Listeria monocytogenes – How This Pathogen Survives in Food-Production Environments?

1Listeria monocytogenes is a Gram-positive facultatively anaerobic microorganism, motile at the temperature range of 22–28°C but non-motile above 30°C, characterized by the growth at temperature range from −0.4°C to 45°C, with an optimum temperature of 37°C. It is able to survive at a relatively low water activity (aW < 0.90) and a broad pH range between 4.6 and 9.5 as well as to tolerate salt conditions up to 20%. These growth conditions made these bacteria able to survive and multiply in adverse environmental conditions which are often present at food production facilities. L. monocytogenes is therefore an important foodborne pathogen responsible the disease called listeriosis, which can appear as sporadic infections or disease outbreaks with significant mortality rate of 20–30% worldwide. Human infection can occur in three forms, depending on the age of the infected person, its immune status, the amount of ingested bacterial cells, and the virulence properties of the strain: severe or mild invasive listeriosis and non-invasive febrile gastroenteritis. Depending on the severity of the illness, symptoms may last from days to several weeks. Mild symptoms may include a fever, muscle aches, nausea, vomiting, and diarrhea. If the more severe form of listeriosis develops, symptoms may include headache, stiff neck, confusion, loss of balance, and convulsions. The disease is especially dangerous for the elderly, pregnant women, unborn babies and immunocompromised people. According to the recent European Food Safety Authority (EFSA) and European Center for Disease Prevention and Control (ECDC) common report for year 2020, a total of 1,876 confirmed cases of invasive listeriosis in humans were noted in the European Union member states, with the notification rate of 0.42 cases per 100,000 population and 97.1% hospitalizations. In the United States, the Centers for Disease Control and Prevention (CDC) estimate that each year about 1,600 persons are infected with L. monocytogenes, with the hospitalization rate of ca. 94%.

2Listeria monocytogenes is a ubiquitous bacterium and has been isolated from soil, water, and feed. It has been demonstrated that the bacteria can survive in the environment for at least 8 weeks. Several investigations have shown that L. monocytogenes is widely distributed in food processing environments where it is able to persist for a long time due to ineffective cleaning and sanitation. Many strains survive in different food processing conditions which are often characterized with a low humidity or oxygen content, and thus becoming a main source of post-processing contamination. Persistence of such strains may be contributed by several external factors as poor hygiene practice or ineffective sanitizers but also by the presence of the specific genes in some L. monocytogenes strains that are responsible for biofilm production.

3As above-mentioned, L. monocytogenes has the ability to grow in a broad range of temperatures (from −0.4°C to 45°C) but also under freezing conditions no significant changes in the live bacteria population was observed. Tolerance to low temperatures resulted in the frequent detection of these bacteria in food products stored under refrigeration conditions. The mechanisms of this phenomenon are complex and involve a decrease in the metabolism of the bacterial cells, changes in cell membrane composition, expression of cold shock proteins, and uptake of cryoprotective compounds from the environment.

4The proper physical condition of the bacterial cell membrane lipids is essential to optimal structural and functional integrity of these membranes. Low temperatures affecting the cell lead to reduced membrane lipid fluidity. In response to this stress factor L. monocytogenes changes the membrane lipid composition toward an increase in the concentration of unsaturated fatty acids, which prevents formation of a gel-like state that may result in leakage of cytoplasmic content. It also creates the optimal membrane fluidity for enzyme activity and transport across the membrane. Furthermore, the rate of intracellular enzyme activity decreases to the necessary minimum, and the cell fitness is improved. For all these purposes the bacteria modulate their genes expression, especially those involved in cell membrane function and synthesis of lipids, carbohydrates and amino acids as well as those involved in biogenesis and motility.

5During exposure to the cold temperature stress conditions, L. monocytogenes responds in different ways. The cells increase the accumulation of glycine betaine and carnitine from the environment by a chill-activated transport system. Both these organic osmolytes are found in high amounts in various foods which may help to promote the survival and growth of L. monocytogenes at lower temperatures. The glycine betaine transporter (Gbu) is an ATP-binding cassette (ABC) transporter that is encoded by the gbu operon, whereas transport of carnitine in response to cold shock is depended on the OpuC ABC transporter, the product of the opuC operon. It has been described that both glycine betaine and carnitine were accumulated much faster by L. monocytogenes at 7°C than at 30°C, and their levels increased several times within the cells when grown at 8°C compared to 37°C.

6The role of sigma factor protein σB (SigB) in adaptation to cold stress in L. monocytogenes has been studied. The protein is stimulated in response to temperature downshift and the sigB-deleted mutant was unable to accumulate solutes such as betaine and carnitine. It has also been shown that the sigma factor contributed to adaptation in a growth phase-dependent manner, since the absence of SigB protein impaired adaptation of stationary-phase cells to grow at the lower temperature and it was also necessary for efficient accumulation of betaine and carnitine by L. monocytogenes as cryoprotectants. Furthermore, it has been also demonstrated that only some cold-induced genes were under sigB control (e.g., opuCA gene encoding OpuCA protein with the ATPase-coupled transmembrane transporter activity), whereas other genes responsible for cold shock may be only partially sigB factor-dependent. On the other hand, showed that σB does not play a key role in survival of L. monocytogenes under low temperature stress conditions. These and other authors also demonstrated that sigB is activated at 4°C in a manner that was independent of the levels of RsbV and RsbW proteins encoded by the respective sigB operon genes. Interestingly, it has been shown that at 4°C there is a significant correlation between the prfA virulence gene regulon responsible for the expression of listeriolysin regulatory protein PrfA and the σB regulon, which moderate the activity of PrfA during the host infection.

7Other mechanisms of adaptation of L. monocytogenes to low temperatures have also been described. One such mechanism is the expression of cold shock-domain family proteins (Csps), which are produced mainly at the temperature range from 4°C to 10°C. Csps are structurally related small proteins (65–70 amino acids long), widely distributed among prokaryotes, with a highly conserved structure, that bind to nucleic acids and regulate the expression of various genes including those involved in stress resistance and virulence, cellular aggregation, and motility in L. monocytogenes. Csps stabilize the nucleic acid conformation and act as molecular chaperone that facilitates replication, transcription, and translation at low temperatures. Among many Csp proteins identified, CspA contributes to resistance of L. monocytogenes to low temperatures.

8Another protein belonging to the cold shock-domain family is a low molecular weight (ca. 18 kDa) ferritin-like protein (Flp), which was detected in much higher levels in L. monocytogenes cultures kept at −20°C and grown at 4°C compared to the bacteria cultured at 37°C. It has been suggested that regulation of Flp synthesis may occur at the transcriptional level since the increase of flp mRNA was detected upon heat and cold shock.

9described the two-component-system histidine kinases that have been involved in growth of L. monocytogenes at low temperatures and play a role in adaptation to cold stress. Two genes, yycGF and lisRK, responsible for the bacterial adaptation to cold stress conditions, were identified. The authors suggested that YycF protein encoded by the yycF gene was more involved in the early stage of cells survival, whereas the lisRK product was rather responsible for a longer cold acclimation.

10Thermal treatment is one of the methods that have been applied in food production and preservation to prevent or limit the growth of pathogenic microorganisms, including L. monocytogenes. However, the efficacy of high temperature used for inactivation of Listeria during food processing may be limited due to their natural resistance to elevated thermal conditions above 45°C. It has been described that the total heat inactivation of L. monocytogenes requires the temperature range from 55°C for 10 min to 65°C for 12 s, respectively. There are several external factors that have an influence on resistance of L. monocytogenes to heat such as bacterial cells’ age, previous growth and stress conditions, composition of food, strain serotype, etc.. Cells in the stationary growth phase are generally more resistant to thermal stress than those in log-phase. It has been shown that some components present in foods or growth media protect the bacterial cells from heat damage either by stimulation of cellular membrane production or expression of stress-related proteins. demonstrated that L. monocytogenes isolates of serotype 1/2a showed a relatively low resistance to elevated temperatures, whereas strains classified to serotypes 1/2b and 4b were more heat-resistant although differences among strains of the same serotypes were also noted.

11In response to elevated temperature the bacterial cells show increased production of heat shock proteins (HSPs). In L. monocytogenes three classes of heat shock-associated genes have been identified. Several genes (grpE, dnaK, dnaJ, groEL, and groES) encode the class I HSPs that act as intracellular chaperones and their expression increases when heat-induced denatured proteins accumulate in the bacterial cytoplasm. The class I HSP genes are controlled by the HrcA repressor which negatively regulates expression of this kind of stress response genes. The main role of this class of heat shock proteins is to stabilize and repair partially denatured proteins and to prevent their intracellular aggregation under heat stress conditions. The class II HSP genes encode general stress proteins whose transcription is dependent on the alternative sigma factor SigB in different growth-inhibiting conditions. Class III heat shock genes, including clpP, clpE, and clpC operons, are negatively regulated by the class III HSP gene regulator CtsR which contains domains that are highly conserved among low GC Gram-positive bacteria, including L. monocytogenes. The ClpC and ClpE proteins possess ATPase activity and are classified to the heat shock protein Clp family of highly conserved molecular chaperones, whereas the serine protease ClpP is a protein possessing proteolytic properties. At increased temperature, McsB kinase, the product of the mcsB gene of the clpC operon, modifies CtsR conformation preventing its binding with gene promoters. As a result, RNA-s32 polymerase binds with promoters leading to gene expression, and CtsR is degraded. It has been shown that ClpC expression is negatively controlled at the transcription level by the cAMP protein receptor PrfA.

12A low pH environment may be present in food that has undergone acidification, one of the methods of food preservation widely applied to dairy products, meat and vegetables, and is primarily the results of fermentation by bacteria either present in the raw food or added as starter cultures. Furthermore, L. monocytogenes meets acid conditions in the gastrointestinal tract of the host. The bacteria are able to survive in a low pH of the environment which is generated by artificially induced acidification during acid sanitation. Low pH increases the concentration of hydrogen protons, which results in the inhibition of microbial growth. Furthermore, it has been shown that low pH not only allows the bacteria to survive but also increases its virulence and provides L. monocytogenes higher protection against other adverse environmental conditions.

13The bacteria are able to adapt to this low pH environment by means of different mechanisms. Pre-exposure of L. monocytogenes to mild acidic pH of 5.5 for 2 h induces the acid tolerance response (ATR), the process in which the bacteria showed increased resistance to lethal acidic, temperature (52°C), salinity (25–30% NaCl) and alcoholic (15%) shocks. These effects were even more evident when the bacteria adapted to acid gradually. also demonstrated that the bacteria exhibited a significant adaptive acid tolerance response following a 1-h exposure to pH 5.5, which is capable of protecting cells from severe acid stress (pH 3.5). It has been also suggested that low pH conditions may have the influence on the selection of L. monocytogenes mutants with increased virulence properties.

14Listeria monocytogenes uses a variety of metabolic and homeostatic mechanisms to maintain the intracellular pH within a range that is optimal for its growth and survival. It is able to increase cytoplasmic buffer capacity through the glutamate decarboxylase (GAD) system or with the action of an internal proton pump. The GAD mechanism is considered as one of the major mechanisms responsible for the maintenance of the intracellular homeostasis. GAD in most L. monocytogenes strains is encoded by five genes, of which three genes (gadD1, gadD2, and gadD3) encode decarboxylases, whereas two other genes (gadT1 and gadT2) are responsible for production of antiporters. All these five genes are organized in three separate genetic loci: gadD1T1, gadT2D2, and gadD3. The glutamate decarboxylase enzyme promotes the irreversible conversion of cytosolic glutamate to a neutral compound, the γ-aminobutyrate (GABA). During the GABA synthesis the intracellular proton level decreases resulted with the subsequent alkalization of the environment and increase of the pH inside of the L. monocytogenes cell. Furthermore, the extracellular GABA excretion leads to the slight neutralization of the pH outside the cell due to the exchange of extracellular glutamate for the more alkaline GABA, and finally the restart of the metabolic pathway.

15Another cell system that protect Gram positive bacteria from low pH is based on the arginine deiminase (ADI) pathway. ADI, with the participation of two other enzymes, carbamoyltransferase and carbamate kinase, all encoded by the arcABC operon, converts external arginine to ornithine which is then extracellularly transported in an energy-independent manner by a membrane-bound antiporter encoded by the arcD gene. The level of ammonia produced as a byproduct of the system combines with intracellular protons to yield NH4+ maintaining the intracellular level of the cytoplasmic pH, thereby protecting the L. monocytogenes cell from adverse acidic extracellular environments. It has been shown that the transcription of arcA and argR genes is both sigma factor protein σB (SigB) and protein receptor PrfA-dependent.

16In addition to GAD and ADI systems, other proton pumps such as F0F1-ATPase have also been suggested as active mechanisms to maintain L. monocytogenes homeostasis in low (mild) pH environments. ATP produced during arginine conversion under ADI mechanisms is used by F0F1-ATPase to generate a proton gradient, enabling H+ expulsion and homeostasis restoration. The enzyme consists of two distinct domains: the membrane domain F0, which is a channel for proton translocation, and the cytoplasmic domain F1, responsible for catalyzing ATP synthesis and hydrolysis.

17The above-described low pH adaptation systems, i.e., ATR, GAD, and proton extrusion (F1F0-ATPase), act at the same time and ensure survival and adaptation to acid stress conditions of L. monocytogenes.

18Listeria monocytogenes also possesses two-component signal transduction system that plays a role in response to environmental stress conditions, including low pH. This system typically contains two genes, lisR and lisK, encoding cytoplasmic response regulator and a membrane-associated histidine kinase sensor, respectively. The LisRK signal transduction system is able to sense pH changes in the environment by histidine kinase, and the response regulator enables the cell to respond by altering the relevant gene expression. It has been shown that a LisRK transposon mutant of L. monocytogenes was more sensitive to low pH than the wild type strain during the logarithmic phase of growth but more acid resistant during stationary phase.

19In food production environment there are several alkaline stress factors, which are sublethal for L. monocytogenes and are mainly associated with the use of detergents and disinfectants. This bacterium has developed many strategies to withstand adverse high pH-related conditions and as a result they become more cross-resistant to subsequent other, usually more severe, stress factors, such as thermal, alkali, and ethanol stresses or cleaning procedures. Alkali adaptation mechanisms of L. monocytogenes may be significant in the persistence of these pathogenic bacteria in food industry equipment and premises in the presence of alkaline-based detergents used in food processing environments.

20Alkali conditions, which may be present in the environment due to the use of detergents and disinfectants, can induce the solubilization of bacterial surface proteins, resulting in exposure of hydrophobic sites of lipids to the extracellular factors. Such conditions may also directly change the structure of the cell membrane by saponification of membrane lipids or alteration of the membrane fatty acids ratio. These changes induce damages that significantly disrupt cell metabolism and structure, preventing effective interactions between bacterial cells and their environment.

21Generally, to resist alkali damage and maintain cytoplasmic pH at optimal values, L. monocytogenes responds in different ways. One of them is increased metabolic production of intracellular acids through deamination of amino acids and fermentation of sugars. The bacteria are also able to induce transporters and enzymes directly responsible for proton retention and cell surface modifications. It has been proved that monovalent cation-proton antiporters are essential to maintain a neutral cytoplasmic pH and, therefore, to allow the bacterial growth under alkaline conditions. In addition, the acidic cell wall polymers such as teichuronic acid and teichuronopeptides contribute to pH homeostasis, and provide a passive barrier to ion flux and elevation of the cytoplasmic buffering capacity.

22A scanning electron microscopy study of L. monocytogenes exposed to sublethal alkaline stress performed by showed that the bacterial cells significantly changed their length, radius and volume that may be associated with increased survival of Listeria in such adverse environments. Furthermore, in alkaline conditions, L. monocytogenes cells develop higher proportions of branched-chain fatty acids, including more anteiso forms that are important in adaptation to high pH.

23Listeria monocytogenes can survive in elevated osmolarity and has the ability to grow even in media supplemented with 12% NaCl and can tolerate adverse salt conditions up to 20%. High NaCl concentrations suppress bacterial growth by decreasing water activity in surrounding environment, enhancing plasmolysis and consequently resulting in decreased intracellular turgor pressure and finally, inhibiting the bacterial amplification. In addition to increasing osmotic pressure, NaCl decreases electrochemical potential across the cell membrane, thus, disturbing ATP production by oxidative phosphorylation. The response of L. monocytogenes to osmotic stress is called osmoadaptation, a biphasic process consisting of primary and secondary response mechanisms. The primary phase of adaptation of the bacteria to elevated osmolarity covers physiological changes of the cells, which maintain their turgor by increasing the uptake of potassium ions (K+) and its counterion, glutamate, into the cell, and then replacing part of the accumulated K+ with low-molecular-weight molecules known as compatible solutes or osmolytes in the second stage of osmoadaptation. L. monocytogenes possesses two K+ transporters, which play a main role in adaptation to high salt concentration: a high affinity KdpABC transporter system, and a low affinity system encoded by the lmo0993 gene.

24Uptake of compatible solutes by L. monocytogenes as a response to elevated osmolarity helps the bacteria to restore turgor pressure and cell volume and stabilize cell protein structure and functions. Several compatible solutes which promote both salt and low temperature tolerances in L. monocytogenes have been identified, including betaine, carnitine, proline, proline betaine, acetylcarnitine, gamma-butyrobetaine, and 3-dimethylsulfoniopropionate. Among them, betaine has the strongest effect on reduced growth under high osmotic conditions. The presence of the osmolytes resulted in an up to 2.6-fold increase in growth rate of salt-stressed L. monocytogenes cells compared to stressed cells without any osmoprotectants.

25It has been described that the main carnitine transport system is encoded by the opuCABCD operon, the glycine betaine by gbuABC, whereas the glycine betaine uptake system depends on the betL gene. The expression of genes encoding betaine, carnitine and proline transporters are transcriptionally regulated by general stress sigma factor σB. In more detail, the product of the opuCABCD operon (OpuCA) releases energy during ATP hydrolysis which is needed for transport of the substrate by a complex consisting of the two transmembrane proteins OpuCB and OpuCD as well as a solute binding protein OpuCC. The products of the remaining genes (betL and gbuABC) are involved in the primary response to the elevated level of NaCl and toleration of L. monocytogenes to a long-term osmolarity, respectively. The strains possessing mutations in these genes showed reduced growth under high osmotic condition. It has been suggested that compatible solutes may play a dual role in osmoregulation process of the bacterial cells: firstly, they are involved in restoring of cell volume and, secondly, they stabilize protein structure and function under these adverse environmental conditions. It has also been shown that the general stress protein Ctc of L. monocytogenes is involved in osmotolerance in the absence of any compatible solutes in the environment.

26Several studies indicated that most osmotolerance-associated genes present in L. monocytogenes have also been activated during other stress environmental conditions such as low temperature and low pH generated during artificial food acidification as well as play a role in virulence in a mouse model. On the other hand, in response to osmotic stress, L. monocytogenes expresses genes other than those associated with osmolyte accumulation, such as csp encoding cold shock-domain family proteins (Csps). These proteins, mainly CspA and CspD, have chaperon activity and facilitate the repair of DNA lesions made by high concentrations of NaCl. Apart from stress survival functions, Csp proteins are also involved in cell regulatory networks, playing a crucial role in the regulation of virulence functions of L. monocytogenes, especially those connected with invasion and listeriolysin (LLO) secretion.

27described that the presence of osmotic conditions in L. monocytogenes environment decreased expression of genes associated with phosphoenolpyruvate (PEP)-dependent phosphotransferase carbohydrate systems (PTS), including those related to uptake of β-glucoside, galactitol, fructose, and cellobiose. This has an influence on a significantly lower growth rate of the bacteria and reduced uptake of carbohydrates under osmotic stress conditions.

28High hydrostatic pressure (HPP) is a food preservation technology used as an alternative to thermal processing. It is widely applied in the meat industry for microbial inactivation of both food spoilage microorganisms and foodborne pathogens, and it is conducted at room temperature, which enhances the safety and shelf life of food. The pressures applied for sterilization depends on the kind of food and potentially present microorganisms and usually ranges between 250 and 700 MPa (mainly 400 and 600 MPa) for a few seconds to 10 min. Primary effects of HPP on bacterial cells are an increase in the permeability of the cell membrane, the disruption of the protein structure and function, and finally, inhibition of the physiological activities of the treated microorganisms.

29It has been reported that HPP causes morphological, structural, physiological, and genetic changes or damages to L. monocytogenes cells. However, several factors influence the resistance of these bacteria to high hydrostatic pressure. Cells in the stationary phase of growth are much more resistant to pressures above 200 MPa than those in the exponential phase. Furthermore, resistance of L. monocytogenes to HPP depends on the strain and the type, composition and matrix of food products. It has also been shown that higher salt concentrations in food may induce uptake of compatible solutes, which in turn stabilizes cells during HPP. It has been shown that pressure-induced damage of the cell membrane has an influence on the Mg2+ leakage from the cell and therefore destabilization of ribosome structure, whereas Ca2+ strengthens the outer membrane of bacterial cells and make the bacteria more resistant to HPP. However, it has been reported that L. monocytogenes treated with HPP up to 550 MPa, resulting in still viable cells, was able to recover and grow during the storage under refrigeration conditions.

30Several genes and mechanisms that may play a role in recovery from HPP damage of L. monocytogenes have been identified. High pressure processing induces expression of genes associated with DNA repair, transcription and translation protein complexes, cell division, general protein secretion system, flagella assemblage and motility, chemotaxis, and membrane and cell wall biosynthesis pathways. On the other hand, HPP suppresses a wide range of energy production and conversion, carbohydrate metabolism and virulence-associated genes. An important aspect of the stress-induced survival is induction of the general stress response mediated by the above-mentioned sigma factor protein σB (SigB) which can activate several protective genes under stressful conditions. However, it has also been suggested that HPP may reduce expression of the sigma factor SigB and part of the sigB regulon.

31It has been shown that HPP also affected genes controlled by the transcription factor CodY, a known global regulator of metabolic genes, including the prfA gene regulon responsible for the expression of listeriolysin regulatory protein PrfA. Another gene induced by HPP is cspL encoding a cold-shock protein, which supports the earlier observations that HPP also induces cross-resistance to other stress factors and conditions like heat, acid, and oxidative stress. Furthermore, mutations in CtsR, a class III stress genes repressor, have also been connected to spontaneous resistance of L. monocytogenes to HPP. Such mutants, characterized by a stable resistance to HPP, showed point mutations, insertions or deletions in the ctsR gene that down-regulated its activity. This feature was connected with increased expression of the clpB, clpC, clpE, and clpP genes, encoding a large protein complex Clp (caseinolytic protein) with both proteolytic and chaperone activities. Clp proteases are able to degradate damaged or denatured proteins forming in the bacterial cells during HPP treatment that are potentially harmful for L. monocytogenes; thus, they increase tolerance of the bacteria to high pressure. However, there is also evidence that isolates which did not have such ctsR and other genetic changes still showed resistance to HPP treatment.

32It has been shown that after HPP treatment L. monocytogenes up-regulates the major PEP-PTS, especially fructose-, mannose-, galactitol-, cellobiose-, and ascorbate-specific, involved in the sugars transport. Furthermore, the cell-division-related genes (divIC, dicIVA, ftsE, and ftsX) were also down-regulated, whereas the peptidoglycan-synthesis genes responsible for cell-wall repair (murG, murC, and pbp2A) were upregulated. Thus, it seems that the bacterial tolerance response to HPP is complex and needs further investigations.

33Pulsed ultraviolet light (PUV) is a non-thermal approach that has a high potential for decontamination of food, water, and air in food production environments. For this purpose, ultra-short duration pulses of an intense broadband emission spectrum that is rich in UV-C light (200–280 nm band) of the highest germicidal efficacy is used. This UV spectrum mediates bacterial inactivation through several mechanisms, including damage to the bacterial cells in the form of pyrimidine dimers and the loss of cytoplasmic contents post-light absorption. The main bactericidal effect of UV-C light is caused by DNA damage as a consequence of the formation of photoproducts such as cyclobutane-pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs), and their Dewar isomers. Furthermore, UV light also has photophysical and photothermal effects on bacterial cells due to the absorption of the high energy light pulses resulting in leakage of cellular content. However, during UV treatment, CDPs are the most common lesions in the bacterial genome, which have the principal effect on the cell development and survival, especially that connected with transcription, DNA replication and cell cycle progression.

34The efficacy of UV treatment in decontamination of food surfaces depends on many factors, such as the kind of food, distance of the product to the light source, energy level given by number and frequency of the light pulses, time applied for the UV treatment, level of contamination, and others. It has been shown that L. monocytogenes is more resistant to UV-C light than other bacterial pathogens, such as E. coli. The presence of organic materials such as food debris on stainless steel surfaces and NaCl content in ultraviolet treated foods have an influence on the efficacy of UV-C radiation on L. monocytogenes due to low ability of this light to penetrate organic substances. Other studies have also shown that the presence of salt in brine increases the amount of UV-C necessary for inactivation of L. monocytogenes in fluid. Similar increased resistance to ultraviolet light was observed for the bacterial cells grown in vitro in a low pH, presence of antibiotics, or disinfectants.

35There is very little information related to detailed molecular mechanisms of UV resistance in L. monocytogenes. It seems that UV-C resistance is not general stress sigma factor protein σB-dependent. Another study of revealed that overall changes in gene expression resulting from ultraviolet treatment of L. monocytogenes 10403S strain were low. However, a number of genes encoding stress proteins, motility and transcriptional regulators were up-regulated by the UV exposure, although no induction of the lmo0588 gene, responsible for deoxyribodipyridimine photolyse activity induced by light was observed. Photolyase, the product of the lmo0588 gene, plays an important role in photoreactivation, i.e., the recovery of bacteria sublethally injured by UV light due to subsequent exposure of visible light. It has been shown that, during photoreactivation, photolyase binds and repairs the pyrimidine DNA lesions using light energy absorbed by its chromophores.

36It has also been described that, among the putative stress response genes located on plasmids of L. monocytogenes, the uvrX gene being a part of the Y-family DNA polymerase, plays a role in response of the cells exposed to UV stress. A similar finding was described for the chromosomally encoded gene uvrA, which was shown to be necessary for the bacterial UV stress survival. Recently, identified several L. monocytogenes plasmids that confer increased UV stress tolerance, although their precise role in this phenomenon needs further investigations.

37Certain heavy metals such as copper, zinc, and iron in trace amounts are essential for bacterial survival and play a role of co-factors for a broad range of L. monocytogenes cellular proteins but the same metals at higher concentrations often become toxic. Other heavy metals (e.g., arsenic and cadmium) are probably not necessary for cellular functions and seem to be toxic in any concentration. However, L. monocytogenes possesses various mechanisms to maintain its cellular heavy metal homeostasis, avoid poisoning, and thus, survive in diverse environmental niches. In this section, resistance to the main toxic heavy metals, i.e., cadmium and arsenic, is discussed.

38It has been reported that approximately 50% or more of tested L. monocytogenes isolates from foods and food processing plants were resistant to cadmium. On the other hand, there is also information that most strains were susceptible to this metal at concentration of 64 μg/ml cadmium sulfate. Resistance to cadmium is encoded by different genetic determinants often located on mobile genetic elements (mainly plasmids). At least five molecular mechanisms have been identified that contribute to cadmium resistance, although resistant strains lacking these genetic determinants have also been identified suggesting that there are yet unidentified means of metal tolerance. The first cadmium molecular resistance gene described in L. monocytogenes was the cadA1 located on plasmid-borne Tn5422 transposon, which is responsible for the activity of efflux P-type ATPase pumps. Few strains harbor Tn5422-associated cadA1 chromosomally. A second putative cadmium resistance sequence, cadA2, was initially detected on the large plasmid pLI100 of L. innocua and then discovered on the approximately 80 kb plasmid pLM80 of L. monocytogenes H7858, a strain implicated in a large listeriosis outbreak in the United States in 1998–1999. It was also found that pLM80-associated cadAC (cadA2) is part of a putative composite transposon that also harbors genes for resistance to benzalkonium chloride. In addition, pLM80 determines resistance to toxic triphenylmethane dyes such as crystal violet and malachite green via the tmr gene. The third cadmium resistance gene, cadA3, is carried on the integrative and conjugative element (ICE) of L. monocytogenes EGD-e. Finally, the cadA4 and cadA5 are on the large chromosomally located Listeria Genomic Island 2 (LGI2) and LGI2-1 islands, respectively. The cadA3, cadA4, and cadA5 genes have so far been identified only on chromosome. It has also been found that LGI2 harbors arsenic resistance cassette comprising of arsD1A1R1D2R2A2B1B2 genes. Comparison of the cadA1-cadA4 sequences revealed that the first three conferred a high level of resistance to cadmium (MIC > 140 μg/ml), whereas cadA4 was responsible for relatively lower resistance levels (MIC < 70 μg/ml). Interestingly, LGI2 genetic element has been mainly connected with L. monocytogenes classified into hypervirulent clones of clonal complexes CC1 and CC2 as well as CC4. Furthermore, strains with multiple cadA variant determinants have been identified. On the other hand, several L. monocytogenes isolates lacking any of the four cadmium resistance determinants were detected, which suggests the presence of one or more unidentified cadmium resistance genes.

39It has been described that the prevalence of the known cadmium resistance molecular determinants was serotype-related: cadA1 was more common in L. monocytogenes isolates of serotypes 1/2a and 1/2b than 4b from food and food-processing environment, while cadA2 was mainly associated with strains of serotype 4b. However, several cadmium-resistant isolates lacking the known cadA determinants were classified to serotype 4b, which suggests that such strains may possess other than cadA-encoded resistance mechanisms. Furthermore, another study showed that cadmium resistance was more common among persistent L. monocytogenes strains, i.e., those repeatedly isolated from foods than among those recovered sporadically.

40One of the most important mechanisms responsible for arsenic resistance in L. monocytogenes appears to be encoded by genes carried on the above-mentioned LGI2, primarily associated with strains of serotype 4b, especially of the hypervirulent clones CC1, CC2, and CC4. Molecular analysis of arsenic-resistant isolates harboring LGI2 revealed that this island was inserted in at least eight different locations, primarily within open reading frames. Strains classified to serotypes 1/2a, 1/2b, and 1/2c rarely carried the LGI2 insert, thus, are usually arsenic-sensitive. It has been shown that the arsenic resistance cassettes contain three (arsRBC) to five (arsRDABC) genes. The products of the arsA (encoding ATPase) and arsB (responsible for a membrane transporter) genes are an ATP-dependent anion pump that exports arsenite out of the bacterial cells. Furthermore, the arsA gene product is also able to act independently as a passive transporter of arsenite. The arsC gene encodes a reductase that is responsible for the conversion of arsenate to arsenite, which is then extruded by ArsA or the ArsA/ArsB complex. The arsA, arsB, and arsC genes are regulated by two regulatory proteins encoded by arsR and arsD. Interestingly, the LGI2 insert present in L. monocytogenes harbors several additional genes, including the putative cadmium resistance determinant cadA4 mentioned above.

41Increased tolerance of L. monocytogenes to disinfectants (biocides) has been deeply studied in relation to quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC), widely applied in food production and health care environments as well as in households due to their effectiveness, low toxicity, and non-corrosive properties. QACs are usually used in concentrations ranging from 200 ppm to 400 ppm on food-contact surfaces; however, some formulations also have 1,000 ppm concentrations. The antimicrobial effectiveness of QACs and other biocides may be affected by the surface structure of the food production equipment to which bacteria are attached, an uneven distribution of disinfectants on the decontaminated surfaces, too high dilution of disinfectants due to presence of water on the equipment surfaces, or the presence of organic pollutants resulting from insufficient cleaning before disinfection. Biofilms, often produced by L. monocytogenes, also have an impact on resistance of these bacteria against QAC disinfectants. Most QACs are aerobically biodegradable and their concentrations in the environment varied resulting in the formation of concentration gradients. This may lead to the generation of selective pressure for adaptation or acquisition of resistance genes of initially susceptible L. monocytogenes strains.

42Quaternary ammonium compounds are active agents that interact with the cytoplasmic membrane of bacteria, including L. monocytogenes, and also able to interact with intracellular targets as well as to bind to DNA. It has been shown that at low concentrations (0.5 to 5 mg/L) they are characterized by bacteriostatic properties whereas, at concentrations of 10–50 mg/L, QACs are bacteriocidal for the same bacteria, depending upon the formulation. However, there are studies showing that several L. monocytogenes isolates were resistant to BC at a concentration of 1,000 mg/L after 24 h of exposure time.

43The action of these biocides involves absorption of QACs by the bacterial cell and penetration of the cell wall, reaction with the cytoplasmic membrane and its disruption, leakage of intracellular components, degradation of proteins and DNA, and finally lysis of bacterial cell wall by autolytic enzymes. It has been shown that the effectiveness of QACs seems to be not different between persistent and non-persistent L. monocytogenes strains isolated from food production environments. However, there are also reports showing that persistent isolates from food processing plants and ecosystems exhibited higher resistance to QACs than non-persistent ones, especially strains classified to serotype 1/2c. Studies of demonstrated that all 50 L. monocytogenes isolates from food tested were resistant to benzalkonium chloride, and more than 50% of these tolerant strains displayed cross-resistance to cadmium. Such correlation between BC and cadmium resistances has been previously reported by other authors, especially among L. monocytogenes of serotypes 1/2a and 1/2b.

44It has been observed that exposure of L. monocytogenes to increasing concentrations of BC resulted in adaptation to higher levels of this and other biocides. These studies demonstrated that repeated exposure to subinhibitory concentrations of QACs and prolonged environmental persistence of tolerant strains may facilitate the development of bacterial resistance to these biocides over time. Thus, selection of BC-tolerant bacteria increases the ability of L. monocytogenes to survive under treatment with higher concentrations of the same biocide. Consequently, this process further contributes to the persistence of L. monocytogenes in the food processing environments.

45The major molecular mechanisms of quaternary ammonium compounds resistance in L. monocytogenes involve several efflux pump systems, including the three-gene cassette bcrABC associated with BC tolerance. These cover two endogenous multidrug efflux pump genes (multidrug resistant Listeria, mdrL, and Listeria drug efflux, lde) of the major facilitator superfamily and efflux pump genes (bcrABC cassette, qacH, emrE, and emrC) located on mobile genetic elements. It has been shown that the two major efflux pump genes, mdrL and lde, have been identified in almost all L. monocytogenes serotypes, and enhanced expression of these two endogenous efflux pumps, especially MdrL, resulted in BC resistance. However, the main role of these gene products is detoxification of macrolides, cefotaxime, heavy metals, and ethidium bromide (mdrL), and fluoroquinolones, ethidium bromide, and acridine orange (lde), respectively.

46Among the four efflux pump genes located on mobile genetic elements, the bcrABC cassette was firstly identified in L. monocytogenes responsible for the multistate listeriosis outbreaks in 1998–1999 in the United States. In most isolates, this cassette is located on the pLM80 plasmid but has also been identified on chromosome. These authors have also demonstrated that, in BAC-tolerant L. monocytogenes from various sources, the bcrABC cassette was present in 98.6% of isolates.

47The qacH efflux pump gene is located on a chromosomally integrated Tn6188 transposon of 5,117 bp in size and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter associated with export of BC in bacteria. The significant expression of qacH-encoded efflux pumps has been shown in the presence of benzalkonium chloride and the qacH deletion mutants had lower tolerance to BC than wild type strains. It has also been described that QacH protein confers higher tolerance to other QACs and ethidium bromide. A study of demonstrated that the majority of Swiss and Finnish L. monocytogenes 1/2c clinical and food isolates resistant to BC were qacH-positive, although a subset of BC-resistant strains lacked genes for efflux pumps currently known to confer BC resistance. Similar observations were described by other authors.

48Other efflux pump genes responsible for the increased tolerance of L. monocytogenes to QAC are emrE and emrC sequences, located on the LGI1 genomic mobile island and pLMST6 plasmid, respectively. The emrE gene was first described in a study on L. monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada in 2008. During this investigation it was found that strains possessing the LGI1 island with the emrE sequence was characterized by a significantly improved bacterial growth in the presence of QACs compared to the adaptation and growth of genetically similar strains but lacking LGI1. Furthermore, the expression of emrE and several other genes on the LGI1 genomic island was induced in the presence of BC, whereas deletion of the emrE gene resulted in reduced bacterial growth and survival in the presence of QACs.

49The emrC QAC resistance gene, carried by plasmid pLMST6, was identified in L. monocytogenes of sequence type ST6 strains, isolated from adults suffering from listeriosis with meningitis. Interestingly, demonstrated that the plasmid pLMST6 was not associated with increased tolerance to benzalkonium chloride, but rather increased tolerance to other types of QAC-based biocides. Furthermore, pLMST6 plasmid had no impact on the sensitivity of L. monocytogenes to non-QAC disinfectants or on resistance of isolates to ampicillin, tetracycline and gentamicin.

50Chlorine-based disinfectants such as sodium hypochlorite, chlorine dioxide gas or aqueous chlorine dioxide are used in food industry to control L. monocytogenes contamination. These chemicals possess fast and strong oxidizing properties and interact with bacterial cell wall membranes, mainly phospholipids, or penetrate directly into the cell wall where they form N-chloro groups that react with the bacterial metabolism due to the interference with key enzymes. The efficacy of chlorine-based disinfectants seems to be bacterial cell age-dependent since younger cultures (24 h) are more resistant than older ones (48 h). Furthermore, in L. monocytogenes biofilms the efficacy of chlorine solutions depends on the material on which the biofilm is formed, e.g., bacteria are more easily destroyed when grown on stainless steel surfaces compared to those grown on polyvinyl chloride or Teflon surfaces.

51The effect of chlorine-based disinfectants against L. monocytogenes also depends on the chemical compounds used. It has been shown that chlorine dioxide is less toxic, more effective at low concentrations and needs a shorter reaction time than chlorine alone. One of the main disadvantages of chlorine-based biocides is the formation of toxic disinfection-by-products, especially when they are dissolved in water containing organic matter which is often the case in food production environments. Such products, including trihalomethanes and haloacetic acids, are potential carcinogens and have been associated with various health problems.

52Acid compounds, like chlorine-based disinfectants, are strong oxidizers and have an effective antibacterial properties. They interfere with cellular phospholipids and cytosolic intracellular material causing irreversible damage (e.g., disruption of proton motive force) and subsequent cells death.

53Listeria monocytogenes is able to attach to a variety of surfaces in food production environments, including stainless steel, polystyrene or glass, and then to form biofilms. This is a serious concern for food safety because biofilm-contaminated food environments may serve as source of pathogenic bacteria for food products and finally for consumers. Bacterial cells in biofilms are embedded in a self-produced matrix of extracellular material, composed of extracellular DNA, proteins, polysaccharides, and other inorganic molecules, called extracellular component matrix (ECM). In the L. monocytogenes biofilm matrix, various extracellular polymeric substances (EPSs) have been identified, with different polysaccharides (mainly teichoic acid), proteins, and extracellular DNA. It has been shown that L. monocytogenes biofilms are strongly influenced by temperature, bacterial strain, incubation time, medium, and the nature of the adhesion surface. and observed that L. monocytogenes was able to form biofilms at 4 and 12°C with higher levels on glass compared to the more hydrophobic stainless steel and polystyrene. Furthermore, in both cases, the production of biofilms was significantly higher at 37°C than at 4°C. These authors suggested that these results were not due to a different cellular physiology but rather to a reduced growth of bacteria. On the other hand, observed biofilm formation at 4°C on different surfaces, with higher levels of biofilm on stainless steel and glass compared to polystyrene. Similar data were presented earlier by who showed that some L. monocytogenes strains were able to adhere in the same way at 4°C and 30°C. Other studies suggested that cold-adapted L. monocytogenes, stored at −20°C for 6 and 24 months, was characterized by increased adhesion and biofilm formation on various abiotic surfaces.

54It has been shown that flagella-mediated motility plays a key role in both initial surface attachment and subsequent biofilm formation by L. monocytogenes and the flaA mutants displayed reduced colonization ability. Since temperature regulates flagella expression in L. monocytogenes, it is clear that this factor has a strong influence on biofilm formation. It has been demonstrated that this pathogen is flagellated and motile at temperatures < 30°C, and not at all or much less flagellated and motile at temperatures above 30°C. However, L. monocytogenes is also able to attach to abiotic surfaces through a flagella-independent binding process, which is not related to a temperature.

55A correlation between L. monocytogenes serotypes or clones and biofilm formation has been investigated but no clear dependence was detected. tested the ability of 98 clinical and food isolates classified to serotypes 1/2a, 1/2b, and 4b to form a biofilm. Most of the strains (63.3%) were classified as weak biofilm producers, whereas the remaining isolates were defined as moderate and strong (9.2% of each) biofilm formers. Interestingly, none of the strains of 4b serotype exhibited strong biofilm formation. It has been also shown that strong biofilm-forming isolates developed biofilm structures within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Using whole-genome sequencing data from 166 environmental and food-related L. monocytogenes biofilm-forming isolates, it has been suggested that serotype-specific differences in biofilm development can be linked to the presence of stress survival islet 1 (SSI-1). In this study, strains of serotype 1/2b, the majority of which contained SSI-1, formed the strongest biofilms, while isolates classified to serotype 4b, which only some of them were SSI-1-positive, were the weakest biofilms producers.

56Investigations performed by on 71 L. monocytogenes of food origin revealed a significant correlation between isolates of lineage I (serotypes l/2b and 4b) but not strains of lineage II (serotypes 1/2a and l/2c) and biofilm formation. However, it was also found that isolates classified to the same clonal lineage produced different levels of biofilms, which may suggest that environmental factors are involved in this process. On the other hand, found a higher biofilm-forming ability for L. monocytogenes isolates of lineage II. A reason for these different results obtained by several authors may be caused by the differences in methods applied and the various strains used for the study.

57Significant differences in gene expression between biofilm-forming and planktonic L. monocytogenes bacterial cells, especially those involved in expression of internalins (InlA and InlC) and listeriolysin O (LLO), have been observed. Isolates with mutations in the inlA gene, which resulted in the reduced length of InlA protein, demonstrated enhanced biofilm forming abilities but a lower virulence potential compared to the strains possessing full-length InlA. Such mutations occur more commonly among food isolates than in strains responsible for human infections.

58found that prfA, the transcriptional activator of virulence genes, promotes biofilm formation in L. monocytogenes. Although in prfA negative mutants the flagellar motility remains intact and the cells are able to attach to abiotic surfaces, they are defective in next stages of biofilm formation.

59investigated the role of the alternative stress sigma factor σB in biofilm formation and revealed that initial attachment of both wild type and mutant L. monocytogenes to the stainless steel surface was the same, but the number of sigB-deficient strain on the surface was significantly lower than the wild type after 48 h or 72 h of incubation.

60Recently, studied a role of the two-component chemotactic system encoded by the cheA/cheY genes, located immediately downstream of the flaA flagellin gene. The cheY knockout mutant showed decreased biofilm formation ability along with reduced cell-surface hydrophobicity compared to wild type strain. Similar results were also obtained by who showed that cheA and cheY are key genes in the formation of L. monocytogenes aggregates in vitro.

61The agrBDCA operon present in L. monocytogenes consists of genes that code for AgrD, an auto-inducing peptide, AgrB, a protein involved in processing the peptide, AgrC, a two-component histidine kinase, and AgrA, a response regulator. It has been shown that agr system, that is involved in quorum sensing, has a strong influence on biofilm formation as mutations in agrA and agrD display reduction in their ability to form biofilms compared to the wild type strains under both static and dynamic conditions. studied the presence and expression of the agrA gene in L. monocytogenes of serotypes 1/2a and 4b, grown at 7°C and 37°C. The authors found that the gene was not detected in strains of serotype 4b, and its transcription level in strains of serotype 1/2a was lower at 7°C compared to 37°C.

62identified 38 genetic loci possibly involved in L. monocytogenes biofilm formation when grown at 35°C. Among them, the D-alanylation pathway genes dltABCD and the phosphate-sensing two component system phoPR were important in this process since the deletion mutants showed decreased ability to produce biofilms. It may suggest that D-alanylation of lipoteichoic acids mediated by the gene products of the dltABCD operon and the phosphate-sensing phoPR system play a significant role for L. monocytogenes to form biofilms.

63A role of autoinducer (AI-2) molecules and the luxS gene in quorum sensing and biofilm production by L. monocytogenes was tested by. The authors revealed that S-ribosylhomocysteinase encoded by luxS catalyzes the hydrolysis of S-ribosylhomocysteine to homocysteine and 4,5-dihydroxy-2,3-pentadione, precursor molecules of AI-2, and thus are involved in biofilm formation. Mutation in luxS led to production of a denser biofilm and better attachment by a luxS-deficient mutant to a glass surface compared to the wild type strain. Furthermore, the culture supernatants of luxS mutants were shown to accumulate S-adenosyl homocysteine and S-ribosyl homocysteine, the AI-2 precursor molecules.

64Persistent L. monocytogenes strains have been defined as isolates (clones) that are repeatedly cultured from the same source or ecological niche over time. Such isolates have indistinguishable molecular background as tested by genome-based approaches, e.g., pulsed-field gel electrophoresis (PFGE) or recently, next generation sequencing (NGS). Persistence of L. monocytogenes is due to different characteristics of such isolates, e.g., tolerance to sanitizers, ability to grow at low temperatures, resistance to heavy metals, or ability to develop biofilm. Persistent isolates present an important challenge to food producers, as they are associated with cross-contamination of food products because they are hardly or not at all eliminated from food production environments.

65Several genetic determinants have been suggested to play a role in persistence of L. monocytogenes; however, the nature of the role of these mechanisms to the persistence phenomenon remains still poorly understood. This may be due to difficulties in creating appropriate conditions under in vitro studies that accurately reflect the natural environment in food production facilities. Overall, there is a lack of conclusive evidence on whether persistent strains are more resistant to particular stress conditions compared to sporadic strains from similar sources.

66Persistent L. monocytogenes strains have been isolated from food processing environments after cleaning and disinfection. The relationship between resistance to various biocides and persistence of certain subtypes of L. monocytogenes in different food processing environments has been studied but no clear correlation was identified. On the other hand, there are investigations that showed that there is a link between resistance to benzalkonium chloride and persistence of some strains, especially those positive for the bcrABC gene cassette.

67A correlation between biofilm formation and persistence of L. monocytogenes in the food production environments has been investigated by several authors. Generally, persistent strains have shown increased biofilm formation in relation to non-persistent strains. It has been also observed that biofilms produced on stainless steel surfaces by persistent strains are thicker than those formed by strains found only sporadically. tested the adherence capability to stainless steel surface of two L. monocytogenes strains with and without persistent ability. It was shown that mean counts of adherent cells over a 24-h period at 25°C were significantly higher for persistent strains. Similar observations were noted by and. Further studies confirmed that persistent L. monocytogenes genotypes were often associated with higher survival and biofilm formation capacity in the presence of sublethal concentrations of benzalkonium chloride. On the other hand, other authors have shown that there were no clear associations between biofilm formation efficiency and persistent or prevalent genotypes.

68In a study of persistent L. monocytogenes of serotype 4b were shown to be extremely resistant to high temperatures and pressure stresses. Resistance to cadmium has been more often noted among persistent clones compared with their sporadically contaminating counterparts. On the other hand, have shown that high frequencies of known cadmium resistance cassettes were almost equally present among both persistent (86%) and presumed non-persistent (83%) L. monocytogenes populations. However, their results suggest that the cadA1 gene was more common in persisters, whereas the cadA4 sequence, which provides lower tolerance to cadmium than cadA1, was only carried in non-persistent isolates.

69Two L. monocytogenes stress survival islets (SSIs) which provide benefits to growth and/or survival under suboptimal or stress conditions, such as low pH (SSI-1), alkaline pH (SSI-2) or oxidative stress conditions (both islets), are usually overexpressed among persistent populations identified in food production environments. Interestingly, it has been suggested that persistent L. monocytogenes may possess a lower virulent potential due to the presence of truncated inlA gene, frequent lack of additional virulence factors such as LIPI-3 and LIPI-4 and the mutations in the prfA gene.

70compared the influence of different stress conditions (10% of NaCl; different concentrations of benzalkonium chloride; and energy sources) on the growth of persistent and sporadic L. monocytogenes strains of food origin and confirmed observations of other authors that there was not a significant difference in growth rate or ability to grow for isolates of persistent strains compared to sporadic strains for any treatments at 37°C.

71Several strategies for L. monocytogenes elimination from food chain have been developed and applied in the food industry. One of them is irradiation processing technology with gamma irradiation. The approach has been shown to be safe and is a proven method used worldwide for food product preservation. Food irradiation involves exposing food to gamma radiation to induce the demise of L. monocytogenes and other bacteria that can cause food poisoning or food spoilage.

72Another method of elimination of the pathogens from food and food production environments is application of ozone, the eco-focused method which is categorized as generally recognized as safe (GRAS). It has been recently shown application of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related L. monocytogenes strains resulted in over 3 log10 CFU/ml reduction of bacterial load after 10 min. Furthermore, a complete inactivation of planktonic cells after 6 h of treatment as well as a significant decrease of the biofilm biomass were observed. Thus, the use of gaseous ozone is a promising method of L. monocytogenes contamination control on both food contact surfaces and on the final products.

73One novel alternative biological method of L. monocytogenes control along the food chain is the use of phages. Phages are considered an effective tool against bacterial pathogens as they only target their specific organism and do not interfere with other microorganisms. This is especially important in the production of fermented foods as they do not have a negative influence on the sensory properties of the final product. There are commercial phage-based products which are successfully applied in food industry to control L. monocytogenes. One of them is ListShield™ (Intralytics, Columbia, MD, United States), a cocktail of six different lytic bacteriophages that is specifically designed for treating foods that are high risk for L. monocytogenes contamination, like ready-to-eat meat (RTE) products. It has been shown that ListShield™ significantly reduced by 82–99% the number of L. monocytogenes in different kinds of RTE food. In the case of smoked salmon, the phages completely eliminated the pathogen in both the naturally contaminated and experimentally contaminated samples without affecting the organoleptic quality of the food. ListShield™ can also be used to eliminate or significantly reduce the levels of L. monocytogenes on non-food contact equipment, surfaces, etc., in food processing plants and other food establishments.

74LISTEXTM P100 (Micreos, Hague, Netherlands) is also approved by the FDA and recommended by EFSA as a phage cocktail product for the reduction of L. monocytogenes on meat and poultry foods during processing or in the final product. The effectiveness of the broad host range bacteriophage P100 present in this products was tested for the reduction of L. monocytogenes in inoculated samples at different temperatures and the maximum decrease of the number of the pathogen was achieved at the level of 4.44 log CFU/g in contaminated food samples compared with the control group. When LISTEX™ P100 was applied on biofilms formed on stainless steel, 3.5–5.4 log CFU/cm2 reductions were observed depending on which of the 21 L. monocytogenes strains were tested. According to the EFSA opinion, LISTEX™ P100 is completely harmless, effective and does not contribute to antibacterial resistance.

75Despite many positive results and recommendations, the routine using of bacteriophages in food production industry and final food products is allowed only in some countries and regulations relate only to individual bacteriophage products. Despite the wide use of LISTEXTM P100 (e.g., in the United States, Canada and Switzerland), its acceptance as a processing aid in multiple countries (including Australia, New Zealand, Israel, Switzerland, Canada even in one EU member, Netherlands), and the previously stated positive opinion of EFSA, the EU has not approved this product for application in food industry. Thus, further studies on the safety and effectiveness of phage-based preparation against L. monocytogenes in foods as well as monitoring of the occurrence of phage-resistant strains in food processing plants are needed.

76Listeria monocytogenes is an important foodborne pathogen responsible for severe sporadic infections or disease outbreaks with high case fatality rates worldwide. These ubiquitous bacteria have been isolated from soil, water, feed, and food production environments, where they can survive and persist for a long time. Resistance of such strains to different food processing conditions is contributed to by several external factors such as poor hygiene practice or ineffective sanitizations, but also by the presence of diverse genetic determinants that are responsible for resistance to extreme temperatures, pH, heavy metals, biocides, and the ability to form biofilms. Although there is much knowledge about the mechanisms of stress responses and resistance to adverse conditions of L. monocytogenes, these pathogenic bacteria are still present in the food production environments and pose a severe threat to consumers. Thus, knowledge and understanding of the mechanisms of L. monocytogenes adaptation to environmental stress factors will have a significant influence on the development of new, efficient, and cost-effective methods of the pathogen control in the food industry which is critical to ensure food production safety.

77JO, BL, and KW conceptualized the idea of the manuscript. BL and KW collected relevant literature. JO drafted the manuscript. All authors reviewed, edited the manuscript, and read and approved the final version of the manuscript.

78The 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.

79All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.