# Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa

Rosa Heydenreich, Alessia I Delbrück, Christina Peternell, Clément Trunet, Alexander Mathys

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## To cite this version:

Rosa Heydenreich, Alessia I Delbrück, Christina Peternell, Clément Trunet, Alexander Mathys. Characterization of high-pressure-treated Bacillus subtilis spore populations using flow cytometry - Shedding light on spore superdormancy at 550 MPa. International Journal of Food Microbiology, 2024, 422, pp.110812. 10.1016/j.ijfoodmicro.2024.110812. hal-05386718

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# Characterization of high-pressure-treated *Bacillus subtilis* spore populations using flow cytometry — Shedding light on spore superdormancy at 550 MPa

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Rosa Heydenreich<sup>a</sup>, Alessia I. Delbrück<sup>a</sup>, Christina Peternell<sup>a</sup>, Clément Trunet<sup>b,**</sup>, Alexander Mathys<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Bacterial spore

Inactivation

Germination

Cell sorting

Superdormant

## ABSTRACT

Mild spore inactivation can be challenging in industry because of the remarkable resistance of bacterial spores. High pressure (HP) can trigger spore germination, which reduces the spore's resistance, and thereby allows mild spore inactivation. However, spore germination is heterogenous. Some slowly germinating or non-germinating spores called superdormant spores remain resistant and can survive. Therefore, superdormant spores need to be characterized to understand the causes of their germination deficiency.

*Bacillus subtilis* spores were pressurized for 50 s – 6 min at a very high pressure (vHP) level of 550 MPa and 60 °C in buffer to trigger germination. For a rapid quantification of the remaining ungerminated superdormant spores, flow cytometry (FCM) analysis was validated using single cell sorting and growth analysis. FCM based on propidium iodide (PI) and SYTO16 can be used for 550 MPa-superdormant spores after short vHP treatments of ≤1 min and post-HP incubation at 37 °C or 60 °C. The need for a post-HP incubation is particular for vHP treatments. The incubation was successful to separate FCM signals from superdormant and germinated spores, thus allowing superdormant spore quantification. The SYTO16 and PI fluorescence levels did not necessarily indicate superdormancy or apparent viability. This highlights the general need for FCM validation for different HP treatment conditions.

The ~7 % of ungerminated, i.e., superdormant, spores were isolated after a vHP treatment (550 MPa, 60 °C, 43–52 s). This allowed the characterization of vHP superdormant spores for the first time. The superdormant spores had a similar dipicolinic acid content as spores of the initial dormant population. Descendants of superdormant spores had a normal vHP germination capacity. The causes of vHP superdormancy were thus unlikely linked to the dipicolinic acid content or a permanent genetic change. Isolated superdormant spores germinated better in a second vHP treatment compared to the initial spore population. This has not been observed for other germination stimuli so far. In addition, the germination capacity of the initial spore population was time-dependent. A vHP germination deficiency can therefore be lost over time and seems to be caused by transient factors. Permanent cellular properties played a minor role as causes of superdormancy under chosen HP treatment conditions.

The study gained new fundamental insights in vHP superdormancy which are of applied interest. Understanding superdormancy helps to efficiently develop a strategy to avoid superdormant spores and hence to inactivate all spores. The development of a mild HP spore germination-inactivation process aims at better preserving the food quality.

<footnote><sup>a</sup> Sustainable Food Processing Laboratory, Institute of Food, Nutrition, and Health, Department of Health Science and Technology, ETH Zurich, Zurich, Switzerland</footnote>

<footnote><sup>b</sup> Univ Brest, INRAE, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, Quimper, France</footnote>

<footnote>* Correspondence to: Alexander Mathys, Schmelzbergstrasse 9, 8092 Zurich, Switzerland.</footnote>

<footnote>** Correspondence to: Clément Trunet, 6 rue de l'Université, 29000 Quimper, France.</footnote>

<footnote>E-mail addresses: clement.trunet@univ-brest.fr (C. Trunet), alexander.mathys@hest.ethz.ch (A. Mathys).</footnote>
# 1. Introduction

Bacterial spores are challenging to inactivate under mild conditions. Bacterial spores are inactive, i.e., dormant, cells formed by the classes Bacilli and Clostridia (De Hoon et al., 2010). Spores are extremely resistant to harsh conditions like drought, heat, or the presence of chemicals and other physical hazards (Setlow, 2006; Setlow and Johnson, 2019). Because of their resistance, spores can withstand mild industrial decontamination treatments like non-thermal high pressure (HP) processing at temperatures of ≤25 °C and pressures of 400–600 MPa (Hu and Balasubramaniam, 2024). HP-treated foods are high-moisture products like fruit juices or fruit puree, meat products, dairy products or ready-to-eat products, for example (Koutsoumanis et al., 2022). If spores are not inactivated and if they can develop into multiplying vegetative cells, they present a risk regarding food safety or food spoilage in these products (Setlow and Johnson, 2019; Wells-Bennik et al., 2016). Heat sterilization at typically ≥115 °C and corresponding temperature holding times effectively inactivates spores but can deteriorate the food quality regarding its nutritional value, texture, or color (Gratz et al., 2021; Jimenez et al., 2023). Therefore, consumers and industry demand less heat-intensive inactivation processes (Jermann et al., 2015).

An HP germination-inactivation process at moderate temperatures (≤80 °C) has been proposed for mild spore inactivation (Black et al., 2007a; Clouston and Wills, 1969). Germination transforms the inactive dormant spores to metabolically active cells and results in a loss of resistance. Due to the loss of resistance, HP-stimulated germination can make spores susceptible to a mild inactivation treatment. The inactivation treatment can be the HP treatment itself or a subsequent treatment, such as mild heating (Black et al., 2007a; Clouston and Wills, 1969).

Under HP, the germination mechanism depends on the pressure level, the pressure-holding time and the temperature (Heydenreich et al., 2023; Reineke et al., 2012). Generally, HP is thought to trigger germination by modifying protein or membrane structures (Black et al., 2007a). In *Bacillus* spores, a moderate high pressure (mHP, 50–300 MPa, 30–50 °C) activates nutrient germinant receptors in the inner membrane. This receptor activation results in calcium dipicolinic acid (Ca-DPA) release from the spore core (Black et al., 2007a). A very high pressure (vHP, 400–600 MPa) causes direct release of Ca-DPA independent of germinant receptors (Reineke et al., 2011). The Ca-DPA release triggers later enzymatic germination events such as the degradation of a spore-protecting layer called the cortex (Reineke et al., 2013). Information on subsequent enzymatic germination steps can be found elsewhere (Christie and Setlow, 2020; Delbrück et al., 2021a; Heydenreich et al., 2023). Cortex hydrolysis seems possible during an mHP treatment (150 MPa, 37 °C) but might happen only after a treatment at vHP (550 MPa, 60 °C) if the post-HP temperature and time allow (Heydenreich et al., 2023). Generally, completion of enzymatic germination steps becomes more unlikely with increasing vHP treatment temperature or time because of enzyme denaturation and spore inactivation (Heydenreich et al., 2023). Compared to vHP, germinated spores are more slowly inactivated at mHP (Reineke et al., 2011; Suklim et al., 2008). The fact that vHP can germinate and inactivate spores without requiring chemical additives makes vHP appealing for food applications compared to other germination stimuli. Other germination stimuli are nutrients like sugars or amino acids, dodecylamine, or exogenous Ca-DPA, for example (Perez-Valdespino et al., 2013; Setlow et al., 2017). An overview of germination stimuli and corresponding germination mechanisms is provided by Christie and Setlow (2020).

Some spores can remain dormant or only germinate very slowly upon exposure to a germination stimulus — so-called superdormant (SD) spores (Ghosh and Setlow, 2009). The SD spores are defined by their decreased germination capacity relative to the other spores within an isogenic spore population. As superdormancy is a relative term, SD spore yields and properties depend on the type and intensity of the applied

germination stimulus (Ghosh et al., 2009). The SD spores hinder the application of a germination-inactivation strategy in industry, as all spores must undergo germination, i.e., release Ca-DPA, for an effective mild inactivation (Reineke et al., 2013). Understanding the mechanisms of superdormancy is key to finding means for tackling SD spores. Therefore, spores that were SD to germination stimuli like mHP, nutrients, Ca-DPA, or dodecylamine have been isolated and characterized (Delbrück et al., 2021b; Ghosh et al., 2009; Ghosh and Setlow, 2009, 2010; Perez-Valdespino et al., 2013; Wei et al., 2010). These authors concluded that there are several, potentially cumulative causes of superdormancy. The causes seemed reversible (transient) or irreversible (permanent).

Considering the high potential of vHP for industrial applications, our goal was to advance vHP germination-inactivation by investigating potential causes of vHP superdormancy. Spores of the model organism for germination, *Bacillus subtilis*, were used. For the characterization of vHP SD spores, method validation and optimization were required. An optimized protocol is presented for the isolation of vHP SD spores using buoyant density centrifugation. Rapid analysis of the vHP SD spore fraction was crucial for an efficient isolation and characterization of SD spores within one day. Therefore, this study validated flow cytometry (FCM) as a powerful tool for fast quantification of the vHP SD spore subpopulation using fluorescence-activated cell sorting (FACS). FACS allowed the isolation of the flow cytometric subpopulations of interest. FACS enabled to study the growth capacity and heat sensitivity of the vHP-treated spore subpopulations of SD, germinated or inactivated spores on a single-cell level. For details on FCM and FACS of HP-treated spores, the reader is referred to Zhang et al. (2020) and Heydenreich et al. (2023). Only mHP-treated spore subpopulations have been characterized using FACS so far (Zhang et al., 2020). In contrast to mHP treatments, the vHP SD spores and germinated spores can show similar FCM signals depending on the vHP and post-HP conditions (Heydenreich et al., 2023). Hence, to ensure that only signals of vHP SD spores are considered for their quantification, validation of FCM for vHP-treatments depending on the post-HP temperature was necessary. Including the results from germinated subpopulations, the present study provides valuable knowledge for the interpretation of FCM spore analysis.

Characteristics of vHP SD spores, like their DPA content and their genetic and temporal stability, were investigated as potential causes of superdormancy. The DPA content was analyzed, since the DPA content seemed to play a critical role in the heat and pressure resistance to 600 MPa and 65 °C (Margosch et al., 2004). In addition, vHP germination is initiated by Ca-DPA release in contrast to mHP or nutrient germination, for example (Reineke et al., 2012). Previous studies showed that neither mHP (150 MPa, 37 °C, 6 min) nor nutrient superdormancy were genetically inherited (Delbrück et al., 2021b; Ghosh and Setlow, 2009). Based on these studies, a permanent genetic cause of vHP superdormancy seemed unlikely but was investigated as the cellular target structures of vHP are different from an mHP or nutrient germination stimulus. Hence, different genes might be relevant depending on the germination stimulus. If these different genes have different replication mechanisms, e.g. are part of mobile transposons for example (Mahillon, 2008), vHP superdormancy could be influenced by a genetic change in contrast to mHP superdormancy. The temporal stability of vHP superdormancy was investigated because it is highly relevant for a potential vHP application if spores can lose their resistance to vHP germination over time. To our knowledge, this study is the first to characterize isolated vHP SD spores and shed light on potential causes of resistance to a vHP germination-inactivation treatment.

# 2. Material and methods

## 2.1. Spore preparation and bacterial strains

The strains PS832 and PS533 originate from the *B. subtilis* strain 168
(Setlow and Setlow, 1996). The strain PS533 carries a plasmid with kanamycin resistance (Fairhead et al., 1993). *B. subtilis* was chosen as it is a non-pathogenic model organism for germination. Hence, it can serve as a surrogate for various pathogenic and non-pathogenic *Bacillus* species. In addition, the vast knowledge on *B. subtilis* provides a strong foundation for the comparison of different germination treatments or mechanisms. As both strains are wild-type-like and germinate similarly under vHP (Supplementary Fig. A.1), validation of FCM using PS832 could be applied to later analysis of PS533 spores. Spores were prepared according to the protocol of Zhang et al. (2020) using Difco sporulation medium agar (pH 7.6), i.e., a standard medium for *B. subtilis* sporulation (Nicholson and Setlow, 1990). After 4 days at 37 °C, spores were harvested, washed 4× with MilliQ water by centrifugation (6000 g, 10 min, 4 °C), and washed 2×/day in the following 7 days. Spores were ≥97 % dormant spores according to FCM or phase contrast microscopy (Kong et al., 2011). Spores were stored at 4 °C in MilliQ water and were washed every two weeks. One spore batch of PS832 and two batches of PS533 were prepared.

## 2.2. High pressure treatment

Spore samples were treated at a vHP of 550 MPa and a temperature of 60 °C, which are optimized conditions for vHP germination treatments (Black et al., 2007b; Reineke et al., 2013). The vHP dwell times chosen for FACS (50 s, 2.5 min, 6 min) allowed analysis of all five subpopulations appearing at 550 MPa and 60 °C between 0 and 20 min (Heydenreich et al., 2023). The vHP dwell time was adapted for each SD spore isolation depending on the spore batch and the spore age to obtain a similar SD spore yield, i.e., to have a similar spore selection pressure. To obtain ~7 % SD spores, PS533 spore batch 1 was treated for 45 s for DPA content analysis, or 43 s one month later for the re-sporulation experiment. PS533 spore batch 2, used for testing the temporal stability of superdormancy, was vHP-treated for 52 s (Fig. 1). The chosen SD spore yield (~7 %) was assumed to be sufficiently low for revealing SD-spore-specific characteristics, based on Ghosh and Setlow (2009). The yield also was sufficiently high for isolation and characterization methods. Spores were treated in 50 mM N-(2-acetamido)-2-amino-ethanesulfonic acid (ACES) buffer (pH 7.0, ThermoFisher, Germany), sterile-filtered with a 0.2 µm polyethersulfone (PES) filter. The spore

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Experimental design of the isolation and characterization of very high pressure (vHP) superdormant (SD) *Bacillus subtilis* PS533 spores corresponding to Sections 2.2, 2.6–2.9.</figcaption></figure>
concentration was 10⁹ CFU/mL, except 10¹⁰ CFU/mL for SD spore isolation. Using cryotube vials (1.5 mL, Nunc A/S, Denmark), 1.5 mL spore suspension was vHP-treated. For simultaneous pressurization of two samples (Sections 2.8 and 2.9), 0.7 mL sample was in each 0.5 mL reaction tube with removed lid (Eppendorf, Germany). A shrink tubing (Nunc CryoFlex Tubing, Nunc A/S, Denmark) was used to seal the vials or tubes. The HP machine (modified Model U111, Unipress, Poland) used propylene glycol (40 % v/v) as pressure-transmitting fluid. The vessel for the spore sample and the control vessel were in a water bath (Huber CC410, Germany) for temperature control. The treatment temperature was monitored inside a dummy tube with 50 mM ACES buffer in the control vessel, similar to Zhang et al. (2020). The same number and type of tubes were used in both vessels. Tubes were pre-cooled on ice for ≥10 min and additionally at -20 °C for 3 min, without liquid-solid phase transition. Both vessels were then loaded with the tubes at the same moment to ensure similar pressure-temperature-time profiles (Supplementary Fig. A.2) in the dummy and sample tubes. As experimental control, an untreated dormant spore sample stayed on ice during the pre-cooling at -20 °C for 3 min and subsequent HP treatment of the other samples. Samples were immediately incubated post-HP at 60 °C for 30 min for FCM or isolation of SD spores. For FACS, aliquots of the untreated or vHP-treated spores were immediately incubated for 2 h 30 min at 37 °C, for 40 min at 60 °C, or were kept on ice for ≥40 min. After post-HP incubation, spores stayed on ice.

## 2.3. Analysis of germination and inactivation by flow cytometry (FCM)

FCM was used for analysis of vHP-treated spore subpopulations, SD spore quantification, and purity check of purified dormant or SD spores. Sterile-filtered MilliQ water (250 µL) with 10⁷ CFU/mL spores was stained by incubation at room temperature in the dark with SYTO16 (final conc. 1.5 µM, 16 min incubation, Molecular Probes, Netherlands) and propidium iodide (PI, final conc. 1.5 µM, 4 min incubation, CAS 25535-16-4, MP Biomedicals, USA). An LSR Fortessa cytometer (BD Biosciences, USA) recorded 15,000 events at a rate of 6000 ± 4000 events/s (excitation: 488 nm (SYTO16) or 561 nm (PI); emission detection: 530/30 nm band-pass filter (SYTO16, green fluorescence) or 610/20 nm band-pass filter (PI, red fluorescence)). Control measurements and the gating scheme for raw data processing were similar to those of Zhang et al. (2020). FCM analysis happened on the day of the HP treatment, or after freezing (−20 °C, ≤2 weeks) and thawing on ice. Results are expressed as mean ± standard deviation.

## 2.4. Fluorescence-activated cell sorting (FACS)

FACS was used to isolate single cells of the FCM subpopulations. FACS was done according to settings from Zhang et al. (2020). In short, 10⁷ CFU/mL spores were kept in 50 mM ACES buffer on ice after post-HP incubation, stained by PI and SYTO16 (Section 2.3), and measured using a BD FACSAriaTM III device (BD Biosciences, United States) with the same optical set-up as for FCM. The event rate was approximately 9000 events/s. The position of FCM subpopulations slightly shifted towards lower SYTO16 signals during data acquisition and the absolute position of FCM subpopulations varied between different days. Gates for cell sorting were therefore adapted to the position of an FCM subpopulation in the moment of cell sorting and were set rather narrow to ensure sorted cells belong to the subpopulation of interest. A single spore was sorted depending on its side scatter, forward scatter, PI fluorescence, and SYTO16 fluorescence in a well of a transparent 96-well plate at room temperature. The PI and SYTO16 signal of a sorted event were recorded to track its position in the FCM plot. Wells contained tryptic soy agar (TSA) or 50 µL 50 mM ACES buffer for growth capacity or heat sensitivity analysis, respectively. Plates were up to 3.5 h at room temperature (24 °C) until all samples had been sorted. 50 mM ACES buffer was stained to check the FCM background position and event rate. The experiment was repeated on ≥3 independent days with ≥1 replicate for

each post-HP incubated sample.

## 2.5. Analysis of the growth capacity and heat sensitivity of single spores

For the analysis of the growth capacity, FACS-sorted cells were incubated on TSA at 37 °C for 48 h. The growth capacity of a subpopulation was expressed as the percentage of colony-forming cells related to the total number of sorted cells of the subpopulation.

For the analysis of the heat sensitivity, the plate of FACS-sorted cells in 50 mM ACES buffer was heated for 20 min in an oven set to 80 °C to inactivate heat-sensitive cells. A non-heated control plate and a sterility control plate were kept at room temperature in the meantime. Then, 200 µL of tryptic soy broth (TSB) medium was added to each well and plates were incubated at 37 °C. The growth capacity was expressed as the percentage of turbidity-causing cells related to the total number of sorted cells of a subpopulation. The optical density at 600 nm (OD600) was measured after 48 h using a Tecan Spark® microplate reader (Tecan Trading AG, Switzerland) to determine the turbidity in each well. The mean OD600-value of the sterility control plate was used as blank. Spores sorted in buffer got in contact with nutrients (TSB) 3–4 h later than those sorted directly on TSA. This did not influence their growth capacity, as growth capacities of non-heated spores were similar in liquid TSB or on solid TSA (Supplementary Table A.2). The HP and post-HP treatment, FACS sorting, and start of cultivation were done within one day resulting in 1–2 multi-well plates per sample. Independent experiments were performed on at least three different days (n ≥ 3).

## 2.6. Purification — isolation of superdormant (SD) spores

The vHP SD spores were separated from germinated spores using buoyant density centrifugation (Delbrück et al., 2021b) as soon as possible after the HP and post-HP treatment. The untreated control and the HP-treated samples were centrifuged (12,000 g, 2 min, 4 °C) in 2 mL tubes. After supernatant removal, spores were incubated in 1 mL sterile-filtered MilliQ water for ~5 min at room temperature for water uptake in the germinated spores. This step was important to increase the difference in density between the ungerminated SD and the germinated spores, facilitate their separation, and yield a high SD spore purity. After centrifugation (12,000 g, 2 min, 4 °C) and water removal, the pellet was suspended in 200 µL 20 % (w/v) Nycodenz® (Serumwerk Bernburg AG, Germany) and carefully layered on 1.8 mL 50 % (w/v) Nycodenz®. A maximum of three HP samples with similar SD spore amounts were pooled on the same 50 % Nycodenz® solution. After the buoyant density centrifugation (13,000 g, 45 min, 4 °C), the upper layers with germinated spores and the supernatant were removed. The more dense, ungerminated dormant or SD spores had pelleted on the bottom and were re-suspended in 100 µL sterile MilliQ water, transferred in fresh tubes with 1 mL 4 °C-cold MilliQ water, and 4× washed using centrifugation (12,000 g, 2 min, 4 °C). The purity of the dormant control or the pool of isolated vHP SD spores was determined using FCM.

## 2.7. Analysis of the spore dipicolinic acid (DPA) content

The DPA content of dormant and SD spores was analyzed using a terbium-DPA fluorescence assay (Fig. 1; Delbrück et al., 2021b; Li et al., 2019). Five to six samples were vHP-treated, and the remaining SD spores were isolated and pooled (see Section 2.6). The isolated SD spores and the dormant control were washed twice with sterile MilliQ water by centrifugation (6000 g, 4 °C, 5 min) to remove external DPA. The spore concentration was set to 1–3 · 10⁸ cells/mL using a counting chamber (depth 0.02 mm, Ref. 0642110, Paul Marienfeld, Germany). To release the DPA from the spore core, 1 mL of each spore suspension was autoclaved in a closed sterile 10 mL plastic tube. As 1 mL is insufficient for the temperature sensor, 100 mL water was used for the temperature sensor of the autoclave (121 °C, 20 min). Autoclaved samples and 1 mL of each non-autoclaved sample were sterile-filtered using a 0.2 µm PES
syringe filter to separate cells and the DPA-containing supernatant. The experiment from HP treatment to supernatant preparation was done within a day and repeated on four days (n = 4). The supernatants (100 µL) were mixed in a black 96-well plate with 100 µL of sterile-filtered 20 mM TbCl₃ solution (CAS: 10042-88-3, Sigma-Aldrich; TbCl₃ was dissolved in 50 mM Trizma buffer (pH 7.5; CAS: 77-86-1 & 1185-53-1, Sigma-Aldrich)). Two wells per sample were incubated at room temperature (24 °C) for 8 min and the fluorescence of the formed [Tb (DPA)₃]³⁻ complex was measured in a microplate reader (excitation: 270 nm, emission: 545 nm, Tecan Spark®, Tecan Trading AG, Switzerland). To determine the DPA concentration, a calibration standard curve (Supplemental Material: Section 4) was established using sterile-filtered DPA (CAS: 499-83-2, Sigma-Aldrich) instead of a spore sample, and the DPA content was calculated (Supplemental Material: Section 4).

## 2.8. Re-sporulation for analysis of genetic stability of superdormancy

Isolated vHP SD spores and the dormant control were germinated, outgrown to vegetative cells, and re-sporulated similarly to Delbrück et al. (2021b), to test the vHP germination capacity of the next spore generation (Fig. 1). The isolated SD spore sample contained the SD spores from 4 vHP-treated samples. The dormant control and the SD spores were heated at 80 °C for 10 min to inactivate traces of viable germinated spores. Spores were germinated and outgrown on TSA plates with 10 µg/mL kanamycin monosulfate (CAS: 25389-94-0, Sigma Aldrich) at 37 °C. The next day, 8 colonies originating from the dormant control and 8 from the vHP SD spores were each used for an overnight TSB culture with 10 µg/mL kanamycin monosulfate. The sporulation and spore preparation were done as described in Section 2.1. Each culture was used for inoculation of one sporulation medium agar plate, resulting in 2 × 8 new dormant spore suspensions with a dormant spore purity of ≥98 % (FCM). Before pooling, the vHP germination homogeneity within the biological replicates was checked using FCM (Supplementary Table A.3). Randomly picked tubes (≥5) were pooled resulting in one spore batch descending from the dormant control and one batch descending from the isolated vHP SD spores. On different days (n = 4), the two batches were simultaneously vHP-treated for 40–60 s, and the remaining SD spores were quantified using FCM for assessment of the germination capacity.

## 2.9. Analysis of temporal stability of superdormancy

The temporal stability of vHP superdormancy was tested by exposing isolated vHP SD spores to a second vHP treatment (Fig. 1). The isolated SD spore sample contained the SD spores from 4 to 5 vHP-treated samples originating from a 1-month-old spore batch. The spore concentrations in the dormant control and the isolated SD spores were adjusted to 10⁹ cells/mL using a counting chamber. Both samples were simultaneously vHP-treated with the same parameters as of the first vHP treatment (550 MPa, 60 °C, 52 s). The second vHP treatment was done on the day of the first HP treatment and one month later after storage at 4 °C in MilliQ water. The remaining SD spores after the second treatment were quantified using FCM. The experiment from first to second vHP treatment and FCM was repeated on three different days (n = 3).

# 3. Results and discussion

## 3.1. Growth capacity and heat sensitivity of high-pressure-treated spore subpopulations

To validate FCM for SD spore quantification, the growth capacity of FCM-detected spores was investigated depending on the vHP treatment time and post-HP conditions. The growth capacity of FCM spore subpopulations was estimated by sorting single spores of each subpopulation depending on their PI and SYTO16 fluorescence in FCM plots and

growing them in 96-well plates. Generally, differences in the growth capacity should reflect effects of the vHP and post-HP treatment only, as all other factors like the sample preparation or the growth medium were similar for all samples.

Without a vHP treatment, the dormant spores appeared in the PI- and SYTO16-negative region R1 due to their intact cortex. They were all culturable (Fig. 2), as expected for the untreated dormant control (Zhang et al., 2020). After treatments of 50 s – 6 min at a vHP of 550 MPa and 60 °C, four to five spore subpopulations were observed in the regions R1–R5 of different PI or SYTO16 fluorescence levels (Fig. 2–4). Expected properties of the spore subpopulations in R1–R5 are described in Fig. 2.

Quantification by FCM relies on counting all events in a region of certain fluorescence levels which is characteristic for one subpopulation. Hence, it is important that different subpopulations show different fluorescence levels to clearly define regions for each subpopulation. It has been recently shown that post-HP incubation at 37 °C or 60 °C is required for the separation of the putative vHP SD spore subpopulation (R1) from other subpopulations in the FCM plots (Heydenreich et al., 2023). An overlap of the SYTO16-negative subpopulation of ungerminated SD spores (R1) and a subpopulation of germinated (R3) spores is a particularity of vHP treatments (Fig. 3: A.1). In contrast to an mHP or nutrient germination treatment, vHP seems to denature cortex-lytic enzymes which prevents SYTO16-uptake in some germinated spores upon post-HP incubation on ice (Black et al., 2007b; Christie and Setlow, 2020; Heydenreich et al., 2023). Post-HP incubation at 37 °C or 60 °C seems to enable the separation of SD and germinated spores, as the incubation might promote the recovery of cortex-lytic enzymes from reversible vHP damages in germinated spores (Heydenreich et al., 2023). Thus, these germinated spores should become SYTO16-positive while the SD spores with an intact cortex stay STYO16-negative. To make sure that post-HP incubation at ≥37 °C separated SD and germinated spores after a short vHP treatment, FCM was validated depending on the post-HP temperature on a subpopulation and single cell level.

The measured growth capacities of sorted spores confirm a good separation of SD and germinated spores in region R1 after a short vHP treatment. After a short vHP treatment of 50 s and subsequent incubation at 37 °C, spores were in region R1 almost all culturable (Fig. 3: B.1–2: 97 ± 2 % culturable). After post-HP incubation on ice, spores in region R1 were similarly well culturable (Fig. 3: A.1–2).

After the short vHP treatment, spores in R1 were heat resistant to 60 °C or 80 °C, as almost all sorted spores were able to grow out despite the heat treatments (Fig. 3: B.3, C.1–2). In contrast, the germinated SYTO16-positive subpopulations in regions R2 and R3 were heat sensitive to 60 °C or 80 °C and lost their growth capacity (Fig. 3: B.3). The heat resistance and high growth capacity of spores in R1, and the fact that spores in R1 appeared in the same region as dormant spores, validate that the spores in region R1 were vHP SD spores (Fig. 3). The high growth capacity of SD spores in R1 (98 ± 3 %) shows a high frequency of SD spore events after 50 s vHP treatment in R1. All culturable spores in R1 were expected to be SD spores as culturable germinated spores must be able to degrade their cortex, i.e. be SYTO16-positive (R2, R3) after incubation at 37 °C. The rare non-culturable events in R1 (2 ± 3 %) could have been i) background events, ii) nutrient SD spores, or iii) non-culturable vHP-inactivated spores with inactivated cortex-lytic enzymes. Background events are data points in the FCM plot that do not originate from spores but originate from noise from the buffer or the instrument (Wang and Hoffman, 2017). Nutrient SD spores are spores that are not or only very slowly germinating upon application of a nutrient germination stimulus like a nutrient-containing agar (Ghosh and Setlow, 2009). Hence, if a spore is not only vHP SD but additionally nutrient SD, the lack of germination on agar would prevent outgrowth and thus the formation of a visible colony. To reduce the number of nutrient SD spores, a nutrient rich agar medium was chosen for an improved nutrient germination. In addition, colonies were counted after 48 h to detect spores that are slow in nutrient germination.
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Flow cytometry and growth analysis of non-high-pressure-(HP)-treated dormant *Bacillus subtilis* PS832 spores after "post-HP" incubation at 37 °C. A representative flow cytometry plot shows propidium iodide (PI) or SYTO16 fluorescence heights (−H) (A.1). A general interpretation of flow cytometric regions based on previous literature is given (Heydenreich et al., 2023). See Supplementary Table A.4 for the event frequency in region R1. In the growth plot, each data point corresponds to a single culturable spore (80–96 spores analyzed in total per day). The growth capacity on tryptic soy agar ("% culturable") is expressed as a mean ± standard deviation of replicates from three different days. SASP: small acid soluble proteins.</figcaption></figure>

After longer vHP treatments of 2.5 min or 6 min, FCM was not suitable for SD spore quantification. Spores in region R1 were barely culturable after 2.5 min and non-culturable after 6 min at a vHP of 550 MPa. Post-HP incubation at 60 °C inactivated all visible subpopulations of 2.5-min- or 6-min-HP-treated spores — except rare events in R1 of the 2.5-min-treated sample (Fig. 4: B.2, D.2). These rare culturable, heat-resistant events in R1 could have been SD spores. Due to the generally low data number in R1 after 2.5 min under pressure (Fig. 4: A.1, B.1), the probability of sorting buffer background events was increased, as background events appear in R1. Most events in R1 could have been therefore non-sporic background events. Alternatively, the unculturable events in R1 after 2.5 min could have originated from the HP-inactivated spore subpopulation with irreversibly inactivated cortex-lytic enzymes (Heydenreich et al., 2023). Due to the higher data number in R1 after 6

min (Fig. 4: C.1, D.1), events in R1 were likely mostly spores instead of non-sporic background events. These spores were HP-inactivated spores.

Regarding the subpopulations R2–R5 of germinated or inactivated spores, the growth capacity depended on the vHP treatment time and post-HP incubation conditions (Figs. 3 and 4). Hence, SYTO16- and PI-fluorescence alone did not directly correlate with the growth capacity of a single spore. However, germinated spores with the highest SYTO16 signals seemed generally better culturable than those with intermediate SYTO16 fluorescence, in the absence of a heat treatment. PI-positive spores were mostly germinated and non-culturable (inactivated). For additional information on the interpretation of FCM subpopulations R2–R5 or SYTO16 levels of HP-treated spores, the reader is referred to the supplementary material, Section 8.

In conclusion, FCM can be used for SD spore quantification after
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Flow cytometry and growth analysis of high-pressure (HP)-treated *Bacillus subtilis* PS832 spores after 50 s at 550 MPa and 60 °C and post-HP incubation at various temperatures (ice, 37 °C, 60 °C). Representative flow cytometry plots show propidium iodide (PI) or SYTO16 fluorescence heights (−H) of spores (A-C.1). See Supplementary Table A.4 for the event frequency in region R1–R4. Growth plots below indicate the growth capacity of 16–24 single spores of each subpopulation R1–R4. To determine the heat sensitivity, spores were heat-treated (80 °C, 20 min) after cell sorting and grown in liquid medium (B.3). The growth capacity on tryptic soy agar or broth ("% culturable") is expressed as a mean ± standard deviation of ≥6 replicates from three different days.</figcaption></figure>

short vHP treatments (≤1 min, 550 MPa, 60 °C) and suitable post-HP incubation conditions (37 °C or 60 °C). For the following vHP SD spore characterization (Sections 3.2–3.5), post-HP incubation at 60 °C was chosen. Using 60 °C, i) the SD and germinated subpopulations were well separated in contrast to post-HP incubation on ice and ii) this separation was faster than at 37 °C post-HP.

## 3.2. Yield and purity of isolated superdormant (SD) spores

The vHP SD spore yield was 6 ± 1 % to 8 ± 1 % after vHP treatment of the initial dormant spore population (n ≥ 4, Fig. 1). The yield depended on the vHP treatment time and the spore batch (Supplementary Fig. A.4). For interpreting the results below, it is worth noting that superdormancy describes a relative spore property. This means the superdormant spore properties depend on the applied germination

stimulus and intensity, here a vHP treatment of 43–52 s, respectively.

Purification removed vHP-germinated spores or traces of spontaneously germinated spores in the vHP-treated or untreated sample, respectively. Purification led to a good purity of 93–99 % ungerminated spores in the isolated vHP SD spore sample (Fig. 1) and 96–99 % in the dormant control samples according to FCM.

## 3.3. DPA content in dormant and superdormant (SD) spores

The DPA content of the dormant control and isolated SD spores was determined using a terbium-DPA fluorescence assay (Fig. 1), as the DPA content might potentially influence superdormancy. The dormant and vHP SD *B. subtilis* PS533 spores contained comparable amounts of DPA of 2.0 ± 0.2·10⁻¹⁶ and 2.1 ± 0.8·10⁻¹⁶ mol/cell, respectively (mean ± standard deviation; n = 4).
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Flow cytometry and growth analysis of high-pressure (HP)-treated *Bacillus subtilis* PS832 spores after 2.5 or 6 min at 550 MPa and 60 °C and post-HP incubation at 37 °C or 60 °C. Representative flow cytometry plots show propidium iodide (PI) or SYTO16 fluorescence heights (—H) of spores (A-D.1). See Supplementary Table A.4 for the event frequency in region R1–R5. Growth plots below indicate the growth capacity of 16–24 single spores of each subpopulation R1–R5. The growth capacity on tryptic soy agar ("% culturable") is expressed as a mean ± standard deviation of ≥6 replicates from three different days.</figcaption></figure>

The DPA contents were within the previously reported range of 2–9·10⁻¹⁶ mol/cell for *B. subtilis* spores (Delbrück et al., 2022; Kort et al., 2005; Li et al., 2019). Similarly to our results, Delbrück et al. (2022) and Ghosh and Setlow (2009) found that the DPA content did not differ remarkably between the initial dormant spore population and nutrient or mHP SD spores (mHP: 150 MPa, 37 °C, 6 min). While mHP and nutrient germination are initiated by receptor activation, germination under vHP is initiated directly by DPA release (see Section 1). DPA channels are involved in the DPA release during germination and also in DPA uptake during sporulation (Christie and Setlow, 2020). Thus, a change in DPA channel proteins or their surroundings could have theoretically resulted in an altered DPA uptake, i.e., DPA content, and an altered DPA release, i.e., vHP superdormancy. Considering that the DPA release is the first critical step in vHP germination but not in mHP or nutrient germination, vHP SD spores could have had an altered DPA content in contrast to mHP and nutrient SD spores.

Another study analyzed the pressure (600 MPa, 80 °C) resistance of different *Bacillus* strains (Li et al., 2019) — in contrast to analyzing

differences between spores of one strain (superdormancy). Li et al. (2019) found that the pressure resistance did not correlate with the DPA content per cell. In conclusion, literature supports that the DPA level is unlikely linked to the cause of vHP (550 MPa, 60 °C) superdormancy.

### 3.4. Genetic stability of superdormancy

A genetic change was evaluated as the cause of vHP superdormancy. The isolated vHP SD spores were germinated and grown in a nutrient-rich medium before re-sporulation to a new dormant spore generation. A dormant spore control was re-sporulated in parallel. The new spore generations were treated at 550 MPa and 60 °C for 40–60 s to compare their germination capacity using FCM (Fig. 1).

After vHP treatment, similar percentages of remaining ungerminated SD spores were measured for the descendants of SD spores or of the dormant control (Fig. 5: A). Hence, descendants of the dormant control and those of SD spores had a similar germination capacity. If a genetic change in the germination machinery caused vHP superdormancy,
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Analysis of the temporal and genetic stability of very high pressure (vHP) superdormancy of *Bacillus subtilis* PS533 spores. Superdormant (SD) spores were isolated after a first vHP treatment of 43 s (A) or 52 s (B) at 550 MPa and 60 °C. The genetic stability (A) was tested by vHP-treatment (550 MPa, 60 °C, 40–60 s) of descendants of isolated SD spores or descendants of the dormant control. The germination capacity indicated by the SD spore yield was analyzed using flow cytometry (mean ± standard deviation; n = 4). To test the temporal stability (B), isolated SD spores were treated together with a dormant control by a second vHP treatment within 12 h after their isolation or 1 month later using the same parameters as of their first vHP treatment (550 MPa, 60 °C, 52 s). The SD spore yield was analyzed (mean ± standard deviation; n ≥ 3). Estimated "<1 %" means the values were below the limit of quantification of the flow cytometer.</figcaption></figure>

descendants of SD spores would have been expected to germinate poorly too. This means these descendants would have shown higher percentages of remaining SD spores after the vHP treatments compared to descendants of the dormant control.

Our results validate that vHP superdormancy is not genetically inherited. It thereby shares a similarity with mHP (150 MPa, 37 °C, 6 min) or nutrient superdormancy (Delbrück et al., 2021b; Ghosh and Setlow, 2009). In contrast to superdormancy, i.e. heterogenous germination within an isogenic population of one strain, spores of different strains or species can likely differ in their (HP) germination capacity due to different (numbers of) genes (e.g., Li et al., 2019; Yu et al., 2023). For example, the copy number of the *spoVA*²*mob* operon in spores of different *Bacillus* species correlated with the HP resistance (600 MPa, 80 °C) of a species (Li et al., 2019; Yu et al., 2023, see also Section 3.5).

### 3.5. Temporal stability of superdormancy

To investigate the temporal stability of superdormancy, isolated vHP SD spores were treated with a second vHP treatment (550 MPa, 60 °C, 52 s) on the day of their isolation or one month later. A dormant control was treated simultaneously in the second vHP treatment for comparison (Fig. 1).

While the SD spore fraction did not germinate during the first vHP treatment, most SD spores germinated during the second vHP treatment, as indicated by a low percentage of remaining SD spores (Fig. 5: B). Hence, vHP superdormancy seemed unstable over time. Partial germination of vHP SD spores was expected from germination kinetics of *B. subtilis* at 550 MPa and 60 °C, showing a proceeding germination after 52 s vHP dwell time (Fig. 5: A; Heydenreich et al., 2023). Surprisingly, the vHP SD spores germinated even better than the dormant control as indicated by the measured remaining SD spore yields (Fig. 5: B). In the vHP SD spore sample, the yield was below the limit of quantification ("<1 %"). In contrast, the SD spore yield of the dormant control was 17 ± 6 % after the vHP treatment on the day of SD spore isolation (Fig. 5: B). Regarding the initial dormant spore population, the first vHP treatment that was done for SD spore isolation yielded only 7 ± 1 % SD

spores (Fig. 1). The dormant control and the initial dormant spore population should theoretically behave similarly under pressure. The different SD yields in these two dormant spore samples were thus probably an effect of different sample volumes or tube types. Smaller tubes were necessary for the parallel treatment of a SD spore and a dormant control sample. These tubes have led to slightly different temperature profiles during the vHP treatment and 2–3 s shorter total treatment times compared to the big tube (Supplementary Fig. A.2: B). A big tube was used for the initial dormant spore population to isolate more vHP SD spores.

Regarding the second vHP treatment one month later, vHP SD spores germinated again better than the dormant control (Fig. 5: B). Interestingly, more spores germinated in the dormant control sample compared to one month before, seen by only 4 ± 2 % SD spores (Fig. 5: B). The date-dependent SD spore yield in the dormant control supports that vHP superdormancy can be unstable over time, i.e., can have transient causes. Accordingly, Ghosh and Setlow (2010) observed an instable superdormancy, as isolated nutrient SD spores lost their superdormancy during storage at 4 °C for >50 days. Isolated mHP SD spores showed similar germination capacities over one week (Delbrück et al., 2021b).

So far, superdormancy or the inability to initiate germination has been reported to be caused by transient or permanent factors (Delbrück et al., 2021b; Ghosh and Setlow, 2010). The transience of superdormancy was supported by the observation that some nutrients-, Ca-DPA-, or mHP-SD spores could germinate shortly after their isolation when exposed to the same germination stimulus as used for their isolation. However, SD spores germinated more slowly or in some cases not at all, compared to the (initial) dormant population (Delbrück et al., 2022; Ghosh and Setlow, 2009; Perez-Valdespino et al., 2013). The non-germinating SD spores suggested not only transient but also permanent causes of superdormancy. The transient, reversible causes of mHP or nutrient superdormancy are not clear. Conformational changes of germinant receptor proteins have been hypothesized as one of the potentially several causes of transient mHP and nutrient superdormancy (Christie and Setlow, 2020; Delbrück et al., 2021b). Permanent factors decreasing the mHP-, nutrient- or Ca-DPA-stimulated germination
efficacy seemed to be the number of certain proteins (Chen et al., 2014; Delbrück et al., 2021a, 2021b, 2022; Doona et al., 2014; Ghosh and Setlow, 2009; Perez-Valdespino et al., 2013). It has been hypothesized, for example, that stochastic variations in the number of germinant receptors could lead to spores with very few germinant receptors and thus a slow nutrient germination rate (Ghosh et al., 2009; Griffiths et al., 2011; Stewart and Setlow, 2013). Later work by Breedijk et al. (2020) suggested a more complex mechanism requiring further molecular components as reason for nutrient germination heterogeneity. These components might be related to the formation of functional germinant receptor protein cluster (germinosomes). Besides the protein level, the spore core's water content as a permanent factor was linked to nutrient superdormancy (Ghosh et al., 2009), but not to mHP superdormancy (Delbrück et al., 2022).

Contrary to previously characterized SD spores, only vHP SD spore germinated better upon re-exposure to vHP, i.e. the same stimulus as used for their isolation (Fig. 5). Permanent, pressure-stable factors therefore played a minor role in causing vHP superdormancy in our study. The improved germination capacity was surprising considering the above-mentioned decreased germination capacities of isolated nutrient or mHP SD spores. Isolated dodecylamine spores also did not germinate better — they germinated similarly as dormant spores when re-exposed to a dodecylamine germination trigger (Perez-Valdespino et al., 2013). Dodecylamine triggers germination by directly releasing DPA like vHP. Permanent factors playing a minor role in vHP superdormancy aligns with analysis of the other permanent properties in our study; both the DPA content and the genetics seemed not clearly changed in SD spores (Sections 3.3 and 3.4). Another permanent factor that should not have changed between the two vHP treatments is the number of certain proteins in a spore. The protein expression levels alone seem consequently unlikely as the main cause of vHP superdormancy. However, the DPA-channel-forming SpoVA proteins have been discussed as an influencing factor on the vHP germination capacity (Doona et al., 2014; Vepachedu et al., 2007). For example, a lower SpoVA protein level in mutant spores correlated with a lower vHP germination rate compared to wildtype *B. subtilis* spores at 550 MPa and 50 °C (Doona et al., 2014). Hence, the SpoVA protein level could theoretically play a role in vHP superdormancy, but its influence might have been too small for detection in our study.

Generally, it is difficult to explain the transient causes of vHP superdormancy on a molecular level, as the mechanism of vHP germination initiation is poorly understood. Germination initiation by DPA release might be an effect of vHP directly on SpoVA channels or on the lipid phase in the inner membrane (Reineke et al., 2013). The causes of vHP superdormancy could be thus related to time-dependent or permanent structural properties of the inner membrane or its embedded proteins. Generally, all cellular structures that help to retain the DPA in the spore core might play a role in vHP superdormancy. It is worth noting that a transient cause of superdormancy is not necessarily reversible. Theoretically, also a transient, irreversible structural modification could influence vHP germination. A vHP can modify or damage protein structures and membranes (see e.g. Section 3.1). Consequently, the increased germination capacity of vHP SD spores could have resulted from reversible or irreversible non-lethal vHP-induced structural changes. These changes could have facilitated germination during the second vHP treatment. Besides vHP-induced modifications, the spore structure can be modified irreversibly over time by maturation of the proteinaceous spore coat (Driks and Eichenberger, 2016). The maturation reinforces the coat by cross-linking of proteins and has been suggested to increase the spore's heat resistance (Camilleri et al., 2019). Furthermore, the coat likely plays a role in HP resistance of *C. botulinum* type E spores to 800 MPa and ≥40 °C (Lenz, 2017). It remains to be explored if coat maturation was linked to the time-dependent, decreasing vHP resistance in the dormant spores in our study. Another transient factor influencing the susceptibility to a vHP germination trigger could be time-dependent structural changes of protein clusters in

the inner membrane and resulting changed membrane properties. For example, germination proteins, such as SpoVA proteins have been shown to move randomly in the inner membrane of dormant spores and transiently interact with other germination proteins (Wen et al., 2022). Furthermore, proteins encoded by the *spoVA*²*mob* operon or their homologs appeared to modulate the inner membrane properties and to increase the pressure and heat resistance in *Bacillus* spp. (Korza et al., 2023; Li et al., 2019 (see Section 3.4); Yu et al., 2023). The *spoVA*²*mob* protein 2Duf has been shown to mediate heat resistance and to influence nutrient germination (Korza et al., 2023). It is not clear yet if it is 2Duf or another *spoVA*²*mob* protein which increased the vHP resistance (Li et al., 2019). Other germination-relevant *spoVA*²*mob* proteins are homologs of SpoVA, i.e., DPA channel proteins. It would be interesting to investigate if and how *spoVA*²*mob* proteins change over time in the membrane and if they are involved in a transient cause of resistance to vHP germination.

A practical implication of the increased germination capacity of vHP SD spores would be that prolonged or repeated vHP treatments could result in the absence of SD spores. However, heat-resistant (SD) *B. subtilis* spores were still detected after 3 vHP cycles (Mok et al., 2022: 500 MPa, 60 °C, 3 × 3 min). After prolonged vHP treatments of 20 min, dormant *B. subtilis* spores were reduced by ~6 log₁₀ units showing the potential of vHP for spore inactivation (Heydenreich et al., 2023; Reineke et al., 2011). Nevertheless, vHP SD spores could be still detected and were not be clearly reduced by additional 10 min at 600 MPa and 60 °C (Reineke et al., 2011). Thus, the SD spore fraction after 20 min might not show a better germination capacity relative to the initial dormant spore population. To become germination-susceptible, this SD spore fraction might need more than additional 10 min under vHP. Alternatively, they might need a certain time without HP, as the isolated vHP SD spores (Fig. 5) were several hours or a month at atmospheric pressure before their second vHP treatment. The previous results imply that the characteristics of the vHP SD spore fraction could strongly depend on the vHP treatment time. These most resistant SD spores, which remained after 20 min vHP, might have been too little concentrated for detection (~0.0001 % = −6 log₁₀ units) in our isolated SD spore fraction. Isolation of such rare events would be relevant for investigating causes of superdormancy. However, the investigation is hampered by the required SD spore amounts for most characterization methods. Notably, plate count was used in the vHP studies above with reductions of 6 log₁₀ units (Heydenreich et al., 2023; Mok et al., 2022; Reineke et al., 2011). Plate count requires nutrient germination before growth on agar. Hence, nutrient-stimulated germination was still functional in at least some vHP SD spores. Similarly, nutrient SD spores germinated normally with other germination stimuli, such as HP or peptidoglycan fragments (Wei et al., 2010). It further suggests that the inability to germinate can be non-permanent since SD spores can be susceptible to other types of germination stimuli.

## 4. Conclusion

This study made it possible to isolate and characterize vHP SD spores for the first time and validated FCM as a powerful method for their quantification. FCM can be used for a rapid SD spore quantification after short vHP treatments (≤1 min, 550 MPa, 60 °C) and a suitable post-HP incubation, e.g., at 37 °C or 60 °C. The need for a post-HP incubation is a particularity for vHP treatments to separate FCM signals from SD and germinated spores. FCM after post-HP incubation has not been investigated on a single cell level in previous FCM studies. Using single cell sorting, the chosen conditions were validated to successfully separate the FCM signals of SD and germinated spores. Interesting for FCM spore analysis in general is that (HP-)germinated spores with the highest SYTO16 signals were generally better culturable than those with intermediate SYTO16 fluorescence. However, SYTO16 did not necessarily indicate culturability. This highlights the general need for FCM validation for different HP treatment conditions.

The characterization of SD spores revealed that the DPA content is
unlikely linked to the cause of vHP superdormancy. Superdormancy to vHP is also unlikely genetically inherited, i.e. caused by a permanent genetic change. Generally, permanent factors appeared to play a minor role in vHP superdormancy under our isolation conditions (550 MPa, 60 °C, 43–52 s, ~7 % SD spores). Contrary to superdormancy to other germination stimuli (nutrients, mHP, Ca-DPA, dodecylamine), only vHP SD spores germinated better upon re-exposure to vHP, i.e. the same stimulus as used for SD spore isolation. Hence, there might be mechanisms to change from a vHP-germination-resistant state, i.e. the SD state, to a germination-susceptible state. This could be highly relevant for applications: if all spores are susceptible to vHP germination, all spores can be inactivated. It remains to be unraveled in future studies if and how we can trick all spores into transitioning to the germination-susceptible state. Once means to tackle superdormancy of the model organism *B. subtilis* are better understood, a species variability and other influencing factors (e.g., exact pressure-temperature-time combination, pH, medium composition, *Delbrück* et al., 2021a) need to be further explored. This is key to potentially applying a vHP germination-inactivation sterilization process to food products with spores and other microorganisms from various food-spoiling or pathogenic species.

The germination-resistant SD spores are the main challenge for the industrial application of a vHP germination-inactivation strategy. By investigating resistance to vHP, our work contributes to the potential development of a mild spore inactivation strategy based on vHP and moderate temperature (<80 °C).

## Funding

We thank the Swiss National Science Foundation SNF for funding this study [Grant number: 31003A_182273].

## CRediT authorship contribution statement

**Rosa Heydenreich:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization.
**Alessia I. Delbrück:** Writing – review & editing, Supervision, Investigation, Conceptualization.
**Christina Peternell:** Writing – review & editing, Investigation.
**Clément Trunet:** Writing – review & editing, Supervision.
**Alexander Mathys:** Writing – review & editing, Supervision, Project administration, Funding acquisition.

## Declaration of competing interest

None.

## Data availability

Raw data of this study are available via the ETH Zurich Research Collection [DOI: 10.3929/ethz-b-000660743].

## Acknowledgments

We thank the Cytometry Facility of the University of Zurich, especially Mario Wickert, for their technical support.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijfoodmicro.2024.110812.

## References

Black, E.P., Setlow, P., Hocking, A.D., Stewart, C.M., Kelly, A.L., Hoover, D.G., 2007a. Response of spores to high-pressure processing. Compr. Rev. Food Sci. Food Saf. 6, 103–119. https://doi.org/10.1111/j.1541-4337.2007.00021.x.

Black, E.P., Wei, J., Atluri, S., Cortezzo, D.E., Koziol-Dube, K., Hoover, D.G., Setlow, P., 2007b. Analysis of factors influencing the rate of germination of spores of *Bacillus subtilis* by very high pressure. J. Appl. Microbiol. 102, 65–76. https://doi.org/10.1111/j.1365-2672.2006.03062.x.

Breedijk, R.M.P., Wen, J., Krishnaswami, V., Bernas, T., Manders, E.M.M., Setlow, P., Vischer, N.O.E., Brul, S., 2020. A live-cell super-resolution technique demonstrated by imaging germinosomes in wild-type bacterial spores. Sci. Rep. 10, 1–12. https://doi.org/10.1038/s41598-020-62377-1.

Camilleri, E., Korza, G., Green, J., Yuan, J., Yongqing, L., Caimano, M.J., Setlow, P., 2019. Properties of aged spores of *Bacillus subtilis*. J. Bacteriol. 201, 1–17. https://doi.org/10.1128/JB.00231-19.

Chen, Y., Ray, W.K., Helm, R.F., Melville, S.B., Popham, D.L., 2014. Levels of germination proteins in *Bacillus subtilis* dormant, superdormant, and germinating spores. PloS One 9, 1–10. https://doi.org/10.1371/journal.pone.0095781.

Christie, G., Setlow, P., 2020. *Bacillus* spore germination: knowns, unknowns and what we need to learn. Cell. Signal. 74, 1–9. https://doi.org/10.1016/j cellsig.2020.109729.

Clouston, J.G., Wills, P.A., 1969. Initiation of germination and inactivation of *Bacillus pumilus* spores by hydrostatic pressure. J. Bacteriol. 97, 684–690. https://doi.org/10.1128/jb.97.2.684-690.1969.

De Hoon, M.J.L., Eichenberger, P., Vitkup, D., 2010. Hierarchical evolution of the bacterial sporulation network. Curr. Biol. 20, R735–R745. https://doi.org/10.1016/j.cub.2010.06.031.

Delbrück, A.I., Zhang, Y., Heydenreich, R., Mathys, A., 2021a. *Bacillus* spore germination at moderate high pressure: a review on underlying mechanisms, influencing factors, and its comparison with nutrient germination. Compr. Rev. Food Sci. Food Saf. 20, 4159–4181. https://doi.org/10.1111/1541-4337.12789.

Delbrück, A.I., Zhang, Y., Hug, V., Trunet, C., Mathys, A., 2021b. Isolation, stability, and characteristics of high-pressure superdormant *Bacillus subtilis* spores. Int. J. Food Microbiol. 343, 1–11. https://doi.org/10.1016/j.ijfoodmicro.2021.109088.

Delbrück, A.I., Tritten, Y., Nanni, P., Heydenreich, R., Mathys, A., 2022. Moderate high-pressure superdormancy in *Bacillus* spores: properties of superdormant spores and proteins potentially influencing moderate high-pressure germination. Appl. Environ. Microbiol. 88, 1–16. https://doi.org/10.1128/aem.02406-21.

Doona, C.J., Ghosh, S., Feeherry, F.F., Ramirez-Peralta, A., Huang, Y., Chen, H., Setlow, P., 2014. High pressure germination of *Bacillus subtilis* spores with alterations in levels and types of germination proteins. J. Appl. Microbiol. 117, 711–720. https://doi.org/10.1111/jam.12557.

Driks, A., Eichenberger, P., 2016. The spore coat. Microbiol. Spectr. 4, 179–200. https://doi.org/10.1128/microbiolspec.tbs-0023-2016.

Fairhead, H., Setlow, B., Setlow, P., 1993. Prevention of DNA damage in spores and *in vitro* by small, acid-soluble proteins from *Bacillus* species. J. Bacteriol. 175, 1367–1374. https://doi.org/10.1128/jb.175.5.1367-1374.1993.

Ghosh, S., Setlow, P., 2009. Isolation and characterization of superdormant spores of *Bacillus* species. J. Bacteriol. 191, 1787–1797. https://doi.org/10.1128/JB.01668-08.

Ghosh, S., Setlow, P., 2010. The preparation, germination properties and stability of superdormant spores of *Bacillus cereus*. J. Appl. Microbiol. 108, 582–590. https://doi.org/10.1111/j.1365-2672.2009.04442.x.

Ghosh, S., Zhang, P., Li, Y.Q., Setlow, P., 2009. Superdormant spores of *Bacillus* species have elevated wet-heat resistance and temperature requirements for heat activation. J. Bacteriol. 191, 5584–5591. https://doi.org/10.1128/JB.00736-09.

Gratz, M., Sevenich, R., Hoppe, T., Schottroff, F., Vlaskovic, N., Belkova, B., Chytilova, L., Filatova, M., Stupak, M., Hajlova, J., Rauh, C., Jaeger, H., 2021. Gentle sterilization of carrot-based purees by high-pressure thermal sterilization and ohmic heating and influence on food processing contaminants and quality attributes. Front. Nutr. 8, 1–20. https://doi.org/10.3389/fnut.2021.643837.

Griffiths, K.K., Zhang, J., Cowan, A.E., Yu, J., Setlow, P., 2011. Germination proteins in the inner membrane of dormant *Bacillus subtilis* spores colocalize in a discrete cluster. Mol. Microbiol. 81, 1061–1077. https://doi.org/10.1111/j.1365-2958.2011.07753.x.

Heydenreich, R., Delbrück, A.I., Mathys, A., 2023. Post-high-pressure temperature and time – overlooked parameters in high pressure treatment of bacterial spores. Int. J. Food Microbiol. 402, 1–12. https://doi.org/10.1016/j.ijfoodmicro.2023.110279.

Hu, H., Balasubramaniam, V.M., 2024. High-pressure processing. In: Encyclopedia of Food Safety. Elsevier, pp. 531–551. https://doi.org/10.1016/B978-0-12-822521-9.00103-9.

Jermann, C., Koutchma, T., Margas, E., Leadley, C., Ros-Polski, V., 2015. Mapping trends in novel and emerging food processing technologies around the world. Innov. Food Sci. Emerg. Technol. 31, 14–27. https://doi.org/10.1016/j.ifset.2015.06.007.

Jimenez, P.S., Bangar, S.P., Suffern, M., Whiteside, W.S., 2023. Understanding retort processing: a review. Food Sci. Nutr. 12, 1545–1563. https://doi.org/10.1002/fnsn.3.3912.

Kong, L., Zhang, P., Wang, G., Yu, J., Setlow, P., Li, Y.Q., 2011. Characterization of bacterial spore germination using phase-contrast and fluorescence microscopy, Raman spectroscopy and optical tweezers. Nat. Protoc. 6, 625–639. https://doi.org/10.1038/nprot.2011.307.

Kort, R., O'Brien, A.C., Van Stokkum, I.H.M., Oomes, S.J.C.M., Crieelaard, W., Hellingwerf, K.J., Brul, S., 2005. Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release. Appl. Environ. Microbiol. 71, 3556–3564. https://doi.org/10.1128/AEM.71.7.3556-3564.2005.

Korza, G., Depratti, S., Fairchild, D., Wicander, J., Kanaan, J., Shames, H., Nichols, F.C., Cowan, A., Brul, S., Setlow, P., 2023. Expression of the 2Duf protein in wild-type *Bacillus subtilis* spores stabilizes inner membrane proteins and increases spore resistance to wet heat and hydrogen peroxide. J. Appl. Microbiol. 134, 1–10. https://doi.org/10.1093/jambio/lxad040.
Koutsoumanis, K., Alvarez-Ordónez, A., Bolton, D., Bover-Cid, S., Chemaly, M., Davies, R., De Cesare, A., Herman, L., Hilbert, F., Lindqvist, R., Nauta, M., Peixe, L., Ru, G., Simmons, M., Skandamis, P., Suffredini, E., Castle, L., Crotta, M., Grob, K., Milana, M.R., Petersen, A., Roig Sagués, A.X., Vinagre Silva, F., Barthélémy, E., Christodoulidou, A., Messens, W., Allende, A., 2022. The efficacy and safety of high-pressure processing of food. EFSA J. 20, 1–195. https://doi.org/10.2903/j.efsa.2022.7128.

Lenz, C.A., 2017. Effect of High Hydrostatic Pressure on *Clostridium botulinum* Type E Endospores. PhD thesis. Technische Universität München, Germany.

Li, Z., Schottroff, F., Simpson, D.J., Gänzle, M.G., 2019. The copy number of the *spoVA2mob* operon determines pressure resistance of *Bacillus* endospores. Appl. Environ. Microbiol. 85, 1–12. https://doi.org/10.1128/AEM.01596-19.

Mahillon, J., 2008. Insertion sequence elements and transposons in *Bacillus* and relatives. In: Applications and Systematics of *Bacillus* and Relatives, pp. 236–253. https://doi.org/10.1002/9780470696743.ch16.

Margosch, D., Gänzle, M.G., Ehrmann, M.A., Vogel, R.F., 2004. Pressure inactivation of *Bacillus* endospores. Appl. Environ. Microbiol. 70, 7321–7328. https://doi.org/10.1128/AEM.70.12.7321-7328.2004.

Mok, J.H., Sun, Y., Pyatkovskyy, T., Hu, X., Sastry, S.K., 2022. Mechanisms of *Bacillus subtilis* spore inactivation by single- and multi-pulse high hydrostatic pressure (MP-HHP). Innov. Food Sci. Emerg. Technol. 81, 1–9. https://doi.org/10.1016/j.ifset.2022.103147.

Nicholson, W.L., Setlow, P., 1990. Sporulation, germination and outgrowth. In: Harwood, C.R., Cutting, S.M. (Eds.), Molecular Biological Methods for *Bacillus*. J. Wiley, pp. 391–450.

Perez-Valdespino, A., Ghosh, S., Cammett, E.P., Kong, L., Li, Y.Q., Setlow, P., 2013. Isolation and characterization of *Bacillus subtilis* spores that are superdormant for germination with dodecylamine or Ca²⁺-dipicolinic acid. J. Appl. Microbiol. 114, 1109–1119. https://doi.org/10.1111/jam.12125.

Reineke, K., Mathys, A., Knorr, D., 2011. The impact of high pressure and temperature on bacterial spores: inactivation mechanisms of *Bacillus subtilis* above 500 MPa. J. Food Sci. 76, M189–M197. https://doi.org/10.1111/j.1750-3841.2011.02066.x.

Reineke, K., Doehner, I., Schlumbach, K., Baier, D., Mathys, A., Knorr, D., 2012. The different pathways of spore germination and inactivation in dependence of pressure and temperature. Innov. Food Sci. Emerg. Technol. 13, 31–41. https://doi.org/10.1016/j.ifset.2011.09.006.

Reineke, K., Schlumbach, K., Baier, D., Mathys, A., Knorr, D., 2013. The release of dipicolinic acid — the rate-limiting step of *Bacillus* endospore inactivation during the high pressure thermal sterilization process. Int. J. Food Microbiol. 162, 55–63. https://doi.org/10.1016/j.ijfoodmicro.2012.12.010.

Setlow, B., Setlow, P., 1996. Role of DNA repair in *Bacillus subtilis* spore resistance. J. Bacteriol. 178, 3486–3495. https://doi.org/10.1128/jb.178.12.3486-3495.1996.

Setlow, P., 2006. Spores of *Bacillus subtilis*: their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 101, 514–525. https://doi.org/10.1111/j.1365-2672.2005.02736.x.

Setlow, P., Johnson, E.A., 2019. Spores and their significance. In: Food Microbiology: Fundamentals and Frontiers. Wiley, pp. 23–63. https://doi.org/10.1128/9781555819972.ch2.

Setlow, P., Wang, S., Li, Y.Q., 2017. Germination of spores of the orders *Bacillales* and *Clostridiales*. Annu. Rev. Microbiol. 71, 459–477. https://doi.org/10.1146/annurev-micro-090816-093558.

Stewart, K.A.V., Setlow, P., 2013. Numbers of individual nutrient germinant receptors and other germination proteins in spores of *Bacillus subtilis*. J. Bacteriol. 195, 3575–3582. https://doi.org/10.1128/JB.00377-13.

Suklim, K., Flick, G.J., Bourne, D.W., Ankenman Granata, L., Eifert, J., Williams, R., Popham, D., Wittman, R., 2008. Pressure-induced germination and inactivation of *Bacillus cereus* spores and their survival in fresh blue crab meat (*Callinectes sapidus*) during storage. J. Aquat. Food Prod. Technol. 17, 322–337. https://doi.org/10.1080/10498850802179776.

Vepachedu, V.R., Hirneisen, K., Hoover, D.G., Setlow, P., 2007. Studies of the release of small molecules during pressure germination of spores of *Bacillus subtilis*. Lett. Appl. Microbiol. 45, 342–348. https://doi.org/10.1111/j.1472-765X.2007.02204.x.

Wang, L., Hoffman, R.A., 2017. Standardization, calibration, and control in flow cytometry. Curr. Protoc. Cytom. 2017, 1.3.1–1.3.27. https://doi.org/10.1002/cpcy.14.

Wei, J., Shah, I.M., Ghosh, S., Dworkin, J., Hoover, D.G., Setlow, P., 2010. Superdormant spores of *Bacillus* species germinate normally with high pressure, peptidoglycan fragments, and bryostatin. J. Bacteriol. 192, 1455–1458. https://doi.org/10.1128/JB.01497-09.

Wells-Bennik, M.H.J., Eijlander, R.T., Den Besten, H.M.W., Berendsen, E.M., Warda, A. K., Krawczyk, A.O., Nierop Groot, M.N., Xiao, Y., Zwietering, M.H., Kuipers, O.P., Abee, T., 2016. Bacterial spores in food: survival, emergence, and outgrowth. Annu. Rev. Food Sci. Technol. 7, 457–482. https://doi.org/10.1146/annurev-food-041715-033144.

Wen, J., Vischer, N.O.E., de Vos, A.L., Manders, E.M.M., Setlow, P., Brul, S., 2022. Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of *Bacillus subtilis* spores. Sci. Rep. 12, 1–12. https://doi.org/10.1038/s41598-022-09147-3.

Yu, B., Kanaan, J., Shames, H., Wicander, J., Aryal, M., Li, Y., Korza, G., Brul, S., Kramer, G., Li, Y.Q., Nichols, F.C., Hao, B., Setlow, P., 2023. Identification and characterization of new proteins crucial for bacterial spore resistance and germination. Front. Microbiol. 14, 1–16. https://doi.org/10.3389/fmicb.2023.1161604.

Zhang, Y., Delbrück, A.I., Off, C.L., Benke, S., Mathys, A., 2020. Flow cytometry combined with single cell sorting to study heterogeneous germination of *Bacillus* spores under high pressure. Front. Microbiol. 10, 1–13. https://doi.org/10.3389/fmicb.2019.03118.