slower than that of untreated spores. The slow germination of individual oxidizing agent-treated spores was due to: (i) 3- to 5-fold longer lag times (T lag ) between germinant addition and initiation of fast release of spores large dipicolinic acid (DPA) depot (ii) 2- to 1-fold longer times (DT release ) for rapid DPA release, once this process had been initiated; and (iii) 3- to 7-fold longer times needed for lysis of spores peptidoglycan cortex. These results indicate that effects of oxidizing agent treatment on subsequent spore germination are on: (i) nutrient germinant receptors in spores inner membrane (ii) components of the DPA release process, possibly SpoVA proteins also in spores inner membrane, or the cortex-lytic enzyme CwlJ; and (iii) the cortex-lytic enzyme SleB, also largely in spores inner membrane. This study further indicates that rapid assays of spore viability based on measurement of DPA release in spore germination can give false-positive readings. Introduction Spores of some Bacillus species can cause food spoilage or foodborne disease, and Bacillus anthracis spores are a potent biological weapon (Setlow and Johnson 212). As a consequence, there is much interest in developing methods to kill such spores and to understand the mechanism(s) of spore killing as well as potential limitations in various spore killing regimens. Spores can be killed by high temperatures and UV or c-radiation, but some regimens for spore inactivation or decontamination use toxic chemicals. Oxidizing agents in particular are used for spore killing, in particular in applied settings, where agents such as hydrogen peroxide (H 2 O 2 ) and sodium hypochlorite are used (Al-Adham et al. 213). The mechanism whereby these latter two agents kill spores has been reasonably well studied, and despite the potential genotoxicity of these agents, they do not kill spores by DNA damage (Young and Setlow 23; Cortezzo et al. 24; Setlow 26; Setlow and Johnson 212). Interestingly, spores treated with either of these agents often germinate, especially if the treatment is not too harsh (Cortezzo et al. 24). However, in most cases, Letters in Applied Microbiology 57, The Society for Applied Microbiology 259

2 Germination of oxidizing agent-treated spores B. Setlow et al. the germinated-treated spores do not outgrow and exhibit minimal metabolism, suggesting that one or more key spore proteins have been severely damaged by these oxidizing agents, as has also been suggested for wet-heat-treated spores of Bacillus species (Coleman et al. 27, 21; Coleman and Setlow 29). Unfortunately, the fact that these oxidizing agent-treated spores do germinate is a potential problem, even if the germinated spores are actually dead, since some rapid assays of spore viability monitor changes that take place in spore germination, in particular, the release of the spore core s large depot of pyridine-2,6-dicarboxylic acid (dipicolinic acid (DPA)) (Yung et al. 27; Yang and Ponce 211). Thus, there is the potential for false-positive readings of spore viability with such assays in spore populations. As noted above, the germination of populations of spores treated with oxidizing agents has been reasonably well studied. However, analysis only of the germination of spore populations can obscure important information, as germination of individuals in spore populations is extremely heterogeneous (Setlow 23; Setlow et al. 212). In recent years a number of techniques have been developed for the analysis of the germination of large numbers of individual spores in populations (Zhang et al. 21a,b; Kong et al. 211). Such techniques have also been applied to the analysis of the germination of Bacillus subtilis spores killed by wet heat, and this analysis has revealed steps in the germination pathway that are especially sensitive to wet heat (Wang et al. 211a,b, 212). In the current work, we have extended this work to compare the kinetic parameters of the germination of B. subtilis spore populations that are either untreated, or treated with H 2 O 2 or hypochlorite to kill approximately 95% of the population, with spore killing measured by the ability to form colonies on nutrient plates. The results of this comparison have given new insight into the effects of oxidizing agents on spores. what step or steps in the germination pathway are slowed by oxidizing agent treatment; and (iii) can the answers to these questions give information on effects of H 2 O 2 and hypochlorite on spores. To initiate attempts to answer the questions posed above, we treated B. subtilis spores with either H 2 O 2 or hypochlorite to get approximately 95% killing (93 97%, with killing defined as the inability of a spore to form a colony on a nutrient plate), inactivated the oxidizing agent, and compared the nutrient germination of individual untreated spores with that of the treated spores, all as described in Methods. As measured by monitoring DPA release, approximately 95% of spores in untreated populations germinated with the nutrient germinant L-valine in approximately 4 min, and the germination behaviour of the untreated spores was unaffected by the procedures used to inactivate either H 2 O 2 or hypochlorite prior to subsequent analysis (Fig. 1a,b). Notably, a large percentage DPA release - % (a) (b) Results and discussion Germination of untreated and oxidizing agent-treated spore populations Previous work has shown that most individual spores in populations killed approximately 9% with H 2 O 2 or hypochlorite germinated to a significant extent, when germination was assessed by release of DPA (Cortezzo et al. 24). However, the germinated-treated spores did not exhibit significant metabolism or outgrowth. Questions that were not previously addressed, however, include: (i) do the oxidizing agent-treated spores that did not germinate in the observation periods used previously eventually germinate (ii) if these treated spores do germinate slowly, Time in minutes Figure 1 (a,b) Germination of untreated and oxidizing agent-treated spore populations. B. subtilis PS533 spore populations that were untreated (a,b) or treated with H 2 O 2 (a) or hypochlorite (b) to get 93 or 97% killing, respectively, were heat activated, germinated, and germination was assessed by monitoring DPA release as described in Methods. The symbols used are: s, untreated spores;, spores treated with catalase or thiosulfate only; and, spores treated with either H 2 O 2 or hypochlorite and then with either catalase (H 2 O 2 )or thiosulfate (hypochlorite). The DPA release measured in this experiment was 3% of the value shown. 26 Letters in Applied Microbiology 57, The Society for Applied Microbiology

3 B. Setlow et al. Germination of oxidizing agent-treated spores of spores in populations that had been killed approximately 95% by H 2 O 2 or hypochlorite also germinated, but took much longer to reach this level of DPA release for the treated spores, up to 3 min for the hypochlorite-treated spores and approximately 2 min for the H 2 O 2 -treated spores (Fig. 1a,b, and data not shown, and see below). The germination of spores in populations killed >999% by hypochlorite or H 2 O 2 was even slower, as only 2 3% of the treated spores germinated in 3 min (data not shown). Together these findings indicate that treatments with H 2 O 2 or hypochlorite killing the great majority of spores in populations does not completely inactivate any key spore germination component, but only slows the action of one or more of these components. However, if the treatment with oxidizing agents is harsh enough, then spore germination, at least on a reasonable time scale, is abolished, and this is also the case for spores treated with wet heat (Coleman et al. 27, 21). Analysis of the germination of multiple individual untreated or oxidizing agent-treated spores While the results with spore populations were useful, previous work has shown that the heterogeneity in spore germination can obscure many of the important kinetic parameters of spore germination (Zhang et al. 21a,b; Kong et al. 211; Setlow et al. 212). Consequently, we monitored the germination of large numbers of individual untreated and H 2 O 2 and hypochlorite-treated spores in spore populations that had been killed 93 and 97%, respectively (Fig. 2). The results of this experiment were relatively similar to those obtained following the germination of untreated and H 2 O 2 and hypochlorite-treated spore populations by monitoring DPA release, as the oxidizing agent-treated spores germinated slower even though 95% of the treated spores did eventually germinate. Analysis of the germination curves of multiple individual spores by DIC microscopy (Fig. 3a c) showed that as expected, the curves for the germination of both the untreated and oxidizing agent-treated individual spores were quite heterogeneous. Previous work has identified a number of key time points in the germination of individual spores of Bacillus species (Zhang et al. 21a,b; Kong et al. 211), as shown for one spore each in Fig. 3a c. Upon addition of nutrient germinants to an individual spore, there is a variable lag period with no obvious change in spores, as spore DPA content remains relatively constant although can decrease somewhat very slowly. However, at some point, rapid DPA release begins, with the time of initiation of this event termed T lag. The point when this rapid DPA release is completed is termed T release, and almost all DPA is released in DT release that is equal to T release T lag. The release of spores DPA and its Germination - % Incubation time (min) Figure 2 Germination of multiple individual untreated and oxidizing agent-treated spores. B. subtilis PS533 spore populations either untreated or oxidizing agent-treated were germinated, heat activated, and the germination of multiple individual spores was assessed by measurement of spores DIC images as described in Methods. Numbers of individual spores examined were (and the symbols used): untreated, 394 ( ); H 2 O 2 treated and killed 93%, 7 (s); and hypochlorite treated and killed 97%, 813 (h). replacement in the spore core by water is accompanied by a decrease in a spore s DIC image intensity of approximately 75%. Following T release, there is a further decrease in spores DIC image intensity due to the hydrolysis of the spores peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and associated core swelling and water uptake. This process ends at T lys, and DT lys, the time for spore cortex hydrolysis equal to T lys T release. It was clear even from a cursory examination of the data for multiple individual untreated and oxidizing agent-treated spores (Fig. 3a c) that the oxidizing agenttreated spores, in particular, those treated with hypochlorite, had much longer T lag values than did untreated spores. Compilation of the kinetic parameters for the germination of individual untreated or oxidizing agent-treated spores showed that as expected, T lag values increased 3- to 5-fold with agent-treated spores compared to untreated spores, values for DT release were 2- to 1-fold higher in the treated spores, and DT lys values were 3- to 7-fold higher in treated spores (Table 1). The results noted above indicate that H 2 O 2 and hypochlorite treatment caused significant damage to one or more spore germination proteins, and this likely indicates some targets for these agents in spores even if these targets are themselves not the lethal targets for these oxidizing agents. One effect of H 2 O 2 and hypochlorite treatment on spore germination kinetics was the 3- to 5-fold increase in T lag values. Previous work has shown that two major variables determining T lag values in spore Letters in Applied Microbiology 57, The Society for Applied Microbiology 261

4 B. Setlow et al. Germination of oxidizing agent-treated spores (a) Normalized intensity (a.u.) 1 Tlag Trelease 2 Tlys Time (min) 4 5 (b) Normalized intensity (a.u.) 1 Tlag Trelease 2 Tlys Time (min) 1 (c) 1 Normalized intensity (a.u.) 12 Tlag Trelease 2 Tlys Time (min) Figure 3 (a c) Germination curves for multiple individual untreated and oxidizing agent-treated spores. The germination of 16 randomly selected individual untreated or oxidizing agent-treated B. subtilis PS533 spores as described in Methods from populations that were either: (a) untreated, (b) H2O2 treated giving 93% killing or (c) hypochlorite treated giving 97% killing were obtained from the DIC microscopic images of single spores as described in Methods. Time points for Tlag, Trelease and Tlys are shown for one spore in each panel of the figure. 262 Letters in Applied Microbiology 57, The Society for Applied Microbiology

5 B. Setlow et al. Germination of oxidizing agent-treated spores Table 1 Kinetic parameters of germination of untreated and oxidizing agent-treated B. subtilis spores* Spores Untreated H 2 O 2 -treated Hypochloritetreated Individual spores Examined 394 (96) 7 (95) 813 (93) (% germinated) Individual spores analysed T lag (min) T release (min) DT release (min) DT lys (min) *Untreated or oxidizing agent-treated B. subtilis PS533 spore populations were heat shocked and germinated, and kinetic parameters for the germination of multiple individual spores were extracted from the data as shown in Fig. 3a c. Spore populations treated with H 2 O 2 or hypochlorite were killed 93 and 97%, respectively. Values for the kinetic germination parameters shown are the average values from the individual spores analysed (all of which germinated) the standard deviations. The germination of individual spores was followed for 6 min (untreated spores), 14 min (H 2 O 2 -treated spores) or 3 min (hypochlorite-treated spores). germination are spores levels of: (i) nutrient germinant receptors (GRs) that recognize and respond to specific nutrient germinants and (ii) the GerD protein essential for proper GR function (Zhang et al. 21a; Wang et al. 211b; Ghosh et al. 212; Ramirez-Peralta et al. 212). GRs and GerD are located in spores inner membrane (IM), and previous work indicates that most oxidizing agents kill spores by damaging the IM such that when spores germinate, this membrane ruptures (Setlow 23; Cortezzo et al. 24). While the specific spore IM damage caused by oxidizing agent treatment is unknown, it has been suggested to be damage to IM proteins, and perhaps damage to GRs and GerD is a reflection of this. H 2 O 2 and hypochlorite treatment also resulted in significant increases in times for the rapid DPA release, DT release, during spore germination. These increases cannot be due to damage to GRs or GerD, as fluctuation in these proteins levels does not alter DT release values (Zhang et al. 21a; Wang et al. 211b). One group of proteins damage to which might result in increased DT release values is the SpoVA proteins involved in DPA movement across the spores IM in germination (Setlow 23; Wang et al. 211b; Setlow et al. 212). Another protein important in determining DT release values is the CwlJ protein, one of Bacillus spores two redundant CLEs that degrade spores PG cortex during germination, as loss of CwlJ results in increases in DT release values up to 15-fold (Peng et al. 29; Setlow et al. 29), and at present, we cannot decide between damage to SpoVA proteins or CwlJ as responsible for the increased DT release values for oxidizing agent-treated spores, although it appears clear (see below) that at least some CwlJ has been inactivated by oxidizing agent treatment. Finally, as DT lys values also increased markedly in oxidizing agent-treated spores, and either CwlJ or the other redundant CLE, SleB, alone is sufficient for normal values of DT lys during spore germination, oxidizing agent treatment must have damaged both CwlJ and SleB significantly. Interestingly, SleB is also largely in spores IM (Chirakkal et al. 22), although CwlJ is located at the spore coat-cortex boundary (Setlow 23) where it will be more accessible to oxidizing agents than IM proteins. While analysis of spore germination kinetics has given new insight into damage caused by oxidizing agent treatment of spores, it is also important to appreciate that these treated spores can germinate well even if most of these spores are actually dead as measured by the ability of a spore to germinate, outgrow and ultimately form a colony. As a consequence, another major conclusion from the current work is that assays of spore germination measuring DPA release are inappropriate for rapid routine measurement of the viability of oxidizing agent-treated spore populations, as such assays can readily give large numbers of false-positive results, as they also will for spores killed by wet heat which also can germinate, albeit slowly, if the wet-heat treatments of spore populations have not been too harsh (Wang et al. 211a, 212). Materials and methods Spores used The B. subtilis strain used in this work was PS533, a prototrophic 168 derivative that carries plasmid pub11 encoding kanamycin resistance (1 mg l 1 ) (Setlow and Setlow 1996). Spores of this strain were prepared on 2x Schaeffer s-glucose plates that were incubated for approximately 3 days at 37 C; following further incubation for 2 3 days at 23 C, the spores were scraped from the plates and purified as described previously (Nicholson and Setlow 199; Paidhungat et al. 2), and stored in water at 4 C. No reduction in spore viability was seen in the approximately 3 months during which experiments with these spores were carried out. All spores used in this work were free (>98%) of growing or sporulating cells, germinated spores and cell debris as seen by phase contrast microscopy. Treatment of spores with H 2 O 2 and sodium hypochlorite Spore treatment with H 2 O 2 or sodium hypochlorite at 23 C to determine killing curves was essentially as described previously (Cortezzo et al. 24), with spores at an optical density at 6 nm (OD 6 ) of 1 in either Letters in Applied Microbiology 57, The Society for Applied Microbiology 263

6 Germination of oxidizing agent-treated spores B. Setlow et al. 5 mmol l 1 KPO 4 buffer plus 25 mol l 1 H 2 O 2 or in 1 mg l 1 sodium hypochlorite prepared in water (measured ph of 11). At various times, aliquots were diluted 1/ 1 in 1 ml of either 5 mmol l 1 KPO 4 buffer (ph 74) with 5 units of beef liver catalase (H 2 O 2 )or1gl 1 Na 2 S 2 O 4 (hypochlorite) and incubated at least 15 min at 23 C to inactivate the oxidizing agents as described previously (Cortezzo et al. 24), and 1 ll aliquots of serial dilutions in sterile water were spotted on LB medium agar plates containing kanamycin (1 mg l 1 ). The plates were incubated for h at 37 C and colonies were counted. Incubation for longer times gave no further increases in colonies. A dead spore was then defined as a spore that does not give rise to a colony in this assay. To obtain large amounts of spores treated with NaOCl, 2 ml of spores was incubated, and at times determined in preliminary experiments that gave approximately 95% killing of spore populations, aliquots were diluted and spore viability was analysed, all as described above. The remainder of the culture was centrifuged, the pellet suspended in 2ml of 1gl 1 Na 2 S 2 O 4, incubated for approximately 15 min at 23 C, washed 3x with water and stored in water at 4 C. Large amounts of H 2 O 2 treated spores were prepared by incubation of spores at 23 C in 5 ml of 5 mmol l 1 KPO 4 (ph 7.4) with spores at an OD 6 of 5. At various times, 1-ml aliquots was placed into a capped test tube, 1 ll of beef liver catalase (approximately 5 units) added on the side of the tube, and the tube was capped and mixed. After incubation for approximately 15 min at 23 C, the sample was washed twice by centrifugation, suspended in 1 ml cold sterile water, and spore viability was determined as described above. The various treated spore samples were stored at 4 C. Monitoring the germination of spore populations Germination of spore populations was preceded by a heat shock (65 C; 3 min) of spores in water followed by cooling on ice. Germination of heat-shocked spores was at an OD 6 of.5 in 2 ll of 25 mmol l 1 K-Hepes buffer (ph 74)-1 mmol l 1 L-valine-5 l mol l 1 terbium (Tb +3 ) chloride, and spore germination was followed in a multiwell fluorescence plate reader, monitoring DPA release by the high fluorescence of Tb-DPA as previously described (Yi and Setlow 21). The total amount of DPA in spores was determined by its fluorescence with Tb after spores DPA was released by boiling for 15 min as described previously (Yi and Setlow 21). Monitoring the germination of individual spores Analysis of the germination of multiple individual spores by DIC microscopy was as described previously (Zhang et al. 21a,b; Kong et al. 211; Wang et al. 212). Briefly, heat-shocked spores prepared as described above (1 ll; approximately 1 8 spores ml 1 in water) were spread on the surface of a microscope coverslip that was then dried in a vacuum desiccator for 5 1 min. The coverslips were then mounted on and sealed to a microscope sample holder kept at 37 C. The DIC microscope was set such that the polarizer and analyser were crossed, and thus, the DIC bias phase was zero. After adding preheated germinant/buffer solution (1 mmol l 1 L-valine-25 mmol l 1 K-Hepes buffer (ph 74)) to spores on the coverslips, a digital CCD camera (16 bit, 1,6 by 1,2 pixels) was used to record the DIC images at a rate of 1 frame per 15 s for 6 12 min. These images were analysed with a computation program in Matlab to locate each spore s position and to calculate the averaged pixel intensity of an area of pixels that covered the DIC image of the whole individual spore. The DIC image intensity of each individual spore was plotted as a function of the incubation time (with a resolution of 15 s); the initial intensity at T (the first DIC image recorded after the addition of the germinant) was normalized to 1, and the intensity at the end of measurements was normalized to zero. Invariably, the latter value had been constant for 1 min at the end of measurements. From the time-lapse DIC image intensity, we can determine the time of completion of the rapid fall of approximately 75% in spore DIC image intensity, which is concomitant with the time of completion of spore DPA release (this time is defined as T release ). DPA release kinetics during germination of individual spores are described by the parameters T lag and DT release, where T lag is the time between the mixing of spores with germinants and the initiation of most DPA release, and DT release = (T release T lag ). We also defined the additional germination parameters, T lys and DT lys, where T lys is the time when hydrolysis of the spore s peptidoglycan cortex is completed as determined by the completion of the fall in the spore s DIC image intensity, and DT lys = (T lys T release ). Acknowledgements This communication is based on work supported by the U.S. Army Department of Defense Multidisciplinary University Research Initiative through the U.S. Army Research Laboratory and the U.S. Army Research Office under contract number W911F References Al-Adham, I., Haddadin, R. and Collier, P. (213) Types of microbicidal and microbiostatic agents. In Principles and Practice of Disinfection, Preservation and Sterilization, 5th 264 Letters in Applied Microbiology 57, The Society for Applied Microbiology

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