Resistance of Listeria monocytogenes to Stress Conditions Encountered in Food and Food Processing Environments

1Along the food chain, bacteria are constantly exposed to a wide range of stress factors, which affect their activity and viability. These stresses are either intrinsic to the food matrix or extrinsic factors intentionally applied to preserve food or imposed onto the organisms upon consumption by the host. L. monocytogenes is an important food-borne pathogen that frequently causes food recalls1,2 and disease outbreaks with significant case numbers and a mortality rate of 20–30%3 worldwide. This organism is known for its ability to survive or even to replicate under a wide range of environmental stress conditions. Resistance to stress supports colonization and persistence of L. monocytogenes in various niches along the food chain and thus formation of reservoirs for contamination. Moreover, it ultimately contributes to the ability of this bacterium to infect humans.

2The stresses encountered by L. monocytogenes in foods include those that are a consequence of various methods of preservation, including traditional ones as acidic pH due to fermentation by e.g., lactic acid bacteria (LAB), and osmotic stress by increased salt concentrations and more contemporary ones as using of growth inhibitors including bacteriocins and other food preservatives. It should be mentioned that bacteriocins, which are small antimicrobial peptides, may either be naturally produced by bacteria used for food fermentation or can be added exogenously as a preserving additive. On the other hand, there are measures of food preservation that are rather technical in nature and are designed either to kill pathogens and spoilage microorganism at the processing stage [thermal treatments and its alternatives high pressure, pulsed electric fields (PEF), radiation] or even to protect foods during their storage (low temperatures/refrigeration, low oxygen concentrations, presence of protective gases in the surrounding atmosphere).

3The same stress may occur on several occasions along the food chain. For example, L. monocytogenes may be exposed to pH and osmotic stress first in the food matrix as consequence of fermentation or food preservation and subsequently in the host gastrointestinal tract. In this respect, it is of importance that resistance to different stresses is interconnected. For example incubation of L. monocytogenes at low temperatures enhances its resistance to high salt concentrations. Likewise, osmotic stress in L. monocytogenes can lead to cross-protection against other causes of injury, including heat, ethanol, acidity, alkalinity, and oxidative stress. This is, at least partially explained by the fact that the stress signal received by the two component systems liaRS, lisRK, cesRK, agrCA, and virRS, which have been demonstrated to play a role in the stress response, converge on the level of SigB, which is the alternative sigma factor σB that controls the general stress response in L. monocytogenes and other Gram-positive bacteria. For L. monocytogenes, SigB has been shown to be involved in the resistance to acidity, osmotic stress, cold and freezing stress, oxidative stress, and high hydrostatic pressure. Appropriate resistance mechanisms are triggered by activation of σB-dependent promoters. The extremely high tolerance to stressful conditions makes L. monocytogenes a major concern in food processing and a suitable model organism to study resistance mechanisms to stress conditions encountered in food and food processing environments.

4Some of the mechanisms (and consequences) of resistance of L. monocytogenes have been expertly reviewed previously. However, most of these reviews focus on osmotic, pH, and temperature stress. With the present review we aim at providing a summary of the current knowledge on resistance and associated mechanisms of L. monocytogenes with a clear focus on stressful conditions that arise from traditional or alternative methods of food processing, preservation and decontamination. While we will touch upon stresses reviewed elsewhere (acidic pH, osmolarity, high and low temperatures, oxidative stress), we will also discuss resistance of L. monocytogenes to other stress conditions that have not gained as much attention (bacteriocins, pulsed or continuous UV radiation or visible light, electrical fields, high pressure). A deeper understanding of the mechanisms used by L. monocytogenes to survive and proliferate in food products may help food specialists to design efficient preservation methods that will extend shelf lives and provide a better protection of consumers against this pathogen while at the same time maintain the sensory and nutritional properties of the food products.

5Thermal treatments and temperature control are strategies that have been applied in food production and preservation for centuries to prevent or limit contamination and outgrowth of food-borne pathogens. However, the efficacy of thermal treatments against L. monocytogenes is limited by the intrinsic ability of this pathogen to survive and actively replicate at temperatures between −0.4 and 45°C.

6Mild thermal treatments (<100°C) are largely applied in food processing in order to inactivate vegetative microbial cells of food-spoilage bacteria and food-borne pathogens. Such treatments ensure food safety and prolonged shelf life as long as food products are properly packed and adequately stored. Despite these benefits, thermal treatments can have a negative impact on the quality of food affecting the nutritional value and sensory properties. However, the main concern with thermal processing of foods remains the ability of sublethally injured pathogenic bacteria to recover and grow during post-processing storage. This is of particular relevance for L. monocytogenes with its ability to grow in a wide temperature range. Although L. monocytogenes does not manifest an extraordinary resistance to high temperatures, it was shown to be more heat tolerant than other non-spore-forming pathogens such as Salmonella and E. coli. Factors that influence the resistance of L. monocytogenes to heat vary among strains, bacterial cells’ age, test and growth conditions, previous environmental stresses, or food components.

7L. monocytogenes has been shown to survive the minimum high-temperature, short-time treatment imposed by U.S. Food and Drug Administration (71.7°C, 15 s) in the case of milk collected from deliberately contaminated cows. Early studies raised the possibility that polymorphonuclear leukocytes present in milk may have a protective effect on L. monocytogenes during heat treatments residing inside these cells. However, subsequent reports showed that, in naturally contaminated milk, L. monocytogenes was not able to resist temperatures greater than 67.5°C combined with a holding time of 16.2 s. Nevertheless, the conditions associated with dairy products seem to influence the resistance of L. monocytogenes to heat treatments. For instance, Casadei and colleagues showed that limited access to essential nutrients in butter and the physical structure of this food could induce a starvation state in L. monocytogenes cells correlated with cross-resistance to other types of stress. In this case L. monocytogenes Scott A grown within this food matrix was four times more resistant to a treatment at 60°C than the same strain grown in TSB broth. Furthermore, L. monocytogenes was shown to survive relatively high temperatures in heat-treated meat, egg products and vegetables such as mushrooms and peas.

8Resistance of L. monocytogenes strains to heat can vary significantly among serotypes. In one study, strains belonging to serotype 1/2a showed relatively low tolerance to heat (up to 2 log CFU/mL), while strains representing serotypes 1/2b and 4b exhibited an extensive variability (from undetectable to 4 log CFU/mL). The highest heat tolerance was recorded for a serotype 7 strain (5 log CFU/mL).

9L. monocytogenes cells exposed to sublethal stresses prior to thermal challenge can become considerably more heat resistant. Shen and colleagues found that exposure of L. monocytogenes to a temperature of 48°C for 30 min led to heat stress adaptation among bacterial cells. Moreover, subjection to this mild stress for a short period of time did not affect the capacity of growing. Salt was also shown to potentiate the ability of L. monocytogenes to withstand thermal treatments. For instance, the D63°C value, which is the time required to kill 90% of bacteria when exposed to the temperature of 63°C, for Scott A strain inoculated in egg products with 10% NaCl increased approximately 6 times in comparison with that of the same strain processed in egg products without salt. This may be due to the protective effect of decreased water activity in the growth medium. Acidity is another factor that can influence bacteria’s thermotolerance. Acid-adaptation in fruit juices was shown to substantially increase the resistance of L. monocytogenes to subsequent heat treatment. Also, L. monocytogenes displays an increased heat tolerance and a significantly increased D60°C value (2.2 min) in stationary compared to exponential growth phase (0.6 min).

10At the molecular level, the response of L. monocytogenes to 48°C involves the expression of genes belonging to specific heat-shock regulons, namely class I and class III heat-shock genes, and genes of the SigB-dependent class II stress response. The upregulation of recA expression, an activator of SOS response implicated in DNA repair could be also observed. Class I heat-shock genes (grpE, dnaK, dnaJ, groEL, and groES) encode for heat-shock proteins (HSPs) that act as intra-cellular chaperones whose expression is increased when denatured proteins accumulate in cytoplasm (Figure 1Ba). The role of HSPs is to stabilize and assemble partially unfolded proteins, preventing their aggregation under stress conditions. Under physiological growth conditions at ambient temperature, expression of class I heat-shock genes is controlled by the HrcA repressor (Figure 1Aa), which in turn is encoded by the first gene of the dnaK operon. Class III heat-shock genes encode for ATP-dependent proteases (ClpC, ClpP, and ClpE) required for degradation of misfolded proteins under stress conditions including high temperature (Figure 1Bb). These proteases are negatively regulated by the CtsR repressor (Figure 1Ab), which is the product of the first gene of the clpC operon. The ClpL protease was recently found to play a considerable role in the elevated temperature tolerance of L. monocytogenes AT3E. This finding was observed upon curing the strain from plasmid pLM58, which harbors clpL, resulting in a strain with reduced heat resistance. Moreover, insertion of clpL increased the resistance of a heat-sensitive L. monocytogenes strain.

11L. monocytogenes is considered a psychrotolerant bacterium due to its ability to grow at temperatures as low as −0.4°C. This tolerance to cold stress is responsible for the frequent detection of L. monocytogenes in refrigerated food products, especially meat, poultry, and seafood. Low temperatures result in decreased metabolic rates and changes in membrane composition, expression of cold shock proteins (Csps), and uptake of cryoprotective compounds.

12The alterations in the membrane in response to cold stress comprise a reduced chain length of fatty acids, an increase in the concentration of unsaturated fatty acids, and altered ratios of iso- and anteiso-branched fatty acids. These changes maintain fluidity of the membrane at low temperatures and prevent formation of a gel-like state that may result in leakage of cytoplasmic content.

13Csps are small proteins (65–70 amino acids long) with a highly conserved structure. They bind to single-stranded nucleic acid molecules via their ribonucleoprotein binding motifs RNP1 and RNP2. This stabilizes the conformation of the nucleic acid and prevents degradation. Thus, Csps act as molecular chaperones that facilitate replication, transcription, and translation at low temperatures. CspA, CspB, and CspD contribute to resistance to low temperatures albeit with different importance. Interestingly, they also seem to be involved in the resistance to osmotic stress. The ferritin-like protein (Flp) was highly induced in response to cold shock suggesting it is involved in response to cold stress. determined the cold shock regulon by genome-wide expression analysis and could show that expresion of 105 and 170 genes was increased during growth on 4°C vs. 37°C in logarithmic- and stationary-phase with an overlap of 30 genes including cspL. Of these 30 genes, many are involved in membrane and cell wall function, lipid metabolism, transcription or translation.

14Another mechanism of L. monocytogenes to counteract cold stress is the import of osmolytes such as glycine betaine, carnitine, γ-butyrobetaine, proline betaine, and 3-dimethylsulphoniopropionate as cryoprotectants. In the above mentioned genome-wide transcriptional analysis, the opuCABCD operon, which encodes a carnitine transporter, and gbuC encoding the substrate binding protein of a glycine betaine transporter showed increased expression in exponential growth phase at 4°C compared to 37°C. Similarly, expression of opuCA and betL were increased after exposure of L. monocytogenes S1 to cold and freezing stress as shown by quantitative RT-PCR. This confirmed previous observations showing that Gbu-mediated betaine uptake improves growth under cold stress and uptake of betaine via BetL and OpuC transport betaine slightly improves cryotolerance. In the same study, OpuC was shown to be the main carnitine transporter, which provided markedly higher resistance to cold stress than betaine uptake.

15Acidification is a method of food preservation widely applied to dairy, meat and vegetable products for centuries and is primarily achieved by fermentation by bacteria either present in the raw food or added as starter cultures. The preserving effect is achieved, on the one hand, by the metabolic end products, which are weak organic acids (e.g., acetate, lactate) that have anti-microbial activity, and, on the other hand, by inhibition of microbial growth at low pH.

16Both planktonic and surface attached cells of L. monocytogenes display adaptive acid tolerance response (ATR), i.e., bacteria pre-exposed to mild acid stress (pH 5.0) showed higher survival to subsequent challenge at a lower pH (3.0) compared to untreated bacteria. The extent of ATR may be influenced by the structural properties of the food matrices. For example, L. monocytogenes grown on the surface of meat product slices formulated with potassium lactate and sodium diacetate exhibited higher resistance to a pH of 1.5 than the same bacteria exposed to the same pH in homogenates of the meat product. Similar observations were made for L. monocytogenes incubated on tomato, lettuce or in culture media for 5 days at 5°C. Bacteria incubated on vegetables were more tolerant to exposure to acidic conditions induced by lactic acid, acetic acid or hydrochloric acid than those kept in tryptic soy broth under the same conditions.

17L. monocytogenes has several mechanisms to maintain its internal pH (pHi) under acid stress (Table 1) including the F0F1-ATPase, the glutamatic acid decarboxylase (GAD; ), and the arginine and agmatine deiminases (ADI and AgDI; ). The F0F1-ATPase is involved in ATR initiation during mild pH stress. The GAD system confers resistance to more severe acidic conditions (pH < 4.5; ) and has also been shown to be activated as result of reduced oxygen availability associated with food atmosphere packaging. It is comprised of two proteins, a cytoplasmic glutamate decarboxylase (GadA or GadB) and a glutamate/GABA antiporter (GadC) located in the cytoplasmic membrane. The role of the GAD system is to increase pHi by converting extracellular glutamate to Γ-aminobutyrate (GABA) in an enzymatic reaction that reduces the intracellular proton concentration. The ADI and AgDI systems are both involved in the response of L. monocytogenes to extreme acidity. ADI imports arginine molecules from the extracellular environment, converting them to ornithine, CO2, ammonia (NH3), and ATP. NH3 is then protonated to ammonium (NH4), which increases pHi. The same is true for AgDI, which converts agmatine into putrescine and NH3.

18Osmotic stress in food is mostly the result of increased concentrations of salts or sugars that are added to improve the sensory properties and as preserving agents to increase the shelf life of seafood, cheese, salami, pickles, jams, or syrups. The presence and concentration of these additives determine water activity and affect bacterial cells by challenging the osmotic balance between cytoplasm and extracellular environment.

19In response to elevated concentrations of salt, L. monocytogenes accumulates osmolytes, known also as compatible solutes, such as carnitine and glycine betaine in the cytoplasm to reduce osmotic pressure and water loss. Besides their property to keep turgor pressure under control, compatible solutes were also shown to stabilize enzymes’ structure and function during stress. This mechanism is mediated by an increased expression of genes encoding for proteins involved in the transport of the respective compatible solutes. These are the main carnitine transport system encoded by opuCABCD operon, the glycine betaine porter II system, encoded by gbuABC, and the sodium-motive-force-dependent glycine betaine uptake system, encoded by betL (; Figure 2). L-carnitine is present in raw meat in relevant quantities, while glycine betaine is found in vegetables (e.g., sugar beet, spinach, cereals). OpuCABCD couples ATP hydrolysis to osmolyte transport across the cytoplasmic membrane. This system is formed of OpuCA that hydrolyses ATP providing the energy for transport of the substrate by a complex consisting of the two transmembrane proteins OpuCB and OpuCD and a solute-binding protein OpuCC. While BetL is involved in the primary response of L. monocytogenes to salt, GbuABC seems to administer the capacity of this bacterium to tolerate such stress during a long-term exposure.

20In response to osmotic stress, L. monocytogenes can also adjust expression levels of genes other than those associated with osmolytes accumulation. For example, growth of L. monocytogenes under salt stress resulted in increased expression of genes for Csps, especially cspA and cspD, promoting also adaptation to cold stress. The chaperone activity of these proteins is thought to facilitate the repair of DNA lesions, since NaCl has been shown to induce DNA breaks. On the other hand, Bae and colleagues showed that presence of salt in the growth medium led to a decreased expression of genes associated with carbohydrate PTS systems in L. monocytogenes including those related to uptake of β-glucoside, galactitol, fructose, and cellobiose. This suggests a possible connection between a significantly lower growth rate and reduced uptake of carbohydrates under osmotic stress.

21While accumulation of compatible solutes plays the main role in L. monocytogenes’ survival to hyper-osmotic shock, a potential response of this bacterium to hypo-osmotic conditions may be mediated by mechanosensitive channels. Bacterial mechanosensitive ion channels regulate turgor pressure by assisting efflux of osmolytes. So far, genes for two putative mechanosensitive channels have been identified in L. monocytogenes. The lmo2064 gene shows significant homology to mscL from E. coli, which encodes for a large-conductance mechanosensitive channel (MscL). Additionally, lmo1013 is similar to mscS of Streptococcus pneumoniae encoding for a small-conductance mechanosensitive channel (MscS). After subjection to osmotic downshock, L. monocytogenes cells have been shown to release almost instantaneously betaine and L-carnitine, which may be linked to the activity of these channels.

22Bacteriocins are antimicrobial peptides produced by a wide range of LAB and are mostly active against Gram-positive bacteria including L. monocytogenes. Bacteriocins are natural and safe food additives for a wide range of food products including fruits, vegetables, dairy products, and meat, that are either produced in situ by LAB used for food fermentation or added exogenously. Most bacteriocins are highly specific for their target organisms and kill their targets by inhibiting growth, disruption of membrane homeostasis and pore formation.

23The only bacteriocin approved as preserving additive in food is nisin, which belongs to the class I bacteriocins and has a broad activity against various Gram-positive bacteria. Nisin is widely used in dairy and meat products with the purpose to inhibit the growth of food-borne pathogens including L. monocytogenes and Clostridium botulinum. The antimicrobial activity of this bacteriocin is mediateded by two mechanisms. Nisin inhibits the cell wall biosynthesis by binding and sequestering lipid II, which is an essential carrier molecule for peptidoglycan building blocks. Moreover, nisin-lipid II complexes form pores in the membrane leading to permeabilization.

24Resistance of L. monocytogenes to nisin has been associated with a series of changes in the cytoplasmic membrane composition aiming to prevent the peptide from crossing this barrier. The studies conducted on nisin-resistant (Nisr) cells noticed a reduction in the content of phospholipids with particular emphasis on phosphatidylglycerol and diphosphatidylglycerol, major components correlated with the interaction between nisin and membrane. In addition, it was indicated an increase in the proportion of straight-chain fatty acids to the detriment of branched-chain fatty acids, changes that result in a less fluid and, in the same time, more rigid cell membrane. The alterations in the cell wall of Nisr strains of L. monocytogenes have been also investigated. The resistance of Nisr cells to the degradation action of lysozyme and their sensitivity to benzylpenicillin and ampicillin suggested compositional changes that occurred at the level of this cellular component. One example could be the D-alanine esterification of teichoic acids. However, a recent transcriptomic analysis of L. monocytogenes survival cells following the exposure to a high nisin concentration reported the downregulation of dltA and dltB, implying that D-alanine residues are not involved in the elevated resistance to this bacteriocin. The study also emphasized the expression regulation of two cell-wall associated genes: downregulation of lmo2714 encoding for a peptidoglycan anchored protein and upregulation of lmo2522 encoding for a cell wall-binding protein with possible implication in nisin tolerance.

25In L. monocytogenes, nisin resistance is directly mediated by VirR, the response regulator of the VirRS two component system previously described to be involved in resistance to stress. However, in the case of nisin an ABC-transporter encoded by virAB seems to be responsible in perception of the stressor instead of the VirS receptor histidine kinase. VirR mediates the resistance to nisin and other stresses of the cell envelop by regulating the dltABCD operon that is responsible for modification of lipoteichoic acids. Other two component systems that were shown to be involved in resistance to nisin are LiaRS and LisRK. Genes/operons and their products regulated by these TCS with a reported role in resistance to nisin are lmo2229, telA, mprF, anrAB, and dltABCD. With the exception of telA, all these genes have a known role in metabolism/biosynthesis of components of the membrane or cell wall. Similar to e.g., resistance to pH or salt stress, nisin resistance can also be induced by other stresses, e.g., increased salt concentrations.

26Recently, a number of class II bacteriocins with activity against L. monocytogenes have been isolated and characterized including pediocins, sakacin P, leucocins, enterococin, mesentericin Y105, garvicin, linocin M18, and others. L. monocytogenes and other bacteria are able to develop resistance to bacteriocins. Natural resistance is observed with a frequency of 1–8% depending on the bacteriocin and the L. monocytogenes strain tested. Consistent with the receptors and mechanisms of action of bacteriocins, resistant strains show altered expression or mutations in certain phosphotransferase systems (PTSs). For instance, a spontaneous leucocin-resistant mutant of L. monocytogenes B73 was lacking a putative IIAB subunit of a mannose PTS. Furthermore, pediocin PA-1-resistant L. monocytogenes 412 mutants overexpressed gene fragments associated with a β-glucoside-specific PTS. Similar results of other studies suggest that resistance of L. monocytogenes to class IIa bacteriocins is correlated with a general mechanism consisting of a lack in EII subunits of mannose PTS and a compensatory upregulation of the β-glucoside PTS genes.

27In recent years, a number of novel technologies are applied by the industry for production and preservation of minimally processed foods and diminish the impact of chemical substances on the environment. Consequently this results in new stress conditions encountered by L. monocytogenes.

28High pressure processing (HPP) is a technology used in food preservation as an alternative to thermal treatments, aiming to destroy food spoilage microorganisms and food-borne pathogens. Depending on the food and spoilage organisms, pressures applied for sterilization are usually between 250 and 700 MPa. Bacterial cells subjected to HPP treatments display morphological and physiological changes that may be reversible depending on pressure and holding time. Primary effects of HPP are an increase in the permeability of the cell membrane, the disruption of the protein structure and function, and, as a consequence, inhibition of the metabolism, replication, and transcription.

29The effect of HPP on survival of L. monocytogenes was tested under various settings in different food products including cheese, fruit juice, jams, whole milk, and RTE cooked meat products. Overall, the results of these studies indicate that resistance of L. monocytogenes to HPP varies depending on the strain. For instance, when pressured with 350 MPa at 20°C, L. monocytogenes EGD-e displayed only 1.0 log CFU/mL reduction and was more resistant to this HPP treatment than LO28 strain (1.8 log CFU/mL reduction) and ScottA strain (3.2 log CFU/mL reduction). In addition, the type, composition and matrix of food products have an impact on the resistance of bacteria to HPP. Vitamins, amino acids, and cations (Ca2+, Mg2+) may have protective effects. For example, Mg2+ is known to stabilize ribosome structure and Ca2+ strengthens the outer membrane. Also, elevated salt concentrations in a food product may induce uptake of compatible solutes, which in turn stabilize cells during HPP. In line with this, a mutant deficient in synthesis of the compatible solute proline showed increased sensitivity to HPP

30The effect of HPP on L. monocytogenes was investigated on the global transcriptomic level by microarray analysis with subsequent RT-PCR on some target genes. This indicated that mRNA levels were reduced globally with increasing intensity and duration of the treatment. Nevertheless, HPP induced expression of genes associated with DNA repair, transcription, translation, cell division, protein secretion, motility, chemotaxis, and membrane and cell wall biosynthesis. On the other hand, reduced expression was observed for genes involved in carbohydrates’ uptake, energy metabolism and virulence. Surprisingly, HPP seemed to reduce expression of the general stress sigma factor SigB and part of the SigB regulon. One of the genes showing highest induction by HPP was cspL encoding a cold-shock protein. This suggests that HPP also induces cross-resistance to other stresses. For example, HPP resistance in semi-skimmed milk was higher than in buffer and the resistant isolate was also more resistant to heat, acid, and oxidative stress.

31Mutations in CtsR, a class III stress genes repressor, have been linked to spontaneous resistance of L. monocytogenes cells to HPP. Mutants with a stable resistance showed point mutations, insertions or deletions in the ctsR gene that negatively affected its activity. This loss in ctsR function in HPP resistant variants of L. monocytogenes was accompanied by increased expression of clpB, clpC, clpE, and clpP. Clp proteases have a clear role in degradation of misfolded or damaged proteins preventing their potentially harmful accumulation in bacterial cells. Since protein denaturation is one of the consequences of HPP treatment increased Clp protease activity is in line with increased HPP tolerance in L. monocytogenes. However, isolation of resistant mutants that do not display these changes indicates that there may be other unknown mechanisms conferring resistance to HPP. Moreover, reported that different levels of HPP resistance among L. monocytogenes strains are not based on ctsR gene mutations.

32L. mononcytogenes ScottA and a spontaneous HPP resistant isolate of this strain were shown to be more resistant to HPP in stationary compared to exponential growth phase. Moreover, it seems that cells in stationary phase of growth do not exhibit the highest resistance to HPP treatment. L. monocytogenes cells found in long-term-survival phase showed even higher HPP tolerance, as transition back to log and stationary phases resulted in less survivors after pressurization. This phenomenon has been attributed to a change in cell morphology from rods to cocci that results in cytoplasmic condensation and, implicitly, reduction of intracellular water activity.

33Another more recent method of food decontamination, included under the umbrella of alternative technologies, is pulsed or continuous UV-light, which kills microorganisms found on the surface of food products as result of cross-contamination occurring during processing procedures such as cutting, slicing or packing. Although approved by the United States and Food and Drug Administration (USFDA) for food application in 1996, the safety of this technology regarding the potential of permanent microbial inactivation still remains under question.

34The bactericidal effect of UV light is caused by DNA damage as a consequence of the formation of photoproducts including cyclobutane-pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs), and their Dewar isomers. Other mechanisms of bacteria inactivation caused by UV light are the photophysical and photothermal effects resulting in leakage of cellular content following the absorption of the high energy light pulses. The efficacy of UV treatment in decontamination of food surfaces depends on a number of factors including the food product, distance and position of the product to the light source, energy level given by number and frequency of the light pulses, level of contamination and others. The potential of UV-C light in L. monocytogenes inactivation was shown to be lower on fruits with smooth surface (apples and pears) compared to fruits with a rougher surface (cantaloupe, strawberry or raspberry). UV light is also used as disinfection procedure to improve hygiene in food processing environments. The presence of organic materials such as food debris on stainless steel surfaces appeared to protect L. monocytogenes cells against UV-C radiation.

35Several studies have been conducted in order to investigate the efficacy of L. monocytogenes inactivation by pulsed UV-light on/within various food matrices. A maximum of inactivation of L. monocytogenes ScottA on the skin side of salmon filets was achieved with 180 pulses of UV light of 5.6 J/cm2 at a distance of 8 cm for 60 s and efficacy was markedly lower on the muscle side. Similarly, the best inactivation rates of the same strain on chicken frankfurters was obtained with 180 pulses in 60 s at UV energy of 1.27/cm2.

36L. monocytogenes has been reported to be more resistant to UV-light than other pathogens, such as E. coli. However, very little is known regarding specific mechanisms of UV resistance in L. monocytogenes. Sublethal challenge with other stresses does not induce cross-resistance to UV light and UV resistance does not seem to depend on SigB, the general stress sigma factor of L. monocytogenes. Global gene expression analysis of the response to both pulsed light (PL) and continuous ultraviolet treatment was conducted in L. monocytogenes 10403S. Although the overall amplitude of the changes in gene expression was low, a number of genes encoding for stress proteins, motility and transcriptional regulators were induced by UV exposure. However, no increased expression was observed for lmo0588. This gene encodes for a (putative) photolyase. This protein plays an important role in photoreactivation, which is the recovery of bacteria sublethally injured by UV light due to subsequent exposure of visible light. During photoreactivation, photolyase binds and repairs the pyrimidine DNA lesions using light energy absorbed by its chromophores. In fact, an increase in viability was observed for a UV-treated L. monocytogenes serotype 1/2b after incubation in daylight for only 90 min followed by storage under dark.

37Pulsed electric fields (PEF) processing is another non-thermal alternative technology for decontamination mainly used in liquid foods processing and thus is not limited to inactivation on the surface of a product. The treatment consists of short, highly intense pulses of electric fields applied to the products in order to achieve the inactivation of unwanted microorganisms. The inactivating effects of PEF are destabilization and, depending on the strength of the PEF, irreversible damage of the cytoplasmic membrane with formation of micropores and leakage of cytoplasmic content. Similar to HPP and UV light but unlike conventional thermal food processing technologies, such as pasteurization, this method is less detrimental to food matrices and better in preserving the sensory and nutritional characteristics of the product. The efficacy of inactivation by PEF is determined by a number of factors related to the process (strength, duration, frequency of the pulses, temperature, etc.), the food product (composition, conductivity, pH, etc.) and the microorganisms to be inactivated (species, growth phase, etc.).

38In general, Gram-positive organisms are believed to be more resistant to PEF than Gram-negative bacteria, presumably due to the thicker cell wall and stiffening (lipo)teichoic acids. For example, L. monocytogenes proved to be more PEF tolerant than Salmonella enteritidis and E. coli when treated in melon and watermelon juices. Thus, PEF alone is probably not the method of choice for inactivation of L. monocytogenes. It has been recommended to combine PEF with other methods such as ozone, mild heat or plants infusions with antimicrobial properties to decontaminate food products at risk for contamination with L. monocytogenes. Low inactivation rates were observed for L. monocytogenes in a Spanish vegetable-based beverage and this was attributed to the neutral pH of the product. In fact, in buffer inactivation rates of L. monocytogenes by PFE were higher at acidic pH. Further data in buffered systems or culture media indicated that resistance to PEF was increased in stationary growth phase and in media with reduced water activities suggesting a cross-resistance with other stresses.

39Besides membrane disruption, PEF was suggested to affect bacterial cells by denaturation of the membrane-bound proteins as result of localized overheating caused by the capacity of the formed pores to conduct electricity. This might imply an involvement of chaperones in the response of L. monocytogenes to PEF. One study compared the expression levels of three major molecular chaperones, namely GroEL, GroES, and DnaJ, in a resistant and a sensitive L. monocytogenes strain treated with a sublethal PFE challenge and found a transient reduction in expression of these chaperones in the sensitive strain.

40Somolinos and colleagues have shown no difference in the resistance to PEF processing between L. monocytogenes EGD-e and its isogenic ΔsigB mutant suggesting that SigB is not involved in the repair mechanism of injured cells as shown for thermal treatment of the same strains. Also, unlike heat challenge, mild acid shock applied to L. monocytogens cells did not increase the resistance to subsequent PEF treatment.

41Under oxidative stress (bacterial) cells encounter high concentrations of oxygen radicals. This disturbs the normal redox state of cells leading to cell death due to the oxidative damage of proteins, lipids and nucleic acids. Bacteria use reduction pathways that repair damage of susceptible amino acids (cysteine and methionine) induced by reactive oxygen (ROS) or reactive chlorine species (RCS). ROS are a group of compounds containing oxygen on different redox states such as hydrogen peroxide, hydroxyl radical or peroxyl radical. In bacteria, these compounds activate enzymes such as superoxide dismutases (SOD), catalases, peroxidases and efflux pumps to counteract oxidative stress.

42Recently, Harter and colleagues revealed the presence of a novel stress survival islet (SSI-2) in L. monocytogenes ST121 and other strains isolated from food and food processing environments. The SSI-2 consists of two genes lin0464 and lin0465 (PfpI protease), that are upregulated after 10 min of exposure to oxidative stress. Lin0464 seems to be a positive gene regulator of lin0465, because the time frame of increased transcription of lin0465 is longer compared to that of lin0464 and because the constitutive expression of lin0464 has no effect on the survival rate in Δlin0465 mutant. Under alkaline or oxidative stress encountered in food processing environments, the expression of both genes offers L. monocytogenes ST121 the possibility to adapt and survive, an independent response mechanism from the alternative sigma factor.

43Even if SigB is the main regulator of stress genes, its role in the oxidative stress resistance is controversial. A number of authors provided experimental data suggesting that, in L. monocytogenes, oxidative stress protection is conferred by σB since ΔsigB mutant cells are sensitive to this stress. Other studies suggested that sigB expression is harmful for stationary-phase L. monocytogenes (EGD-e and 10403S) cells grown aerobically, under oxidative stress conditions mediated by hydrogen peroxide. Furthermore, ΔsigB mutant proved, besides oxidative stress resistance, a stronger catalase activity upon addition of 30% H2O2, compared to the wild type. Interestingly, no difference was observed in the transcription of the catalase gene between the ΔsigB mutant and the wild type. All these discrepancies within the role of sigB in oxidative stress response may be explained by variation between strains, oxidative agents tested, differences in growth phase, and oxygen tension of the culture.

44In L. monocytogenes, the resistance to oxidative stress was also correlated with biofilm formation. Four genes related to oxidative stress, kat, perR (peroxide operon regulator), sigB and recA (recombinase A) were upregulated in a Δsod mutant, which produced more ROS than the wild-type L. monocytogenes 4b G. Also, a ΔperR L. monocytogenes mutant showed increased sensitivity to hydrogen peroxide stress. Moreover, catalase activity in these cells increased to a toxic level resulting in smaller colonies and changes in cell morphology compared to the wild type.

45The anti-oxidative kat gene acts synergistically with sod gene (superoxide dismutase), both being involved in the protection against toxic effects of hydrogen peroxide and superoxide anion radicals. The sodA gene encodes for MnSOD, a cytosolic SOD enzyme which uses manganese in the catalytic reactions. Archambaud and colleagues reported that, during stationary phase, L. monocytogenes MnSOD activity is downregulated by phosphorylation at serine/threonine residues. MnSOD activity increases only when dephosphorylation is performed, condition that facilitates its secretion in the bacterial culture media via SecA2 pathway.

46Other genes involved in the response to oxidative stress are fri, gltB, and gltC. Based on its iron-binding activity, fri-encoded ferritin detoxifies oxidative agents. Huang and colleagues introduced a role of gltB and gltC gene products in oxidative stress and L. monocytogenes biofilm formation. GltC is a member of LysR-type transcriptional regulator and gltB encodes for a glutamate synthase regulated by GltC. Experiments with gltB and gltC mutants revealed a reduced ability to form biofilm and an increased sensitivity to oxidative stress.

47L. monocytogenes is able to use diverse mechanisms to survive various stress conditions encountered in food matrices. This explains the efforts made by scientists to understand these mechanisms in order to develop more efficient methods to reduce L. monocytogenes occurrence in food and food related environments. With the present review, we aim at providing an overview of the current knowledge on food-related stress and stress resistance of L. monocytogenes. As observed for many other organisms, L. monocytogenes employs different survival mechanisms for the same stress or use the same mechanism for different stresses (heat-shock genes are expressed when L. monocytogenes is subjected to heat stress, HPP or PEF; cold-shock genes are expressed and osmolytes transport systems are activated when L. monocytogenes encounters cold or osmotic stress). However, compared to other organisms, the large number of mechanisms also increases the possibilities of this organism for cross-resistance.

48The ongoing trend toward healthier, minimally processed food products with unaltered sensory and nutritional properties demands new strategies for food preservation, while no compromises are accepted for food safety. Alternative treatments (e.g., high pressure, pulsed electrical field, UV light), have yielded promising results, but their application often allows to L. monocytogenes recovery. However, current data also suggest that combinations of these techniques with e.g., natural preserving additives such as bacteriocins may be feasible solutions. Nevertheless, the effects on and resistance of L. monocytogenes to such combinations of stresses need to be investigated.

49FB and LG-G drafted the manuscript and all authors revised and contributed to its final version.

50The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.