Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens

Authors

  • G. Wang,

    1. Department of Physics, East Carolina University, Greenville, NC, USA
    Current affiliation:
    1. Laboratory of Biophysics, Guangxi Academy of Sciences, Nanning, China
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  • D. Paredes-Sabja,

    1. Departments of Biomedical Sciences and Microbiology, Oregon State University, Corvallis, OR, USA
    2. Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas,Laboratorio de Mecanismos de Patogénesis Bacteriana, Universidad Andres Bello, Santiago, Chile
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  • M.R. Sarker,

    1. Departments of Biomedical Sciences and Microbiology, Oregon State University, Corvallis, OR, USA
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  • C. Green,

    1. Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA
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  • P. Setlow,

    1. Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA
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  • Y-q. Li

    Corresponding author
    • Department of Physics, East Carolina University, Greenville, NC, USA
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Correspondence

Yong-qing Li, Department of Physics, East Carolina University, Greenville, NC 27858-4353, USA. E-mail: liy@ecu.edu

Abstract

Aim

To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens.

Methods and Results

Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl. perfringens with various germinants. When incubated in water at 90–100°C for 10–30 min, more than 90% of spores were inactivated but 50–80% retained their Ca2+-dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640–1680 cm−1 (amide I) and 1230–1340 cm−1 (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of Tlag, ΔTrelease and ΔTlys, during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl. perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer Tlag and ΔTrelease values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased Tlag, ΔTrelease and ΔTlys values, as did wet heat-treated sleC spores germinating with dodecylamine.

Conclusions

(i) Some proteins important in Cl. perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat.

Significance and Impact of the Study

This study provides information on the germination of individual Cl. perfringens spores and improves the understanding of effects of wet heat treatment on spores.

Introduction

Spores of bacteria of Bacillus and Clostridium species are dormant and extremely resistant to environmental stress factors including heat, desiccation, radiation and many toxic chemicals (Setlow 2006). These properties enable such spores to survive for extremely long periods in the environment and in the absence of nutrients. Spores of these species are often present in foodstuffs, and some of their growing forms can cause food spoilage or foodborne disease (Gerhardt and Marquis1989; Setlow 2003; Setlow and Johnson 2012). Consequently, there has been much work on understanding the mechanism of spores' extreme resistance, in particular to wet heat, which is the treatment used most commonly to destroy spores. Factors that cause spore wet heat resistance include the: (i) high level of dipicolinic acid (DPA) and its associated divalent cations (generally Ca2+, giving CaDPA) in the spore's central core; (ii) spore core's low water content; (iii) types and amounts of divalent cations associated with DPA; and (iv) protection of spore DNA by its saturation with a group of α/β-type small, acid-soluble spore proteins (SASPs) (Gerhardt and Marquis1989; Setlow 2006). The levels of Mn and the maturation of released spores during sporulation also affect spore wet heat resistance (Ghosh et al. 2011; Sanchez-Salas et al. 2011).

In contrast to the significant understanding of the mechanisms of spore resistance to wet heat, there is much less knowledge of how wet heat kills spores. Under normal conditions, DNA damage and oxidative reactions are not involved in spore wet heat killing (Setlow 2006), but both protein denaturation and enzyme inactivation are associated with spore killing by wet heat (Coleman et al. 2007; Subramanian et al. 2007; Zhang et al. 2010a). However, protein denaturation has not been shown to specifically cause wet heat killing of spores, and specific proteins whose inactivation causes spore wet heat killing have not been identified. Wet heat treatment often causes the release of the spore core's huge (c. 20% of core dry wt) CaDPA depot, presumably due to rupture of the spore's inner membrane permeability barrier. However, significant spore protein denaturation takes place during wet heat treatment, and at least some denaturation precedes CaDPA release (Zhang et al. 2010a, 2011). High levels of wet heat-killed spores of a number of Bacillus species and Clostridium botulinum also retain CaDPA if heating is not too extreme (Coleman et al. 2007, 2010; Stringer et al. 2009). The wet heat-killed Bacillus spores that retain CaDPA can also germinate, albeit slowly, although are blocked in spore outgrowth perhaps due to inactivation of one or more key metabolic enzymes. However, wet heat-killed Bacillus spores that have lost CaDPA do not germinate (Coleman et al. 2007, 2010). The germination of wet heat-treated Cl. botulinum and Clostridium perfringens spores is also slowed, and a significant percentage of these spores that do germinate are also blocked in outgrowth indicating they are dead (Duncan et al. 1972; Stringer et al. 2009, 2011; Márquez-González et al. 2012). However, a heat treatment giving more than a 4-log kill did not significantly affect the germination kinetics of Clostridium difficile spores (Rodriguez-Palacios and LeJeune 2011). In addition, inclusion of lysozyme in recovery medium plates can increase recoveries of wet heat-treated Cl. botulinum spores, perhaps by promoting spore germination (Duncan et al. 1972; Stringer et al. 1997), although this is not the case for spores of Bacillus subtilis (Coleman et al. 2007). It appears possible then that analysis of specific germination defects in wet heat-killed spores may help to identify one or more spore proteins that are inactivated by wet heat treatment, and whose inactivation is associated with spore death. The analysis of the mechanism of wet heat killing of spores of Bacillus species has been especially facilitated by examining the germination behaviour of multiple individual wet heat-treated spores of Bacillus species using techniques such as Raman spectroscopy and differential interference contrast (DIC) microscopy (Wang et al. 2011b).

The bacterium Cl. perfringens is one of the most common causes of foodborne illness in the United States and the high heat resistance of the spores of this species can allow them to survive a cooking process and ultimately germinate, grow and cause illness (Duncan et al. 1972; Orsburn et al. 2008; Márquez-González et al. 2012). Clostridium perfringens spores generally exhibit higher wet heat resistance than spores of Bacillus species, with c. 100°C generally required to efficiently kill Cl. perfringens spores (Raju and Sarker 2005; Orsburn et al. 2008). However, factors important in the wet heat resistance of spores of Bacillus species, including levels of DPA and divalent metal ions, the protoplast-to-sporoplast ratio and SASPs are also important in resistance of Cl. perfringens spores to wet heat (Raju et al. 2007; Orsburn et al. 2008). Germination of wet heat-treated Cl. perfringens spore populations has been examined (Duncan et al. 1972; Márquez-González et al. 2012). However, the kinetics of germination of multiple individual wet heat-treated Cl. perfringens spores of Cl. perfringens has not been studied, and such a study might facilitate the elucidation of the mechanism of Cl. perfringens spore killing by wet heat (Wang et al. 2011b). Germination of spores of both Bacillus and Clostridium species is heterogeneous and many of the same types of proteins play important roles in the germination of spores of both genera, including germinant receptors (GRs) to recognize and respond to many germinants, SpoVA proteins encoded by the spoVA operon and almost certainly involved in CaDPA release in spore germination, and cortex-lytic enzymes (CLEs) that degrade the spores' peptidoglycan cortex (Setlow 2003; Paredes-Sabja et al. 2009a,b, 2010; Wang et al. 2011a; Setlow and Johnson 2012). The denaturation of any of these key proteins would likely affect spore germination kinetics and perhaps also the viability of spores after wet heat treatment. Consequently, by comparing the germination behaviour of both untreated and wet heat-treated spores with a variety of germinants, it might be possible to identify proteins to which wet heat damage reduces rates of spore germination.

The germination kinetics of spore populations is usually monitored by measuring the optical density at 600 nm of spore cultures, which falls c. 40% upon release of spores' CaDPA depot, one of the early events in spore germination, and falls a total of 60% upon completion of germination (Setlow 2003). However, the kinetics of germination of individual spores cannot be determined from these population measurements because of the heterogeneity in rates of germination of individual spores (Zhang et al. 2010b; Kong et al. 2011; Wang et al. 2011a). A number of techniques have been developed for examining the germination of individual spores, including Raman spectroscopy to follow individual spores' CaDPA levels, DIC or phase-contrast microscopy to follow the germination of many hundreds of individual spores simultaneously (Zhang et al. 2010b; Kong et al. 2011), and measuring the time to generate measurable turbidity in microtitre wells containing growth medium and seeded with c. 1 spore/well (Billon et al. 1997; Stringer et al. 1997, 2005, 2009, 2011; Smelt et al. 2008; Ter Beek et al. 2011). Raman spectroscopy is widely used in biochemical studies, as this technique has high sensitivity and responds rapidly to subtle changes in molecular structure (Wang et al. 2011b; Zhang et al. 2011).When Raman spectroscopy is combined with confocal microscopy and optical tweezers, the resultant laser tweezers Raman spectroscopy (LTRS) allows the nondestructive, noninvasive detection of biochemical processes in physiological solutions (Zhang et al. 2010b; Kong et al. 2011). In this work, we have used DIC microscopy and LTRS to characterize the germination of individual untreated and wet heat-treated wild-type Cl. perfringens spores as well as Cl. perfringens spores lacking their only CLE, SleC, with a number of different germinants. The results of these analyses indicate that some key protein or proteins involved in spore germination are likely damaged during wet heat treatment of spores of Cl. perfringens, generally quite similar to what has been found with spores of Bacillus species (Coleman et al. 2007, 2010).

Materials and methods

Bacterial strains and spore preparation

Clostridium perfringens SM101 derivatives used in this study were the following: (i) MRS101 (Δcpe) (wild-type) lacking the Cl. perfringens enterotoxin (CPE) (Sarker et al. 1999); and (ii) DPS121 (ΔcpeΔsleC), a derivative of strain MRS101 lacking CPE and Cl. perfringens spores' only essential CLE SleC. The DPS121 strain was constructed by introduction of the ΔsleC suicide vector (pDP66) into Cl. perfringens strain MRS101 by electroporation, and a chloramphenicol and tetracycline-resistant ΔcpeΔsleC mutant was isolated as described (Paredes-Sabja et al. 2009b). The identity of the ΔcpeΔsleC strain DPS121 was confirmed by PCR and Southern blot analyses.

Clostridium perfringens sporulating cultures were prepared as previously described (Paredes-Sabja et al. 2009a,b). Briefly, aliquots of an overnight fluid thioglycolate culture were inoculated into 10 ml of Duncan–Strong sporulation medium (Duncan and Strong 1968), followed by incubation for 24 h at 37°C, and the presence of spores was confirmed by phase-contrast microscopy. Large amounts of spores were prepared by scaling up the latter procedure and spores were cleaned by repeated centrifugation and washing with sterile distilled water until spore suspensions were more than 99% free of sporulating cells, cell debris and germinated spores. Purified spores were suspended at a final optical density at 600 nm (OD600) of c. 6 and stored at −20°C until use.

Wet heat treatment of spores and measurement of spore viability

Clostridium perfringens spores were incubated in distilled water at c. 3 × 106 spores ml−1 at 95 or 100°C for 10–60 min, followed by cooling at 25°C for c. 30 min prior to subsequent spectroscopic and germination analyses. The viability of wet heat-treated spores that were incubated in phosphate buffer saline at c. 6 × 106 spores ml−1 at 90 or 100°C for 10–60 min was measured by plating serial dilutions onto brain heart infusion agar plates, and colonies were counted after 24 h of incubation at 37°C under anaerobic conditions (Raju and Sarker 2005; Raju et al. 2007). Incubation for longer times gave no increase in numbers of colonies.

Analysis of individual spores by Raman spectroscopy

Raman spectra of individual Cl. perfringens spores were obtained by LTRS (Kong et al. 2011). The untreated or wet heat-treated spores were suspended in 25 mmol l−1 Na-Hepes buffer (pH 7·4) at room temperature. An individual spore was captured with optical tweezers using a 780 nm laser beam, its Raman spectrum excited by the same laser beam was recorded with a spectrograph and a charge-coupled device (CCD), and measurements were made on c. 100 individual spores. The CaDPA content of individual spores was determined by the intensity of their major CaDPA-specific Raman band at 1017 cm−1, and this value was calibrated with a solution of 60 mmol l−1 CaDPA. The number of spores that retained or lost their CaDPA was determined by the presence of the CaDPA-specific band at 1017 cm−1 in their Raman spectra. The Raman spectra of c. 100 individual spores that retained or lost their CaDPA, respectively, were averaged to analyse the spectral changes because of wet heat treatment.

Spore germination

Clostridium perfringens spores were germinated with: (i) 100 mmol l−1 KCl in 25 mmol l−1 Na-Hepes buffer (pH 7·4) at 30°C; (ii) 50 mmol l−1 L-asparagine in 25 mmol l−1 Na-Hepes buffer (pH 7·4) at 30°C; (iii) 60 mmol l−1 CaDPA (made to pH 7·4 with NaOH) at 40°C; or (iv) 0·8 mmol l−1 dodecylaminein 25 mmol l−1 Na-Hepes buffer (pH 7·4) at 45°C. Except for dodecylamine germination, unless noted otherwise, spores in water were routinely heat-activated prior to germination by a 10-min incubation at 80°C and subsequently cooled at 25°C for 5 min prior to germination experiments.

Monitoring spore germination

The germination of multiple individual Cl. perfringens spores was monitored simultaneously by Raman spectroscopy and DIC microscopy or analysed by DIC microscopy alone as described (Zhang et al. 2010b). Briefly, c. 1 μl of heat-activated spores (c. 108 spores ml−1 in water) was spread on and fixed to the surface of a microscope coverslip. The coverslip with the adhered spores was then mounted on and sealed to a DIC microscope sample holder kept at a constant temperature. Preheated germinant/buffer solution was then added to the spores on the coverslip, and a digital CCD camera was used to record the DIC images at intervals of 12 s for 60–90 min. These images were analysed to locate each spore's position and to calculate the summed pixel intensity of each individual spore on the DIC image. The DIC image intensity of each individual spore was plotted as a function of the incubation time (with a resolution of 12 s) and the initial intensity was normalized to 1 and the intensity at the end of measurements was normalized to zero. From the time-lapse DIC image intensity, we can determine the time of completion of the rapid fall of c. 75% in spore DIC image intensity, which is concomitant with the time of completion of spore CaDPA release, as confirmed by simultaneous Raman spectroscopy and DIC microscopy on individual spores as shown in the current work (see 'Results') and previously (Zhang et al. 2010b). The parameters Tlag, ΔTrelease, Tlys and ΔTlys were used to describe the CaDPA release kinetics during germination of individual spores. Tlag is the time between the mixing of spores with germinants and the initiation of the rapid release of most CaDPA, Trelease is the time of completion of rapid CaDPA release and ΔTrelease = (Trelease − Tlag). Tlys is the time when spore cortex hydrolysis is completed as determined by the completion of the fall in wild-type spores' DIC image intensity, and ΔTlys = (Tlys − Trelease). We also used a parameter, Ilag, which was defined as the intensity of a spore's DIC image at Tlag, to describe the germination of individual spores. The per cent germination of spores in response to different treatments was calculated by counting the number of germinated spores at various times and dividing by the number of dormant spores at the time of mixing of spores with germinants, with ≥250 individual spores analysed in each experiment. For wet heat-treated spores, only the spores retaining CaDPA were treated as dormant spores, because wet heat-treated spores that have released their CaDPA do not germinate (data not shown), as also found with wet heat-treated spores of Bacillus species (Coleman et al. 2007, 2010; Wang et al. 2011b).

Results

Spore killing and DPA content during wet heat treatment

Wet heat treatment is routinely used for inactivation of spores of Clostridium species. Indeed, c. 30% and more than 95% of wild-type Cl. perfringens(MRS101) spores were killed upon incubation for 20 min in water at 90 and 100°C, and longer incubation at these temperatures led to even more spore inactivation (Fig. 1a). However, even at these temperatures, many of the killed spores retained CaDPA, as spores incubated at 100°C for 10–20 min were inactivated 97–99% but the majority of these spores retained their CaDPA (Fig. 1b). Even incubation for 30 min at 100°C resulted in loss of CaDPA by only c. 70% of spores, despite these spores being killed ≥99% (Fig. 1a,b). These results indicate that while incubation at 100°C for 10–30 min inactivates most Cl. perfringens spores, many of these killed spores still retained CaDPA.

Figure 1.

Effects of wet heat treatment on Clostridium perfringens spore viability and Ca2+-dipicolinic acid (CaDPA) release. (a) Wild-type Cl. perfringens spores (strain MRS101) were incubated at 90°C (■) or 100°C (●), and at various times spore viability was quantified as described in 'Materials and methods'. (b) Wild-type spores were incubated at either 95°C (▼) or 100°C (●) for various times, and the percentages of individual spores that retained CaDPA were determined by Raman spectroscopy of c. 100 individual spores as described in 'Materials and methods'.

Raman spectra of wet heat-treated individual Clostridium perfringens spores

As noted previously, significant percentages of wet heat-killed Cl. perfringens spores retained CaDPA as was shown by Raman spectroscopy of individual spores (Fig. 2a). In the Raman spectrum of unheated spores (Fig. 2a, curve 1), bands at 662, 825, 1017, 1397 and 1572 cm−1are because of CaDPA (Zhang et al. 2010a; Wang et al. 2011a,b), the band at 1004 cm−1 is owing to phenylalanine in spore protein, and the bands at 1650 and 1665 cm−1 are associated with α-helical and irregular structures of the protein amide I (peptide bond C=O stretch), respectively (Kitagawa and Hirota 2002; Coleman et al. 2007). The average intensity of the CaDPA-specific 1017 cm−1 Raman band from the untreated wild-type Cl. perfringens spores was similar to that of the wet-heated treated spores that retained CaDPA, which indicated that the CaDPA levels in these spores were 377 ± 87 attomol/spore (Fig. 2a, curves 1,2). In contrast, the Raman spectrum of the wet heat-treated spores that had lost their CaDPA exhibited no CaDPA-specific bands, as expected (Fig. 2a, curve 3).

Figure 2.

Raman spectra of individual wild-type Clostridium perfringens spores with or without wet heat treatment. All spectra shown were averaged over 100 individual spores. (a) Average Raman spectrum of spores: (1) without heat treatment; (2) that retained Ca2+-dipicolinic acid (CaDPA) following wet heat treatment at 100°C for 15 min; and (3) that lost CaDPA following wet heat treatment at 100°C for 15 min. (b) Average Raman spectra of the enlarged 1200–1380 cm−1 region of the spectra in (a) show the protein amide III bands. (c) Average Raman spectra of the enlarged 1620–1700 cm−1 region of the spectra shown in (a) show the amide I band of proteins with α-helical or irregular structures.

Raman spectra can also give significant information about the state of proteins in spores, and Fig. 2b,c show spores' Raman spectra in the 1230–1360 cm−1 (amide III of proteins) and 1640–1680 cm−1 (amide I of proteins) regions, respectively. In curves 1 and 2 of the 1640–1680 cm−1 region (Fig. 2c), the Raman spectra of the unheated spores and the heat-treated spores which retained CaDPA looked very similar, although there was a slight decrease in the ratio of the 1655 cm−1 band intensity over that at 1665 cm−1 in the wet heat-treated spores (Fig. 2c; compare vertical dashed lines in curves 1 and 2). The intensity of the Raman band at 1655 cm−1 was even more reduced and the band at 1665 cm−1 became more prominent in the wet heat-treated spores that had lost CaDPA (Fig. 2c, compare curves 2 and 3), indicating that much of the spore proteins' structure had changed from an α-helical to an irregular and presumably denatured state concomitant with or soon after CaDPA release during wet heat treatment, as seen previously in Bacillus spores(Coleman et al. 2007; Zhang et al. 2010a, 2011; Wang et al. 2011a). The change in spore protein structure because of wet heat treatment could also be seen in the amide III band (Fig. 2b). The intensity of the 1280 cm−1 band (α-helix) from untreated spores (curve 1) was reduced and shifted to those of the 1250 cm−1 (random coil) and 1238 cm−1 (β-sheet) bands of the heat-treated spores that had lost their CaDPA (curve 3), indicating that much of the spore proteins' structure changed from an α-helical to an irregular denatured state upon severe wet heat treatment (Chen et al. 1973; Maiti et al. 2004).

KCl germination of wet heat-treated individual Clostridium perfringens spores

To examine the germination kinetics of individual wet heat-treated spores. We obtained wet heat-treated Cl. perfringens spores that were largely inactivated but retained CaDPA and germinated them with KCl via the GerKA-KC germinant receptor (GR)-dependent pathway (Paredes-Sabja et al. 2010). More than 90% of spores heated for 20 min at 90–100°C that retained their CaDPA germinated with 100 or 2 mmol l−1 KCl within 60–90 min (Fig. 3a,b). However, with spores treated at 100°C for 30 min, ≤30% of the spores that retained CaDPA germinated in 60–90 min. In addition, although the rates of germination of spores treated at 90–100°C for ≤20 min were similar to that of the untreated spores (Fig. 3a–d), the average values for Tlag, ΔTrelease and ΔTlys were 1·4 to1·9-fold higher in spores heated at 100°C for 20 min (Table 1). The increase in Tlag was undoubtedly even higher with spores heated at 100°C for 30 min, in particular because so many of these spores that retained CaDPA did not even germinate in 60 min (Fig. 3a). In addition, the average value of Ilag decreased from 75% to c. 65% in the most severely wet heat-treated spores. In contrast to the good germination of wild-type spores with KCl, most Cl. perfringens spores lacking their only essential CLE, sleC, did not germinate with KCl (Table 1). In addition those individual sleC spores that did germinate with KCl exhibited longer average Tlag and ΔTrelease values than wild-type spores, although the sleC spores exhibited no obvious cortex hydrolysis, as expected (Paredes-Sabja et al. 2009b) (Table 1).

Figure 3.

KCl germination of untreated and wet heat-treated wild-type Clostridium perfringens spores. (a, b) Untreated spores (■) and spores wet heat-treated at 90°C for 20 min (●), 95°C for 20 min (▲), 100°C for 20 min (▼), and 100°C for 30 min (♦) were heat-activated, germinated with (a) 100 mmol l−1 or (b) 2 mmol l−1 KCl and the germination of ≥250 individual spores was monitored by differential interference contrast (DIC) microscopy as described in Methods. (c, d) The 100 mmol l−1 KCl germination of ten individual spores that were either (c) untreated or (d) wet heat-treated at 100°C for 20 min was followed by DIC microscopy as described in Methods. The arrows in (c) and (d) denote the Tlag, Trelease and Tlys points for one individual spore.

Table 1. Mean values and standard deviations of germination parameters of untreated and wet heat-treated Clostridium perfringens sporesa
Strain and germinantTreatmentTlag (min)Trelease (min)Tlys (min)ΔTrelease (min)ΔTlys (min)Ilag (%)Germination (%)
  1. a

    Untreated or wet heat-treated Cl. perfringens spores were germinated with various germinants. The measurement time was 60 min for germination of MRS101 (wild-type) spores with 100 mmol l−1 KCl and 90 min for all other germinations, and values for various germination parameters were calculated from data on c. 30 individual spores. The per cent germination for the various spore preparations was determined by analysis of the germination of 250–400 spores that retained CaDPA after wet heat treatment.

  2. b

    The P-values for the observed differences in the values of Tlag, ΔTrelease and ΔTlys between untreated MRS101 spores and wet heat-treated spores (100°C for 20 min) germinated with KCl, L-asparagine, CaDPA and dodecylamine were calculated as P < 0·01 using the two sample t-test.

  3. c

    nc –The values of Tlys and ΔTlys for dodecylamine germination of MRS101 spores and all germinations of DPS121 (sleC) spores were not calculated because of the absence of periods of obvious cortex hydrolysis in these germinating spores, and c. 30 individual spores were examined.

MRS101

100 mmol l−1

KCl

None11·0 ± 5·312·9 ± 5·416·8 ± 5·41·9 ± 0·53·9 ± 2·475·1 ± 12·494·5
90°C 20 min14·2 ± 5·715·8 ± 5·724·9 ± 8·01·7 ± 0·29·1 ± 3·965·5 ± 7·594·8
95°C 20 min12·6 ± 3·914·4 ± 3·920·2 ± 4·82·2 ± 1·17·9 ± 2·969·4 ± 9·196·1
100°C 20 min17·1 ± 7·4b18·9 ± 7·527·2 ± 9·52·6 ± 1·4b7·4 ± 3·5b65·0 ± 12·191·9

DPS121 (sleC)

100 mmol l−1

KCl

None29·9 ± 16·430·8 ± 16·8ncc2·9 ± 1·3nc61·0 ± 13·312·7

MRS101

Asparagine

None27·2 ± 21·228·8 ± 21·231·0 ± 21·81·6 ± 0·62·2 ± 1·880·8 ± 10·242·1
95°C 20 min45·6 ± 25·847·4 ± 25·852·1 ± 24·81·8 ± 0·74·7 ± 4·677·9 ± 7·131·5
100°C 20 min48·8 ± 25·651·3 ± 25·257·8 ± 24·42·5 ± 1·16·5 ± 6·075·4 ± 7·131·6

MRS101

CaDPA

None23·7 ± 20·826·1 ± 21·128·0 ± 21·02·4 ± 0·61·9 ± 1·084·1 ± 7·458·0
95°C 20 min36·2 ± 26·439·0 ± 26·441·9 ± 26·32·7 ± 0·63·0 ± 2·983·2 ± 8·640·8
100°C 20 min46·5 ± 25·151·7 ± 24·556·6 ± 23·55·2 ± 3·25·0 ± 4·272·3 ± 10·611·5

DPS121

CaDPA

None      6·9

DPS121

Buffer only

None      2·5
MRS101 dodecylamineNone13·0 ± 11·415·2 ± 12·1nc2·6 ± 1·1nc61·4 ± 14·983·5
95°C 20 min40·3 ± 17·643·2 ± 17·7nc3·0 ± 0·8nc44·6 ± 8·614·7
100°C 20 min40·2 ± 23·843·4 ± 24·4nc3·3 ± 0·9nc34·0 ± ±8·110·4
DPS121, dodecylamineNone16·6 ± 13·219·5 ± 14·3nc2·9 ± 1·4nc62·1 ± 18·683·7
90°C 10 min37·3 ± 21·640·9 ± 22·3nc3·6 ± 1·8nc47·6 ± 13·625·1

To be sure that CaDPA release also paralleled the initial fall of c. 75% in spores' DIC intensity even during germination of wet heat-treated spores, germination of individual wet heat-treated spores was simultaneously monitored by Raman spectroscopy and DIC microscopy during KCl germination (Fig. 4a,b). As seen previously with untreated spores (Wang et al. 2011a), the time of complete CaDPA release, Trelease, corresponded well with the end of the rapid fall in DIC image intensity during germination of the wet heat-treated spores.

Figure 4.

KCl germination of individual wet heat-treated wild-type Clostridium perfringens spores monitored simultaneously by Raman spectroscopy and differential interference contrast (DIC) microscopy. The KCl (100 mmol l−1) germination of individual wild-type Cl. perfringens spores treated at 95°C for 20 min was simultaneously monitored by Raman spectroscopy (a) and DIC microscopy (b), with analyses carried out every 30 s for Raman spectroscopy and every 15 s for DIC microscopy. The times of Tlag, and Trelease are labelled for one spore in a.

l-asparagine germination of wet heat-treated Clostridium perfringens spores

In addition to KCl, Cl. perfringens spores were germinated with l-asparagine, which also triggers Cl. perfringens spore germination through the GerKA-KC GR. While l-asparagine was a less effective germinant than KCl, wet heat treatment also decreased rates and extents of spore germination with l-asparagine (Fig. 5a–d, Table 1). In particular, with spores treated at 100°C for 20 min, average values of Tlag, ΔTrelease and ΔTlys for germination with l-asparagine were increased 1·5- to 3-fold compared with values for the untreated spores (Table 1).

Figure 5.

l-Asparagine germination of untreated and wet heat-treated wild-type Clostridium perfringens spores. (a) Untreated spores (■) and spores wet heat-treated at 90°C for 20 min (●), 95°C for 20 min (▲), 100°C for 20 min (▼), and 100°C for 30 min (♦) were heat-activated, germinated with l-asparagine and the germination of ≥250 individual spores was monitored by differential interference contrast (DIC) microscopy as described in 'Materials and methods'. (b–d) The l-asparagine germination of 10 individual spores that were either (b) untreated, (c) wet heat-treated at 95°C for 20 min or (d) wet heat-treated at 100°C for 20 min was followed by DIC microscopy as described in 'Materials and methods'.

Germination of wet heat-treated Clostridium perfringens spores with exogenous CaDPA

In addition to germinants such as l-asparagine and KCl that trigger germination via GRs, exogenous CaDPA also triggers Cl. perfringens spore germination, perhaps at least in part in a GR-dependent process (Paredes-Sabja et al. 2009b). About 60% of untreated wild-type Cl. perfringens spores germinated in 90 min with CaDPA (Fig. 6a; Table 1). In contrast, most sleCspores did not germinate with exogenous CaDPA, presumably because of the absence of SleC, although a small percentage of these spores released CaDPA even when incubated in the absence of any germinant (Table 1). As expected, the germination of wild-type Cl. perfringens spores decreased markedly when the spores were subjected to increasingly severe wet heat treatments (Fig. 6; Table 1). The kinetic parameters of the CaDPA germination of individual wet heat-treated wild-type Cl. perfringens spores were also significantly changed, as wet heat treatment increased values of Tlag, ΔTrelease and ΔTlys 2 to 2·5-fold (Table 1).

Figure 6.

Ca2+-dipicolinic acid (CaDPA) germination of untreated and wet heat-treated wild-type Clostridium perfringens MRS101 spores.(a) Untreated spores (■) and spores wet heat-treated at 95°C for 20 min (●), 100°C for 20 min (▲), or 100°C for 30 min (♦) were heat-activated, germinated with CaDPA and the germination of ≥250 individual spores was monitored by differential interference contrast (DIC) microscopy as described in 'Materials and methods'. (b, c) The CaDPA germination of ten individual spores that were either (b) untreated, or (c) wet heat-treated at 100°C for 20 min was followed by DIC microscopy as described in 'Materials and methods'.

Germination of wet heat-treated Clostridium perfringens spores with dodecylamine

An additional germinant for Cl. perfringens spores is the cationic surfactant dodecylamine that also germinates spores at least partially in a non-GR-dependent manner, perhaps through direct effects on one or more CaDPA channel proteins in spores' inner membrane (Setlow et al. 2003; Vepachedu and Setlow 2007; Paredes-Sabja et al. 2008).Dodecylamine germination of untreated wild-type and sleC spores was almost identical, including extremely similar average Tlag, Trelease and ΔTreleasevalues (Table 1). However, wet heat treatment significantly slowed the dodecylamine germination of both wild-type and sleC spores, as average Tlag values were increased 2·5- to 3·0-fold and the Ilag was decreased up to almost twofold in wet heat-treated spores, although average ΔTrelease values were almost unaffected (Table 1).These effects of wet heat on dodecylamine germination are certainly consistent with dodecylamine triggering spore germination by acting upon a protein as noted previously. It was also notable that minimal cortex hydrolysis was observed during dodecylamine germination, even with wild-type spores (Table 1).

Discussion

The work in this article allows a number of conclusions about the effects of wet heat treatment on spores of Cl. perfringens. These conclusions include the following: (i) Release of CaDPA took place well after spore killing by wet heat. This indicates that spore killing by wet heat is not because of rupture of spores' inner membrane permeability barrier. (ii) CaDPA loss during wet heat treatment was an all or none phenomenon, as no spores with intermediate CaDPA levels were seen after wet heat treatment. (iii) Notable alterations in spore protein structure, presumably denaturation, preceded CaDPA release during wet heat treatment, consistent with the idea that inactivation of one or more specific spore proteins is the reason that wet heat kills spores. (iv) Massive protein denaturation rapidly followed CaDPA release during wet heat treatment, although well after spores were already dead. The proteins denatured in this process are most likely soluble core proteins, and presumably the rapid denaturation following CaDPA release is because loss of CaDPA results in an immediate rise in core water content thus eliminating the protection of core proteins against wet heat because of the dormant spores' normally low water content (Gerhardt and Marquis 1989).With spores of Bacillus species, the low core water content is actually maintained during wet heat treatment as long as CaDPA is retained in the spore core (Coleman et al. 2007, 2010). (v) Wet heat treatment significantly increased the Tlag times for spore germination with all germinants tested, including compounds such as KCl and L-asparagine that act via a GR or GRs, as well as CaDPA and dodecylamine that may act only partially if at all via GRs. This effect of wet heat treatment on Tlag values was likely much larger than shown, because in a number of cases, the wet heat treatment greatly decreased the percentage of spores that germinated in the 60–90 min observation period. (vi) Times for ΔTrelease and ΔTlys during spore germination were also increased up to threefold by wet heat treatment, although cortex lysis was not observed during germination with dodecylamine. (vii) Ilag values were consistently lower for wet heat-treated spores germinating with any germinant tested.

Notably, all of the conclusions noted earlier are identical to those made in studies of effects of wet heat treatment on spores of various Bacillus species (Coleman et al. 2007; Zhang et al. 2010a, 2011; Wang et al. 2011b). In addition, significant protein denaturation has been seen to accompany wet heat killing of Clostridium tyrobutyricum spores (Subramanian et al. 2007), and it has been suggested that damage to germination proteins is one factor in the wet heat killing of Cl. botulinum spores (Stringer et al. 1997). Detailed studies of the effects of wet heat on individual Cl. botulinum spores has also shown that wet heat kills spores without obligatory release of CaDPA and also increases the lag time between nutrient germinant addition and CaDPA release (Stringer et al. 1997, 2009, 2011). Thus, in most regards, the effects of wet heat treatment on spores of Bacillus and Clostridium species appear rather similar overall, although can differ in degree.

In addition to the similarities in the findings for wet heat-treated spores of Cl. perfringens and spores of Cl. botulinum and Bacillus species, there were also several observations that were new to Cl. perfringens spores as follows. (i) Values of Ilag for wet heat-treated spores germinating with dodecylamine decreased much more with Cl. perfringens spores than with spores of Bacillus subtilis (Wang et al. 2011b). However, neither the significance nor the cause of this effect is clear. (ii) Cl. perfringens spore germination with either KCl or CaDPA was greatly decreased by a sleC mutation. The reason for this effect is also not clear, but perhaps SleC action is essential for rapid CaDPA release from Cl. perfringens spores during KCl germination. Indeed, loss of the B. subtilis CLE CwlJ increases ΔTrelease times in nutrient germination up to 15-fold (Peng et al. 2009; Zhang et al. 2012), although why this is the case is not clear. The absence of CaDPA germination of the sleC Cl. perfringens spores also suggests that CaDPA may directly activate SleC, much as CaDPA activates CwlJ action in CaDPA germination of spores of Bacillus species (Peng et al. 2009; Setlow et al. 2009). However, it is also possible that in Cl. perfringens spores CaDPA activates the spore protease CspB that is essential to generate the active form of SleC early in spore germination (Paredes-Sabja et al. 2009c).

The major goal of the work described in this communication was to determine the effects of wet heat treatment on Cl. perfringens spore germination to gain a better understanding of how wet heat kills Cl. perfringens spores. The fact that there were significant alterations in spore protein structure prior to CaDPA release during wet heat treatment is certainly consistent with wet heat killing Cl. perfringens spores by protein damage, although does not prove this. However, the increased values of Tlag, ΔTrelease and ΔTlys in the germination of wet heat-treated spores that retained CaDPA but were largely dead is difficult to explain except as caused by damage, perhaps lethal damage to one or more key germination proteins by wet heat. There are three steps in the germination of spores of Bacillus and Clostridium species that require known proteins (Setlow 2003; Paredes-Sabja et al. 2010): (i) recognition of nutrient germinants by GRs; (ii) release of CaDPA perhaps through channels composed at least in part of SpoVA proteins; and (iii) hydrolysis of the spore's peptidoglycan cortex by the CLEs CwlJ and/or SleB in Bacillus species and by SleC in Cl. perfringens. Loss of the activity of any proteins that are essential for these steps could block germination, and it is possible that one or more unknown proteins play some role in signal transduction during the germination process. An obvious question then is which of these possible proteins is inactivated by wet heat leading to decreased rates of spore germination? While inactivation of one or more GR proteins would likely compromise KCl and l-asparagine germination, GRs are almost certainly not essential for dodecylamine germination of Cl. perfringens spores, and perhaps not for CaDPA germination as well. Consequently, as dodecylamine germination in particular was greatly decreased by wet heat treatment, it seems unlikely that inactivation of a GR protein alone could be the reason that wet heat treatment decreases rates of spore germination. A second possibility is that it is through inactivation of SleC or CspB or both that wet heat treatment decreases spores' germination rate. Indeed, with B. subtilis spores there is evidence that at least the CLE CwlJ is rather sensitive to wet heat (Atrih and Foster 2001; Wang et al. 2011b), and as noted previously, lysozyme can recover spores of Cl. botulinum (but not spores of B. subtilis) that appear killed by wet heat. However, this cannot be a complete explanation for the decreased rates of germination of wet heat-treated Cl. perfringens spores, because CaDPA release during dodecylamine germination of sleC Cl. perfringens spores is even faster than from wild-type spores (Paredes-Sabja et al. 2009b). The only remaining known germination proteins in spores of Cl. perfringens are those involved in the CaDPA release process itself, with the SpoVA proteins being the only known proteins thought to be involved in CaDPA release during spore germination (Paredes-Sabja et al. 2010; Li et al. 2012). Certainly, if one or more of these proteins, a putative CaDPA channel itself or the mechanism whereby such a channel's opening is triggered, is inactivated by wet heat, these damaged spores would perhaps not germinate with any germinant. Indeed, the fact that ΔTrelease values are increased in the germination of wet heat-treated spores with all germinants is consistent with the CaDPA release process being a key target of wet heat damage. Interestingly, in B. subtilis there is evidence that alterations in SpoVA protein levels as well as specific point mutations in spoVA genes also affect values of Tlag significantly, and with both GR-dependent and GR-independent germinants including dodecylamine (Wang et al. 2011c). While the CaDPA release process during germination is certainly an additional attractive target for wet heat damage that inactivates Cl. perfringens spores, the available evidence is by no means overwhelming that this is the crucial target for wet heat killing of spores. At present, the composition and gating of the structure that releases CaDPA during spore germination is not known. Perhaps, this latter knowledge will allow more informed analyses of the effects wet heat treatment of CaDPA release during bacterial spore germination.

Acknowledgements

This work was supported by a grant from the Army Research Office (Y.Q.L./P.S.) and by a Multi-University Research Initiative award through the US Army Research Laboratory and the Army Research Office under contract number W911NF-09-1-0286 (M.R.S., P.S., Y.Q.L.). D.P.-S. was supported by a grant from MECESUP UAB0802.

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