**6. Cell-to-cell communication in** *Salmonella* **biofilms (quorum sensing)**

It has been thoroughly suggested that bacterial cells communicate by releasing and sensing small diffusible signal molecules, in a process commonly known as quorum sensing (QS) (Miller & Bassler, 2001; Smith et al., 2004; Whitehead et al., 2001). Through cell-to-cell signaling mechanisms, bacteria modulate their own behaviour and also respond to signal produced by other species (Ryan & Dow, 2008). QS involves a density-dependent recognition of signaling molecules (autoinducers, AIs), resulting in modulation of gene expression (Bassler, 1999). Gram-negative bacteria primarily use a variety of *N*acylhomoserine lactones (AHLs) as AI (autoinducer-1, AI-1), while Gram-positive bacteria

only under LB but not under ATM conditions. Stafford & Hughes (2007) showed that the conserved flagellar regulon gene *flhE*, while it is not required for flagella production or swimming, appeared to play a role in flagella-dependent swarming and biofilm formation on PVC. Kim & Wei (2009) noticed that flagellar assemply was important during biofilm formation on PVC in different (meat, poultry and produce) broths and on stainless steel and

Colanic acid, a capsular extracellular polysaccharide, essential for *S*. Typhimurium biofilm development on epithelial cells was found not to be required for *Salmonella* biofilm formation on abiotic surfaces (Ledeboer & Jones, 2005; Prouty & Gunn, 2003). Solano et al. (2002) showed that colonic acid was important to form a tight pellicle under LB conditions, while it was dispensable under ATM conditions. De Rezende et al. (2005) purified another capsular polysaccharide (CP) from extracellular matrix of multiresistant *S*. Typhimurium DT104 which was found to be important for biofilm formation on polystyrene centrifuge tubes and was detected at both 25°C and 37°C. This was comprised principally of glucose and mannose, with galactose as a minor constituent. Malcova et al. (2008) confirmed the importance of this capsular polysaccharide in the biofilm formation capacity of strains unable to produce either curli fimbriae or cellulose. Due to mucoid and brown appearance on Congo Red agar plates,

However, other capsular polysaccharides can be present in the extracellular biofilm matrix of *Salmonella* strains (de Rezende et al., 2005; Gibson et al., 2006; White et al., 2003), and the exact composition depends upon the environmental conditions in which the biofilms are formed (Prouty & Gunn, 2003). Another component of the EPS matrix of *Salmonella* bile-induced biofilms, the O-antigen (O-ag) capsule, while it was found to be crucial for *S*. Typhimurium and *S*. Typhi biofilm development on gallstones, this was not necessary for adhesion and biofilm formation on glass and plastic (Crawford et al., 2008). The formation of this O-ag capsule was also found to be important for survival during desiccation stress (Gibson et al., 2006). Anriany et al. (2006) highlighted the importance of an integral lipopolysaccharide (LPS), at both the O-antigen and core polysaccharide levels, in the modulation of curli protein and cellulose production, as well as in biofilm formation, thereby adding another potential component to the complex regulatory system which governs multicellular behavior in *S*. Typhimurium. Mireles et al. (2001) observed that for *S*. Typhimurium LT2, all of the LPS mutants examined were able to form a biofilm on polyvinyl chloride (PVC) but none were able to attach to a hydrophilic surface such as glass. Kim & Wei (2009) noticed that a *rfbA* mutant of *S*. Typhimurium DT104, showing an aberrant LPS profile, was impaired in rdar expression, pellicle formation, biofilm forming capability on PVC in meat, poultry and produce broths and

**6. Cell-to-cell communication in** *Salmonella* **biofilms (quorum sensing)** 

It has been thoroughly suggested that bacterial cells communicate by releasing and sensing small diffusible signal molecules, in a process commonly known as quorum sensing (QS) (Miller & Bassler, 2001; Smith et al., 2004; Whitehead et al., 2001). Through cell-to-cell signaling mechanisms, bacteria modulate their own behaviour and also respond to signal produced by other species (Ryan & Dow, 2008). QS involves a density-dependent recognition of signaling molecules (autoinducers, AIs), resulting in modulation of gene expression (Bassler, 1999). Gram-negative bacteria primarily use a variety of *N*acylhomoserine lactones (AHLs) as AI (autoinducer-1, AI-1), while Gram-positive bacteria

their morphotype was designated as sbam (smooth, brown and mucoid).

biofilm formation on stainless steel and glass.

glass in LB broth.

use a variety of autoinducing polypeptides (AIPs). AHLs are synthesized and recognized by QS circuits composed of LuxI and LuxR homologues, respectively (Whitehead et al., 2001). Both AHLs and AIPs are highly specific to the species that produce them. A third QS system is proposed to be universal, allowing interspecies communication, and is based on the enzyme LuxS which is in part responsible for the production of a furanone-like compound, called autoinducer-2 (AI-2) (Schauder et al., 2001).

Bacteria use QS communication circuits to regulate a diverse array of physiological activities, such as genetic competence, pathogenicity (virulence), motility, sporulation, bioluminescence and production of antimicrobial substances (Miller & Bassler, 2001). Yet, a growing body of evidence demonstrates that QS also contributes to biofilm formation by many different species (Annous et al., 2009; Davies et al., 1998; Irie & Parsek, 2008; Lazar, 2011). As biofilms typically contain high concentration of cells, autoinducer (AI) activity and QS regulation of gene expression have been proposed as essential components of biofilm physiology (Kjelleberg & Molin, 2002; Parsek & Greenberg, 2005).

To date, three QS systems have been identified in *S. enterica* and are thought to be mainly implicated in the regulation of virulence (SdiA, luxS/AI-2 and AI-3/epinephrine/ norepinephrine signaling system) (Boyen et al., 2009; Walters & Sperandio, 2006). Firstly, the LuxR homologue SdiA has been characterized in *Salmonella*, but there does not appear to be a corresponding signal-generating enzyme similar to LuxI in this species (Ahmer et al., 1998). Since *Salmonella* does not possess a luxI homologue, it cannot produce its own AHLs (Ahmer, 2004). However, *Salmonella* SdiA can detect AHLs produced by a variety of bacterial species, leading to the suggestion that SdiA can be used in interspecies communication within a mixed-species community (Michael et al., 2001; Smith & Ahmer 2003). Till now, SdiA is known to activate the expression of the *rck* operon and the *srgE* gene (Ahmer et al., 1998; Smith & Ahmer, 2003). In contrast to the function of SdiA in *E. coli* adherence to HEp-2 epithelial cells and also biofilm formation on polystyrene (Lee et al., 2009; Sharma et al., 2010), no direct link between SdiA and *Salmonella* biofilms has been reported. Interestingly, Chorianopoulos et al. (2010) demonstrated that cell-free culture supernatant (CFS) of the psychrotrophic spoilage bacterium *Hafnei alvei*, containing AHLs among other unknown metabolites, negatively influenced the early stage of biofilm formation by *S.* Enteritidis on stainless steel. Similarly, Dheilly et al. (2010) reported the inhibitory activity of CFS from the marine bacterium *Pseudoalteromonas* sp. strain 3J6 against biofilm formation on glass flow cells by *S. enterica* and other Gram-negative bacteria. Taking into account that *Salmonella* possess SdiA, a receptor of AHLs which may be produced by resident flora on food-contact surfaces (Michael et al., 2001; Smith & Ahmer, 2003; Soares & Ahmer, 2011), the effect of AHLs on biofilm formation by this pathogen in multispecies real food processing environments needs to be further studied.

The second QS system of *Salmonella* uses the LuxS enzyme for the synthesis of AI-2 (Schauder et al., 2001; Soni et al., 2008). The Lsr ABC transporter is known to be involved in the detection and transport of AI-2 into the cell (Taga et al., 2001), while the *rbs* transporter has recently been suggested as an alternative AI-2 uptake system (Jesudhasan et al., 2010). A *S.* Typhimurium *luxS* deletion mutant was impaired in biofilm formation on polystyrene (De Keersmaecker et al., 2005; Jesudhasan et al., 2010). However, this phenotype could not be complemented by extracellular addition of QS signal molecules, suggesting that AI-2 is not the actual signal involved in *Salmonella* biofilm formation (De Keersmaecker et al., 2005). To this direction, Kint et al. (2010) analyzed additional *luxS* mutants for their biofilm phenotype. Interestingly, a *luxS* kanamycin insertion mutant and a partial deletion mutant,

Attachment and Biofilm Formation by Salmonella in Food Processing Environments 169

occurring simultaneously. The challenge becomes more intriguing given that microflora on inadequately cleaned and disinfected food processing surfaces is a complex community,

Undoubtedly, a clearer understanding of the factors which influence microbial attachment to abiotic surfaces could provide the information necessary to modify processes in food processing environments in order to reduce microbial persistence and therefore reduce the contamination of food products. For instance, the understanding of bacterial attachment to solid surfaces, such as stainless steel, may help in the future development of surfaces with no or reduced attachment, or in developing an effective sanitation programme and thus reducing the potential contamination of processed products by spoilage or/and pathogenic bacteria. Undoubtedly, the ability to recognize how *Salmonella* attach to food-contact surfaces and form biofilms on them is an important area of focus, since a better understanding of this ability may provide valuable ways towards the elimination of this pathogenic bacterium from food processing environments and eventually lead to reduced *Salmonella*-associated human illness.

Authors would like to acknowledge European Union project ProSafeBeef (ref. Food-CT-2006-36241) within the 6th Framework Programme for the financial support of some of the

Ahmer, B.M.M. (2004). Cell-to-cell signalling in *Escherichia coli* and *Salmonella enterica*.

Ahmer, B.M.; van Reeuwijk, J.; Timmers, C.D.; Valentine, P.J. & Heffron, F. (1998). *Salmonella* 

Annous, B.A.; Fratamico, P.M & Smith, J.L. (2009). Quorum sensing in biofilms: why bacteria behave the way they do. *Journal of Food Science*, Vol.74, No.1., pp. R24-R37 Anriany, Y.; Sahu, S.N.; Wessels, K.R.; McCann, L.M. & Joseph, S.W. (2006). Alteration of the

Asséré, A.; Oulahal, N. & Carpentier, B. (2008). Comparative evaluation of methods for

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*typhimurium* encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. *Journal of Bacteriology*,

rugose phenotype in *waaG* and *ddhC* mutants of *Salmonella enterica* serovar Typhimurium DT104 is associated with inverse production of curli and cellulose. *Applied and Environmental Microbiology*, Vol.72, No.7, (July 2006), pp. 5002-5012 Arnold, J.W. & Yates, I.E. (2009). Interventions for control of *Salmonella*: clearance of

microbial growth from rubber picker fingers. *Poultry Science*, Vol.88, No.6, (June

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contrary to the laboratory studied pure-species biofilms.

studies on *Salmonella* biofilms performed on our lab.

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2009), pp. 1292-1298

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1692-1702

**8. Acknowledgement** 

**9. References** 

that only lacked the 3′ part of the *luxS* coding sequence, were found to be able to form mature wild-type biofilms on polystyrene, despite the fact that these strains were unable to produce AI-2. These authors concluded that a small regulatory RNA molecule, MicA, encoded in the *luxS* adjacent genomic region, rather than LuxS itself, infuences *S*. Typhimurium biofilm formation phenotype. On the other hand, Prouty et al. (2002) showed that a *S*. Typhimurium *luxS* insertion mutant formed scattered biofilm on gallstones with little apparent EPS even after 14 days of incubation. Yoon & Sofos (2008) showed that biofilm formation by *S.* Thompson on stainless steel, under monoculture conditions (72 h at 25°C), was similar between AI-2 positive and negative strains. Altogether, these results demonstrate that the relationship between biofilm formation and the presence of an active LuxS system and AI-2 in *S. enterica* is not clear and further research is needed.

The third QS system of *Salmonella* uses the two component system PreA/B (Bearson & Bearson 2008; Merighi et al., 2006). PreA/B is similar to the *luxS*-dependent two component QseB/QseC of enterohemorrhagic *E. coli*, which has been shown to sense the QS signal AI-3, as well the eukaryotic hormones epinephrine and norepinephrine (Sperandio et al., 2002; Walters & Sperandio, 2006). In *S*. Typhimurium, the histidine sensor kinase QseC, which is able to detect norepinephrine, has been implicated in the regulation of virulence traits, such as motility and *in vivo* competitive fitness in pigs (Bearson & Bearson, 2008). Even though the role of AI-3/epinephrine/norepinephrine signaling system in the formation of biofilm by *Salmonella* is still unknown, given that motility is usually an important biofilm determinant in many bacterial species, it is quite possible that this third QS system may also affect *Salmonella* biofilm formation.
