**5. The action of the essential oils on quorum sensing system and biofilm formation**

EOs can act also on QS systems that coordinate the whole system of pathogenicity of bacteria [30, 69] (**Figure 2**). This property is of noticeable interest, due to the continuous research for new therapeutic and antibacterial agents, which could

**175**

*Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

concurrently act in no toxic manner and without encouraging the development and emergence of resistant bacterial strains [45]. EOs can work on one or more events regulating the entire quorum sensing activity of microorganisms. Summarily, bacterial QS may be inhibited through different mechanisms. Their action against Gram-negative bacteria can be mainly expressed in three basic steps: a first step can block the "upstream" mechanism through the synthesis of AHL; a second mechanism may act further downstream, blocking the AHL transport and/or secretion. If bacteria still manage to produce AHLs and these molecules are still transported and secreted outside, other EO or their components could in any case be able to "capture" these molecules, effectively preventing cell-cell communication between bacteria. Other EOs can therefore act by exhibiting an antagonistic action with respect to AHLs or operating an inhibitory effect downstream of AHL receptor binding [49]. The versatility of action of EOs depends essentially on their chemical composition and the presence of functional groups. EOs containing largely terpenes (*p*-cymene, limonene, terpinene, sabinene, and pinenes) as well as some oxygenated components (for instance, camphor and camphone, borneol and bornyl acetate, 1,8 cineole, α-pinene, and verbenonone) generally do not exhibit a so strong antibacterial activity, which is just more manifest against Gram-positive bacteria. Further than the composition, the antimicrobial activity of EOs is also due to their concentration. In fact, depending on such element, EOs or their components can operate in a different manner on one or more factors that affect the mechanisms of cell-cell communication among bacteria. Thus, some EOs, also at low concentration, are capable to impede the chemical activity of those enzymes involved in the production of energy for the survival and growth of bacteria or, at higher concentration, even to disaggregate and denature microbial proteins [30, 70]. Subinhibitory concentrations of clove EO, tested on *P. aeruginosa* and *Aeromonas hydrophila*, were capable to significantly reduce the las- and rhlregulated virulence factors such as LasB, total protease, chitinase and pyocyanin production, swimming motility, and exopolysaccharide production. The biofilmforming capability of these two strains was also reduced in a concentrationdependent manner at all tested sub-MIC values [71]. Peppermint EO at

sub-minimum inhibitory concentrations (sub-MICs) strongly can interfere with the production of AHL-regulated virulence factors and biofilm formation in *P. aeruginosa* and *A. hydrophila*. Such effect is mainly due to the presence of menthol, which interferes with QS systems of various Gram-negative pathogens, acting essentially on the las and pqs QS systems [72]. Different bacterial strains are used to test the potential inhibitory effect of essential oils on QS. Apart from the well-known models (*Vibrio harveyi*, *P. aeruginosa*, *S. aureus*, *E. coli*) more recently *Chromobacter violaceum*, in particular the mutant strain CV026, has been also used with this scope. This strain can provide, through the production or not of its purple pigment violacein, directly linked to QS, useful information about the capability of a substance to act or do not act as quorum-quenching agent, respectively. Through the use of such approach, Szabo and co-workers [73] studying several EOs ascertained that, among some EOs, rose, geranium, lavender, and rosemary EOs were the most potent QS inhibitors. Eucalyptus and citrus oils were moderately active, while the chamomile, orange, and juniper EOs which did not show any were ineffective. In several cases, the synergistic effect of more components can enhance the capability of an EO to inhibit the mechanism of communication among bacteria. Khan and co-workers evaluated the capability of different EOs and of their main components to act as quorum-quenching agents. Their study evidenced that clove essential oil showed promising anti-QS activity, followed in activity by cinnamon, lavender, and peppermint oils, and that eugenol, the major constituent of clove oil, could not exhibit anti-QS activity [74]. In other cases, the effectiveness of EOs is related both

#### *Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

*Essential Oils - Oils of Nature*

**4. Mechanisms of essential oils on microorganisms**

As above indicated, the mechanisms which allow EOs to damage bacteria are largely dependent on their composition. Usually, antimicrobial activity can originate from a flow of reactions implicating the total bacterial cell; this is essentially due to the fact that, since the EOs are composed of many groups of chemical compounds, these last act in different ways [30]. Generally, Gram-positive bacteria and Gram-negative bacteria are differently susceptible to the action of EOs, due to the structural differences of their cell wall of these two groups of bacteria. The higher susceptibility of Gram-positive bacteria is caused mainly by the presence of peptidoglycan within their cell wall, which allows more easily the hydrophobic molecules to have access within the cell, acting therein with cytoplasm [30]. The cell wall of Gram-negative bacteria shows an outer membrane, composed of a double layer of phospholipids linked to the peptidoglycan layer by lipopolysaccharides. This allows these bacteria to exhibit greater resistance to the penetration of essential oils and/or their components; in fact, some hydrophobic molecules can be capable to enter into the cell, only through the access given by the porins, proteins that form water-filled channels distributed all over the cell wall. The different compositions of cell wall let it that Gram-negative bacteria are even more resistant to hydrophobic antibiotics [30, 67]. The mechanism through which the EOs or their components act on microbial cell is well known: it includes one or more simultaneous actions, ranging from cell wall degradation, to the damage caused to the cytoplasmic membrane and membrane proteins, as well as to a reduction of the proton-motive force until to damage to the ATP synthesis mechanism. Lipophilic character of EO compounds allows them to penetrate the cell membrane and remain between the phospholipids and/or affect the synthesis of membrane lipids, with a consequent change of membrane structure and with an alteration of its permeability. In addition, EOs can affect directly also the morphology of bacterial cell, altering it even irreversibly, to cause the complete destruction of the entire microbial cell scaffolding [30, 68] (**Figure 2**).

**5. The action of the essential oils on quorum sensing system and biofilm** 

EOs can act also on QS systems that coordinate the whole system of pathogenicity of bacteria [30, 69] (**Figure 2**). This property is of noticeable interest, due to the continuous research for new therapeutic and antibacterial agents, which could

**174**

**formation**

*Effect of essential oils on microbial cells (modified from [65]).*

**Figure 2.**

concurrently act in no toxic manner and without encouraging the development and emergence of resistant bacterial strains [45]. EOs can work on one or more events regulating the entire quorum sensing activity of microorganisms. Summarily, bacterial QS may be inhibited through different mechanisms. Their action against Gram-negative bacteria can be mainly expressed in three basic steps: a first step can block the "upstream" mechanism through the synthesis of AHL; a second mechanism may act further downstream, blocking the AHL transport and/or secretion. If bacteria still manage to produce AHLs and these molecules are still transported and secreted outside, other EO or their components could in any case be able to "capture" these molecules, effectively preventing cell-cell communication between bacteria. Other EOs can therefore act by exhibiting an antagonistic action with respect to AHLs or operating an inhibitory effect downstream of AHL receptor binding [49]. The versatility of action of EOs depends essentially on their chemical composition and the presence of functional groups. EOs containing largely terpenes (*p*-cymene, limonene, terpinene, sabinene, and pinenes) as well as some oxygenated components (for instance, camphor and camphone, borneol and bornyl acetate, 1,8 cineole, α-pinene, and verbenonone) generally do not exhibit a so strong antibacterial activity, which is just more manifest against Gram-positive bacteria. Further than the composition, the antimicrobial activity of EOs is also due to their concentration. In fact, depending on such element, EOs or their components can operate in a different manner on one or more factors that affect the mechanisms of cell-cell communication among bacteria. Thus, some EOs, also at low concentration, are capable to impede the chemical activity of those enzymes involved in the production of energy for the survival and growth of bacteria or, at higher concentration, even to disaggregate and denature microbial proteins [30, 70]. Subinhibitory concentrations of clove EO, tested on *P. aeruginosa* and *Aeromonas hydrophila*, were capable to significantly reduce the las- and rhlregulated virulence factors such as LasB, total protease, chitinase and pyocyanin production, swimming motility, and exopolysaccharide production. The biofilmforming capability of these two strains was also reduced in a concentrationdependent manner at all tested sub-MIC values [71]. Peppermint EO at sub-minimum inhibitory concentrations (sub-MICs) strongly can interfere with the production of AHL-regulated virulence factors and biofilm formation in *P. aeruginosa* and *A. hydrophila*. Such effect is mainly due to the presence of menthol, which interferes with QS systems of various Gram-negative pathogens, acting essentially on the las and pqs QS systems [72]. Different bacterial strains are used to test the potential inhibitory effect of essential oils on QS. Apart from the well-known models (*Vibrio harveyi*, *P. aeruginosa*, *S. aureus*, *E. coli*) more recently *Chromobacter violaceum*, in particular the mutant strain CV026, has been also used with this scope. This strain can provide, through the production or not of its purple pigment violacein, directly linked to QS, useful information about the capability of a substance to act or do not act as quorum-quenching agent, respectively. Through the use of such approach, Szabo and co-workers [73] studying several EOs ascertained that, among some EOs, rose, geranium, lavender, and rosemary EOs were the most potent QS inhibitors. Eucalyptus and citrus oils were moderately active, while the chamomile, orange, and juniper EOs which did not show any were ineffective. In several cases, the synergistic effect of more components can enhance the capability of an EO to inhibit the mechanism of communication among bacteria. Khan and co-workers evaluated the capability of different EOs and of their main components to act as quorum-quenching agents. Their study evidenced that clove essential oil showed promising anti-QS activity, followed in activity by cinnamon, lavender, and peppermint oils, and that eugenol, the major constituent of clove oil, could not exhibit anti-QS activity [74]. In other cases, the effectiveness of EOs is related both

to their composition and to the bacterium of reference; thus, an EO can act as mixture, better than a singular component on a specific bacterium; therefore, one or more components can act better than parent EOs against another bacterium. The effect of clary sage, juniper, lemon, and marjoram EOs and their major components on the formation of bacterial and yeast biofilms and on the inhibition of AHLmediated QS, evaluated using *Bacillus cereus*, *Pichia anomala*, *Pseudomonas putida*, and a mix of bacteria containing also *E. coli*, demonstrated that marjoram EO inhibited all these tester strains. However, all components exhibited more strength in limiting the biofilm capacity of *B. cereus* than the parent EOs. Lemon EO was capable to inhibit *E. coli* and mixed-culture biofilms; on the other hand, cinnamon was effective against the mixed forms [75]. Conversely, the entire EO of tangerine (*Citrus reticulata*) is capable to inhibit the *P. aeruginosa* biofilm formation more than its main component limonene, by an inhibition of the QS autoinducer production and elastase activity [76]. This also highlights how, within a same genus, not all the species show the same biological activity. Thus, the EO of *C. reticulata* (tangerine) can be more active in inhibiting the QS system; on the other hand, the EO recovered from orange (*Citrus sinensis*) can be completely ineffective [73]. Some terpenoids, for example, thymol, carvacrol, linalool, menthol, geraniol, linalyl acetate, citronellal, and piperitone, have antibacterial activity mediated by their functional group. Carvacrol is one of the most active components present in different EOs, in particular from Labiatae. Its spectrum of activity is much wide. At sublethal concentrations (<0.5 mM), it is capable to inhibit the formation of biofilms of *C. violaceum*, *Salmonella enterica* subsp. *typhimurium*, and *S. aureus*, while it does not exhibit effects on the formation of *P. aeruginosa* biofilms. In all cases, this concentration seems to not have effects on total bacterial numbers, indicating that carvacrol bactericidal effect could not be also linked to its inhibitory effect on biofilm formation. Sub-MIC concentrations of carvacrol could reduce the expression of cviI (a gene coding for the N-acyl-l-homoserine lactone synthase) and decrease the production of violacein and the activity of chitinase (both regulated by quorum sensing) at concentrations coinciding with carvacrol's inhibiting effect on biofilm formation. These results indicate that carvacrol activity in inhibition of biofilm formation might be also related to the disruption of quorum sensing [77]. Thymol, one of the main constituents of *Thymus vulgaris* EO, can affect (at the same manner of the parent EO) not only the AHL production (acting thus in the blockage of the communication system among bacteria), but it also can suppress flagella gene transcription (reducing the mRNA level of flagella gene), the bacterial motility, and finally the formation of biofilm [78]. Cinnamaldehyde, another widely diffused component, present, for example, in cinnamon EO, can show different mechanisms of action. The use of 60 μΜ cinnamaldehyde can decrease down to 55% the bioluminescence of *V. harveyi BB886*, which is induced by *3-*hydroxy-C4-HSL, and from 60 to 100% that of *V. harvevi* BB170 (mediated by AI-2). This indicates, once again, that the activity of EOs, like all other phytochemicals, can be dependent even on the strain used within the same species [30, 65, 79, 80], further than on the QS molecule involved. Another study showed that cinnamaldehyde particularly directs its action toward the short-chain AHL synthase (RhlI) and inhibits AHL production by RhlI [81]. Also cinnamaldehyde analogs and derivatives are capable to inhibit AI-2-based QS system of *V. harveyi* in a dose-dependent manner [82] and are effective against AI-2-regulated QS of *Vibrio* spp. too [83]. Three other cinnamaldehyde analogs, *trans*-2-nonenal, *trans*-3-decen-2-one, and *trans*-3-nonen-2-one, can interfere with AI-2 QS in different manner. In *Vibrio* spp., *trans*-2-nonenal and *trans*-3-decen-2-one inhibit the AI-2-based QS system by reducing the DNAbinding ability of LuxR, causing a decrease in the production of QS-regulated virulence functions such as biofilm formation, matrix production, and protease

**177**

biofilm and to invade Caco-2 cells.

*Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

production [83]. Therefore, some compounds, such as *p*-anisaldehyde can act as AHL mimics, inhibiting the production of violacein by *C. violaceum* [84]. Eugenol inhibits QS in pathogenic bacteria; this was shown, for example, by Zhou and co-workers [85], evaluating the reduction of violacein production in *C. violaceum* after contact with eugenol. This molecule is also capable to affect lasB and pqsA in *E. coli*. This suggests an inhibitory action of eugenol on Las and pseudomonas quinolone signal (PQS)-controlled transcription. The action of eugenol on pathogenic bacteria at subinhibitory concentrations also considerably translates into a reduction in the QS-regulated production of some molecules/enzymes (elastase, protease, chitinase, pyocyanin, and exopolysaccharides) with a concurrently decreased formation of biofilm EPS in *P. aeruginosa* PAO1 [86]. In the Gram-positive

pathogen, *S. aureus*, eugenol exhibited also antivirulence property acting on bacterial capability to produce exotoxin, through the repression of the agrA transcription [86]. Some EOs can effectively act both in preventing the biofilm formation and in disrupting the preformed biofilm. The EOs obtained from *Pogostemon heyneanus* and *Cinnamomum tamala* are capable to reduce the extracellular polymeric substance (EPS) and the synthesis of the two factors of the biofilm assemblage built by methicillin-resistant *S. aureus* (MRSA) strains. These EOs are also effective in reducing some virulence factors, such as staphyloxanthin and hemolysin. In silico docking studies demonstrated that (E)-nerolidol showed better

binding affinity toward the enzyme dehydroxysqualene synthase of MRSA which is responsible for the synthesis of staphyloxanthin [87]. Different ratios between two components present in an EO can provide a different effectiveness of the EO as a QS inhibitor. Two among five EOs of *Lippia alba*, in particular one containing a greater prevalence of geranial/neral (the two isomers of the octa-2,6-dienal citral) and the other with an higher limonene/carvone content, were the most effective QS inhibitors and also had small effects on cell growth [88]. The activity of EOs on the cell-cell mechanism of communication could depend also on the chemical organization of one or some of their main components. The (+)-enantiomers of carvone, limonene, and borneol are potentially capable to increase the production of violacein and pyocyanin in *C. violaceum* and *P. aeruginosa*, respectively, while their levorotary analogs inhibit such production [84]. Among phenols present in the EOs, eugenol at subinhibitory concentrations is capable of inhibiting the production of virulence factors, involving production of violacein and pyocyanin, synthesis and expression of elastase, and finally the organization of the biofilm. In fact, using two *E. coli* biosensors, MG4/pKDT17 and pEAL08-2, Zhou and co-workers demonstrated that also this compound could act of one or more QS systems, in particular on las and pqs QS systems [85]. The process applied for the extraction of the EO may affect its biological activity indeed. The inhibitory effect of *Citrus medica* L. var. *sarcodactylis* EO obtained by hydrodistillation extraction (HE), microwaveassisted hydrodistillation extraction (MHE), and ultra-microwave-assisted hydro

distillation extraction (UMHE) on biofilm formation by *S. aureus* and *S. typhimurium* was significantly higher than that of the essential oil obtained by standard extraction. This could also be related to the different chemical compositions of the different EOs, which elements can differ in terms of quality and rate [89]. Therefore, the diurnal variation can affect the chemistry of the essential oils, affecting their biological properties, including the capability to inhibit the biofilm [90]. New EOs are exhibiting interesting action against the formation of biofilm by microorganisms. *Cannabis sativa* EO is receiving particular attention because, further than other well-known properties, it showed a certain capability to attenuate the virulence of *Listeria monocytogenes* [91], with downregulation of flagella motility genes and of the regulatory gene prfA and a decreasing ability to form

#### *Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

*Essential Oils - Oils of Nature*

to their composition and to the bacterium of reference; thus, an EO can act as mixture, better than a singular component on a specific bacterium; therefore, one or more components can act better than parent EOs against another bacterium. The effect of clary sage, juniper, lemon, and marjoram EOs and their major components on the formation of bacterial and yeast biofilms and on the inhibition of AHLmediated QS, evaluated using *Bacillus cereus*, *Pichia anomala*, *Pseudomonas putida*, and a mix of bacteria containing also *E. coli*, demonstrated that marjoram EO inhibited all these tester strains. However, all components exhibited more strength in limiting the biofilm capacity of *B. cereus* than the parent EOs. Lemon EO was capable to inhibit *E. coli* and mixed-culture biofilms; on the other hand, cinnamon was effective against the mixed forms [75]. Conversely, the entire EO of tangerine (*Citrus reticulata*) is capable to inhibit the *P. aeruginosa* biofilm formation more than its main component limonene, by an inhibition of the QS autoinducer production and elastase activity [76]. This also highlights how, within a same genus, not all the species show the same biological activity. Thus, the EO of *C. reticulata* (tangerine) can be more active in inhibiting the QS system; on the other hand, the EO recovered from orange (*Citrus sinensis*) can be completely ineffective [73]. Some terpenoids, for example, thymol, carvacrol, linalool, menthol, geraniol, linalyl acetate, citronellal, and piperitone, have antibacterial activity mediated by their functional group. Carvacrol is one of the most active components present in different EOs, in particular from Labiatae. Its spectrum of activity is much wide. At sublethal concentrations

(<0.5 mM), it is capable to inhibit the formation of biofilms of *C. violaceum*, *Salmonella enterica* subsp. *typhimurium*, and *S. aureus*, while it does not exhibit effects on the formation of *P. aeruginosa* biofilms. In all cases, this concentration seems to not have effects on total bacterial numbers, indicating that carvacrol bactericidal effect could not be also linked to its inhibitory effect on biofilm formation. Sub-MIC concentrations of carvacrol could reduce the expression of cviI (a gene coding for the N-acyl-l-homoserine lactone synthase) and decrease the production of violacein and the activity of chitinase (both regulated by quorum sensing) at concentrations coinciding with carvacrol's inhibiting effect on biofilm formation. These results indicate that carvacrol activity in inhibition of biofilm formation might be also related to the disruption of quorum sensing [77]. Thymol, one of the main constituents of *Thymus vulgaris* EO, can affect (at the same manner of the parent EO) not only the AHL production (acting thus in the blockage of the communication system among bacteria), but it also can suppress flagella gene transcription (reducing the mRNA level of flagella gene), the bacterial motility, and finally the formation of biofilm [78]. Cinnamaldehyde, another widely diffused component, present, for example, in cinnamon EO, can show different mechanisms of action. The use of 60 μΜ cinnamaldehyde can decrease down to 55% the bioluminescence of *V. harveyi BB886*, which is induced by *3-*hydroxy-C4-HSL, and from 60 to 100% that of *V. harvevi* BB170 (mediated by AI-2). This indicates, once again, that the activity of EOs, like all other phytochemicals, can be dependent even on the strain used within the same species [30, 65, 79, 80], further than on the QS molecule involved. Another study showed that cinnamaldehyde particularly directs its action toward the short-chain AHL synthase (RhlI) and inhibits AHL production by RhlI [81]. Also cinnamaldehyde analogs and derivatives are capable to inhibit AI-2-based QS system of *V. harveyi* in a dose-dependent manner [82] and are effective against AI-2-regulated QS of *Vibrio* spp. too [83]. Three other cinnamaldehyde analogs, *trans*-2-nonenal, *trans*-3-decen-2-one, and *trans*-3-nonen-2-one, can interfere with AI-2 QS in different manner. In *Vibrio* spp., *trans*-2-nonenal and *trans*-3-decen-2-one inhibit the AI-2-based QS system by reducing the DNAbinding ability of LuxR, causing a decrease in the production of QS-regulated virulence functions such as biofilm formation, matrix production, and protease

**176**

production [83]. Therefore, some compounds, such as *p*-anisaldehyde can act as AHL mimics, inhibiting the production of violacein by *C. violaceum* [84]. Eugenol inhibits QS in pathogenic bacteria; this was shown, for example, by Zhou and co-workers [85], evaluating the reduction of violacein production in *C. violaceum* after contact with eugenol. This molecule is also capable to affect lasB and pqsA in *E. coli*. This suggests an inhibitory action of eugenol on Las and pseudomonas quinolone signal (PQS)-controlled transcription. The action of eugenol on pathogenic bacteria at subinhibitory concentrations also considerably translates into a reduction in the QS-regulated production of some molecules/enzymes (elastase, protease, chitinase, pyocyanin, and exopolysaccharides) with a concurrently decreased formation of biofilm EPS in *P. aeruginosa* PAO1 [86]. In the Gram-positive pathogen, *S. aureus*, eugenol exhibited also antivirulence property acting on bacterial capability to produce exotoxin, through the repression of the agrA transcription [86]. Some EOs can effectively act both in preventing the biofilm formation and in disrupting the preformed biofilm. The EOs obtained from *Pogostemon heyneanus* and *Cinnamomum tamala* are capable to reduce the extracellular polymeric substance (EPS) and the synthesis of the two factors of the biofilm assemblage built by methicillin-resistant *S. aureus* (MRSA) strains. These EOs are also effective in reducing some virulence factors, such as staphyloxanthin and hemolysin. In silico docking studies demonstrated that (E)-nerolidol showed better binding affinity toward the enzyme dehydroxysqualene synthase of MRSA which is responsible for the synthesis of staphyloxanthin [87]. Different ratios between two components present in an EO can provide a different effectiveness of the EO as a QS inhibitor. Two among five EOs of *Lippia alba*, in particular one containing a greater prevalence of geranial/neral (the two isomers of the octa-2,6-dienal citral) and the other with an higher limonene/carvone content, were the most effective QS inhibitors and also had small effects on cell growth [88]. The activity of EOs on the cell-cell mechanism of communication could depend also on the chemical organization of one or some of their main components. The (+)-enantiomers of carvone, limonene, and borneol are potentially capable to increase the production of violacein and pyocyanin in *C. violaceum* and *P. aeruginosa*, respectively, while their levorotary analogs inhibit such production [84]. Among phenols present in the EOs, eugenol at subinhibitory concentrations is capable of inhibiting the production of virulence factors, involving production of violacein and pyocyanin, synthesis and expression of elastase, and finally the organization of the biofilm. In fact, using two *E. coli* biosensors, MG4/pKDT17 and pEAL08-2, Zhou and co-workers demonstrated that also this compound could act of one or more QS systems, in particular on las and pqs QS systems [85]. The process applied for the extraction of the EO may affect its biological activity indeed. The inhibitory effect of *Citrus medica* L. var. *sarcodactylis* EO obtained by hydrodistillation extraction (HE), microwaveassisted hydrodistillation extraction (MHE), and ultra-microwave-assisted hydro distillation extraction (UMHE) on biofilm formation by *S. aureus* and *S. typhimurium* was significantly higher than that of the essential oil obtained by standard extraction. This could also be related to the different chemical compositions of the different EOs, which elements can differ in terms of quality and rate [89]. Therefore, the diurnal variation can affect the chemistry of the essential oils, affecting their biological properties, including the capability to inhibit the biofilm [90]. New EOs are exhibiting interesting action against the formation of biofilm by microorganisms. *Cannabis sativa* EO is receiving particular attention because, further than other well-known properties, it showed a certain capability to attenuate the virulence of *Listeria monocytogenes* [91], with downregulation of flagella motility genes and of the regulatory gene prfA and a decreasing ability to form biofilm and to invade Caco-2 cells.
