Adaptive Tolerance of Listeria monocytogenes to Chemical Oxidants: Comparative Analysis of Transcriptomic Studies

ABSTRACT

Listeria monocytogenes is a foodborne pathogen that poses significant challenges to food safety and public health due to its ability to adapt to harsh environments, particularly those found in food processing facilities. This review explores the global transcriptional responses of L. monocytogenes to various chemical oxidants, including hydrogen peroxide, chlorine dioxide, ozone, and plasma‐activated water. By comparing the transcriptomic data of multiple studies, we identified the differentially expressed genes associated with key cellular processes, including oxidative stress responses, cell envelope biosynthesis, metabolic adaptation, efflux mechanisms, and virulence regulation. This review demonstrates that L. monocytogenes employs distinct gene expression patterns to resist disinfectant stress, primarily by upregulating efflux pumps, reactive oxygen species detoxification mechanisms, and DNA repair pathways as well as modulating central metabolism. Several disinfection treatments commonly affect the key genes related to peptidoglycan biosynthesis, cell envelope, cell division, glycolysis, oxidative stress response, and chemotaxis. Although oxidizing agents induce widely conserved gene expression patterns, other treatments trigger unique responses. However, interpretation of different study findings is restricted by methodological inconsistencies, including variations in treatment conditions, media, bacterial states, and transcriptomic techniques. These variations and nonuniform fold change thresholds for differentially expressed genes complicate the comparison of different studies. Therefore, standardized frameworks are necessary to elucidate the adaptive responses of L. monocytogenes and refine its disinfection methods in food processing.

1. Introduction

Listeria monocytogenes, a resilient foodborne pathogen commonly found in soil, water, vegetation, livestock, and excrement, can contaminate food across the supply chain (Rodriguez et al. 2021). Its ability to thrive under extreme acidic, alkaline, cold, and dry conditions makes its control challenging. This pathogen transitions from a saprophytic lifestyle at 20–25°C to a pathogenic state at 37°C by repressing flagellar gene expression (Pizarro‐Cerdá and Cossart 2019). It also forms biofilms and adheres to diverse surfaces, which contributes to its persistence in food and clinical settings (Bechtel and Gibbons 2021). Surveillance studies have reported its presence in dairy products, seafood, meat, and produce (Gérard et al. 2020; Y. Liu et al. 2020; Zakrzewski et al. 2024).

Food industry uses chemical antimicrobials, such as chlorine, ozone (O₃), and peracetic acid (Simpson and Mitch 2022), typically in combination with alkaline or acidic cleaning processes (Simpson and Mitch 2022) to prevent contamination. Additionally, physical methods, such as ultraviolet and high‐pressure processing methods, are used to decrease the microbial load (Sharma et al. 2022). However, bacteria develop tolerance to these measures. Antibiotic resistance remains a major public health concern, as multidrug‐resistant “superbugs” are emerging faster than new antibiotics can be developed (Fatima et al. 2023; Salam et al. 2023). Acquired tolerance is particularly concerning, as it enables previously susceptible bacteria to resist treatment via horizontal gene transfer and chromosomal gene mutations. These changes often reduce the antibiotic uptake by altering or downregulating the porin proteins (Mancuso et al. 2021).

Chemical oxidants differ from antibiotics in that they inflict widespread damage rather than targeting specific sites (Griffiths 2005). Resistance to chemical oxidants is better understood as increased tolerance. Exposure conditions are crucial for examining tolerance. L. monocytogenes count is reduced by 1000‐fold (3 log reduction) by 0.5 mg/L hypochlorous acid in 10 s in the absence of chlorine interference (Gongora et al. 2024). However, for vegetable wash water with 800 mg/L chemical oxygen demand (indicating oxidizable matter), a 2‐min contact with 50 mg/L hypochlorous acid is essential to achieve the same L. monocytogenes reduction (Van Haute et al. 2013). Comparatively, organic materials produce less potent chloramines that only cause sublethal stress, potentially inducing adaptive responses (Pfaller et al. 2021).

Sublethal exposure to oxidants affects antimicrobial resistance. In Pseudomonas spp., such exposure is associated with increased antibiotic resistance (Tong et al. 2021). Chlorine dioxide (ClO₂)‐treated wastewater shows a high prevalence of bacteria with tolerance genes (S.‐S. Liu et al. 2018), possibly due to enhanced horizontal gene transfer via natural transformation (Jin et al. 2020). Chlorination increases the membrane permeability and reactive oxygen species (ROS) levels and activates the salt overly sensitive (SOS) responses, facilitating the mutation and spread of tolerance genes (W. Zhao et al. 2025). Despite the widespread use of oxidants, L. monocytogenes frequently survives in food processing environments. Its resilience is due to its ability to colonize microniches and exhibit temporary adaptive tolerance via biofilm formation, decreased metabolism, and increased efflux pump activity (Tong et al. 2021; van Dijk and Verbrugh 2022). However, the specific molecular mechanisms underlying adaptive tolerance remain unclear. Recent transcriptomic studies have revealed the L. monocytogenes responses to oxidative stress at the gene level (Fernández‐Gómez et al. 2023; Kastbjerg et al. 2010; Siderakou et al. 2022).

Proper L. monocytogenes contamination control is crucial to prevent foodborne outbreaks and serious health consequences in the food industry. In healthcare, L. monocytogenes infects individuals with weak immune systems, including expectant mothers, necessitating the development of effective preventive strategies. This review discusses previous reports on the global transcriptional responses of L. monocytogenes to chemical oxidants to understand the molecular mechanisms underlying its adaptation and survival. Additionally, this review highlights the L. monocytogenes survival strategies under chemical oxidant stress to facilitate the development of suitable strategies to prevent Listeria persistence in various industries, including food processing.

2. Selection of Disinfection Treatments for Review

We conducted a thorough, albeit nonsystematic, literature search to identify the studies on L. monocytogenes transcriptomic responses to chemical oxidants. Our search spanned Google Scholar, PubMed, Scopus, and Web of Science using combinations of keywords, such as “Listeria monocytogenes,” “transcriptome,” “RNA‐seq,” “disinfectants,” “chemical oxidants,” “oxidative stress,” “hydrogen peroxide,” “ozone,” and “chlorine.” We carefully reviewed the titles, abstracts, and full‐texts of the articles to identify relevant studies on bacterial responses to disinfectants. We extracted gene expression data as reported by the original authors without reprocessing the raw data from the main text, supplementary materials, figures, and tables and consulted raw datasets, when available, to support our findings. Although no formal systematic review protocol was followed, our approach was designed to be as thorough and focused as possible within the scope of this review.

We selected disinfection treatments based on the availability of published transcriptome data for L. monocytogenes. This review focuses on the global transcriptional responses of L. monocytogenes to various disinfectants, particularly oxidizing agents such as hydrogen peroxide (H₂O₂), ClO₂, ozone (O₃), and plasma‐activated water (PAW). We also discuss additional treatments, including benzethonium chloride (BZT), lactic acid (LA), pulsed magnetic fields (PMF), and high‐pressure processing (HPP). While our primary focus is on oxidizing agents, we also provide a comparative analysis of nonoxidizing chemical disinfectants (e.g., BZT and LA) and physical treatments (e.g., PMF and HPP). This comparative perspective is crucial for distinguishing the transcriptional responses specific to oxidative stress from general stress responses triggered by various treatment types. Details regarding the strains, treatment conditions, and transcriptome analysis methods used in the reviewed studies are summarized in Table 1. Further discussion of the transcriptomic effects of BZT, LA, PMF, and HPP is included in the Supporting Information Data. By establishing a comparative baseline, this review aims to clarify the unique adaptive mechanisms that L. monocytogenes employs to survive oxidative stress, thereby enhancing our understanding of how this pathogen responds to different disinfection strategies.

TABLE 1.

Disinfection treatments used to compare the transcriptomic responses of Listeria monocytogenes in this review.

Disinfection treatments	Treatment conditions	L. mono. strains	Bacterial reduction	Transcriptome method	Total DEGs [fold change (FC)]	Reference
Benzethonium chloride (BZT)	L. monocytogenes was grown in the tryptic soy broth (TSB) in the presence of 4 ppm BZT at 14°C until the early stationary phase	6179, serotype 1/2a	0.16 Abs 610 nm (0.49 to 0.33)	Microarray	92 (FC ≥7.5)	Fox et al. (2011)
			Not recorded	RNA‐seq	600 (FC ≥ 4)	Casey et al. (2014)
Lactic acid (LA)	L. monocytogenes strains were exposed to 1% lactic acid (pH 3.4) in TSB at 20°C for 30 min		Not recorded	RNA‐seq	2280 (log2 FC > 1)	Cortes et al. (2020)
		R479a, serotype 1/2a	Not recorded	RNA‐seq	2151 (log2 FC >1)
Hydrogen peroxide (H2O2)	L. monocytogenes strains were exposed to 0.01% hydrogen peroxide in TSB at 20°C for 30 min	6179, serotype 1/2a	Not recorded	RNA‐seq	2280 (log2 FC > 1)	Cortes et al. (2020)
		R479a, serotype 1/2a	Not recorded	RNA‐seq	2151 (log2 FC >1)
Chlorine dioxide (ClO2)	L. monocytogenes in the brain heart infusion (BHI) broth was exposed to 300 mg/L aqueous ClO2 at 37°C and 230 rpm for 15 min		0.33 ± 0.37 log CFU/mL reduction	Microarray	340 (FC ≥ 1.5)	Pleitner et al. (2014)
Ozone (O3)	L. monocytogenes (250 µL; > 6.5 log CFU/g) was inoculated on the surface of tomato fruits and treated with O3 at 2 µg/g tomato fruits (moderate dose) at 23°C for 1 h	ATCC 19115, serotype 4b	1.075 ± 0.3 log reduction	RNA‐seq	283 (PCC ≥ 0.9999) a	Shu et al. (2021)
Plasma‐activated water (PAW)	Stationary‐phase L. monocytogenes cells were suspended in Ringer solution and treated for 5 min at room temperature with PAW generated using a plasma power of 36 W and an activation time of 30 min, and stored for 24 h	ULE1265, serotype 1/2c	2.2 ± 0.1 log10 CFU/mL reduction	RNA‐seq	399 (log2 FC > 1)	Fernández‐Gómez et al. (2023)
Pulsed magnetic field (PMF)	L. monocytogenes in phosphate‐buffered saline (PBS) was exposed to PMF at 8 T intensity with 20 pulses at room temperature	ATCC 19111, serotype 1/2a	9.6% (90.4% reduction)	RNA‐seq	588 (FC >2)	Qian et al. (2020)
High hydrostatic pressure processing (HPP)	L. monocytogenes in the tryptone soy yeast extract (TSYE) broth was subjected to HPP at 400 and 600 MPa and 15°C for 5 min	S2542, serotype 4b	> 3 log CFU/mL reduction	Microarray	NA b	Bowman et al. (2010)

^a PCC, Pearson's correlation coefficient.

^b NA, not available.

2.1. Differentially Expressed Gene Identification Under the Selected Disinfection Treatments

Differentially expressed genes (DEGs) were meticulously identified and extracted from the article text, tables, and supplementary figures of the respective published research articles on BZT (Fox et al. 2011; Casey et al. 2014), LA, H₂O₂ (Cortes et al. 2020), ClO₂ (Pleitner et al. 2014), O₃ (Shu et al. 2021), PAW (Fernández‐Gómez et al. 2023), PMF (Qian et al. 2020), and HPP (Bowman et al. 2008). Each gene upregulated or downregulated by the selected disinfection treatments was logged into a Microsoft Excel file (Microsoft Corporation, Redmond, WA, USA) and organized accordingly. Transcriptome data of the L. monocytogenes American Type Culture Collection (ATCC) 19115 strain under O₃ treatment were aligned and annotated against the reference genome of L. monocytogenes serovar 1/2a (strain ATCC BAA‐679/EGD‐e) using the Geneious Prime 2024.0.5 software (Kearse et al. 2012). Functional categories of each gene were determined using the UniProt Gene Ontology annotation tool with the prokaryotic slimming set (https://www.uniprot.org/), with L. monocytogenes strain ATCC BAA‐679/EGD‐e as the reference organism (Tables S1–S9; Figure 1). An online tool for Venn diagrams (https://www.interactivenn.net) (Heberle et al. 2015) was used to merge the gene lists for individual disinfection treatments and identify the genes affected by multiple disinfection treatments (Figure 2). Furthermore, a heat map was generated using Microsoft Excel to illustrate the effects of various disinfection methods on specific genes (Figure S1).

FIGURE 1.

Differentially expressed gene (DEG) distribution among different role categories. Total number of upregulated (↑) and downregulated (↓) genes in each role category is indicated. Each cell represents the number of Listeria monocytogenes genes differentially expressed in response to various disinfection treatments: benzethonium chloride (BZT), chlorine dioxide (ClO₂), hydrogen peroxide (H₂O₂), hydrostatic pressure processing (HPP), lactic acid (LA), ozone (O₃), plasma‐activated water (PAW), and pulsed magnetic field (PMF).

FIGURE 2.

Comparative transcriptome profiles of L. monocytogenes under various disinfection treatments. A Venn diagram illustrates the total number of DEGs in L. monocytogenes under various disinfection treatments. (A) Oxidizing agents (hydrogen peroxide [H₂O₂], chlorine dioxide [ClO₂], ozone [O₃], and plasma‐activated water [PAW]). (B) Other disinfectants (benzethonium chloride [BZT], lactic acid [LA], pulsed magnetic field [PMF], and hydrostatic pressure processing [HPP]).

3. Transcriptional Responses of L. monocytogenes to Various Disinfection Treatments

As mentioned above, the common bactericidal mechanism of most disinfection treatments involves the destruction of the cell membrane, resulting in the leakage of intracellular substances and DNA damage in L. monocytogenes. Additionally, disinfectants disrupt the intracellular ROS balance in L. monocytogenes by inhibiting the antioxidant enzyme activity, leading to bacterial inactivation (H. Li et al. 2021). However, their effects on the genes and pathways in L. monocytogenes vary substantially, depending on the specific disinfectant used and pathogen responses. Repeated disinfectant exposure in the food industry induces selective pressure, enhancing the tolerance to these compounds, triggering the development of antibiotic resistance, and increasing the fitness of L. monocytogenes (Bland et al. 2022). Genetic mechanisms underlying the adaptive tolerance to disinfectants involve changes in the expression levels of various regulatory genes and pathways necessary for stress adaptation and tolerance (Schulz et al. 2023). Therefore, a deeper understanding of the transcriptional responses of L. monocytogenes to various disinfectants is essential for the development of effective disinfection approaches.

3.1. Chlorine Dioxide (ClO₂)

ClO₂ is a powerful and rapid oxidizing agent. It is used in both gaseous and aqueous food systems to effectively reduce the counts of foodborne pathogens, including L. monocytogenes (Contreras‐Soto et al. 2021; Pleitner et al. 2014). This low‐molecular‐weight oxidative disinfectant targets almost every biomolecule, including nucleic acids, amino acids, peptides, proteins, and lipids (Fukuzaki 2006), and multiple cell wall regions, causing cytoplasmic membrane disruption and peroxidation, enzyme inhibition (Maillard and Pascoe 2024), bacterial genome destabilization, reduced energy production, protein synthesis disruption, and eventual cell death (Imlay 2003; Kohanski et al. 2010; Sazykin and Sazykina 2023).

Pleitner et al. (2014) conducted whole‐genome microarray analysis to characterize the transcriptional changes in L. monocytogenes 10403S when vegetative cells in the brain heart infusion broth were exposed to 300 mg/L ClO₂ for 15 min. Along with a 0.33 ± 0.37 log CFU/mL reduction in L. monocytogenes count, ClO₂ exposure resulted in the differential expression of 340 genes, of which 113 and 16 genes belonged to sigB and CtsR regulons, respectively, implicating both the general and protein‐specific stress response pathways. In total, levels of 223 genes related to carbohydrate and glycerol metabolism, protein quality control, hemostasis, phosphotransferase system (PTS), quorum sensing (QS), and several stress response genes (mainly oxidative stress response genes) were upregulated. In contrast, chemotaxis and flagellar gene levels were downregulated (Figure 1). Oxidative stress responses of L. monocytogenes to ClO₂ involve the increased expression levels of several redox‐associated genes, including superoxide dismutase A (sodA), organic hydroperoxide resistance (ohr)‐A, lmo2230, and global stress regulator (spxA and spxH) genes. These genes are crucial to maintain the redox balance and manage oxidative damage. Additionally, upregulation of the DNA repair (e.g., fri and deoC), protein quality control (clpB, clpC, clpP, and clpX), and heat shock repressor (ctsR) gene levels indicates that the bacteria actively repair the damaged proteins to reduce the cellular stress. Taken together, these findings suggest that L. monocytogenes combats ClO₂‐induced oxidative stress by enhancing the systems involved in redox homeostasis, DNA repair, and protein maintenance (Pleitner et al. 2014).

3.2. Hydrogen Peroxide

Oxidizing agents, reactants involved in redox reactions, gain electrons and oxidize other substances. H₂O₂ is an oxidizing agent generating hydroxyl radicals (•OH) through various processes, such as the Fenton reaction that involves H₂O₂ decomposition into oxygen and water, accompanied by considerable heat release (Andrés et al. 2022; Manso et al. 2020). H₂O₂ is generally recognized as safe (21 CFR 184.1366) for use as a food disinfectant, provided that any residual H₂O₂ is eliminated using suitable physical and chemical methods during food processing (Robinson and D'Amico 2021). H₂O₂ inactivates bacteria by generating radicals, causing oxidative damage to their cells, targeting the peptidoglycan wall and cell membrane, disrupting the nucleic acid structure, denaturing the proteins and lipids, and ultimately leading to cell death (Harter et al. 2017; Manso et al. 2020).

Along with evaluating the responses to LA, Cortes et al (2020) recorded the transcriptional responses of pathogens to oxidative stress by exposing L. monocytogenes strains 6179 and R479a to 0.01% H₂O₂ in tryptic soy broth (TSB) at 20°C for 30 min. Only a few genes (154 and 73 DEGs in 6179 and R479a, respectively) showed differential expression under H₂O₂ conditions compared to those showing differential expression under LA conditions. Levels of several genes related to the oxidative stress response and DNA repair were upregulated, whereas those of genes involved in cell wall and glycolytic processes were downregulated (Figure 1). At the molecular level, L. monocytogenes relies on conserved antioxidant defense mechanisms to mitigate H₂O₂‐induced oxidative damage. These include ohrA, ohrR, and lmo2230, which contribute to the detoxification of peroxides. Gene sodA converts superoxide radicals into H₂O_2, which is neutralized by catalases and peroxidases (Winterbourn 2020). Levels of several DNA repair genes (dinB, uvrA, uvrB, recA, exoA, addA, and addB) are upregulated in response to oxidative DNA damage. This repair activity is partially coordinated by the LexA‐regulated SOS response, in which DNA damage induces RecA‐mediated LexA autoproteolysis, thereby increasing SOS gene repression to promote DNA repair and cell survival (Cory et al. 2024). A low H₂O₂ concentration and interaction with TSB components may possibly suppress gene expression changes, as noted by Cortes et al. (2020). Nevertheless, upregulation of oxidative stress gene levels suggests a conserved bacterial strategy for redox detoxification and DNA damage repair.

3.3. Ozone (O₃)

O₃, an allotrope of oxygen, appears colorless to pale blue and is soluble in water (Chuwa et al. 2020). It is a potent oxidizing agent with a chlorine‐like odor, extensively used as a disinfectant in the food industry (Botondi et al. 2021). Molecular O₃ and the free radicals (O^•─, O₂ ^•─, O₃ ^•─, ^•OH, ^•HO₂, HO₃ ^•, and H₂O₂) generated by its decomposition inactivate bacteria through complex processes targeting multiple cellular components (Epelle et al. 2023). These include cell membrane lysis, resulting from the presence of unsaturated lipids and peptidoglycans in the cell envelope, and leakage of cellular components, such as nucleic acids, proteins, and enzymes, from the cytoplasm (Khadre et al. 2001). Gaseous and aqueous forms of O₃ effectively inactivate L. monocytogenes in various foods, including lettuce (Ölmez and Akbas 2009), spinach (Wani et al. 2015), apple juice (Song et al. 2015), apples, strawberries, and cantaloupes (Rodgers et al. 2004).

Shu et al. (2021) evaluated the antimicrobial efficacy of O₃ at different doses (1, 2, and 3 µg O₃/g of fruit) and exposure periods (1, 2, and 3 h) against L. monocytogenes on the surface of tomato fruits. They found that a moderate O₃ dose (2 µg O₃/g of fruit) effectively reduced the L. monocytogenes count (1.1 ± 0.3 log reduction) within a short exposure time of 1 h. Transcriptional profile of L. monocytogenes at this O₃ dose revealed the differential expression of 283 genes, including 121 upregulated and 161 downregulated genes. Levels of genes related to general and oxidative stress responses, cell division, virulence, and chemotaxis were upregulated. In contrast, levels of several genes with unknown functions and those related to metabolism and transport systems were downregulated (Figure 1). O₃ exposure triggered complex transcriptional responses, including the upregulation and downregulation of stress‐associated genes, in L. monocytogenes. Levels of the oxidative stress gene sodA were upregulated, whereas those of trxB (thioredoxin reductase) and prli42 (membrane‐associated stress protein) were downregulated. This indicates the selective modulation of pathways related to redox and membrane stress. Furthermore, the upregulation of cold shock protein (Csp)‐D and osmC levels indicated a response to cold shock and osmotic stress. Increased cheR, flgE, and mogR levels indicated enhanced chemotaxis and flagellar regulation, supporting adaptive motility under oxidative stress conditions. These gene activation and repression patterns suggest that L. monocytogenes prioritizes responses that enhance stress tolerance during transport and metabolic processes in response to moderate O₃ doses (Shu et al. 2021).

3.4. Plasma‐Activated Water

Nonthermal plasma, particularly PAW, effectively disinfects L. monocytogenes (Jyung et al. 2023; Rothwell et al. 2022). PAW is generated by treating water with a plasma discharge (ionized gas stream) that produces reactive species (Y.‐M. Zhao et al. 2020). ROS generated in PAW contain potent oxidizing agents, including ^•OH, H₂O₂, atomic oxygen (O), superoxide anions (O₂ ^•─), and O₃. ROS in PAW induce oxidative stress in microbes and inactivate them by damaging the redox state of the antioxidants (Z. Zhang et al. 2013), penetrating the cell membrane by promoting lipid oxidation (Joshi et al. 2011), damaging the cell structure by destroying the intramolecular bonds of the peptidoglycan layer (Yusupov et al. 2013), interfering with metabolic activities, and eventually causing cell death (Wong et al. 2023). Fernández‐Gómez et al. (2023) analyzed the transcriptomic responses of L. monocytogenes ULE1265 (serotype 1/2c), which were harvested from a stationary‐phase culture, resuspended in Ringer solution, treated with PAW (generated with 36 W discharge power and 30‐min activation time) for 5 min, and stored for 24 h. This PAW disinfection treatment for 5 min resulted in a 2.2 ± 0.1 log₁₀ CFU/mL reduction in L. monocytogenes count. Moreover, 399 genes were differentially expressed, with 178 being upregulated and 221 being downregulated. Expression levels of metabolism‐related genes, especially the cobalamin‐dependent gene cluster (CDGC) associated with ethanolamine (EA) and 1,2‐propanediol (PD) utilization, internal surface protein, chemotaxis, and QS genes, were increased. In contrast, levels of genes related to the cell envelope and PTS were decreased (Figure 1). PAW treatment altered the expression levels of several genes related to virulence and general stress responses, many of which were regulated by the alternative sigma factor, SigB. Notably, the glutamate decarboxylase (GAD) system, crucial for acid stress responses, was upregulated. However, no significant changes in the genes typically associated with the oxidative stress responses were observed under the tested conditions. These results suggest that L. monocytogenes responds to PAW by remodeling carbon metabolism, PTS, and CDGC (Fernández‐Gómez et al. 2023).

4. Comparative Analysis of L. monocytogenes Transcriptional Profiles Under Different Disinfection Treatment Conditions

Comparison of the gene expression patterns of L. monocytogenes under different treatment conditions provides insights into the behavioral mechanisms of the pathogen under various stresses, improving our understanding of the gene regulatory networks and pathways involved in specific biological processes (Q. Wang et al. 2022). Comparative transcriptome analysis revealed the distinct effects of different disinfection treatments on the genes responsible for key biological processes in L. monocytogenes (Figure 2). Most gene regulation mechanisms were specific to a particular treatment or shared by only two treatments. Few genes associated with peptidoglycan biosynthesis (murA, murE, mreB, and glmS), cell division (filamentous temperature‐sensitive [fts]‐E, ftsX, ftsW, and actA), glycolysis (gpmA and gpmI), glycerol metabolism (glpD and glpK), nucleic acid metabolism (guaA), protein synthesis (rpsP and rpiB), PTS (bglP), cold shock response (cspD), oxidative stress response (sodA), chemotaxis (cheR), flagellar assembly (fliN and flgE), and virulence (inlA) were commonly affected by at least three different disinfection treatments, as shown in Figure 2 and Figure S1.

4.1. Cell Wall/Membrane Synthesis and Integrity

L. monocytogenes exhibits a cell wall composed of cross‐linked peptidoglycans with multiple layers of teichoic and lipoteichoic acids (Vollmer and Seligman 2010). The cell wall plays a crucial role in maintaining the integrity of the bacterium, with high internal osmotic pressure defining the cell shape, protecting against mechanical stress, and serving as an anchor for various proteins (Pucciarelli et al. 2007). Most disinfectants disrupt the bacterial cell membrane, which is closely associated with the cell wall, thereby compromising the cell wall integrity (Brauge et al. 2020). Transcriptome analysis of L. monocytogenes exposed to various disinfection treatments revealed that these treatments affect multiple genes associated with peptidoglycan and wall teichoic acid (WTA) biosynthesis, cell envelope, and cell division.

4.1.1. Peptidoglycan Biosynthesis

The bacterial cell wall is a primary disinfection target. The peptidoglycan biosynthesis pathway is essential to maintain the cell wall integrity and is tightly regulated in L. monocytogenes (Fox et al. 2011). Alterations in this pathway affect the cell surface properties, cell wall integrity, stress tolerance, and overall fitness of L. monocytogenes. Oxidizing agents H₂O₂ (khpA), O₃ (glmS and murO), and ClO₂ (glmU, murE, and mreB) inhibit the regulation of several peptidoglycan pathway genes (Figure S1). The murA levels are upregulated in L. monocytogenes following O₃ treatment. Peptidoglycan biosynthesis occurs in the cytoplasm by converting UDP‐N‐acetylglucosamine to UDP‐N‐acetylmuramic acid and lipids I and II through the actions of MurA, MurB, MurCDE, MurF, MraY, and MurG (Schulz et al. 2022). GlmS, GlmM, GlmU, and GlmR catalyze a four‐step process to convert fructose‐6‐phosphate to UDP‐N‐acetylglucosamine, which is essential for peptidoglycan biosynthesis (Schulz et al. 2022).

4.1.2. WTA Biosynthesis

WTAs are anionic glycopolymers covalently attached to the peptidoglycan layers of Gram‐positive bacteria (S. Brown et al. 2013). These polymers play crucial roles in determining the cell shape, regulating cell division, and influencing other essential physiological features of Gram‐positive bacteria. Teichoic acid glycerol (tag genes) encodes the proteins involved in WTA biosynthesis. Levels of genes involved in teichoic acid biosynthesis, namely gtcA, dltA, dltB, dltC, and dltD, are downregulated after ClO₂ and H₂O₂ treatments (Table S1).

4.1.3. Cell Division Genes

Cell division is a crucial process in living organisms. Bacteria use binary fission to generate offspring identical to the parent cell (Casiraghi et al. 2020). To ensure correct cell division, parent cells coordinate various processes, including overall biomass increase, membrane synthesis, chromosome duplication, and cell wall production (Silber et al. 2020). These processes are mediated by a protein complex, known as the divisome, which regulates cell division (Mahone and Goley 2020). fts genes in L. monocytogenes show different responses to various disinfection treatments. ClO₂ downregulates ftsE, ftsX, ftsW, and ftsL levels, but not ftsH levels. O₃ and H₂O₂ suppress the regulation of sepF, LMOf2365_2563, and ftsW. Moreover, levels of yneA, which encodes the cell division suppressor protein YneA, are upregulated in response to H₂O₂. The products of these fts genes are essential for creating the septum and initiating cell division in bacteria (Han et al. 2021).

L. monocytogenes modifies its cell wall composition and thickness in response to stress, which affects its susceptibility to disinfectants (Cotter and Hill 2003). Downregulation of peptidoglycan (mur and glm) and WTA (dlt) biosynthesis gene levels in L. monocytogenes upon exposure to ClO₂, H₂O₂, and O₃ indicates a stress response during which the bacterium conserves energy by limiting cell wall synthesis. This results in weakened cell walls and increased membrane permeability, making the pathogen more susceptible to oxidizing agents (Schulz et al. 2022).

4.2. Metabolic Pathway Genes

Central metabolic pathways actively produce stored biological energy and generate vital metabolic precursors necessary to build the fundamental cellular components of all living organisms (Romano and Conway 1996). Gene transcription operates in conjunction with cell metabolism, with both mutually regulating each other (Bian et al. 2022). Bacterial metabolic processes are the primary targets of disinfection treatments that cause L. monocytogenes to adjust its metabolism to survive under the disinfection stress (Bland et al. 2022). Disinfection treatments affect various metabolic pathways, including respiration, energy, carbohydrate, lipid, nucleic acid, and amino acid metabolism pathways.

4.2.1. Respiration and Energy Metabolism

Cellular respiration is crucial for generating energy and maintaining the electron balance in cells, particularly under stress. Aerobic organisms generate energy via cellular respiration, which involves oxidizing an electron donor, such as glucose, and reducing an electron acceptor, such as oxygen, resulting in the production of carbon dioxide. Electron exchange occurs via the electron transport chain to produce ATP, the primary energy currency for cells (Fuhrmann 2021). L. monocytogenes exhibits flexible respirofermentative metabolism, allowing it to thrive in diverse environments using various carbon sources (Rivera‐Lugo et al. 2022). Cellular respiration maintains the redox balance, regenerates nicotinamide adenine dinucleotide (NAD⁺), and produces a proton motive force. The Menaquinone (MK) biosynthetic pathway is essential for both aerobic and anaerobic respiration (Smith et al. 2023). Various genes involved in the electron transport chain, NAD⁺ regeneration, and ATP synthesis are differentially regulated under ClO₂ and O₃ treatments (Table S2; Supporting Information: Data S2.2.1), suggesting that energy metabolic pathways are responsive to oxidative stress.

4.2.1.1. NAD⁺ Regeneration and ATP Synthesis

NAD⁺ is a crucial small molecule facilitating electron and proton transfer during enzymatic reactions (Chen et al. 2024). It also plays a vital role in cellular energy metabolism by reducing NAD⁺ to NADH (reduced form of NAD) in multiple metabolic signaling pathways, including glycolysis and tricarboxylic acid (TCA) cycle (Xiao et al. 2018). NAD⁺ acts as a coenzyme in vital processes, including glycolysis, oxidative phosphorylation, protein deacetylation, DNA repair, and Ca²⁺ activation. Expression of the NADPH dehydrogenase gene namA and the glutamine‐dependent NAD⁺ synthetase gene nadE was suppressed by O₃. Exposure to ClO₂ increases the expression of atpC in L. monocytogenes. ATP synthases are multi‐subunit enzyme complexes found in bacterial cell membranes (Guo et al. 2019). They use transmembrane electrochemical proton motive force to generate ATP from ADP and inorganic phosphate (Pi) (Meyrat and von Ballmoos 2019). Downregulation of NADPH dehydrogenase and NAD⁺ synthetase gene levels by O₃ suggests a potential reduction in NADPH and ATP synthesis. This causes an imbalance in the NADH/NAD⁺ ratio within the cells, affecting central carbon and energy metabolism (Zhang et al. 2014). These expression patterns indicate the stress‐associated shifts in redox‐related pathways.

4.2.2. Carbohydrate Metabolism

Carbohydrate metabolism encompasses all biochemical processes responsible for the formation, breakdown, and conversion of carbohydrates to provide a steady energy source for living cells (Chandel 2021). Among the other carbohydrates, glucose, fructose, and galactose play vital roles in several metabolic pathways. The disinfection treatments differentially expressed several carbohydrate metabolic pathways, including glycolysis, the pentose phosphate pathway (PPP), and genes associated with acetoin biosynthesis, pyruvate fermentation, rhamnose and lactose metabolism, as well as glycosidase activity (Table S2; Supporting Information: Data S2.2.2).

4.2.2.1. Glycolysis

Glycolysis is a central metabolic pathway that converts glucose to pyruvate or lactate, producing ATP, NADH, or water through various enzymatic reactions to generate energy (Chaudhry and Varacallo 2024). Among the disinfection treatments against L. monocytogenes, ClO₂ and PAW treatments upregulated several glycolytic pathway gene levels, whereas O₃ treatment downregulated them (Supporting Information: Data S2.2.2). The major glycolytic genes upregulated by disinfection treatments included gpmA (ClO₂), ccpN (H₂O₂), cggR (PAW), eno (PAW), and pgk (PAW), whereas the downregulated genes included gpmA (O₃), gpmI (O₃), pgi (ClO₂), and pgmB (O₃). In addition to generating ATP and NADH, glycolysis produces precursor metabolites for several metabolic pathways, including the peptidoglycan biosynthesis pathway, which is essential for cell wall integrity and tolerance to cell wall‐targeting antimicrobial agents (Fuhrmann 2021; Reed et al. 2015). Upregulation of glycolytic gene levels by disinfection treatments allows L. monocytogenes to efficiently utilize the available carbohydrates, ensuring a constant supply of energy and metabolic intermediates necessary for cell wall regeneration, energy production, and other stress response mechanisms. However, glycolysis disruption by ClO₂ treatment possibly induces cell death (Ranjbar et al. 2020).

4.2.2.2. Pentose Phosphate Pathway

PPP is a glucose oxidation pathway operating alongside glycolysis (Wamelink et al. 2008). It produces ribose 5‐phosphate and NADPH, redirecting carbon to the glycolytic or gluconeogenic pathways. Ribose 5‐phosphate is used to create nucleotides, whereas NADPH maintains the redox balance and facilitates biosynthetic activities, including the production of tetrahydrofolate, deoxyribonucleotides, proline, and fatty acids (Kim et al. 2023). Transcript levels of PPP enzymes, including tal1, lmo2674, and lmo0342, are increased in L. monocytogenes in response to ClO₂ treatment. O₃ treatment also increases the expression levels of the PPP enzyme fsa and decreases those of tkt and tktC in L. monocytogenes. PPP plays crucial roles in maintaining the balance of cellular redox levels and supporting biosynthesis (TeSlaa et al. 2023). These transcriptional changes suggest treatment‐specific effects on carbon allocation via PPP. PPP interruption influences the cell wall, alters the oxygen radical sensitivity, reduces nucleotide synthesis, and affects biofilm formation (Kim et al. 2023).

4.2.2.3. TCA Cycle

L. monocytogenes has a distinct and specialized metabolism because of its incomplete TCA cycle (Schauer et al. 2010). It lacks the α‐ketoglutarate dehydrogenase enzyme, essential for converting α‐ketoglutarate to succinyl‐coenzyme A (CoA). This absence restricts the pathogen's capacity to fully utilize the TCA cycle for energy production and biosynthesis (Whiteley et al. 2017). L. monocytogenes compensates for this by using alternative pathways, including glycolysis, PPP, and pyruvate carboxylation pathways, to produce the intermediates necessary for metabolism and energy production (Schär et al. 2010; Schauer et al. 2010). This metabolic flexibility is crucial for the survival and virulence of bacteria within host cells (Fuchs et al. 2012). H₂O₂ treatment upregulates the expression levels of citB, citC, and citZ genes in L. monocytogenes (Table S2). Genes citB, citC, and citZ, which encode aconitase, isocitrate dehydrogenase, and citrate synthetase, respectively, are associated with the regulation of the TCA cycle and central metabolism. Oxidative stress due to H₂O₂ induces metabolic changes in bacteria, including increased pyruvate production and reduced pyruvate dehydrogenase (PDH) activity, resulting in altered metabolic flux and elevated pyruvate levels under stress (Bignucolo et al. 2013).

4.2.2.4. Pyruvate Metabolism

Pyruvate is an essential metabolite that connects critical metabolic pathways, including glycolysis, gluconeogenesis, TCA cycle, amino acid biosynthesis, and fatty acid biosynthesis (FAB) (Luo et al. 2023). The initial substrate for the gluconeogenic pathway is pyruvate, which can be utilized to produce glucose (Brissac et al. 2015). H₂O₂ and ClO₂ disinfection treatments exert different effects on the expression levels of genes involved in pyruvate metabolism. Levels of genes associated with PDH complexes, pdhA, pdhB, pdhC, and pdhD, and the pyruvate formate‐lyase gene pflA are downregulated by H₂O₂ treatment. In contrast, ClO₂ upregulates pdhC, pdhD, and pyruvate carboxylase (PYC) gene pycA levels. Pyruvate metabolism, primarily through the function of the PDH complex and PYC, plays a vital role in carbon metabolism in L. monocytogenes. The PDH complex catalyzes the oxidative decarboxylation of pyruvate to acetyl‐CoA, which connects glycolysis to the TCA cycle (Fuchs et al. 2012). L. monocytogenes exhibits an incomplete TCA cycle, making PYC‐catalyzed carboxylation of pyruvate essential for oxaloacetate production (Schär et al. 2010). These observations suggest potential shifts in the carbon flux under oxidative stress.

4.2.2.5. Other Carbohydrate Metabolic Processes

Disinfection treatments further altered the expression of L. monocytogenes genes involved in other carbohydrate metabolic processes, such as fructose metabolism (fba upregulated by O₃; fruA upregulated by PAW), lactose metabolism (ldh downregulated by PAW), rhamnose metabolism (lmo1082 downregulated by ClO₂), and chitin degradation (lmo1883 upregulated by ClO₂; chbG downregulated by O₃). During disinfection, L. monocytogenes cells enter a state of stress, requiring increased intake of carbohydrates as catabolic products for cell repair. Downregulation of genes related to carbohydrate metabolism suggests that disinfection methods directly restrict the ability of bacteria to absorb carbohydrates from their surroundings. This further affects the ability of bacteria to use their breakdown products for cellular maintenance, potentially limiting access to metabolic intermediates necessary for their survival (Qian et al. 2020).

4.2.3. Lipid Metabolism

In addition to carbohydrate metabolism, the disinfection treatment affected several genes related to FAB and glycerol metabolism (Table S2). Lipids are the primary components of the bacterial cytoplasmic membrane. They form a phospholipid bilayer structure and can be categorized into several chemical classes varying in molecular size and isomeric form (Strahl and Errington 2017). The precursor molecules of lipids are primarily obtained from central metabolism. Lipid metabolism is crucial for the ability of a bacterium to survive and multiply within host cells. Disinfection with H₂O₂, ClO₂, O₃, and PAW affected FAB and glycerol metabolism differently (Table S2; Supporting Information: Data S2.2.3).

4.2.3.1. Fatty Acid Biosynthesis

The FAB pathway is a potential target for disinfection treatments, including antibiotics, because it plays a vital role in creating phospholipid membranes, lipoproteins, and lipopolysaccharides (Y.‐M. Zhang et al. 2006). Phospholipids are key components of the membrane lipids that form lipid bilayers. They create a protective barrier for cells and facilitate cellular processes within subcellular compartments (Dai et al. 2021). Phosphatidic acid is produced through two consecutive acylation reactions of glycerol‐3‐phosphate using acyl‐acyl carrier protein (ACP) produced by the bacterial type II fatty acid synthesis system (FASII). This substance is a precursor of all phospholipid species (Yao and Rock 2017). Listeria spp. initiate the first reaction of the FASII pathway by combining fatty acid thioesters with different acyl chain lengths with a malonyl‐ACP (Soares da Costa et al. 2017). Exposure of L. monocytogenes to PAW upregulates the levels of FASII pathway genes accB and fabI. In contrast, ClO₂ downregulates the levels of FAB gene acpA, fabG, fabH, and lmo0970 in L. monocytogenes. These transcriptional changes indicate alterations in membrane lipid synthesis in response to oxidizing agents. FASII initiation, elongation, and acyltransferase modules work together to synthesize fatty acids in appropriate quantities and structures (Parsons and Rock 2013).

4.2.3.2. Glycerol Metabolism

Glycerol plays a crucial role in L. monocytogenes virulence and biofilm formation, serving as a significant carbon source that is metabolized through pathways involving glycerol kinase (GlpK) and glycerol‐3‐phosphate dehydrogenase (GlpD) (Joseph et al. 2008). Bacteria use glycerol dehydrogenase to convert glycerol into dihydroxyacetone (Dha). L. monocytogenes possesses two Dha kinase systems, which are part of a PEP‐dependent phosphorylation system for Dha utilization and are connected to the PPP (Monniot et al. 2012). Expression levels of genes involved in glycerol metabolism in L. monocytogenes are upregulated (isp and LMOf2365_1834) and downregulated (ispE, ispD, dhaK1, LMOf2365_1310, LMOf2365_0375, LMOf2365_2202, LMOf2365_2487, and LMOf2365_2806) after O₃ treatment. Other chemical oxidants, H₂O₂ (dhaI, dhaK, and dhaM) and ClO₂ (glpD, glpK, and lmo0347), upregulated, whereas PAW (glpD and glpK) downregulated glycerol metabolism gene levels in L. monocytogenes. These changes in gene expression suggest that glycerol utilization is differentially regulated in response to varying levels of oxidative stress.

4.2.4. Nucleic Acid Metabolism

Disinfection also significantly affected the expression of genes involved in nucleotide biosynthesis. Nucleotides are crucial for various cellular processes such as DNA replication, energy storage, virulence, and signaling and are continuously synthesized de novo in all cells (Goncheva et al. 2022). Treatment with H₂O₂ increased the expression of the xpt gene, whereas ClO₂ decreased xpt expression in L. monocytogenes. The xpt gene encodes xanthine phosphoribosyltransferase, which is involved in purine salvage pathways (Glaser et al. 2001). This enzyme converts xanthine, a nucleic acid breakdown product, into xanthosine 5'‐monophosphate, which allows the pathogen to reuse xanthine for RNA or DNA synthesis. Levels of the guaA gene, which encodes a guanosine monophosphate (GMP) synthase, are decreased after ClO₂ treatment (Table S2). This gene is responsible for converting inosinic acid to GMP (Bennett et al. 2007). Similar to glycerol metabolism, levels of several genes related to nucleic acid metabolism in L. monocytogenes are upregulated (purA, mrnC, nrdD, nudL, xerC, gmk, carB, and tenA) or downregulated (purL, purE, recF, adaB, dinB, gyrB, holB, cysK, rnhC, dprA, LMOf2365_1336, LMOf2365_1861, LMOf2365_2183, LMOf2365_2186, LMOf2365_2656, and LMOf2365_1822) by O₃ treatment. These transcriptional shifts indicate treatment‐specific effects on nucleotide synthesis, salvage, and DNA repair under stress conditions.

4.2.5. Amino Acid Metabolism

Amino acids are essential for protein synthesis in microorganisms and play crucial roles in regulating peptidoglycan metabolism, maintaining cell structure, promoting bacterial growth, facilitating biofilm formation, and protecting against environmental hazards (Miyamoto and Homma 2021). ClO₂ treatment upregulates the expression levels of gcvT, gcvH, and gcvPB (Table S2). Excess glycine hinders the growth of several bacteria (Minami et al. 2004). The glycine cleavage system is a multienzyme system, catalyzing the oxidative cleavage of glycine to form 5,10‐methylene‐tetrahydrofolate, a crucial one‐carbon precursor for various biosynthetic processes, along with serine, thymidine, and purines (M. J. Brown et al. 2014). The GCS pathway also contributes to energy metabolism in bacterial cells by generating NADH (Kikuchi et al. 2008). Upregulation of GCS gene levels by ClO₂ treatment increases the metabolic fitness of L. monocytogenes (M. J. Brown et al. 2014).

Some genes associated with the GAD pathway are differentially expressed in L. monocytogenes in response to disinfection. ClO₂ and PAW treatments upregulate the expression levels of gadB and gadC in L. monocytogenes. The GAD pathway functions as a central mechanism for acid tolerance, facilitating microbial survival at low pH (Sezgin and Tekin 2023). This pathway transports extracellular glutamate into the cell and converts it to γ‐aminobutyrate (GABA), thereby increasing the intracellular pH under acid stress to maintain the intracellular pH homeostasis (C. Wang et al. 2018). GAD pathway gene expression changes in L. monocytogenes by ClO₂ and PAW treatments primarily affect its acid tolerance and intracellular pH homeostasis, further impacting metabolism, stress response, and survival (Feehily and Karatzas 2013).

4.3. Metabolic Enzymes

L. monocytogenes resists disinfectants via biofilm formation (Skowron et al. 2018), detoxification using efflux pumps (Jiang et al. 2016), oxidative stress responses (Ogunleye et al. 2024), and adaptive metabolic flexibility (Wiktorczyk‐Kapischke et al. 2021). Metabolic flexibility allows L. monocytogenes to use various carbon sources (e.g., such as glycerol and amino acids), depending on the environmental conditions (Schauer et al. 2010). Metabolic enzymes facilitate this adaptability, enabling the bacterium to switch between metabolic pathways, thereby enhancing its survival upon disinfectant exposure. Understanding these enzymes can aid in the development of novel methods to control L. monocytogenes infections in the food industry. The major metabolic enzymes exhibiting differential expression in response to various disinfection treatments (H₂O₂, ClO₂, O₃, and PAW) are oxidoreductases, peptidases, transferases, and hydrolases (Table S3; Supporting Information: Data S2.3).

4.3.1. Oxidoreductases

Oxidoreductases are a class of enzymes facilitating oxidation–reduction or redox reactions by transferring electrons between molecules (Nord 2009). In these reactions, one substrate is oxidized and loses electrons, whereas the other is reduced and gains electrons. Common oxidoreductases include dehydrogenases, which remove hydrogen atoms (and thus electrons) from substrates, oxidases, which transfer electrons to oxygen molecules, and reductases, which catalyze the reduction of a substrate by adding electrons (Cárdenas‐Moreno et al. 2023). These enzymes play critical roles in various biological processes, including cellular respiration, metabolism, and ROS detoxification. Oxidizing agents H₂O₂ (ywnB), ClO₂ (lmo0521, lmo1789, lmo0669, lmo0613, lmo2572, lmo0761, lmo1433, lmo2426, and lmo2573), and O₃ (morA and LMOf2365_0583) upregulate the expression levels of oxidoreductase activity genes. However, O₃ (ssuE and bsaA) and PAW (adhE) decrease the expression levels of specific oxidoreductase genes in L. monocytogenes. Increased expression of oxidoreductases protects the cells against damage from ROS and other oxidizing agents (Ingram 2018). These enzymes detoxify harmful oxidants and maintain the redox balance within the cells (Cheng et al. 2017).

4.3.2. Transferases

Transferases facilitate the transfer of functional groups from donors to acceptor molecules. These transferases can be categorized into several subclasses based on their functional groups, including methyltransferases, N‐acetyltransferases, glutathione S‐transferases, sulfur transferases, transaminases, phosphotransferases, acyltransferases, glycosyltransferases, selenotransferases, and molybdenum‐ or tungsten‐transferases based on the functional groups they transfer (McDonald and Tipton 2023). Transferases in bacteria play essential roles in various functions, including cellular detoxification (Allocati et al. 2009), biosynthesis pathways of disaccharides, oligosaccharides, and polysaccharides; protein glycosylation (J. Schmid et al. 2016), antimicrobial drug resistance; and bacterial capsule polymerization (Litschko et al. 2024). H₂O₂ treatment increases the expression of specific transferase genes, including lmo0602 and lmo0652. In contrast, O₃ treatment resulted in both an increase (LMOf2365_0152) and a decrease (ackA, dacA, and LMOf2365_0447) in the expression of some transferase genes. Some transferases in the bacterial antioxidant defense system protect against oxidative stress by neutralizing ROS and other oxidizing agents (Allocati et al. 2009). The elevated expression of transferase genes induced by the oxidizing agents H₂O₂ and O₃ suggests that the bacteria actively attempt to neutralize or modify the disinfectant molecules. These alterations in expression may indicate a shift in the cellular processing of metabolites or detoxification responses to oxidative stress.

4.3.3. Hydrolases

Hydrolases are enzymes that catalyze the cleavage of chemical bonds (e.g., C–C, C–O, C–N, and C–S) by adding –H or –OH groups from H₂O across the bond (Kermasha and Eskin 2021). These hydrolytic enzymes participate in various cellular processes, including DNA repair, RNA processing, protein degradation, cell wall growth and remodeling, cell division, autolysis, peptidoglycan recycling, detoxification, and regulation of cell signaling pathways (Do et al. 2020; Nardini et al. 1999; Qi et al. 2022). Expression levels of specific hydrolase activity genes (yocD, bds1, and lmo2266) in L. monocytogenes are upregulated in response to H₂O₂. ClO₂ and O₃ treatments both upregulate (ClO₂: lmo1577, lmo0869, lmo2603, lmo0268, lmo0907, lmo1254, lmo0764, lmo0261, and lmo1728; O₃: aguA) and downregulate (ClO₂: lmo0998, lmo2450, and lmo1240; O₃: LMOf2365_0277, LMOf2365_0523, and LMOf2365_0341) specific hydrolase activity gene levels in L. monocytogenes. Peptidoglycan hydrolases play a role in cell‐wall remodeling and turnover. These enzymes may help to modify the cell wall structure to reduce disinfectant penetration during stress (Lee and Huang 2013). Some hydrolases may directly inactivate or modify disinfectant molecules, helping bacteria detoxify and survive during treatment. These changes suggest potential alterations in the cell wall or detoxification response to oxidative stress (F. Zhang and Cheng 2022).

Peptidases, also known as proteases, catalyze the breakdown of peptide bonds through a process called hydrolysis. By cleaving these bonds, peptidases convert long protein molecules into smaller peptides or individual amino acids, which are essential for various biological processes, such as protein maturation, recycling, and cellular regulation (Potempa and Pike 2004). They are crucial for bacterial growth, metabolism, and adaptation to various environments. These proteolytic enzymes have diverse structures, perform different biological roles, and can be found in the cytoplasm, attached to the cell membrane, or released into the surroundings (Nguyen et al. 2019). The O₃ and PAW treatments showed varied effects on the expression of specific peptidase activity genes in L. monocytogenes, with some genes being upregulated (O₃: pepT; PAW: iap) and others being downregulated (O₃: srtA, LMOf2365_0284, LMOf2365_1945, and LMOf2365_0569; PAW: lspA). In contrast, ClO₂ (lmo1354, lmo1578, lmo1611, LMOf6854_0307, and LMOf6854_0278) upregulated the expression of specific genes. As peptidases may play an active role in breaking down and utilizing proteins, the differential expression of genes encoding these enzymes following disinfection indicates that bacteria may be attempting to manage damaged or misfolded proteins under challenging conditions.

4.4. Small Molecule Biosynthesis

Synthesis of biomolecules is a multistage process involving enzymatic conversion or combination of simple compounds to produce macromolecules or more intricate products in living organisms (Abdel‐Aziz et al. 2017). This process of small molecule biosynthesis is crucial for bacteria as it allows the synthesis of various compounds necessary for their survival, growth, and interactions with the environment. Bacteria synthesize many small molecules, including vitamins, minerals, cofactors, organic acids, ketones, and alcohols (Coupat et al. 2008; França et al. 2014). Disinfection treatments (H₂O₂, ClO₂, O₃, and PAW) affect various small molecule biosynthesis pathways, such as cobalamin, EA, and PD utilization, pantothenate, MK, molybdenum cofactor, and heme biosynthesis, and myo‐inositol catabolic pathways (Table S4; Supporting Information: Data S2.4).

4.4.1. Cobalamin Biosynthesis and EA and PD Utilization

Cobalamin (vitamin B12) serves as a cofactor for enzymes crucial for various metabolic processes, such as methylation reactions in amino acid metabolism (e.g., methionine synthase) (Matthews 2009), rearrangements in carbon metabolism (e.g., methylmalonyl‐CoA mutase) (Roth et al. 1996), reduction reactions in nucleotide synthesis (e.g., ribonucleotide reductase) (Booker and Stubbe 1993), and fermentation reactions during anaerobic fermentation (e.g., ethanol to acetate) (Buckel and Thauer 2013). Cobalamin biosynthesis is a complex and tightly regulated process involving approximately 30 enzyme‐mediated steps. It is also influenced by environmental factors, such as oxygen and cobalt availability (Raux et al. 2000). Levels of cobalamin metabolism gene cluster cob‐cbi and EA and PD utilization operons (eut and pdu, respectively), which are part of CDGC, were upregulated by PAW. Levels of PD gene pduX, cobalamin biosynthesis genes cbiD and cbiH, and eut genes eutB, eutC, eutE, eutH, and eutT were also upregulated. In contrast, except for cbiG levels, cobC, cobJ, cobK, cobA, and LMOf2365_1212 levels were downregulated by O₃. The eut operon uses EA as the carbon and nitrogen source, whereas the pdu operon is involved in PD metabolism. The enzymes involved in these pathways rely on cofactors derived from cobalamin, which are produced de novo through cob and cbi (Anast et al. 2020). CDGC activation enhances competitive fitness against commensal bacteria in the gastrointestinal tract and other environments. Sublethal exposure to specific disinfection treatments activates the CDGC signaling pathways, potentially increasing the expression levels of operons linked to host cell internalization and stress adaptation. This response further increases the virulence of persistent bacterial strains (Fernández‐Gómez et al. 2023).

4.4.2. Pantothenate Biosynthesis

Pantothenate (vitamin B5) is a precursor of CoA, essential for many metabolic processes in bacteria (Leonardi and Jackowski 2007). CoA is a vital cofactor for cell growth and integral to numerous metabolic processes, including phospholipid metabolism, FAS and fatty acid breakdown, nonribosomal peptide biosynthesis, and the TCA cycle (Spry et al. 2008). Levels of panB, the coax gene required for pantothenate biosynthesis and CoA, are downregulated by O₃ treatment (Table S3). Pantothenate biosynthesis is linked to other metabolic pathways, including branched‐chain amino acid biosynthesis and nucleotide metabolism pathways (Leonardi and Jackowski 2007; Primerano and Burns 1983). Downregulation of several amino acid and nucleotide metabolism gene levels in L. monocytogenes by O₃ disinfection is correlated with the downregulation of the levels of genes involved in pantothenate and CoA biosynthesis (Tables S2 and S3). This suggests that the oxidizing agent O₃ inhibits numerous metabolic processes essential for the survival of L. monocytogenes through chain reactions.

4.4.3. MK Biosynthesis

MK (vitamin K2) plays a vital role in prokaryotes by facilitating electron transfer, ATP production, oxidative phosphorylation, and endospore formation (Das et al. 1989; Hiratsuka et al. 2008). MK is the primary component of the bacterial cell membrane and plays a crucial role in the aerobic and anaerobic respiration of many Gram‐positive bacteria. It facilitates the transfer of electrons between the enzymes, NADH dehydrogenase I and II, succinate dehydrogenase, cytochrome bc1–aa3 supercomplex, F₀F₁ ATP synthase, cytochrome bd oxidase, nitrate, and fumarate reductase (Boersch et al. 2018; Kurosu and Begari 2010). The L. monocytogenes genes involved in MK biosynthesis, menD and menH, were upregulated by O₃, whereas menB and menG were downregulated by O₃ and PAW, respectively. These mixed responses indicate complex regulation of the respiratory chain during oxidative stress. As MK plays a central role in redox balance, changes in its biosynthesis affect electron transport and cellular energy status. MK biosynthesis is crucial for the survival of Gram‐positive bacteria (Truglio et al. 2003), and inhibition of MK production leads to bactericidal effects, irrespective of the growth phase (Boersch et al. 2018).

4.4.4. Molybdenum Cofactor Biosynthesis

Mo is a trace element found in molybdoenzymes and plays a crucial role in essential cellular functions, including energy generation, detoxification reactions, and anaerobic fermentation (Zhong et al. 2020). The molybdenum cofactor (Moco) biosynthesis pathway is conserved in both prokaryotes and eukaryotes (Leimkühler 2020). In bacteria, the pathway consists of four main steps: (i) formation of cyclic pyranopterin monophosphate (cPMP) from GTP, (ii) insertion of two sulfur atoms into cPMP to form molybdopterin (MPT), (iii) insertion of molybdenum into MPT to form Moco, and (iv) additional modification of Moco to form dinucleotide variants (specific to bacteria) (Mendel and Leimkühler 2015). This process involves the interactions of multiple proteins and requires iron, ATP, and copper. The key enzymes/proteins involved include MoaA and MoaC for cPMP formation; MoaD, MoaE, and MoeB for MPT synthesis; and MogA and MoeA for molybdenum insertion (Hasnat et al. 2024). MPT synthase catalytic subunit moeE and MPT molybdenum transferase moeA were upregulated and downregulated by O₃, respectively. Molybdoenzymes in bacteria are primarily regulated by anaerobiosis and require iron. The transcription factor fumarate and nitrate reduction (FNR) requires a [4Fe‐4S] cluster for binding (Hasnat et al. 2024). MoaA contains a [4Fe‐4S] cluster and directly connects the Moco biosynthesis pathway to the formation of iron‐sulfur (Fe‐S) clusters (Yokoyama and Leimkühler 2015). A feedback regulation mechanism controls the transcription of the moa operon genes. When MPT accumulates in the cells, it triggers a molybdenum cofactor riboswitch. This mechanism allows the bacteria to adapt to environmental changes (Regulski et al. 2008). The increased moeE expression in L. monocytogenes in response to O₃ potentially enhances its tolerance to oxidative stress (Cai et al. 2023).

4.4.5. Heme Catabolism

Heme is a complex ring composed of porphyrins and iron. It is a redox‐active component essential for the function of several cellular proteins (Choby and Skaar 2016). Hemoproteins rely on heme and are involved in energy production and immune responses. Heme plays a crucial role in cellular respiration and acts as an electron carrier in the electron transport chain (Shimizu et al. 2019). It is also essential for several cellular functions, including signaling, cellular differentiation, and miRNA processing (Choby and Skaar 2016; Faller et al. 2007). Levels of lmo2133, which encodes an isomer‐type heme‐degrading monooxygenase, are upregulated by ClO₂. In contrast, levels of the isdG gene (iron‐containing steroidogenic enzyme) are downregulated by O₃. Heme oxygenase catalyzes heme degradation to form biliverdin in the presence of an electron donor, releasing iron, which allows L. monocytogenes to utilize the host heme as an iron source (Duong et al. 2014). Heme oxygenase plays crucial roles in maintaining iron homeostasis and protecting against oxidative stress by breaking down heme (Alavi et al. 2018).

4.5. Transcription and Translation

Transcription is the fundamental process of copying genetic information from DNA to RNA, producing proteins essential for biological functions (Boyle 2005). RNA polymerase (RNAP) is the key enzyme responsible for this process, which involves initiation, elongation, and termination. In bacteria, a single RNAP enzyme performs all transcriptional functions, tightly regulating the process to allow the cells to adapt to environmental changes and effectively manage gene expression (Wilson et al. 2018). O₃ treatment of L. monocytogenes downregulated the levels of RNAP subunit rpoB, transcription factors tsf and LMOf2365_1316, and other components of the transcription machinery, including trmL, rny, and codY (Table S5).

Bacteria use mRNA transcribed from DNA to produce proteins through a process known as translation. This process occurs in the ribosome and involves several key steps, including initiation, elongation, termination, and recycling (Arenz and Wilson 2016). Ribosomal biogenesis is an intricate process involving ribosome assembly that is crucial for protein synthesis (Davis and Williamson 2017). Disinfection treatments differentially regulate the genes associated with ribosomal biogenesis and translation. O₃ and ClO₂ treatments differentially affect the expression levels of translational machinery genes in L. monocytogenes, upregulating some genes (O₃: rpmI, rpiB, and rnaJ; ClO₂: rplY, def, mecA, groEL, groES, rsgA1, lmo2511, lmo1502, lmo1218, and lmo1703) and downregulating others (O₃: rsmE, rpmG, rpsB, and mecA; ClO₂: rplM, rpmF2, rpmG2, rpmH, rpsB, rpsJ, rsmG, bipA, ychF, gatA, lepA, metG, and mnmE). In contrast, H₂O₂ and PAW treatments downregulated the levels of specific genes (rpsP and rnjB, respectively).

Ribosomal biogenesis and translation are crucial processes ensuring the synthesis of proteins essential for the cellular functions and survival of bacteria. Roller et al. (2016) demonstrated the correlations between the number of ribosomal RNA operons (rrn) and growth rate and stability. They reported that, when the rrn copy number doubles, the maximum reproductive rate of bacteria also doubles, with the rate and yield of protein production reflecting the general trend in maximum growth rate and efficiency (Roller et al. 2016). Increased expression levels of ribosomal genes in L. monocytogenes following disinfection lead to enhanced ribosome production. This is crucial for protein synthesis and cellular repair, enabling the pathogen to rapidly produce proteins vital for stress responses. Transcriptional changes in ribosomal genes possibly reflect the cellular responses to stress, including protein synthesis adjustments, to support L. monocytogenes survival and repair under disinfection stress.

4.6. Transport Genes

L. monocytogenes harbors several transporters playing crucial roles in various cellular processes and facilitating its adaptation to different environments, thereby contributing to its pathogenicity and survival. The major transporters affected by disinfection treatments include multidrug efflux transporters, phosphoenolpyruvate‐dependent PTS, metal ion transporters, and amino acid and protein transporters. Among these transporters, several genes belonging to the PTS system were differentially expressed in response to the reviewed disinfection treatments (Table S6; Supporting Information: Data S2.6).

4.6.1. Phosphotransferase System

PTS, a sugar‐specific transport system that simultaneously phosphorylates and transports various sugars into the cell, is crucial for carbohydrate transport and metabolism (Casey et al. 2014). Levels of lmo0781, lmo0915, lmo1255, lmo2000, lmo2373, lmo2650, and lmo1254, and several other genes belonging to the IIA component of the mannose/fructose/sorbose family PTS are upregulated by ClO₂ treatment. In contrast, H₂O₂ (bglC, bglH, and lmo0785) and O₃ (celB, LMOf2365_0537, LMOf2365_0660, LMOf2365_0798, LMOf2365_0937, LMOf2365_2023, LMOf2365_2344, LMOf2365_2621, LMOf2365_2664, and LMOf2365_2774) reduce the expression levels of several PTS system‐related genes. PAW treatment decreases the expression levels of PTS‐type galactitol (gatA, gatB, and gatC), cellobiose (catA, catB, and catC), and mannose (manY and manR) genes. Carbon sources fructose, galactitol (sugar alcohol derived from galactose hydrogenation), and cellobiose (disaccharide produced by cellulose hydrolysis) are essential precursors for cell wall biosynthesis in L. monocytogenes (Popowska et al. 2012). PTS downregulation under PAW treatment restricts the absorption and use of these carbohydrates, allowing the pathogen to conserve energy and resources for other stress response mechanisms.

4.6.2. Multidrug Efflux Transporters

Bacterial multidrug efflux transporters contribute to the ability of bacteria to resist and expel toxic compounds, including disinfectants, antibiotics, and heavy metals. Tolerance to quaternary ammonium compounds (QACs) and antibiotics mediated by multidrug efflux transporters has been reported in L. monocytogenes (Mata et al. 2000; Romanova et al. 2006). Levels of ebrA and qacC are upregulated in response to PAW treatment. QacC is a small multidrug resistance family efflux transporter that is a part of the bcrABC cassette, which primarily confers tolerance to QACs in L. monocytogenes (He et al. 2022). Upregulation of multidrug efflux transporter gene (qacH, qacC, and ebrA) levels by BZT indicates an adaptive mechanism to actively expel the disinfectant molecules from bacterial cells, thereby reducing their intracellular accumulation and toxicity.

4.6.3. Metal Ion Transporters

Metal ion transporters in bacteria play crucial roles in various cellular processes, including the maintenance of intracellular metal ion homeostasis, regulation of gene expression, response to oxidative stress, and virulence (Abrantes et al. 2011). Both mntB and mntR levels are upregulated in response to PAW treatment. H₂O₂ downregulates the expression of mntH. Manganese is involved in ROS detoxification and is essential for the oxidative stress responses in bacteria (Juttukonda and Skaar 2015). Mn transporters regulate the levels of Mn inside cells and shield the bacteria from oxidative stress (Jakubovics and Jenkinson 2001). The increased expression of manganese transporter genes in L. monocytogenes following PAW treatment indicates enhanced manganese uptake, which is crucial for the oxidative stress response, acid tolerance, and other stress adaptation mechanisms in L. monocytogenes (Juttukonda and Skaar 2015).

4.6.4. Amino Acid and Protein Transporters

Bacteria use different transport mechanisms to uptake amino acids and oligopeptides. These include ATP‐binding cassette (ABC) transporters, which are divided into subfamilies, such as polar and hydrophobic amino acid transport families (Hosie and Poole 2001), and sodium‐dependent and independent systems (Landick et al. 1985). ABC transporters use ATP hydrolysis to transport solutes across cellular membranes. Recent studies have identified six transporters facilitating the uptake of amino acids and oligopeptides in L. monocytogenes, thereby aiding the survival and replication of this bacterium (Joseph et al. 2006; Schauer et al. 2010). In L. monocytogenes, H₂O₂ increases the expression levels of bilEA, bilEB, and pbuX. Simultaneously, secA1 and gadC levels were elevated by ClO₂ and PAW, respectively. Levels of yrhG and dtpT decreased in response to H₂O₂ and PAW, respectively. L. monocytogenes BilE protein, encoded by bilEA and bilEB, resembles an ABC importer, and contributes to virulence and stress responses by transporting QACs (Ruiz et al. 2016). PAW treatment decreases the pH levels, indicating increased acidity due to the reactive oxygen and nitrogen species interacting with water molecules to generate protons (H+) (Jirešová et al. 2022). The gadC gene encodes a glutamate/GABA antiporter in bacteria, which is essential for the glutamate‐dependent acid resistance system (Tsai et al. 2013). GadC promotes the exchange of deprotonated glutamate (Glu^–) for protonated GABA⁺ across the cell membrane, effectively expelling protons and preventing their influx into the cell. Through the upregulation of gadC levels by PAW, L. monocytogenes enhances its ability to thrive under acidic conditions by maintaining an intracellular pH balance.

4.7. Stress Responses

Stress refers to the genomic, proteomic, and environmental changes reducing the growth rate and survival of an organism (Vorob'eva 2004). Sensing and responding to environmental signals are crucial for the adaptability and survival of bacterial pathogens (Guerreiro et al. 2020). Foodborne pathogens adjust their cellular processes to survive and grow under stress conditions (Sibanda and Buys 2022). L. monocytogenes encounters various physical and chemical challenges impeding its growth and survival throughout the food chain. This pathogen is primarily known for its ability to persist and thrive in food production environments (Sibanda and Buys 2022). Notably, enhanced QAC tolerance is observed in L. monocytogenes isolates persisting for over 4 years in such environments (Aase et al. 2000). Many genes involved in stress responses, including those encoding the general stress response proteins, cold shock proteins (Csps), and heat shock proteins (Hsps), and those related to temperature, osmotic, cell envelope stress, oxidative stress, antibiotics, radiation, DNA damage, signal peptide processing, protein quality control, and homeostasis, were differentially regulated by the reviewed disinfection treatments (Table S7; Supporting Information: Data S2.7).

4.7.1. General Stress Responses

General stress response proteins comprise a group of proteins that allow the bacteria to withstand various types of stress, including oxidative, acidic, and osmotic stress. Stressosome is a large bacterial protein complex that detects environmental stress and activates a general stress response (Martinez‐Bond et al. 2025). Upon stressosome activation, RsbT is released, which activates σB, a crucial regulator of the stress response managing over 300 genes related to stress resilience and virulence (Guerreiro et al. 2020). O₃ treatment reduces prli42 gene expression, which encodes the stressosome‐associated protein Prli42, in L. monocytogenes. This membrane‐anchored miniprotein is crucial for σB activation and essential for bacterial growth and survival under oxidative stress. Prli42 conveys the environmental stress signals to the stressomes (Impens et al. 2017).

4.7.2. Temperature Stress Responses

Disinfection treatments regulate several cold‐ and heat‐shock proteins in different ways. Csps are highly conserved small nucleic acid‐binding proteins involved in stress tolerance mechanisms (Muchaamba et al. 2021). Csp genes cspA, cspB, and cspD are induced upon exposure to various stresses, including cold, osmotic, oxidative, and desiccation. Csp gene levels in L. monocytogenes are upregulated in response to ClO₂ (cspB, cspD, and ltrC) and O₃ (cspD) treatment. Csps possibly counteract the harmful effects of stress by preserving cellular functions, such as DNA replication, transcription, and translation, thereby ensuring cell survival and growth (B. Schmid et al. 2009). Csps also play a role in biofilm formation and motility, which are critical for the persistence and transmission of L. monocytogenes in food‐processing environments (Eshwar et al. 2017). Similarly, heat shock response is a crucial process allowing the bacteria to survive and adapt to harsh environmental conditions. In response to temperature changes, heat shock response triggers the production of several proteins, known as Hsps (Maleki et al. 2016). Hsps primarily assist in proper protein folding and participate in the repair or degradation of misfolded proteins and harmful aggregates. When temperatures suddenly increase, Hsps rapidly accumulate in the cells. After the organism adapts, these protein levels decrease but remain elevated above their original baseline, facilitating bacterial growth even under suboptimal conditions (Roncarati and Scarlato 2017). ClO₂ increases the expression levels of Hsp‐related genes dnaK and grpE. Upregulation of Csp and Hsp levels in L. monocytogenes represents a stress adaptation mechanism that enhances the ability of the pathogen to survive and grow under the specific treatment conditions (B. Schmid et al. 2009).

4.7.3. Oxidative Stress Responses

Disinfectants often generate ROS and activate genes to combat oxidative stress, such as superoxide dismutase (SOD) and catalase. Increased expression of sigB‐dependent genes by oxidizing agents H₂O_2, O_3, and ClO₂ enables L. monocytogenes to activate genes protecting it from acid and oxidative stress. For instance, ClO₂ exposure increases the expression levels of oxidative stress response genes sodA, ohrA, spxA, spxH, lmo0906, and lmo0983 in L. monocytogenes, enabling it to combat oxidative stress. Other disinfection treatments, such as H₂O₂ (ohrR, ohrA, sodA, and gpo) and O₃ (sodA), also increase the levels of oxidative response genes. Levels of the sodA gene, which encodes SOD, are consistently upregulated by all oxidizing agents. SOD is an antioxidant enzyme playing an essential role in protecting cells from oxidative damage (Zheng et al. 2023). SOD catalyzes the conversion of superoxide (O₂•−) into oxygen and H₂O₂, transforming the highly reactive superoxide radical into less harmful substances (Manso et al. 2020). The increased expression of oxidative stress response genes enables L. monocytogenes to counteract the detrimental effects of oxidative damage caused by ROS (Bucur et al. 2018) from disinfectants, such as H₂O₂, O₃, and ClO₂, thereby improving its survival under oxidative stress.

4.7.4. Responses to Toxic Substances

Bacteria can adapt to chemical stress induced by toxic substances. Excessive or improper disinfectant application decreases their susceptibility and increases their tolerance (Rozman et al. 2021; van Dijk and Verbrugh 2022). Bacterial regulatory networks typically exhibit distinct changes in response to physical or chemical stresses, such as insufficient nutrient availability, elevated temperatures, disinfectants, and antibiotics (LeRoux et al. 2015). Danger sensing is the ability to identify external substances posing a threat to the organism. Levels of phosphate regulator gene phoU (ClO₂), arsenic resistance gene lmo2230 (H₂O₂ and ClO₂), and toxin resistance genes lmo2570 (ClO₂) and lmo1708 (ClO₂) are upregulated in L. monocytogenes in response to disinfection. In contrast, PAW downregulates the phoU and toxin resistance gene telA1 levels. The two‐component regulatory system is crucial for signal transduction, allowing bacteria to sense and respond to changes in various environmental conditions (Casado et al. 2022). In bacteria, the two‐component regulatory system governs the phosphate (Pho) regulon, with PhoU as its key negative regulator (Santos‐Beneit 2015). PhoU is a crucial regulator of persister cell development. Its inactivation leads to the loss of persisters, resulting in increased sensitivity to antibiotics and stress (Y. Li and Zhang 2007). Escherichia coli PhoU mutant cannot suppress the metabolic processes necessary for persister formation, thereby showing increased susceptibility (Y. Li and Zhang 2007).

4.7.5. DNA Damage (DNA Repair) Responses

Bacteria respond to agents causing DNA damage by activating the SOS response, which contributes to DNA repair (Arcari et al. 2020). Disinfection treatments H₂O₂ (dinB, uvrA, uvrB, recA, lexA, exoA, addA, addB, lmo0157, lmo2222, lmo2050, and lmo1639) and ClO₂ (fri, deoC, and lmo1514). RecA plays a crucial role in DNA repair and acts as a key activator of the SOS response in L. monocytogenes (Van Der Veen et al. 2010). RecA filaments bind to single‐stranded DNA, triggering the self‐cleavage of LexA, an SOS regulon repressor. Cleaved LexA is further processed by ClpXP bacterial proteases, which activate the SOS pathway (Cohn et al. 2011). This leads to a temporary halt in cell division, allowing DNA repair (Walker 1996). Nucleotide excision repair (NER) is another highly conserved multistep DNA recognition and repair process mediated by the UvrABC repair system (Truglio et al. 2004). UvrA detects damage and forms the heterotrimeric complex, UvrA2UvrB, with UvrB (Theis et al. 2000). UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision, and repair synthesis (Verhoeven et al. 2002). LexA and DnaA proteins regulate the transcription of uvrB (NER system), dinJ (antitoxin component of a type II toxin‐antitoxin system), and recN (DNA double‐strand break repair) in E. coli, thereby influencing the SOS regulon (Wurihan et al. 2018). SOS response of L. monocytogenes plays a role in its survival under various stressors, further contributing to its persistence in the environment (Van Der Veen et al. 2010).

4.7.6. Protein Quality Control and Homeostasis

In addition to inducing DNA damage, disinfection stress leads to the accumulation of damaged proteins, which must be eliminated by the cellular proteolytic systems. L. monocytogenes uses stress regulators and response genes to manage oxidative stress. Most stress response genes are regulated by SigB factors, including ctsR, which represses the caseinolytic protease (clp) operon under optimal conditions. CLP proteins, including both chaperones and proteases, are activated as a survival strategy under temperature and stress conditions (Manso et al. 2020). Clp ATPases are a large family of related proteins playing key housekeeping roles, including in protein reactivation, remodeling, and targeting of specific proteins for degradation by ClpP peptidase (Lemos and Burne 2002). ClO₂ (clpB, clpC, clpP, clpX, and ctsR) and PAW (clpP) increase the expression levels of Clp protease genes. ClpP is a tetradecameric peptidase degrading misfolded proteins aided by chaperones, such as ClpC and ClpX (Baker and Sauer 2012). The ClpXP complex plays crucial roles in protein quality control, SOS responses, and virulence in L. monocytogenes (Balogh et al. 2022). ClpX, a hexameric ATPase associated with diverse cellular activities (AAA+) and ATP‐driven chaperone, identifies protein substrates and channels unfolded peptide chains into the tetradecameric barrel of the ClpP serine protease for breakdown (Baker and Sauer 2012). L. monocytogenes possesses two ClpP isoforms, ClpP1 and ClpP2, which form a heterooligomeric complex with enhanced proteolytic activity. ClpP1 enhances degradation efficiency, whereas ClpP2 facilitates ClpX binding and functions as a substrate gatekeeper (Balogh et al. 2022). Regulation of clp genes is mediated by CtsR, which represses stress response gene expression (Wiktorczyk‐Kapischke et al. 2021).

4.8. Chemotaxis and Motility

Bacterial chemotaxis is a phenomenon by which bacteria sense changes in the chemical composition of their environment via chemoreceptors and migrate toward a favorable environment (Bren and Eisenbach 2000). Motility is a crucial feature allowing the bacteria to search for nutrients and avoid toxic compounds. Bacteria have to adapt to various stressors, including antibiotics and disinfectants, to survive (Piskovsky and Oliveira 2023). Particularly, disinfection significantly alters the regulation of several key genes involved in chemotaxis and flagellar biosynthesis (Table S8; Supporting Information: Data S2.8).

4.8.1. Chemotaxis

Chemotaxis plays vital roles in various biological processes, including stress responses, biofilm formation, QS, bacterial pathogenesis, and host infection (Karmakar 2021). Methyltransferase enzyme CheR plays a crucial role in chemotaxis, enabling bacteria to sense and respond to their environment by modifying the chemotaxis receptors. It regulates the chemotaxis pathway by modulating the methylation state of methyl‐accepting chemotaxis proteins in response to chemotactic gradients. O₃ and PAW treatments increase the expression levels of cheR in L. monocytogenes. Upregulation of chemotactic gene levels by O₃ and PAW treatment helps L. monocytogenes to navigate toward more favorable environments for survival. In contrast, ClO₂ treatment decreases the expression levels of chemotactic genes lmo1699 and lmo0693 in L. monocytogenes, limiting its potential to sense and respond to the stress caused by these treatments.

4.8.2. Bacterial Flagellum Biogenesis

Flagella are motile chemotactic devices facilitating bacterial survival, adaptation, and pathogenesis (O'Neil and Marquis 2006). O₃ (flgE and mogR) and PAW (fliH, flgE, and fliI) treatments increase the expression levels of genes responsible for flagellar assembly. Conversely, ClO₂ (flgD, fliK, and fliQ) and H₂O₂ (fliP) treatments decrease the expression levels of flagellar genes. O₃ treatment also suppresses the expression of specific genes (motB, fliN, flgB, fliE, and fliF) in L. monocytogenes. Many chemotaxis genes, such as cheA, cheY, and methyl‐accepting chemotaxis protein genes, coordinate with the flagellar assembly genes to support bacterial motility. Y. Li and Zhang (2007) identified a correlation between the increased expression levels of several genes related to flagella and chemotaxis and the absence of the negative regulator, PhoU, in E. coli. Consistently, downregulation of phoU levels by PAW treatment increased the expression levels of chemotaxis and flagellar genes. Conversely, upregulation of phoU genes by ClO₂ decreased these gene levels. These patterns suggest regulatory interactions between PhoU and motility genes that affect bacterial adaptation and energy expenditure under stress. Y. Li and Zhang (2007) further proposed that the loss of the negative regulator PhoU increases cellular metabolic activity, thereby enhancing susceptibility to various antibiotics and stressors.

Motility and flagellar gene expression levels in L. monocytogenes are strongly regulated by temperature. These gene levels are generally upregulated at environmental temperatures (≤ 30°C) and repressed at 37°C by the transcriptional repressor MogR (Ivy et al. 2010; Shen and Higgins 2006). This repression is halted at low temperatures by the anti‐repressor, GmaR, which acts as a thermosensor but becomes inactive or aggregates at high temperatures (Kamp and Higgins 2009, 2011). In this review, we compared disinfection treatments across various temperatures, ranging from 20°C (H₂O₂) to 37°C (ClO₂). Therefore, the variations in flagellar gene expression patterns indicated here reflect both the disinfection treatment and baseline regulatory influence of the incubation temperature.

4.9. QS and Virulence‐Related Genes

QS is a bacterial signaling mechanism enabling cell–cell communication within and between species by producing, detecting, and responding to the extracellular signaling molecules, known as autoinducers (AIs) (Ismail et al. 2016). This mechanism enables bacteria to regulate their gene expression, metabolic activity, and behaviors in response to variations in population density, thereby inducing biofilm formation (Preda and Săndulescu 2019; Rutherford and Bassler 2012). Expression levels of virulence factors, such as adhesins, internalins, surface proteins (e.g., ActA), siderophores, hemolysins (e.g., listeriolysin O), phospholipases, and toxins, and survival within host cells significantly influence the severity of L. monocytogenes infection (Coelho et al. 2018). Disinfection procedures influence multiple QS‐ and virulence‐related genes (Table S9; Supporting Information: Data S2.9), indicating their potential effects on bacterial communication and pathogenic traits.

4.9.1. Quorum Sensing

QS is an intercellular communication mechanism regulating gene expression across cellular processes (Waters and Bassler 2005). It is essential for bacterial adaptation and survival under different environmental conditions during infection, processing, and transmission (Banerji et al. 2022). PAW increases the levels of accessory gene regulator (agr)‐B, whereas ClO₂ upregulates the levels of luxS, which synthesizes AI‐2. The agr system is crucial for both biofilm formation and virulence and facilitates adaptation to changing environments (Zetzmann et al. 2016). In L. monocytogenes, agrB is essential for processing the precursor peptide from agrD to the mature autoinducing peptide (Rieu et al. 2007). Autoinducing peptide activates histidine kinase AgrC, which phosphorylates and activates response regulator AgrA, thereby regulating the genes associated with biofilm formation and virulence (Riedel et al. 2009). The agrBDCA operon is autoregulated, exhibiting higher expression levels during biofilm growth than during the planktonic or early attachment phases (Zetzmann et al. 2016). luxS encodes the LuxS enzyme, which is involved in the synthesis of AI‐2, a universal signaling molecule sensed by various bacterial species, facilitating interspecies communication. Communication enables bacteria to coordinate behaviors, such as biofilm formation, virulence, and antibiotic resistance (Wang et al. 2019). PAW and ClO₂ treatments upregulate the QS gene (agrB and luxS) levels, enhancing biofilm formation. This biofilm protects L. monocytogenes from disinfectants by forming a physical barrier and modifying the internal microenvironment.

4.9.2. Virulence‐Related Genes

The prfA virulence gene cluster (pVGC) is the primary pathogenicity island in L. monocytogenes, comprising prfA, plcA, hly, mpl, actA, and plcB. Levels of pVGC and other virulence factor genes are decreased by O₃ (comGB, ESAT‐6‐like secretion system [ess]‐A, essC, lmaC, plcA, actA, LMOf2365_0374, LMOf2365_0909, LMOf2365_1977, and LMOf2365_2034) and PAW (actA). In contrast, H₂O₂ (lhrC and lisK), PAW (inlA and iap), and O₃ (lysR, lmaD, and epsC) increase specific gene levels in L. monocytogenes. essA and essC are part of the ESS or type VII secretion system in Gram‐positive bacteria. This system is crucial for the identification and secretion of proteins enhancing virulence and bacterial competition (Zoltner et al. 2016; Burts et al. 2005). The ActA protein of L. monocytogenes plays a crucial role as a virulence factor. Actin polymerization is initiated on the bacterial surface, forming an actin tail that enables bacteria to move within and between the host cells (Pillich et al. 2017). lisK encodes the LisK protein, a histidine kinase in the LisRK two‐component system, in L. monocytogenes. This protein is crucial for the adaptation of organisms to environmental stresses, such as ethanol and oxidative stress (Alejandro‐Navarreto and Freitag 2024). Virulence gene expression changes enhance disinfection resistance by improving the stress tolerance, suppressing biofilm formation, and altering the cell surface properties in Gram‐positive pathogens (Frees et al. 2004; Rouhi et al. 2024; Kastbjerg et al. 2010). These varying transcriptional responses show that disinfection treatments either inhibit or stimulate virulence‐related pathways, influencing the ability of L. monocytogenes to persist under, adapt to, or withstand stress, depending on the environmental conditions.

Virulence gene expression in L. monocytogenes is regulated by temperature‐dependent mechanisms. Levels of genes within pVGC, including actA, plcA, and hly, are generally upregulated at 37°C but remain low at low temperatures. This control is mediated by a thermosensitive RNA structure in the 5′‐untranslated region of prfA, which unfolds at 37°C to allow translation (Johansson et al. 2002; Loh et al. 2012). Consequently, ClO₂ treatment at 37°C induces a greater baseline expression of virulence genes compared to that at low temperatures (e.g., H₂O₂ at 20°C; PAW at room temperature). This partially explains the variability in virulence gene expression under different treatments.

5. Limitations and Future Considerations

This review summarizes the overall genetic activity of L. monocytogenes under different disinfection treatments. Gene expression changes in L. monocytogenes in response to different disinfectants provide valuable insights into the cellular mechanisms of this foodborne pathogen under stress conditions. However, the limited number of studies on L. monocytogenes responses to disinfection treatments makes it difficult to conclude that this pathogen responds solely in the discussed manner. Moreover, this review article has several limitations resulting from the types of disinfection treatments selected for comparison. One major issue is the variability in the treatment conditions (Table 1) applied to L. monocytogenes, which complicates the interpretation of results and identification of specific patterns related to particular conditions. Additionally, most discussed studies did not document the nature of the bacteria (bactericidal, bacteriostatic, or exhibiting reduced growth) during RNA isolation for transcriptomic analyses. For instance, populations of L. monocytogenes after treatment with BZT (Casey et al. 2014), LA, and H₂O₂ (Cortes et al. 2020) were not recorded. Moreover, the substrate or medium used for L. monocytogenes disinfection varied among the selected studies. For example, O₃ was applied to the surface of tomatoes, whereas BZT, LA, H₂O₂, and HPP were applied to L. monocytogenes suspended in TSB or the tryptone soy yeast extract broth. ClO₂ and PMF were applied to L. monocytogenes suspended in the brain heart infusion broth and phosphate‐buffered saline, respectively.

Each experiment included in our analysis was conducted under different temperature conditions, which may have influenced the growth kinetics and stress responses of L. monocytogenes (Poimenidou et al. 2023). Temperature is a critical factor affecting both the efficacy of chemical oxidants and the physiological resilience of bacterial cells (Gélinas et al. 1984; Manso et al. 2020). Additionally, different L. monocytogenes strains from multiple serotypes, such as 1/2a, 1/2c, and 4b, have been used in various studies (Table 1). Considering the well‐documented genetic and phenotypic diversity of L. monocytogenes strains, including differences in their stress tolerance and virulence, this variability further complicates the interpretation of the results (Nightingale et al. 2005; Muchaamba et al. 2022). However, it is important to note that only a small number of strains were evaluated in the studies discussed, which limits how well the findings apply to the broader L. monocytogenes population. Collectively, these differences make it difficult to draw definitive conclusions regarding the typical responses of L. monocytogenes to the chemical oxidants discussed in this review.

In addition to the disinfection treatments and bacterial conditions, the methods used for transcriptome analysis, such as RNA‐sequencing and microarray analysis, also varied among the included studies. Moreover, the fold change (FC) threshold, an important factor to accurately compare the transcriptomic responses of L. monocytogenes under different treatment conditions, varied across studies. For example, DEGs in L. monocytogenes treated with BZT were analyzed using FC thresholds ≥ 7.5 (Fox et al. 2011) and ≥ 4 (Casey et al. 2014). In contrast, with LA, H₂O₂, and PAW treatments, DEGs were identified using log2 FC > 1. Studies using low FC thresholds identified many DEGs, including those showing minor expression changes. Conversely, studies with high FC thresholds only identified the genes exhibiting significant expression changes. These varying FC cutoff points affected the results, impacting their accurate comparison and interpretation.

Results interpretation under different experimental conditions requires careful analysis and assessment. Investigation of a specific technique under bactericidal, bacteriostatic, and reduced growth conditions can reveal the differences in gene expression. Thorough analysis is necessary when significant variations are observed. For instance, comparative analysis of their behaviors in biofilm‐forming environments and suspensions can provide insights into bacterial adaptive responses. Such meticulous analysis is crucial for understanding complex microbial behaviors and guiding future research on bacterial growth control.

Another limitation is that most transcriptomic studies of L. monocytogenes under oxidative stress conditions, as mentioned in this paper, use short exposure times—often ranging from 5 min to 1 h, which primarily capture the bacterium's initial adaptive responses rather than its fully developed physiological states and do not address the persistence of these transcriptional changes. These early responses involve the rapid upregulation of general stress response genes, such as those controlled by alternative sigma factors (e.g., sigB), chaperones, and noncoding RNAs, which help reduce immediate damage and prepare the cell for survival (Cortes et al. 2020; Anast and Schmitz‐Esser 2020). However, these transcriptional changes are usually temporary and might not accurately reflect the long‐term regulatory adjustments, metabolic reprogramming, or structural changes needed for sustained stress tolerance (Bren et al. 2023; Kratz and Banerjee 2024). Furthermore, the transient activation of these stress responses could lead to cross‐protection, where sublethal exposure to an oxidant preadapts the bacterium to tolerate subsequent, unrelated stresses (e.g., acid, osmotic, or thermal stress) that it encounters later (Ferreira et al. 2003; Lou and Yousef 1997). Additionally, many stress‐induced gene expression changes are reversible; the final physiological state of the cell depends on the duration and intensity of the stress, as well as post‐transcriptional, translational, and post‐translational regulatory layers (Roemhild et al. 2022; Papadimitriou et al. 2016). Therefore, relying on short‐term transcriptome data may overemphasize early responses while underestimating the complexity of ongoing adaptation mechanisms, highlighting the need for integrated time‐course and multiomics studies to fully understand the stress physiology of L. monocytogenes.

Furthermore, it is essential to acknowledge that transcriptomic data alone do not necessarily confirm the functional significance of gene expression changes. Although stress triggers rapid and widespread changes in gene transcription (e.g., via SigB), the actual protein levels or subsequent metabolic activity often do not correlate with mRNA abundance due to post‐transcriptional, translational, and post‐translational regulation (Sibanda and Buys 2022; Chaturongakul et al. 2008). While proteomic studies on L. monocytogenes under oxidative stress are currently limited, some research suggests that there may be a poor correlation between mRNA levels and corresponding protein expression under stress conditions (Johansson and Freitag 2019). This includes regulation by RNA‐binding proteins (such as Hfq), small RNAs, mRNA stability, translation efficiency, and protein degradation rates (Seixas et al. 2025). Additionally, extrinsic biological noise and environmental fluctuations further contribute to the disconnect between transcript and protein levels. As a result, relying solely on transcriptomic data may misrepresent the actual physiological state of L. monocytogenes under stress. This underscores the importance of integrating proteomic analyses to achieve a comprehensive understanding of bacterial adaptation and survival mechanisms (Bowman et al. 2012; Chaturongakul et al. 2008). Combining data from transcriptomic studies with proteomic and functional assays would offer a more accurate depiction of the pathogen's survival strategies.

6. Conclusion

L. monocytogenes can adapt to and survive under disinfection stress, posing significant challenges for the food industry. This review highlights its complex transcriptional responses to various chemical oxidants. Oxidizing agents (e.g., ClO₂, H₂O₂, and O₃) induce strong oxidative stress responses (e.g., sodA and ohrA) and activate DNA repair systems (recA and uvrABC) in L. monocytogenes. Nonoxidizing treatments (e.g., BZT and HPP) upregulate the cell wall biosynthesis gene (murA and glmS) levels, hindering disinfectant penetration. Interestingly, sublethal exposure to some disinfectants unintentionally increases the virulence (inlA and lisK) and QS (agrB and luxS) gene levels, enhancing the adaptive tolerance of L. monocytogenes. Changes in efflux pumps (qacC and ebrA) and metabolic shifts (e.g., activation of PPP and glycerol utilization) further highlight the adaptability of this pathogen. However, variations in experimental conditions (e.g., disinfectant concentration and bacterial physiological state), transcriptomic analysis methods, and DEG thresholds across studies limit the comparability of the data. To enhance the disinfection efficacy, future studies should use standardized methodologies to investigate the pathogen responses under different conditions and states, including biofilm‐forming environments and the planktonic (suspended) state. A deeper understanding of the underlying molecular mechanisms is critical to develop effective control measures against L. monocytogenes contamination in food processing environments and prevent listeriosis.

Author Contributions

Nagendran Rajalingam: conceptualization, investigation, methodology, writing–original draft, software, formal analysis, data curation, writing–review and editing. Sam Van Haute: funding acquisition, writing–review and editing, validation, visualization, project administration, supervision, resources, formal analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting Material: crf370260‐sup‐0001‐SuppMat.docx

Supporting Table and FIgure: crf370260‐sup‐0002‐SuppMat.docx

Rajalingam, N. , and Van Haute S.. 2025. “Adaptive Tolerance of Listeria monocytogenes to Chemical Oxidants: Comparative Analysis of Transcriptomic Studies.” Comprehensive Reviews in Food Science and Food Safety 24, no. 5: 24, e70260. 10.1111/1541-4337.70260