# Persistence of vegetative and sporulated forms of Clostridium perfringens exposed to air at different relative humidities

Léa Savard, Sylvie Moundanga, Stéphane Guyot, Narjes Mtimet, Olivier

Firmesse, Sébastien Dupont, Laurent Beney

► To cite this version:

Léa Savard, Sylvie Moundanga, Stéphane Guyot, Narjes Mtimet, Olivier Firmesse, et al.. Persistence of vegetative and sporulated forms of Clostridium perfringens exposed to air at different relative humidities. Food Microbiology, 2026, 135, pp.104968. 10.1016/j.fm.2025.104968. hal-05329926

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# Persistence of vegetative and sporulated forms of *Clostridium perfringens* exposed to air at different relative humidities

Léa Savard<sup>a</sup>, Sylvie Moundanga<sup>a</sup>, Stéphane Guyot<sup>a</sup>, Narjes Mtimet<sup>b,c</sup>, Olivier Firmesse<sup>b</sup>, Sébastien Dupont<sup>a</sup>, Laurent Beney<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Foodborne pathogen
Slaughterhouse
Spores
Persistence
Dehydration
Resistance
Oxidative stress

## ABSTRACT

C. perfringens, an anaerobic bacterium, is a common cause of food poisoning that can persist on surfaces in slaughterhouses. However, the mechanisms governing its survival in such environments – characterised by variations in relative air humidity (RAH) – remain poorly understood. This study aimed to evaluate the effect of air exposure on C. perfringens survival and to identify the mechanisms responsible for its inactivation. Vegetative cells and spores of C. perfringens were deposited on inert surfaces and exposed to different RAH (11 %, 43 %, 75 %, 100 %) under both aerobic and anaerobic conditions, to assess the contributions of osmotic and oxidative effects induced by dehydration to cell death. At low RAH, more than 99 % of vegetative cells were inactivated within one day, regardless of oxygen presence. Epifluorescence microscopy and flow cytometry analyses revealed that dehydration and rehydration disrupted membrane integrity, contributing to inactivation through lethal mechanical damage. At 100 % RAH, vegetative cells survived over 3 days under aerobic conditions (>1 %) and over 30 days under anaerobic conditions (>0.003 %). The composition of the dehydration medium had little effect on cell survival. In contrast, spores were much more resistant, with around 10 % survival after two months of stress in presence of oxygen, without any significant effect of dehydration. These results highlight the potential of exploiting RAH fluctuations to develop control strategies targeting C. perfringens vegetative cells. However, the extreme resilience of spores confirms the need for specific and targeted decontamination methods to eliminate them effectively.

## 1. Introduction

The meat industry plays a key role in the global economy and continues to face significant food safety challenges. In 2022, global meat production reached approximately 361 million tonnes, including 64 million tonnes in Europe, with chicken, pork and beef being the most widely produced meats (FAO, 2024). Microbial contamination, which can occur at different stages of the food chain, remains one of the main health and economic risks in this sector.

Among the microorganisms responsible for these contaminations, *Clostridium perfringens* was one of the most frequently reported pathogens in the European Union in 2023, with toxins from the bacterium responsible for 140 foodborne outbreaks (European Food Safety Authority, 2024). This Gram-positive bacterium is classified into seven

toxinotypes (A to G) based on the production of six major toxins: alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), iota-toxin (ITX), *Clostridium perfringens* enterotoxin (CPE) and necrotic enteritis B-like toxin (NetB) (Rood et al., 2018). C. perfringens causes many human diseases, including gastroenteritis, necrotic enteritis, gas gangrene and antibiotic-associated diarrhoea. Strains capable of producing enterotoxins are of particular concern, as they are responsible for human food poisoning (García et al., 2022). These anaerobic bacteria have the ability to sporulate, allowing them to withstand harsh environmental conditions such as extreme temperatures, high osmotic pressure, chemicals, radiation and pH fluctuations (Li and McClane, 2006a, 2006b; Paredes-Sabja et al., 2008; Sarker et al., 2000). Strains with the enterotoxin gene located on the chromosome (C-cpe) show higher resistance than other strains (Abbona and Stagnitta, 2016;

<footnote><sup>a</sup> Université Bourgogne Europe, Institut Agro, INRAE, UMR PAM, F-21000 Dijon, France</footnote>

<footnote><sup>b</sup> Staphylococcus, Bacillus & Clostridium (SBCL) Unit, Laboratory for Food Safety, University Paris-Est, French Agency for Food, Environmental and Occupational Health & Safety (ANSES), 94700 Maisons-Alfort, France</footnote>

<footnote><sup>c</sup> The National Veterinary School of Alfort, 94700, Maisons-Alfort, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: Laurent.Beney@u-bourgogne.fr (L. Beney).</footnote>
Orsburn et al., 2008; Sarker et al., 2000). Thanks to its spore form, these ubiquitous bacteria can therefore be found in a variety of environments, including soil, wastewater, faeces and food (García et al., 2019; Petit et al., 1999). Naturally present in vegetative and spore forms in the digestive tract of humans and healthy animals (Uzal et al., 2014), *C. perfringens* can contaminate slaughterhouses, during animal evisceration through contact with tools, work surfaces or carcasses (Álvarez-Pérez et al., 2018; Hong et al., 2023). Cross-contamination in this environment can also lead to persistence of this bacterium (Jiang et al., 2022). Once introduced into meat products, *C. perfringens* spores may germinate and multiply if storage conditions are not controlled, thereby increasing the risk of foodborne outbreaks (ANSES, 2017; García et al., 2019). As a result, particular attention is paid to the implementation of various slaughter steps, as well as on rigorous cleaning and disinfection procedures in slaughterhouses.

Slaughterhouses, characterised by significant variations in temperature and relative air humidity (RAH), constitute complex environments that may influence the persistence of *C. perfringens*. In the digestive tract, mainly in the colon, these bacteria are in a quite stable environment and thrive in anaerobic, nutrient-rich, and humid conditions at around 37 °C, which is ideal for their survival and proliferation. If intestinal content is spilled during carcass handling, the bacteria may be projected onto inert surfaces or onto the carcass surface, where environmental conditions differ substantially. On inert surfaces, cells are directly exposed to the ambient atmospheric conditions, with or without nutrients, whereas on a carcass they are located in a limit layer corresponding to the balance between atmospheric conditions and the physicochemical parameters of the carcass rich in nutrients. Under low RAH conditions, vegetative cells or spores may be dehydrated, resulting in increased contact with gaseous oxygen. This phenomenon is likely to induce oxidative stress, not only because of this direct exposure to oxygen in the air - whose molecular concentration is about 30 times higher than in water - but also because of the slowing down, or even stoppage, of detoxification systems for reactive radical species (França et al., 2007; Laguerre et al., 2017). Conversely, in more humid environments, such as microdroplets, oxygen is present in dissolved form, which limits its diffusion and availability to cells, potentially offering them some protection against oxidative damage.

The classification of *C. perfringens* as an anaerobe remains somewhat ambiguous: it is variably described in the literature as a “strict anaerobe” (Briolat and Reysset, 2002; Jean et al., 2004), an “obligate anaerobe” (Lu and Imlay, 2021; Trinh et al., 2000), an “anaerobe” (Mehdizadeh Gohari et al., 2021; Stine et al., 2005), or even an “aerotolerant anaerobe” (Briolat and Reysset, 2002; Charlebois et al., 2017; Jean et al., 2004; Labbe and Juneja, 2017), depending on experimental conditions and the criteria used by different authors. This inconsistency reflects physiological nuances, particularly the distinction between the bacterium's ability to grow versus merely survive under oxygen exposure. Indeed, several studies have reported a strong sensitivity to oxygen. For example, Al-Qadiri et al. (2015) found that vegetative cells did not survive for more than 2 weeks at 7 °C on meat or in BHI when exposed to oxygen. It has also been demonstrated that oxygen exposure rapidly halted bacterial growth in liquid culture and induced rapid death of the vegetative cells within just over 1 h of exposure in a nutrient-rich medium lacking antioxidants (Trinh et al., 2000). However, *C. perfringens* has also been shown to survive for up to 5 day at 22 °C on meat under aerobic conditions (Al-Qadiri et al., 2015), and even to grow in the presence of oxygen at temperatures ranging from 15 °C to 42 °C on meat (Juneja et al., 1994). These findings suggest that this bacterium may display a limited but significant tolerance to oxygen, at least under specific environmental conditions. To our knowledge, this bacterium's ability to persist on inert surfaces in contact with air has not been fully investigated. A study has reported that *C. perfringens* spores are not sensitive to wet (85.7–90.3 % RAH) or dry (45.1–48.4 % RAH) conditions after two weeks of exposure on vegetable surfaces (Stine et al., 2005), suggesting that RAH has a limited effect on spore persistence. No

data are currently available for vegetative cells.

When a cell is exposed to air, it can undergo a dehydration process, the intensity of which varies according to the difference between its own water activity, that is generally closed to 1 in physiological conditions, and the RAH, ranging from 100 % (air saturated with water vapor) to 0 % (fully desiccated). This process constitutes a complex environmental perturbation that combines both hydric imbalance and increased contact with air (Dupont et al., 2014). Therefore, desiccation in air potentially generates two main classes of damages to anaerobic cells, with structural and mechanical effects related to osmosis and change in molecular hydration, and oxidative effects related to increased contact with oxygen (Dupont et al., 2014; Greffe and Michiels, 2020). The cumulative effects of oxidative stress induced by contact with atmospheric oxygen and the dehydrating effect of low relative air humidity are still largely unknown for *C. perfringens*.

In the present study, in order to understand the effect of gaseous oxygen on this bacterium, the persistence of vegetative cells and spores in a liquid medium under aerobic and anaerobic conditions was first evaluated and compared with the effect of a chemical oxidant commonly used in industrial environments, hydrogen peroxide ($H_2O_2$). The effect of contact with air at different RAH (11 %, 43 % and 75 %) was then assessed, under both aerobic and anaerobic conditions, in order to distinguish the specific effects of dehydration and to identify the mechanisms involved. Additionally, the contribution of the composition of the suspension medium to the survival of vegetative cells was examined. Any damage caused by these stress conditions, particularly to the plasma membrane, was analysed using fluorescence microscopy and flow cytometry.

## 2. Material and methods

### 2.1. Bacterial strain and growth conditions

*C. perfringens* strain 17SBCL19, classified as toxigenotype F and C-cpe, was used in this study. It was isolated during a foodborne outbreak in France in 2017 and kindly provided by ANSES (Maisons-Alfort, France). *C. perfringens* was cultured on Columbia agar supplemented with 5 % sheep blood (COS, Thermo Fisher Scientific, reference PB5039A, Ilkirch, France) for 24 h at 37 °C using Gaspak EZ Anaerobic $H_2CO_3$ (Grosseron, reference 260678, Couëron, France) in anaerobic jars (Merck, France). Pre-culture was carried out by adding one colony of this strain to 10 ml of thioglycollate broth with resazurin (Biokar, reference BK017HA, France), followed by incubation for 24 h at 37 °C under anaerobic conditions. Culture was then initiated by adding 100 µl of the pre-culture to 9.9 ml of thioglycollate broth with resazurin, followed by incubation for 6 h at 37 °C under anaerobic conditions. After incubation, the vegetative cells were at the end of exponential growth.

### 2.2. Production of bacterial spores

A fresh colony of *C. perfringens* was added to 10 ml of Tryptone-Peptone-Glucose (TPG) medium (protocol provided by ANSES, Maisons-Alfort; tryptone 50 g/l [Thermo Fisher Scientific, reference LP0042B], peptone 5 g/l [Thermo Fisher Scientific, reference LP0037B], glucose 4 g/l [Sigma-Aldrich, reference G7021, Saint-Quentin Fallavier, France], sodium thioglycollate 1 g/l [Sigma-Aldrich, reference T0632], dilution in PBS [Phosphate Buffer Saline, Sigma-Aldrich, reference P4417], pH 7.5). The bacterial suspension was incubated for 24 h at 37 °C under anaerobic conditions. 100 µl of this pre-culture was added to 9.9 ml of TPG and incubated for 6 h at 37 °C under anaerobic conditions. 300 µl of the culture was then removed and placed in a 50 ml Falcon tube containing 30 ml of TPG sporulation medium (TPG medium, 1,1 ml/l $MnSO_4$ at 10 g/l [Manganese (II) sulfate monohydrate, Sigma-Aldrich, reference M7634], 1 ml/l $CaCl_2$ at 10 g/l [Calcium chloride anhydrous, Merck, reference 2388.1000, Germany], pH 7.5) and a bile salt, taurocholate (sodium taurocholate hydrate, Sigma-Aldrich,
reference 86339-1G), at a final concentration of 500 µM. This bacterial suspension was incubated for 3 days at 37 °C under anaerobic conditions. The presence of spores at the end of the incubation period was checked by phase-contrast microscopy (Primo Star, Zeiss, Germany).

The spore suspension was purified by three centrifugations (15,000×g, 10 min at 4 °C) and three successive washes with sterile distilled water. The spores were then concentrated 10 times and stored in sterile distilled water at 4 °C for a maximum of two weeks before use.

## 2.3. Dehydration and rehydration processes

### 2.3.1. Presentation of the dehydration system

Dehydration of the bacterial suspensions was carried out in hermetically sealed plastic boxes (28 × 22 × 8 cm) containing saturated saline solutions at a controlled water activity (a<sub>w</sub>) value, which allowed the RAH in the box to be regulated (Lemetais et al., 2012). The a<sub>w</sub> of the saline solution was adjusted according to the desired RAH in the box, while the a<sub>w</sub> of the sample was equilibrated to the relative humidity of the gas phase. The saline solutions used were lithium chloride (LiCl, Alfa Aesar, reference 10515, USA), potassium carbonate (K<sub>2</sub>CO<sub>3</sub>, Prolabo, reference 26719.362, France) and sodium chloride (NaCl, Thermo Fisher Scientific, reference BP358-1, France) to achieve a<sub>w</sub> values of 0.11, 0.43 and 0.75 respectively. A a<sub>w</sub> value of 1, corresponding to the liquid state, was also tested by adding sterile distilled water to the bottom of a sealed box. The water activity of the saturated salt solutions was checked using an a<sub>w</sub> meter (AQUA LAB Series 3 TE, Munich, Germany). The samples and a thermo-hygrometer (Otio, Villeurbanne, France), used to control the maintenance of the desired humidity, were placed on a grid above the saturated saline solution in each sealed box. The sealed boxes were kept at room temperature (around 24 °C) in the dark.

To determine the mechanical effect of dehydration on the persistence of C. perfringens, the experiment was also carried out under anaerobic conditions. Hermetically sealed boxes containing saturated saline solutions were prepared in the absence of oxygen in an anaerobic chamber (BACTRON600-2, Sheldon Manufacturing, Oregon, USA) containing a gas mixture of 5 % H<sub>2</sub>, 10 % CO<sub>2</sub>, 85 % N<sub>2</sub>.

### 2.3.2. Samples dehydration and rehydration

#### 2.3.2.1. Dehydration and rehydration of vegetative cell samples.
Vegetative cell suspensions were centrifuged (5000×g, 5 min at 20 °C) and then concentrated 10 times in PBS (nutrient-free medium) or thioglycollate broth (nutrient-rich medium). The cell concentration was determined by the colony forming unit (CFU) method and ranged from 8.5 x 10<sup>-8</sup>–10<sup>9</sup> CFU/ml. A 10 µl drop of this suspension was placed in a 35 × 10 mm Petri dish (Falcon, reference 351008, France), which was then placed on a grid at 11 %, 43 %, 75 % and 100 % RAH for a specified period of time. After drying, the cells were harvested by adding 500 µl of PBS to the dehydrated drop, followed by 20 aspirations and discharges with a pipette. The cell suspension was then collected in a 1.5 ml Eppendorf tube. This step was performed twice. The entire experiment was carried out aerobically.

For the part of the experiment carried out under anaerobic conditions, the media used were first degassed with CO<sub>2</sub>. The oxygen concentration in the media was measured before the start of the experiment using a specific sensor (Micro Fibre Optic Oxygen Transmitter, Microx 4 Trace, PreSens, Regensburg, Germany). Cell recovery was carried out in the anaerobic chamber.

#### 2.3.2.2. Dehydration and rehydration of spore samples.
The concentration of spores was determined by the CFU method before and after heat treatment (80 °C for 10 min) and was about 10<sup>8</sup> CFU/ml. This heat treatment ensured the complete inactivation of residual vegetative cells in the sample (Park and Rafii, 2018). As with vegetative cells, a 10 µl

drop of spores stored in water was placed in a 35 × 10 mm Petri dish, which was then placed at 11 %, 43 %, 75 % and 100 % RAH for a given period of time. Spore recovery was then carried out in the same way as for vegetative cells, except that the drop exposed to controlled RAH was resuspended in sterile distilled water instead of PBS. The whole experiment was carried out under aerobic conditions.

C. perfringens spores were also exposed to these RAH in degassed sterile distilled water under anaerobic conditions. The oxygen concentration was checked with the sensor mentioned in section 2.3.2.1. Spore recovery was carried out in the anaerobic chamber.

#### 2.3.2.3. Microscopic observation of the effect of RAH on samples.
At the end of each experiment, the rehydrated cell suspensions were centrifuged (5000×g, 5 min at 4 °C), concentrated at least 10 times in distilled water for the spores and in PBS for the vegetative cells. They were then observed by phase-contrast microscopy.

## 2.4. Oxidative treatment with hydrogen peroxide

### 2.4.1. Treatment of vegetative cells

The following procedure is based on the method described by Setlow and Setlow (1993). From a fresh culture of vegetative cells prepared under the above conditions, the bacterial suspension was centrifuged (5000×g, 5 min at 4 °C) and the supernatant removed. The pellet was resuspended in 100 µl PBS. This step was repeated twice. Decimal dilutions of this bacterial suspension were made and then plated on COS agar. Petri dishes were incubated for 24 h at 37 °C under anaerobic conditions. At the same time, 10 µl of the previous suspension was added to 1 ml of PBS containing 4M H<sub>2</sub>O<sub>2</sub> (30 % hydrogen peroxide solution, Sigma-Aldrich, reference 31642). After 2, 5 or 10 min of treatment at room temperature, 10 µl were taken and added to 1 ml of PBS containing 5000 U of catalase (bovine liver catalase, Sigma-Aldrich, reference C30) to stop the action of the H<sub>2</sub>O<sub>2</sub>. This amount of catalase is sufficient to degrade H<sub>2</sub>O<sub>2</sub> in less than 5 s (Setlow and Setlow, 1993). Treatments without H<sub>2</sub>O<sub>2</sub> and without catalase, and with catalase only, were used as controls.

### 2.4.2. Treatment of spores

The same protocol as for vegetative cells was used to treat C. perfringens spores with 4M H<sub>2</sub>O<sub>2</sub> for 5, 10, 20 or 30 min at room temperature. The initial spore concentration was estimated before and after heat treatment (80 °C for 10 min) using the CFU method.

## 2.5. Bacterial cultivability measurement

Cultivability was estimated using the CFU method. After recovery, vegetative cells were serially diluted in PBS, and 100 µL of appropriate dilutions were plated on COS agar. For spores, decimal dilutions were made on the bacterial suspension before and after heat treatment at 80 °C for 10 min and plated on COS agar. In both cases, colonies were counted after incubation for 24 h at 37 °C under anaerobic conditions. The results obtained were expressed as CFU/ml and related to the initial inoculum concentration to calculate the percentage cultivability. The method's detection threshold representing the minimum concentration of cultivable cells was 0.003 % for vegetative cells and 0.03 % for spores for section 2.3. For section 2.4, the detection limits were 0.1 % for vegetative cells and spores. There was no significant difference between before and after heat treatment meaning that germination had not occurred. Therefore, only the results after heat treatment are presented in this article.
## 2.6. Characterisation of inactivation mechanisms

### 2.6.1. Fluorescence microscopy

**2.6.1.1. Preparation of dehydrated and rehydrated samples.** After the vegetative cells had been exposed to different RAH during 24 h and resuspended in 1 ml of PBS, they were centrifuged at 5000×g for 5 min at 20 °C. The supernatant was removed and the pellet was resuspended in 100 µl of PBS. Samples were stained with 1 µl of propidium iodide (20 mM, Invitrogen, reference L34856B, California, USA), followed by a 5-min incubation in the dark at room temperature. Propidium iodide (PI) is a red-fluorescent nucleic acid dye that intercalates into DNA and is commonly used to identify dead cells due to its ability to penetrate cells with damaged plasma membranes (Stiefel et al., 2015).

**2.6.1.2. Preparation of dehydrated samples.** From a culture of *C. perfringens* vegetative cells maintained in PBS at a concentration of approximately 10⁸ CFU/ml, a 1 ml volume of cell suspension was transferred to an Eppendorf tube. A 1 µl volume of PI was then added (final concentration = 20 µM) and the mixture homogenised for a few seconds. A 10 µl drop was placed on a microscope slide and incubated for 24 h at 24 °C in the dark under three conditions of RAH: 11 %, 43 % or 75 %. 10 µl of the bacterial suspension stained with PI were also kept at 100 % RAH as a control. After incubation, a coverslip was placed on the sample for microscopic observation.

**2.6.1.3. Image acquisition.** Microscopic observations were performed using a Zeiss Axiolab 5, Bio-TL/FL, equipped with an Axiocam 208 color camera and a 100 × magnification objective. The excitation source was at 470 nm and the emission filter, corresponding to PI emission wavelength (Stiefel et al., 2015), was at 645/40 nm. Images were acquired using Zeiss Zen 3.9 software, within 15 min after the samples had been prepared. Bright-field and fluorescence images were recorded for each sample. For each condition, four images were analysed per sample. Bacterial enumeration was performed directly from the acquired images, with the total number of counted cells ranging from 100 to 300.

### 2.6.2. Flow cytometry

**2.6.2.1. Preparation of samples.** After the vegetative cells had been exposed to different RAH during 24 h and resuspended in 1 ml of PBS, they were centrifuged at 5000×g for 5 min at 20 °C. The supernatant was removed and the pellet resuspended in 10 ml of PBS to obtain a final concentration of approximately 10⁶ CFU/mL. A 1 ml volume of this cell suspension was taken and transferred to an Eppendorf tube. To assess cellular metabolic activity, the marker CFDA (5-(and-6)-Carboxyfluorescein Diacetate, Invitrogen, reference C195) was added at a final concentration of 5 µM, then the tube was incubated at 37 °C for 15 min in the dark. PI was then added at a final concentration of 2 µM, followed by incubation for 5 min at room temperature, also in the dark.

**2.6.2.2. Acquisition.** Flow cytometry analyses were performed using an Attune NxT cytometer (Thermo Fisher Scientific). Both CFDA and PI were excited using a 488 nm laser. The PI signal was detected using a 695/40 nm band-pass filter, while the CFDA signal was detected using a 530/30 nm band-pass filter. For each sample, 20,000 events were recorded and analysed using the Attune™ Cytometric software (version 5.3.0). Doublets and cellular aggregates were excluded from the analysis based on FSC and SSC signal characteristics. Viable cells in late exponential phase and vegetative cells treated with 70 % ethanol for 10 min were stained with PI and CFDA, and used as controls.

## 2.7. Statistical analysis

All experiments were performed in three to nine independent

biological replicates, with data presented as mean ± standard deviation. Statistical analyses were performed using R studio (version 2023.06.0). The normality of data distributions was assessed for each stress condition using the shapiro.test function (package: stats). When normality was confirmed ($p > 0.05$), homogeneity of variances was tested using the bartlett.test function (stats). If variances were homogeneous ($p > 0.05$), an ANOVA (anova, stats) followed by a Student-Newman-Keuls test (SNK.test, package: agricolae) was performed to identify significantly different groups. If normality was not met, a non-parametric Kruskal-Wallis test (kruskal.test, stats) was applied, followed by Dunn's post hoc test (dunn.test, package: dunn.test) without correction for multiple comparisons. For all tests, differences were considered statistically significant at $p < 0.05$.

## 3. Results

### 3.1. Effect of contact with oxygen in a liquid medium on *C. perfringens* survival

To assess the effect of contact with oxygen on *C. perfringens*, the survival of vegetative cells and spores was monitored over several days in a nutrient-free liquid medium (PBS) under both aerobic and anaerobic conditions, and compared with stress induced by an oxidising agent, hydrogen peroxide.

Vegetative cells were able to survive for more than 3 day at 100 % RAH in the presence of oxygen (Fig. 1A). However, the presence of oxygen has a significant effect on the survival of vegetative cells over time. Indeed, under aerobic conditions, cell survival reached the limit of detection (0.003 %) in less than 10 days, whereas under anaerobic conditions, their survival lasted for over 30 days (0.0034 %, Fig. 1B). The vegetative cells of *C. perfringens* are therefore capable of surviving for several days in contact with dissolved oxygen and in the absence of nutrients. Observations of the samples by phase-contrast microscopy showed no sporulation of the vegetative cells after storage in liquid medium at the various measurement times.

The results of the survival of *C. perfringens* vegetative cells exposed to H₂O₂ are shown in Fig. 1E. The addition of catalase to stop the effect of H₂O₂ did not affect the survival of *C. perfringens* vegetative cells (data not shown). H₂O₂ treatment had a rapid and significant effect on the survival of *C. perfringens* cells. After 2 min of exposure, cell inactivation reached the limit of detection (0.1 %). This demonstrated a high sensitivity of vegetative cells to H₂O₂-induced oxidation, in contrast to exposure to dissolved oxygen, where vegetative cells were able to survive for several days.

The results for spores are presented in Fig. 1C, D and 1F. The spores proved to be quite resistant to oxygen exposure unlike vegetative cells and were able to survive for several weeks (Fig. 1C), with a loss of around 89 % of the population after two months. Under anaerobic conditions (Fig. 1D), there were no significant differences in survival over time. According to these results, dissolved oxygen seems to have caused a loss of cultivability of *C. perfringens* spores over time.

As with vegetative cells, the addition of catalase did not affect the survival of *C. perfringens* spores (data not shown). The results of the treatments with H₂O₂ are shown in Fig. 1F. Spores not treated with H₂O₂ showed stable survival throughout the experiment, with no significant reduction in the number of viable spores. In contrast, treatment with H₂O₂ resulted in a rapid reduction in the number of spores. After 10 min of exposure, a reduction in survival of approximately 87 % was observed. After 20 min of treatment, the detection limit of 0.1 % was reached. No significant difference in inactivation was observed between the control condition and the spores treated for 5 min, suggesting a limited effect of H₂O₂ treatment over short periods. Like vegetative cells, *C. perfringens* spores were sensitive to the oxidising agent hydrogen peroxide, but were able to survive prolonged exposure to dissolved oxygen.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Survival of *C. perfringens* vegetative cells and spores in a nutrient-free liquid medium under aerobic, anaerobic, or oxidative stress conditions. A: Cultivability of vegetative cells maintained in PBS under aerobic conditions; B: Cultivability of vegetative cells maintained in PBS under anaerobic conditions; C: Cultivability of spores maintained in distilled water under aerobic conditions; D: Cultivability of spores maintained in distilled water under anaerobic conditions; E: Cultivability of vegetative cells exposed to hydrogen peroxide; F: Cultivability of spores exposed to hydrogen peroxide. Catalase was added at the end of each treatment to neutralize hydrogen peroxide. The control consisted of vegetative cells or spores not exposed to hydrogen peroxide but treated with catalase. The grey line symbolises the limit of detection. Letters (a, b) indicate treatments that are significantly different (p < 0.05).</figcaption></figure>

## 3.2. Effect of RAH levels on *C. perfringens* survival

In order to evaluate the effect of contact with air, and to assess the respective roles of the mechanical and oxidative effects of dehydration in the survival of *C. perfringens*, vegetative cells and spores were exposed to various RAH under aerobic and anaerobic conditions in a nutrient-free liquid medium (PBS for vegetative cells and distilled water for spores) for a few days to a few weeks.

The detection limit was reached on the first day of dehydration of vegetative cells in PBS at 11 % and 43 % RAH, both under aerobic and anaerobic conditions, which means that the survival rate was less than 0.003 % (Fig. 2A and B). At 75 % RAH in aerobic conditions, the survival of vegetative cells was very low from the first day (less than 0.01 %), and the detection limit was reached at the next measurement point. A rapid decline in survival was also observed at 75 % RAH in anaerobic conditions, but less intensely than in aerobic conditions, with a mortality rate of more than 99.99 % at day 9. It should be noted that the dehydrated and rehydrated samples observed by phase-contrast microscopy did not show any initiation of the sporulation process of the vegetative cells. Comparing with results described in section 3.1, vegetative cells in a

liquid medium survived significantly longer than dehydrated cells, both under aerobic and anaerobic conditions.

These results showed that *C. perfringens* vegetative cells are strongly affected by dehydration in a nutrient-free medium. At 11 % and 43 % RAH, the mechanical effect of dehydration was responsible for cell death. At 75 % RAH, it appears that dehydration mainly exerts a mechanical effect on the survival of vegetative cells, with a small contribution of oxidation in this process.

*C. perfringens* spores were exposed to different RAH for several weeks in distilled water. The results are presented in Fig. 2C and D. In contrast to vegetative cells, spores were more resistant to changes in RAH, with a survival rate of over 10 % and 3 % after two months of stress in presence and absence of oxygen respectively. Spores exposed to a 43 % RAH in aerobic conditions had a higher survival rate (43 %) after 57 days than those maintained at 75 % or 100 % RAH (around 11 % survival). However, survival at 11 % RAH did not differ significantly from that observed at the other humidity levels at the end of the experiment (57 days). Thus, the spores proved to be quite resistant to dehydration and were able to survive for several weeks under varying RAH conditions. In anaerobic conditions (Fig. 2D), there were no significant differences
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Survival of *C. perfringens* vegetative cells and spores in a nutrient-free liquid medium equilibrated at different RAH. A: Cultivability of vegetative cells in PBS equilibrated at different RAH under aerobic conditions; B: Cultivability of vegetative cells in PBS equilibrated at different RAH under anaerobic conditions; C: Cultivability of spores in distilled water equilibrated at different RAH under aerobic conditions; D: Cultivability of spores in distilled water equilibrated at different RAH under anaerobic conditions. The data at 100 % RAH are the same as those presented in Fig. 1A, B, 1C and 1D. The grey line symbolises the limit of detection. Letters (a, b, c, d, e) indicate treatments that are significantly different (p < 0.05).</figcaption></figure>

over time for a given humidity. However, there was a significant difference in survival between 11 % and 43 % RAH. From 21 days of stress, 11 % RAH was significantly different from the 100 % RAH until the end of the experiment, suggesting a lethal effect on the spores during intense dehydration under anaerobic conditions. The results didn't clearly demonstrate a potential effect of dehydration level on the survival of *C. perfringens* spores, except at 11 % RAH under anaerobic conditions, where a mechanical or a structural effect could be involved.

### 3.3. Contribution of nutrient-rich medium composition to the survival of vegetative cells in contact with air

In order to assess the effect of medium composition on resistance to dehydration, vegetative cells of *C. perfringens* were placed in their growth medium (thioglycollate broth) at different RAH for several days (Fig. 3). The limit of detection was reached on the first day of stress for cells maintained at 43 % RAH in presence and absence of oxygen. At 11 % RAH, survival was slightly prolonged compared to the nutrient-free

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Survival of *C. perfringens* vegetative cells in a nutrient-rich medium equilibrated at different RAH. A: Cultivability of vegetative cells equilibrated at different RAH under aerobic conditions; B: Cultivability of vegetative cells equilibrated at different RAH under anaerobic conditions. The grey line symbolises the limit of detection. Letters (a, b, c, d) indicate treatments that are significantly different (p < 0.05).</figcaption></figure>

medium (Fig. 2B) in absence of oxygen. At 75 % RAH, a rapid decrease in survival was observed, whereas at 100 % RAH, vegetative cells showed better survival over a longer period. No significant difference in survival was observed at 75 % RAH between nutrient-rich and nutrient-free conditions, regardless of oxygen presence (statistical data not shown). As with the nutrient-free medium (Fig. 2A and B), vegetative cells survived longer at 100 % RAH under anaerobic conditions. Thus, there was no significant protective effect of the nutrient-rich environment on the survival of vegetative cells in contact with air, even though it contained antioxidants (L-cystine and sodium thioglycolate).

### 3.4. Understanding the mechanisms involved in the inactivation of vegetative cells

As evaluation of survival to dehydration by the UFC method necessarily implies both desiccation and rehydration, the integrity of cells through a permeabilization test was assessed after dehydration to different RAH for 24 h and after subsequent rehydration.

The effect of dehydration alone was evaluated on *C. perfringens* vegetative cells. Vegetative cells were stained with PI, dehydrated for 1 day at different RAH, and then observed by fluorescence microscopy. The results, presented in Fig. 4, showed significant differences between the dehydration conditions at 11 % and 43 % RAH, as well as between 11 % and 75 % RAH. It was observed that nearly all vegetative cells were stained with PI following dehydration at 43 % RAH, whereas approximately 52 % were stained at 75 % RAH and only about 7 % at 11 % RAH. In addition, no significant differences were observed between the 100 % RAH control condition and the dehydrated samples at 11 % and 75 % RAH. Approximately 14 % of vegetative cells were stained with PI at 100 % RAH, indicating membrane permeabilization in a fraction of the population, which could be related to the loss of cultivability observed in Fig. 1A. Vegetative cells appeared smaller under 11 % RAH compared to 43 % and 75 % RAH, indicating potential cell shrinkage associated with dehydration. The observed background fluorescence likely results from an increase in probe concentration due to water removal during dehydration.

Fluorescence microscopy results for samples that have undergone a dehydration-rehydration cycle are presented in Fig. 5. Vegetative cells dehydrated at 11 % and 43 % RAH during 24 h and subsequently
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Effect of dehydration at different RAH on the permeabilization of the plasma membrane of *C. perfringens* vegetative cells. A: Percentage of PI-positive vegetative cells calculated from four fluorescence microscopy images. Letters (a, b, c) indicate treatments that are significantly different (p < 0.05). B: Light (1) and fluorescence (2) microscopy images of vegetative cells dehydrated at different RAH, stained with PI.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Effect of dehydration at different RAH and rehydration on the permeabilization of the plasma membrane of *C. perfringens* vegetative cells. A: Percentage of PI-positive vegetative cells calculated from four fluorescence microscopy images. Letters (a, b) indicate treatments that are significantly different (p < 0.05). B: Light (1) and fluorescence (2) microscopy images of vegetative cells dehydrated at different RAH and rehydrated, stained with PI.</figcaption></figure>

rehydrated with PBS were all stained with PI, indicating compromised membrane integrity. At 75 % RAH, nearly 93 % of cells were PI-positive. In contrast, only a few cells showed PI staining at 100 % RAH. These

findings indicate that dehydration during 24 h followed by rapid rehydration damages the plasma membrane of vegetative cells, leading to cell death. These observations were consistent with the CFU results.
Thus, the results presented in Figs. 4 and 5 would suggest a role of rehydration in plasma membrane permeabilization. At 43 % RAH, membrane permeabilization would appear to result mainly from dehydration and/or maintenance for 24 h in this condition, whereas at 75 % RAH both dehydration (and maintaining this humidity) and rehydration would contribute equally. Likewise, at 11 % RAH, membrane permeabilization would be induced by both the dehydration process (and its maintenance for 24 h) and subsequent rehydration.

As microscope investigation cannot be applied to a huge number of cells, flow cytometry with double staining was used to assess the permeabilization of *C. perfringens* suspensions after a dehydration-rehydration cycle. Flow cytometry results (Fig. 6) also confirmed plasma membrane permeabilization following dehydration and subsequent rehydration of vegetative cells. Indeed, more than 94 % of the cells were PI-positive and CFDA-negative when dehydrated, indicating loss of viability. Under humid conditions (100 % RAH), 85 % of the vegetative cells were stained with CFDA, confirming their viability. These observations confirm the results previously obtained by CFU counts and fluorescence microscopy. Furthermore, the very low proportion of cells stained with both CFDA and PI indicated an absence of an intermediate physiological state where residual enzymatic activity would have persisted despite membrane damage. This suggested that cells had undergone severe stress, likely leading directly to irreversible damage or death.

## 4. Discussion

This study aimed to investigate the influence of dehydration at different levels of RAH on the persistence of *C. perfringens*, considering

its two main physiological forms: vegetative cells and spores. Our results revealed different survival rates depending on the bacterium's physiological state. In the literature, two main causes have been reported regarding the mechanisms by which dehydration may affect bacterial survival. First, a mechanical effect related to osmosis and cell shrinkage could compromise cellular integrity due to deformation and aggregation of membranes (Dupont et al., 2014; Zoz et al., 2021), lead to a concentration of intracellular solutes, and a net decrease in the hydration of vital molecular assemblies (Bremer and Krämer, 2019; Greffe and Michiels, 2020; Potts, 1994). Second, an oxidative effect, linked to enzyme dysfunction and increased contact with atmospheric oxygen (Dupont et al., 2014), could exacerbate cellular damage – particularly in an anaerobic bacterium such as *C. perfringens*. The experiments conducted in this study were designed to address these two causes. The effect of reactive oxygen species (ROS) generated by chemical agents such as hydrogen peroxide was also investigated to further explore the oxidative sensitivity of *C. perfringens*.

### 4.1. Mechanical stress induced by RAH changes and its effect on *C. perfringens* persistence

Vegetative cells of *C. perfringens* proved to be extremely sensitive to dehydration. After just one day of exposure to 11 % or 43 % RAH, cell survival fell below the detection limit (<0.003 %). At 75 % RAH, a small fraction of the population (~0.007 %) remained viable for one day, while at 100 % RAH, vegetative cells persisted for several days (about 1.6 % on day 3) (Fig. 2A), highlighting the protective effect of high humidity. No evidence of sporulation was observed under any of the tested conditions, suggesting that this survival strategy was not

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Flow cytometry analysis of *C. perfringens* vegetative cells dehydrated at different RAH and rehydrated, stained with PI and CFDA. Q1: PI+/CFDA-; Q2: PI+/CFDA+; Q3: PI-/CFDA-; Q4: PI-/CFDA+. A: Percentage of stained cells in the different regions. Calculated from three biological replicates; B: Representative flow cytometry plots for the three biological replicates of vegetative cells exposed to different RAH.</figcaption></figure>
triggered by dehydration.

Flow cytometry and fluorescence microscopy confirmed plasma membrane permeabilization following dehydration during 24 h and rehydration (Figs. 4–6). PI staining revealed compromised membranes, while the absence of PI+/CFDA + double staining in flow cytometry results suggested the membrane damage induced by dehydration and rehydration was irreversible and rapidly led to cell death. At 11 % RAH, PI did not stain all vegetative cells of *C. perfringens* (Fig. 4). It is possible that a portion of the cells retained membranes sufficiently intact to prevent PI entry. Moreover, the formation of a glassy intracellular state under very low humidity could stabilize cellular structures and limit membrane damage. Indeed, it has been suggested that at this level of RAH, cell wall stiffening could help prevent rupture of the plasma membrane (Zoz, 2016). Additionally, the entry or diffusion of PI into the cell may be restricted by this glassy intracellular condition. Indeed, the reduced water availability could hinder PI diffusion and alter its interaction with cellular components, particularly nucleic acids, thereby affecting PI binding and fluorescence. Thus, during rehydration, water may facilitate the entry of PI through the membrane permeabilized by dehydration, enabling its binding to DNA. Figs. 5 and 6 appear to support this hypothesis, showing detectable fluorescence when cells are dehydrated at 11 % RAH for 24 h, then rehydrated and stained with PI. Otherwise, at 11 % RAH the size of the vegetative cells appeared smaller compared to other humidities (Fig. 4B), highlighting cell shrinkage linked to dehydration. A reduction in cell volume of around 80 % had already been observed in *Listeria monocytogenes* at this RAH (Zoz, 2016). These results confirm that decreasing air humidity leads to cell inactivation: the more severe the dehydration stress, the greater the mechanical damage.

In addition to the dehydrating effect on vegetative cell inactivation, the rehydration phase also plays a critical role in cell mortality, in particular by the speed at which it is carried out (Lang et al., 2016; Zoz et al., 2016). Dehydration during 24 h and subsequent rehydration were shown to disrupt the plasma membrane of vegetative cells, as demonstrated by the microscopy results (Figs. 4 and 5). The contribution of each step may vary with RAH: dehydration – and its maintenance for 24 h – could be the main contributor at 43 % RAH, dehydration and/or rehydration might play a role at 11 % RAH, and both phases appear to contribute equally at 75 % RAH. Intermediate relative humidities are of particular interest, as a previous study reported high mortality in *L. monocytogenes* cells maintained at 43 % and 68 % RAH during a short exposure time (90 min) and subsequently rehydrated with PBS (Zoz et al., 2016). High-amplitude humidity fluctuations can induce substantial cell volume changes, exerting mechanical stress on the plasma membrane. This stress promotes membrane deformation and internalization, leading to vesicle formation. Upon rehydration, the reduced membrane surface area compromises cellular integrity, resulting in membrane permeabilization (Dupont et al., 2010). Moreover, instantaneous rehydration - such as the one carried out during our study - would lead to significant rupture of the plasma membrane due to the rapid influx of water into dehydrated cells, whereas progressive rehydration could favour better cell survival as shown in a previous study (Zoz et al., 2016). These results confirm the potential of air humidity fluctuations to maintain surface hygiene (Zoz et al., 2021).

In contrast, spores demonstrated high resistance to dehydration and maintained viability over time, with a slightly higher survival rate at 43 % RAH (43 %) compared with 75 % RAH (11 %) and 100 % RAH (12 %) after two months under aerobic conditions (Fig. 2C). This could suggest a possible protective role of moderate dehydration. However, this pattern was not observed at 11 % RAH, making it difficult to establish a clear effect of dehydration. In the study of Murrell and Scott (1966), spore resistance was found to be highest at water activity levels between 0.2 and 0.4 when faced with heat treatment. This hydration range is thought to stabilize the internal structure of the spore. Conversely, under very low water activity conditions, thermal resistance decreased, likely due to dehydration-induced impairment of stabilizing molecules, such

as proteins. When water activity is higher (>0.4), spore resistance decreases, potentially due to increased molecular mobility and facilitation of heat-induced denaturation processes (Murrell and Scott, 1966). Dehydration and rehydration have been shown to alter spore dimensions, with dehydration causing shrinkage, followed by a biphasic rehydration process involving a rapid swelling due to water diffusion into the coat and cortex, and a slower swelling associated with water diffusion into the spore core (Westphal et al., 2003). The loss of water during dehydration appeared to affect the spore's DNA, leading to structural damage (Fairhead et al., 1994). Indeed, under severe dehydration, structural damage has been involved in spore death. For instance, Munakata et al. (1997) demonstrated that extreme dehydration under high vacuum (<1 Pa, ~0 % RAH) induced DNA double-strand breaks and DNA-protein crosslinks. They hypothesized that the removal of water molecules, which normally surround and stabilize DNA, leads to destabilization and damage, potentially lethal if repair pathways are compromised (Munakata et al., 1997).

The difference in sensitivity to the mechanical effect of dehydration in air between *C. perfringens* spores and vegetative cells can be explained by the distinct structures of these two physiological forms. Spores, which have a multi-layered protective envelope, are intrinsically resistant to environmental stresses (Setlow, 2006). Vegetative cells, on the other hand, lack this protection and are more vulnerable to the deleterious effects of dehydration.

The resistance of *C. perfringens* spores to dehydration in our results is consistent with the observations from a previous study, which reported prolonged persistence of these spores in both dry and wet environments over a two-week period (Stine et al., 2005). Compared to spores of other sporulating bacteria including aerobic species, *C. perfringens* spores appeared more sensitive to dehydration. Indeed, a study showed that the viability of *Bacillus stearothermophilus* spores remained unchanged after one year of storage at different RAH (0 %, 20 %, 44 % and 66 %), whereas those of *Bacillus subtilis* showed a reduction of only 0.5 log after 1 year at room temperature and 0 % RAH (Reich and Morien, 1982). The structure and composition of the spore could explain the observed differences in persistence following dehydration between these species. The α/β-type small acid-soluble spore proteins (SASP), which bind to DNA, have been identified in *B. subtilis* as key factors for spore resistance to desiccation (Fairhead et al., 1994). One hypothesis could be that the SASPs in *C. perfringens* may be less effective in protecting DNA from dehydration-induced damage. This may be explained by the fact that *C. perfringens* possesses fewer ssp genes encoding SASPs compared to *B. subtilis* (Raju et al., 2006).

## 4.2. Effect of oxygen exposure induced by RAH changes on the persistence of C. perfringens

The results of this study provided insights into the effects of oxygen exposure during dehydration on vegetative cells and spores of *C. perfringens*. The focus on oxygen contact was motivated by the anaerobic nature of the bacterium. Under humid conditions (100 % RAH), vegetative cells demonstrated the capacity to survive for at least three days (1.6 %) in the presence of oxygen at 24 °C (Fig. 1A), and for at least 30 days in absence of oxygen (0.0034 %) (Fig. 1B). These results, observed in the absence of osmotic stress, suggest a long-term oxidative effect on vegetative cells, while short-term survival appears feasible regardless of oxygen exposure. Notably, at least 80 % of vegetative cells lose cultivability within the first day (Fig. 1A et 1B), regardless of oxygen availability, suggesting that nutrient deprivation could be a key contributor to cell death. During dehydration, no significant oxygen effect was observed on vegetative cells, since all cells were inactivated at 11 % and 43 % RAH. Although oxidative stress may contribute, the mechanical effects of dehydration appear to dominate. At 75 % RAH, the observed inactivation appeared to result from a combination of mechanical and oxidative stresses (Fig. 2A and B). Furthermore, the composition of the stress medium did not influence vegetative cell
survival during air exposure, even when antioxidant-rich nutrient medium was used, indicating that mechanical stress prevails (Fig. 3). Overall, *C. perfringens* can be positioned as an anaerobic bacterium displaying some level of oxygen tolerance, but it is clearly unable to survive like an aerobic organism.

In contrast to vegetative cells, spores were not affected by oxygen to the same extent. At 100 % RAH, there was a gradual inactivation of spores over time without any detectable germination under aerobic conditions (Fig. 1C), but these results were not significantly different from those obtained in anaerobic conditions (Fig. 1D, statistical data not shown), suggesting that oxygen had no role to play. There was no difference in inactivation during dehydration in the presence or absence of oxygen, except at 11 % RAH after two months of stress. Indeed, under anaerobic conditions, a mechanical effect of dehydration was observed at this RAH, with ~4 % survival (Fig. 2D). However, this effect was not observed in the presence of oxygen (35 % survival, Fig. 2C), making it impossible to conclude on a coupled effect of the mechanical effect and the oxidative effect of dehydration. The effect observed at 11 % RAH under anaerobic conditions remains difficult to interpret. A major difference between the aerobic and anaerobic experiments is the CO2 concentration, which is substantially higher in the anaerobic chamber (10 % compared to 0.04 % in air (Iz, 2022)). This high CO2 level may have caused acidification of the medium. Notably, this destructive effect on spores under anaerobic conditions is observed only at 11 % RAH. This is the condition in which spores are most dehydrated and in a solid state, directly exposed to the gaseous environment. At higher humidities, no significant differences were observed between aerobic and anaerobic conditions at the 57-day measurement point and under these humidity conditions, the spores were not as dehydrated as they were at 11 % RAH. It has been shown that acidification had a detrimental effect on spore preservation over three months, in contrast to a higher pH (Li and McClane, 2006b), although these results were observed in a liquid medium.

### 4.3. Effect of oxidative stress induced by hydrogen peroxide on the survival of C. perfringens

The effect of oxidative stress was further investigated through treatments involving H₂O₂, a widely used cleaning and disinfection agent known to induce the production of ROS. The form and reactivity of oxygen greatly influence its impact on microbial cells. Contact with dissolved oxygen, typically present in aqueous environments, involves molecular oxygen, which is relatively non-reactive under normal conditions. Although it can cause some oxidative stress, its effects are generally limited by the bacterial antioxidant defences. In contrast, H₂O₂ is a far more potent oxidising agent, notably because it can generate highly reactive species, for example hydroxyl radicals, through reactions such as the Fenton reaction (Winterbourn, 1995). These ROS are extremely reactive, and can cause extensive and irreversible damage to essential cellular components like DNA, proteins, and lipids (Imlay, 2003; Mishra and Imlay, 2012), leading to rapid inactivation.

This mechanistic difference explains why exposure to H₂O₂ is significantly more lethal to *C. perfringens* than simple oxygen exposure. In our experiments, vegetative cells were extremely sensitive to oxidative stress, as evidenced by their complete inactivation following a short treatment with 15 % H₂O₂ (Fig. 1E). This is consistent with previous studies. Indeed, it was shown that the survival rate of vegetative cells from a toxinotype F strain was 25.3 % after exposure to a 5 % H₂O₂ solution for 10 min (Hu et al., 2021). In the presence of a 10 % H₂O₂ solution during 10 min, the survival of vegetative cells of this bacterium decreased to 1.8 % (Charlebois et al., 2017). These results suggest that *C. perfringens* vegetative cells can tolerate low concentrations of H₂O₂ for short durations, but their resistance rapidly decreases at higher concentrations or longer exposures.

This high sensitivity may be attributed to the limited antioxidant defence systems in *C. perfringens*. Unlike aerobic or aerotolerant

bacteria, this anaerobe lacks efficient enzymatic systems such as catalase (Boyanova et al., 2024). However, some protective mechanisms have been identified for example: (i) rubrerythrine, which plays a key role in detoxifying hydrogen peroxide (Geissmann et al., 1999; Morvan et al., 2021); and (ii) superoxide dismutase, which catalyses the dismutation of superoxide anions into hydrogen peroxide and molecular oxygen (Geissmann et al., 1999; Hu et al., 2021; Jean et al., 2004; Lu and Imlay, 2021). Despite their presence, these systems appear insufficient to prevent oxidative damage in our tested conditions.

Spores of *C. perfringens* displayed greater resistance than vegetative cells, but they were not immune to this oxidative stress. Short treatments (<10 min) with 15 % H₂O₂ had limited effects on spore survival (>10 %). However, prolonged exposure (20 min) resulted in a significant 99.9 % reduction in spores counts (Fig. 1F). This positions *C. perfringens* spores as moderately resistant to this stress. Indeed, *C. perfringens* spores appear to be more sensitive to H₂O₂ than *B. subtilis* spores, which require a 50-min exposure to 15 % H₂O₂ to achieve only a 90 % reduction in viability (Setlow and Setlow, 1993). In contrast, *Clostridioides difficile* spores are fully inactivated following 20 min of exposure to a 10 % H₂O₂ solution (Edwards et al., 2016), suggesting greater sensitivity to oxidative treatment. These differences in resistance profiles among spore-forming bacteria likely stem from structural and biochemical differences in their spore composition and internal detoxification systems. *C. perfringens*, being more aerotolerant than *C. difficile*, may possess a more effective arsenal of oxidative defences at the spore level, though still inferior to that of aerobic species like *B. subtilis*. Taken together, these observations position *C. perfringens* as exhibiting an intermediate level of resistance to hyperoxidative disinfectants, characterized by high susceptibility in its vegetative state and moderate resistance in its spore form.

While our study used liquid hydrogen peroxide primarily to assess the role of oxidative stress in *C. perfringens* inactivation, industrial applications must consider the corrosive effects of this disinfectant on metal surfaces and equipment. Interestingly, a study showed that vaporized hydrogen peroxide, applied in an industrial sterilization process, effectively inactivated spores of bacteria such as *Geobacillus stearothermophilus* and *Bacillus atrophaeus* (McEvoy et al., 2023). This process could help minimize corrosion of metal surfaces, and it might be relevant to evaluate its efficacy on *C. perfringens* spores to determine whether it is comparable to liquid hydrogen peroxide.

## 5. Conclusion

This study provides new insights into the impact of air exposure on the survival of *C. perfringens*, with a particular focus on the mechanical and oxidative stresses associated with dehydration at varying levels of RAH. The results demonstrated that vegetative cells of *C. perfringens* are highly sensitive to dehydration, regardless of oxygen presence, indicating that the mechanical stress associated with water loss is the primary cause of inactivation at low RAH (11 % and 43 %), while an additional oxidative effect may contribute at 75 % RAH. Rapid rehydration also played a key role in membrane permeabilization likely due to abrupt cell swelling following dehydration-induced shrinkage. Flow cytometry and fluorescence microscopy confirmed that membrane integrity was compromised under dehydration and rehydration, correlating with a marked loss in cultivability. Despite being an anaerobic bacterium, *C. perfringens* was able to survive for several days under aerobic conditions at high RAH, indicating that oxygen exposure alone does not prevent its persistence. This contrasts with extremely oxygen sensitive (EOS) bacteria and highlights a potential risk of contamination on surfaces in slaughterhouses, even in the presence of oxygen. Spores, in contrast, remained highly resistant to dehydration, with only limited cultivability loss over extended periods. The lack of significant RAH effects on spore inactivation underscores their remarkable resilience and the difficulty of achieving complete destruction.

These results indicate that controlled dehydration-rehydration
cycles can inactivate *C. perfringens* vegetative cells and thus represent a potential avenue for developing novel decontamination strategies. In practice, however, slaughterhouses cannot maintain the very low humidity levels (<20 % RAH) that produced the strongest inactivation in the laboratory, and spores—and, to a lesser extent, vegetative cells—remain viable at the higher humidities typically encountered in industry. Although published data on RAH values in slaughter facilities are lacking, air temperatures of 20–25 °C have been reported in pig slaughter areas (Álvarez-Pérez et al., 2018), matching the one used in our study (24 °C); under similarly humid conditions (~100 % RAH), vegetative cells could survive a few days, whereas even moderate drying (~75 % RAH) should markedly reduce their persistence on working surfaces. By contrast, spores are likely to withstand a wide range of RAH and therefore constitute the principal long-term contamination risk. This underscores the need for tailored sanitation measures. Chemical agents such as hydrogen peroxide, which showed effective inactivation, particularly on vegetative cells, justify their use in industrial sanitation protocols.

## CRediT authorship contribution statement

**Léa Savard:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. **Sylvie Moundanga:** Methodology, Investigation. **Stéphane Guyot:** Writing – review & editing, Methodology, Conceptualization. **Narjes Mtimet:** Methodology. **Olivier Firmesse:** Resources, Methodology, Funding acquisition. **Sébastien Dupont:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Laurent Beney:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

## Funding

This work was supported by the French National Research Agency (ANR) bearing the following reference "ANR-21-CE21-0007".

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

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