**6. High hydrostatic pressure**

osmolytes from the external environment to restore osmotic balance within cells. The solute-mediated osmoprotection stimulates the growth of cells subjected to high salt concentrations. Deletions of these osmolyte transporters reduce the growth of *Listeria* under conditions of hyperosmolarity [14, 30, 35]. In addition to previously mentioned compatible solutes (glycine, betaine, and carnitine), proline is important for the survival under hyperosmolarity conditions [36]. σB factor, as an important part of the overall stress response of *L. monocytogenes,* mediates the expression of *ctc* gene and the use of betaine and carnitine as

In response to osmotic stress, two genes involved in cell envelope modification have been identified: *lmo2085*, a putative peptidoglycan-linked protein, and *lmo1078*, a putative UDPglucose phosphorylase that catalyzes the formation of UDP-glucose, a precursor of mem-

A further mechanism of osmotic adaptation is the modification of genetic expression leading to an increased or a decreased synthesis of several proteins. Salt-shock proteins are rapidly induced and overexpressed for a short time period, being similar to those induced in coldshock response (CSPs and CAPs). Among CSPs induced in *L. monocytogenes,* there are two general stress response proteins, DnaK that acts as a heat-shock protein stabilizing cellular proteins and Ctc that is involved in high osmolarity resistance in the lack of osmoprotectants, such as glycine, betaine, and carnitine, in the medium [38]. Additional stress response proteins, including ClpC (an ATPase), ClpP (a protease), and HtrA (a protease), are essential for osmotic and acid stress adaptation in *L. monocytogenes* [39]. HtrA may play a role in degrading misfolded proteins and is beneath LisRK control, a two-component regulatory system impor-

Desiccation tolerance defines the bacteria's aptitude to survive for extended periods on a surface, deficient of nutrients and water. As so, *L. monocytogenes* desiccation tolerance is most likely associated with the ability to persist in food production surfaces and consequently cross-contaminate food products [40]. The low aw resulting from high osmolarity decreases turgor pressure in a bacterial cell inhibiting bacterial growth [41]. Drying and addition of salt or sugar are traditional methods to lower food aw and therefore enhance its prolonged shelf life. *L. monocytogenes* grows optimally at aw ≥ 0.97, although it may survive in foods with low aw [42]. When compared to other common infectious foodborne pathogens, *L. monocytogenes* does not appear to grow at aw < 0.90 but it can survive in these conditions, particularly under refrigeration, for long periods. To date, existing information regarding *L. monocytogenes* desiccation survival is limited and primarily focuses on factors influencing the survival to osmotic stress [40, 43–46]. Strains of serotypes 1/2c and 1/2b were the most tolerant to desiccation, followed by 4b and 1/2a [47]. Hansen and Vogel [46] showed the protective effect of osmoadaptation and also the formation of biofilms on

osmoprotectors.

94 Listeria Monocytogenes

brane glycolipids and of the cell wall [37].

tant for osmoregulation [36].

the desiccation survival.

**5. Desiccation stress response**

A high hydrostatic pressure (HHP) represents the application of pressure in the range of 50–1000 MPa, though the inactivation of vegetative cells of bacterial species is typically reached from 300 to 700 MPa, and bacterial spores inactivation demands higher pressure levels up to 1000 MPa [48]. However, depending on the pressure level, HHP treatments can fully inactivate bacteria or impose sublethal injuries. For pressures up to 400 MPa, the integrity of Gram-positive bacterial cells and metabolic activity are maintained, with very limited cell destruction [49]. Over the last years, it has been stated that *L. monocytogenes* is potentially capable of recovering culturability following HHP exposure [49–52]. Physiological studies have also demonstrated that increasing pressure levels results in an accelerated decline of metabolic indicators, such as the activity of the LmrP membrane transport system [53]. These findings suggest that bacteria exposed to HHP are unable to grow due to cell injury, but yet can mount a nonspecific response to high pressure. A proportion of the cell population is able to maintain cellular activity of some kind after HHP, demonstrating the capacity to cellular repair and regrow, when adequate conditions are available [49].

To date, little research has been conducted regarding the mechanisms of bacterial adaptation and resistance to high pressure. Wemekamp-Kamphuis et al. [54] demonstrated that one of the responses that enable *Listeria* survival upon HHP treatment results from induction of the general stress response mediated by σB. *L. monocytogenes sigB* deletion mutant was more susceptible to HHP exposure than the wild type, while induction of σB resulted in an increased HHP protection relative to the untreated control strain.

Several pressure-induced proteins have been increasingly synthesized when compared to the synthesis of other control proteins at atmospheric pressure [55]. *L. monocytogenes* has shown to actively express many genes as a response to high pressure, but some functional categories appear more affected than others. Genes that tend to be expressed at higher levels under high pressure are genes encoding for transport and binding, signal transduction and chemotaxis, cellular processes, transcriptional regulators, metabolism, and protein fate [56]. The stabilization and maintenance of the bacteria cell is at high focus, showed by the significant regulation of ribosomes and proteins, together with components involved in the cell envelope and the septal ring. It is assumed that the activation of genes involved in the lipid and peptidoglycan biosynthetic pathways is connected to this function. Upregulation of genes associated with generalized repair and maintenance has been proved, where the activation of cold- and heatshock genes is an example for this [57, 58]. When high pressure demands more energy to be used on repair, energy production and conversion is suppressed. The repression of several energy production/conversion, carbohydrate, and other carbon compound catabolic genes may represent a diminishment of catabolism in cells imposed by HPP treatments. This can be seen by the pressure-induced switch from active growth to a cell repair state, the stationary phase, resulting in a decreased growth rate [59].

Several genes associated with cell formation and shape, as well as synthesis or reassembly of cell-wall constituents, in particular peptidoglycan and fatty acids, were observed to have an increased expression. Because of this, genes involved in such functions can be considered as very central in the response to high pressure. It is presumed that *L. monocytogenes* increases both cell division and cell-envelope-associated gene expression aiming to replace damaged components and thus compensate membrane and wall damages [59].

**Involvement Proteins/function Ref.**

*Class III stress-response regulator, a transcription repressor.*

*transcription.*

**SigmaB (σB)**

**CtsR**

**HrcA**

**InlB**

*membrane.*

**PC-PLC**

**Mpl**

**HtrA**

**Bsh**

**ClpC**

**ClpP**

*condition.*

*macrophages and thus virulence.*

*transcription repressor.*

*the E-cadherin.*

*General stress transcription factor.*

*Positive regulatory factor A, central virulence regulator of virulence gene* 

The Impact of Environmental Stresses in the Virulence Traits of *Listeria monocytogenes* Relevant…

*Heat regulation at controlling inverted repeat of chaperone expression elements. A* 

*Internalin A, surface protein that mediates entry into cells expressing its receptor,* 

*Internalin B, surface protein that mediates entry into cells expressing one of the receptors gC1qR, HGF-SF, Met, and the glycosaminoglycanes (GAGs).*

*Listeriolysin O, hemolysin required for vacuole escape by lysis of the phagosome* 

*Phospholipase activated by proteolytic cleavage involving Mpl or by cellular* 

*proteases. Required for the lysis of the double-membrane vacuole.*

*Hexose phosphate transporter required for intracytosolic proliferation.*

*Bile salt hydrolase involved in the intestinal and hepatic phases of listeriosis.*

*Serine protease involved in proteolysis and required for growth under stress* 

*ATPase protein promoting early bacterial escape from the phagosome of* 

*Actin assembly-inducing protein, involved in cell-to-cell spread*.

*Serine protease involved in acid and osmotic stress response.*

*Metalloprotease required for the maturation of PC-PLC.*

[68]

97

http://dx.doi.org/10.5772/intechopen.76287

[69]

[70]

[71]

[65]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

Regulation **PrfA**

Attachment and invasion **InlA**

Lysis of vacuoles **LLO**

Intracellular multiplication **Hpt**

Cell-to-cell spread **ActA**

Environmental stress response

and virulence

Cell membranes damage by HPP may possibly be a main cause of inactivation or death in Gram-negative bacteria, but it is fallacious to admit that in Gram-positive bacteria. Cell membrane and wall stabilization in the stationary growth phase do provide a protective effect against HPP, being a major factor for the survival of HPP-induced damage [60]. Beyond cell envelope damage, HPP interferes within the nascent septal ring formation along with other associated cell-wall formation and chromosome segregation processes [59].

#### **7. Stress impact on** *L. monocytogenes* **virulence**

*L. monocytogenes* has a profound ability to adapt to unfavorable stressful environments, switching from a saprophyte to an intracellular pathogen capable of causing serious infection to the host [61]. In this transformation, σB dominates both in the external environment and during gastrointestinal transit, while positive regulatory factor A (PrfA) plays a central role on the intracellular infection. In concert with PrfA, σB activates the transcription of several *L. monocytogenes* virulence genes: (1) *bsh*, encoding bile salt hydrolase, essential in gastrointestinal colonization prior to invasion; (2) *inlA*, encoding internalin A, mediates entry into human intestinal epithelial cells; and (3) *gadA*, encoding part of the glutamate decarboxylase system, crucial for acid survival [62]. σB also contributes to the transcriptional activation of *prfA*, encoding PrfA, a central virulence regulator of virulence gene expression in *L. monocytogenes* [63].

PrfA-dependent virulence gene cluster or LIPI-1 (*Listeria* pathogenicity island 1) encodes most virulence factors involved in the pathogenic infectious cycle. This chromosomal locus comprises the following genes: (1) *hly*, encoding listeriolysin O (LLO), a pore-forming toxin crucial in the escape from phagocytic vacuoles; (2) *plcA* and *plcB*, encoding two phospholipases C which cooperate with LLO in the escape from bacterial phagosomes; (3) *mpl*, encoding a metalloprotease implicated in the maturation of proenzyme pro-PlcB; (4) *actA*, encoding ActA protein involved in the intra- and intercellular motility of the bacteria; and (5) *prfA*, encoding PrfA, a transcriptional activator of LIPI-1 genes [64]. The expression of additional genes dispersed on the chromosome may be PrfA-regulated, as the internalin locus *inlAB* [65], the genes encoding internalins InlA and InlB cell-wall-anchored proteins which induce *Listeria* phagocytosis [66].

Following the complete genome sequencing of several *L. monocytogenes* strains, an increasing number of virulence-related proteins are being identified and their specific involvement during infectious stages deciphered (**Table 1**).

In addition to other factors, the infectious potential of *L. monocytogenes* is conditioned by the environmental conditions prior to host invasion. A correlation between stress response and virulence seems to exist and associates strains having more effective stress response mechanisms to being also more virulent [84]. Early studies by Durst [84] and Wood and Woodbine [85] demonstrated that cold storage may enhance virulence of some strains because the


increased expression. Because of this, genes involved in such functions can be considered as very central in the response to high pressure. It is presumed that *L. monocytogenes* increases both cell division and cell-envelope-associated gene expression aiming to replace damaged

Cell membranes damage by HPP may possibly be a main cause of inactivation or death in Gram-negative bacteria, but it is fallacious to admit that in Gram-positive bacteria. Cell membrane and wall stabilization in the stationary growth phase do provide a protective effect against HPP, being a major factor for the survival of HPP-induced damage [60]. Beyond cell envelope damage, HPP interferes within the nascent septal ring formation along with other

*L. monocytogenes* has a profound ability to adapt to unfavorable stressful environments, switching from a saprophyte to an intracellular pathogen capable of causing serious infection to the host [61]. In this transformation, σB dominates both in the external environment and during gastrointestinal transit, while positive regulatory factor A (PrfA) plays a central role on the intracellular infection. In concert with PrfA, σB activates the transcription of several *L. monocytogenes* virulence genes: (1) *bsh*, encoding bile salt hydrolase, essential in gastrointestinal colonization prior to invasion; (2) *inlA*, encoding internalin A, mediates entry into human intestinal epithelial cells; and (3) *gadA*, encoding part of the glutamate decarboxylase system, crucial for acid survival [62]. σB also contributes to the transcriptional activation of *prfA*, encoding PrfA, a central virulence regulator of virulence gene expression in *L. monocytogenes* [63].

PrfA-dependent virulence gene cluster or LIPI-1 (*Listeria* pathogenicity island 1) encodes most virulence factors involved in the pathogenic infectious cycle. This chromosomal locus comprises the following genes: (1) *hly*, encoding listeriolysin O (LLO), a pore-forming toxin crucial in the escape from phagocytic vacuoles; (2) *plcA* and *plcB*, encoding two phospholipases C which cooperate with LLO in the escape from bacterial phagosomes; (3) *mpl*, encoding a metalloprotease implicated in the maturation of proenzyme pro-PlcB; (4) *actA*, encoding ActA protein involved in the intra- and intercellular motility of the bacteria; and (5) *prfA*, encoding PrfA, a transcriptional activator of LIPI-1 genes [64]. The expression of additional genes dispersed on the chromosome may be PrfA-regulated, as the internalin locus *inlAB* [65], the genes encoding internalins InlA and InlB cell-wall-anchored proteins which induce *Listeria* phagocytosis [66]. Following the complete genome sequencing of several *L. monocytogenes* strains, an increasing number of virulence-related proteins are being identified and their specific involvement dur-

In addition to other factors, the infectious potential of *L. monocytogenes* is conditioned by the environmental conditions prior to host invasion. A correlation between stress response and virulence seems to exist and associates strains having more effective stress response mechanisms to being also more virulent [84]. Early studies by Durst [84] and Wood and Woodbine [85] demonstrated that cold storage may enhance virulence of some strains because the

components and thus compensate membrane and wall damages [59].

96 Listeria Monocytogenes

associated cell-wall formation and chromosome segregation processes [59].

**7. Stress impact on** *L. monocytogenes* **virulence**

ing infectious stages deciphered (**Table 1**).


Garner et al. [93] reported an intensified invasiveness of *L. monocytogenes* for Caco-2 cells when grown at 7°C rather than at 37°C, and, for both temperatures, the invasion ability was greater in cells grown at pH 7.4 compared to growth at pH 5.5. A growth temperature of 37°C, pH 7.4, in the presence of NaCl or sodium lactate, enhanced *L. monocytogenes* invasiveness; however, the pre-exposure to gastric fluid (pH 4.5), even for as short as 10 s, substantially reduced its invasion. These findings intimate that listerial virulence-associated characteristics seem to be affected by specific food properties (e.g., the presence of organic acids or salt). The authors further showed that *L. monocytogenes* growth phase affects its ability to invade Caco-2 cells. The invasion by logphase cells was 9.5-fold lower than invasion by stationary-phase cells, corroborating other studies which demonstrate that exposure of *L. monocytogenes* to different environmental conditions can change invasiveness and virulence [93]. Accordingly, the increased stationary-phase invasiveness also coincides with stationary-phase induction of σB activity [90]. In stationary-phase cells, *inlA* expression is regulated in a σB-dependent manner, and growth phase-dependent effects on invasion appear independent of PrfA [94, 95], contributing to *inlA* transcription [96].

The Impact of Environmental Stresses in the Virulence Traits of *Listeria monocytogenes* Relevant…

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99

Complementary studies demonstrate that *L. monocytogenes* pathogenicity requires an adaptive acid tolerance response, so the ability to survive gastric acid fluid and to invade host cells is related to ATR activation [30, 89, 97]. This finding is supported by the fact that the glutamate decarboxylase (GAD) system, as the ATR most important component, is required for listerial survival in the gastric environment, and also LisRK deletion, a two-component system involved in acid resistance regulation, caused a dramatic reduction in virulence [29, 98].

A further prerequisite for *L. monocytogenes* infection depends on the ability to counteract conditions of elevated osmolarity in the gastrointestinal tract. As mentioned in Section 2.1, the carnitine uptake system (OpuC) is directly linked to osmotic stress resistance of *L. monocytogenes* and to its ability to reach and proliferate in the liver and spleen [17]. Carnitine (produced from the desquamation of the gastrointestinal epithelial layer) was formerly proved to act as a crucial osmoprotectant, facilitating growth in this gastrointestinal environment, once changing the carnitine transported OpuC resulted in a significant reduction in *Listeria* ability to colonize the upper small intestine and cause subsequent systemic infection [99, 100]. A supporting study by Wemekamp-Kamphuis et al. [17] demonstrated that a triple mutant, defective in all three compatible uptake systems (BetL, Gbu, and OpuC), showed a similar phenotype to that of a single opuC mutant, mutually revealing a decreased ability to cause systemic infection relative to the parent. Those were clear evidences that *betL* and *gbu* do not play a significant role in *L. monocytogenes* pathogenesis and that it is the carnitine uptake system that most induces listerial virulence. In addition, Joseph et al. [101] also identified OpuCA and OpuCB as being induced intracellularly. Since the contribution of each transporter is dependent on the external environment, there are occurrences

when each system is tailored for optimal effects within a certain environmental niche.

Over the last years, novel trends in food production tend to preserve the natural flavor and texture of products using minimal processing. Non-thermal food preservation usually allows a significant microbial reduction, and mounting evidence also demonstrates that the conditions applied by alternative technologies may influence bacterial virulence [102]. The application of HHP has been shown not to induce mutations in the internal genes, *inlA* and *inlB*, implicated in the adhesion and internalization of *L. monocytogenes* in human cells. However, when the effect of HPP on the *ctsR* gene is observed, a reduction in virulence potential of

**Table 1.** Stress response and virulence-associated proteins in *Listeria monocytogenes* (adapted from reference [67]).

pathogen virulence rather increases when grown under refrigeration than at optimal growth temperature. By contrast, virulence gene expression was reported to be downregulated at temperatures below 30°C, besides PrfA is only formed at 37°C [85]. According to Loh et al. [86], the expression of *prfA* is nearly 16-times higher at 37°C compared to that at 30°C, and imperceptible in cells cultivated at 20°C. The specific pathogenicity of LLO can be fully recovered in less than 24 h by incubating refrigerated cells at 37°C [87]. This virulence recovery after heat shock reinforces the importance of eliminating *L. monocytogenes* from minimally processed ready-to-eat foods held at refrigeration temperatures for long periods.

Low pH and high salt content are common factors often found in foods contaminated with *L. monocytogenes* [89]. Even though at these conditions, the growth of most foodborne and spoilage bacteria is restricted, *L. monocytogenes* is capable of surviving and even grow in such environments; long-term adaptation to these sublethal stress conditions results in altered virulence [88].

Conte et al. [31, 89] demonstrated that short-term exposure (1 h) of *L. monocytogenes* to a sublethal acidic environment (pH 5.1) not only increased its invasiveness to the human colon adenocarcinoma cell line Caco-2 but also increased the ability of *L. monocytogenes* to survive and proliferate in macrophage-like cells, suggesting that exposure to a low pH (e.g., in the human stomach) may enhance listerial overall virulence. In addition, LLO excreted by virulent *L. monocytogenes* showed a maximal activity at pH 4.0–5.0. In another study, the exposure of *L. monocytogenes* to acidic shock has induced the transcription of two important virulence genes (*inlA* and *bsh*) [90]. Conversely, a study by Rieu et al. [91] reported a decrease in virulence gene transcription after 5 h at pH 4.0 achieved with acetic acid. This conflicting finding may be sustained by the use of organic acids since they might be more harmful to the bacteria. Some weak organic acids enhance pathogenicity of the bacterium, while others reduce it, as the secretion of LLO is increased by citrate, acetate, and lactate, whereas sorbate inhibited this hemolysin [92]. This knowledge would be important for the selection of acidulants to be used in different foods.

Garner et al. [93] reported an intensified invasiveness of *L. monocytogenes* for Caco-2 cells when grown at 7°C rather than at 37°C, and, for both temperatures, the invasion ability was greater in cells grown at pH 7.4 compared to growth at pH 5.5. A growth temperature of 37°C, pH 7.4, in the presence of NaCl or sodium lactate, enhanced *L. monocytogenes* invasiveness; however, the pre-exposure to gastric fluid (pH 4.5), even for as short as 10 s, substantially reduced its invasion. These findings intimate that listerial virulence-associated characteristics seem to be affected by specific food properties (e.g., the presence of organic acids or salt). The authors further showed that *L. monocytogenes* growth phase affects its ability to invade Caco-2 cells. The invasion by logphase cells was 9.5-fold lower than invasion by stationary-phase cells, corroborating other studies which demonstrate that exposure of *L. monocytogenes* to different environmental conditions can change invasiveness and virulence [93]. Accordingly, the increased stationary-phase invasiveness also coincides with stationary-phase induction of σB activity [90]. In stationary-phase cells, *inlA* expression is regulated in a σB-dependent manner, and growth phase-dependent effects on invasion appear independent of PrfA [94, 95], contributing to *inlA* transcription [96].

Complementary studies demonstrate that *L. monocytogenes* pathogenicity requires an adaptive acid tolerance response, so the ability to survive gastric acid fluid and to invade host cells is related to ATR activation [30, 89, 97]. This finding is supported by the fact that the glutamate decarboxylase (GAD) system, as the ATR most important component, is required for listerial survival in the gastric environment, and also LisRK deletion, a two-component system involved in acid resistance regulation, caused a dramatic reduction in virulence [29, 98].

pathogen virulence rather increases when grown under refrigeration than at optimal growth temperature. By contrast, virulence gene expression was reported to be downregulated at temperatures below 30°C, besides PrfA is only formed at 37°C [85]. According to Loh et al. [86], the expression of *prfA* is nearly 16-times higher at 37°C compared to that at 30°C, and imperceptible in cells cultivated at 20°C. The specific pathogenicity of LLO can be fully recovered in less than 24 h by incubating refrigerated cells at 37°C [87]. This virulence recovery after heat shock reinforces the importance of eliminating *L. monocytogenes* from minimally

**Table 1.** Stress response and virulence-associated proteins in *Listeria monocytogenes* (adapted from reference [67]).

**Involvement Proteins/function Ref.**

*Chaperone heat-shock proteins encoded by the dnaK operon and required for* 

*Chaperone proteins which regulate HrcA posttranscriptionally.*

*The glutamate decarboxylase system, involved in acid stress response.*

*Glycine betaine transport system I, involved in osmotic stress response.*

*Glycine betaine transport system II, involved in osmotic stress response.*

*Carnitine transport system, involved in cold and osmotic stress response.*

[22]

[23]

[29]

[82]

[15]

[83]

**DnaKJ**

98 Listeria Monocytogenes

**GAD**

**BetL**

**Gbu**

**OpuC**

*phagocytosis.*

**GroES, GroEL**

Low pH and high salt content are common factors often found in foods contaminated with *L. monocytogenes* [89]. Even though at these conditions, the growth of most foodborne and spoilage bacteria is restricted, *L. monocytogenes* is capable of surviving and even grow in such environments; long-term adaptation to these sublethal stress conditions results in altered virulence [88]. Conte et al. [31, 89] demonstrated that short-term exposure (1 h) of *L. monocytogenes* to a sublethal acidic environment (pH 5.1) not only increased its invasiveness to the human colon adenocarcinoma cell line Caco-2 but also increased the ability of *L. monocytogenes* to survive and proliferate in macrophage-like cells, suggesting that exposure to a low pH (e.g., in the human stomach) may enhance listerial overall virulence. In addition, LLO excreted by virulent *L. monocytogenes* showed a maximal activity at pH 4.0–5.0. In another study, the exposure of *L. monocytogenes* to acidic shock has induced the transcription of two important virulence genes (*inlA* and *bsh*) [90]. Conversely, a study by Rieu et al. [91] reported a decrease in virulence gene transcription after 5 h at pH 4.0 achieved with acetic acid. This conflicting finding may be sustained by the use of organic acids since they might be more harmful to the bacteria. Some weak organic acids enhance pathogenicity of the bacterium, while others reduce it, as the secretion of LLO is increased by citrate, acetate, and lactate, whereas sorbate inhibited this hemolysin [92]. This knowledge would be important for the selection of acidulants to be used in different foods.

processed ready-to-eat foods held at refrigeration temperatures for long periods.

A further prerequisite for *L. monocytogenes* infection depends on the ability to counteract conditions of elevated osmolarity in the gastrointestinal tract. As mentioned in Section 2.1, the carnitine uptake system (OpuC) is directly linked to osmotic stress resistance of *L. monocytogenes* and to its ability to reach and proliferate in the liver and spleen [17]. Carnitine (produced from the desquamation of the gastrointestinal epithelial layer) was formerly proved to act as a crucial osmoprotectant, facilitating growth in this gastrointestinal environment, once changing the carnitine transported OpuC resulted in a significant reduction in *Listeria* ability to colonize the upper small intestine and cause subsequent systemic infection [99, 100]. A supporting study by Wemekamp-Kamphuis et al. [17] demonstrated that a triple mutant, defective in all three compatible uptake systems (BetL, Gbu, and OpuC), showed a similar phenotype to that of a single opuC mutant, mutually revealing a decreased ability to cause systemic infection relative to the parent. Those were clear evidences that *betL* and *gbu* do not play a significant role in *L. monocytogenes* pathogenesis and that it is the carnitine uptake system that most induces listerial virulence. In addition, Joseph et al. [101] also identified OpuCA and OpuCB as being induced intracellularly. Since the contribution of each transporter is dependent on the external environment, there are occurrences when each system is tailored for optimal effects within a certain environmental niche.

Over the last years, novel trends in food production tend to preserve the natural flavor and texture of products using minimal processing. Non-thermal food preservation usually allows a significant microbial reduction, and mounting evidence also demonstrates that the conditions applied by alternative technologies may influence bacterial virulence [102]. The application of HHP has been shown not to induce mutations in the internal genes, *inlA* and *inlB*, implicated in the adhesion and internalization of *L. monocytogenes* in human cells. However, when the effect of HPP on the *ctsR* gene is observed, a reduction in virulence potential of surviving cells was noted. Likewise, virulence and reduced motility may be the result of a mutation in this gene corresponding to the loss of a single amino acid. This suppression could be related to a high-pressure tolerance [70, 103].

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#### **8. Conclusions**

Exposure of *L. monocytogenes* to sublethal environmental stresses can enhance its survival to subsequent lethal conditions and additionally induce the expression of the organism's virulence genes. Therefore, exposure of *L. monocytogenes* to food-associated stresses such as high salt concentrations or low temperatures during refrigerated storage may result in increased virulence and thus a higher risk for listeriosis. Any strain of *L. monocytogenes* present in food is actually considered equally pathogenic. However, results from several studies support the idea that the heterogeneity among strains regarding the response to stress and virulence potential should be considered, once responses to food matrix and storage conditions are often strain specific.

Although significant advances in our understanding on stress response and virulence potential have been achieved in the last years, there is still a need to fulfill knowledge gaps on molecular mechanisms behind *L. monocytogenes* response to stress and virulence. Further studies on the influence of food matrix on stress tolerance and virulence potential of different strains, recovered from foods and from patients, are needed. This information can be further used by regulators to refine previous risk assessments and also in the definition of control measures by the food industry.

#### **Acknowledgements**

This work was supported by National Funds from FCT—Fundação para a Ciência e a Tecnologia through project UID/Multi/50016/2013. Publication in open access was co-financed by the project NORTE-01-0246-FEDER-000011, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Financial support for author Sofia Pereira was provided by ESF—European Social Fund, under the PORTUGAL 2020 Partnership Agreement, through doctoral fellowship NORTE-08-5369-FSE-000007\_BD\_1.

#### **Author details**

Sofia Araújo Pereira, Ângela Alves, Vânia Ferreira and Paula Cristina Maia Teixeira\*

\*Address all correspondence to: pcteixeira@porto.ucp.pt

Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Porto, Portugal
