**2. Bacterial attachment to surfaces and advantages of the biofilm lifestyle**

For most of the history of microbiology, microorganisms have primarily been characterised as planktonic, freely suspended cells and described on the basis of their growth characteristics in nutritionally rich culture media. Although this traditional way of culturing bacteria in liquid media has been instrumental in the study of microbial pathogenesis and enlightening as to some of the amazing facets of microbial physiology, pure culture planktonic growth is rarely how bacteria exist in nature. On the contrary, direct observation of wide of variety of natural habitats has shown that the majority of microbes persist attached to surfaces within a structured biofilm ecosystem and not as free-floating organisms (Costerton et al., 1987, 1995; Kolter & Greenberg, 2006; Verstraeten et al., 2008).

The data on which this theory is predicated came mostly from natural aquatic ecosystems, in which direct microscopic observations together with direct quantitative recovery techniques showed unequivocally that more than 99.9% of the bacteria grow as biofilms on a wide variety of surfaces. The diversity and distribution of salmonellae in fresh water biofilms has also been recently shown (Sha et al., 2011). Moreover, it is becoming clear that these natural assemblages of bacteria within the biofilm matrix function as a cooperative consortium, in a relatively complex and coordinated manner (James et al., 1995; Moons et al., 2009; Wuertz et al., 2004). Nowadays, besides natural aquatic systems, it is well established that biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical devices and also industrial systems, such as pharmaceutical industries, oil drilling, paper production, waste water treatment and food processing (Hall-Stoodley et al., 2004). Thus, examples of this bacterial lifestyle are abundant in daily life: the slimy material that covers flower vases, pipelines, submerged rocks, and even the surface of teeth (Marsh, 2005; Wimpenny, 2009).

Biofilm formation occurs through sequential steps in which the initial attachment of planktonic bacteria to a solid surface is followed by their subsequent proliferation and

are usually inhabited by numerous different species in close proximity (Wimpenny et al., 2000). Spatial and metabolic interactions between species contribute to the organization of multispecies biofilms, and the production of a dynamic local environment (Goller & Romeo, 2008; Tolker-Nielsen & Molin, 2000). Indeed, cell-to-cell signalling and interspecies interactions have been demonstrated to play a key role in cell attachment and detachment from biofilms, as well as in the resistance of biofilm community members against antimicrobial treatments (Annous et al., 2009; Burmølle et al., 2006; Irie & Parsek, 2008; Nadell et al., 2008; Remis et al., 2010). Mixed-species biofilms are usually more stable than mono-species biofilms, while biofilm formation by *Salmonella* has also been shown to be influenced by either the natural *in situ* presence of other species, or just their metabolic byproducts (Chorianopoulos et al., 2010; Girennavar et al., 2008; Habimana et al., 2010b; Jones

In this chapter, we review up-to-date available voluminous literature on the attachment and biofilm formation by *Salmonella* strains on abiotic surfaces, simulating those encountered in food processing areas (section 4). Before this, the advantages of biofilm lifestyle for microorganisms are briefly discussed (section 2), together with the serious negative implications of biofilm formation for the food industry (section 3). Major molecular components building up *Salmonella* biofilm matrix are then reported (section 5). Finally, we review available knowledge on the influence of cell-to-cell communication (quorum

**2. Bacterial attachment to surfaces and advantages of the biofilm lifestyle** 

For most of the history of microbiology, microorganisms have primarily been characterised as planktonic, freely suspended cells and described on the basis of their growth characteristics in nutritionally rich culture media. Although this traditional way of culturing bacteria in liquid media has been instrumental in the study of microbial pathogenesis and enlightening as to some of the amazing facets of microbial physiology, pure culture planktonic growth is rarely how bacteria exist in nature. On the contrary, direct observation of wide of variety of natural habitats has shown that the majority of microbes persist attached to surfaces within a structured biofilm ecosystem and not as free-floating organisms (Costerton et al., 1987, 1995; Kolter & Greenberg, 2006; Verstraeten et al., 2008). The data on which this theory is predicated came mostly from natural aquatic ecosystems, in which direct microscopic observations together with direct quantitative recovery techniques showed unequivocally that more than 99.9% of the bacteria grow as biofilms on a wide variety of surfaces. The diversity and distribution of salmonellae in fresh water biofilms has also been recently shown (Sha et al., 2011). Moreover, it is becoming clear that these natural assemblages of bacteria within the biofilm matrix function as a cooperative consortium, in a relatively complex and coordinated manner (James et al., 1995; Moons et al., 2009; Wuertz et al., 2004). Nowadays, besides natural aquatic systems, it is well established that biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical devices and also industrial systems, such as pharmaceutical industries, oil drilling, paper production, waste water treatment and food processing (Hall-Stoodley et al., 2004). Thus, examples of this bacterial lifestyle are abundant in daily life: the slimy material that covers flower vases, pipelines, submerged rocks, and even the surface of teeth (Marsh, 2005; Wimpenny, 2009). Biofilm formation occurs through sequential steps in which the initial attachment of planktonic bacteria to a solid surface is followed by their subsequent proliferation and

& Bradshaw, 1997; Prouty et al., 2002; Soni et al., 2008).

sensing) on the establishment of *Salmonella* biofilms (section 6).

accumulation in multilayer cell clusters, and the final formation of the bacterial community enclosed in a self-produced polymeric matrix (Goller & Romeo, 2008; Lasa, 2006; O'Toole et al., 2000; Palmer et al., 2007; Rickard et al., 2003). The initial interaction between solid surface and bacterial cell envelope appears to be mediated by a complex array of chemical and physical interactions, with each affected by the chemical and physical environment to which the bacterial cell and the surface are currently or recently exposed (Palmer et al., 2007). Mature biofilms are highly organized ecosystems in which water channels are dispersed and can provide passages for the exchange of nutrients, metabolites and waste products (Stoodley et al., 2002). Once the biofilm structure has developed, some bacteria are released into the liquid medium, in order to colonize new surfaces, probably when surrounding conditions become less favourable (Gilbert et al., 1993; Hall-Stoodley & Stoodley, 2002, 2005; Klausen et al., 2006).

According to Darwin's theory of evolution, the only true driving force behind the course of action of any organism is reproductive fitness. Outside of the laboratory bacteria rarely, if ever, find themselves in an environment as nutrient rich as culture media, and in these conditions, there are a number of fitness advantages imparted by the biofilm mode of growth (Jefferson, 2004). The process of biofilm formation is believed to begin when bacteria sense certain environmental parameters (extracellular signals) that trigger the transition from planktonic growth to life on a surface (Lopez et al., 2010). Currently, four potential incentives behind the formation of biofilms by bacteria are considered: (i) protection from the harmful environment (as a stress response mechanism), (ii) sequestration to a nutrient rich area, (iii) utilization of cooperative benefits (through metabolic cooperativity), and (iv) acquisition of new genetic traits (Davey & O'Toole, 2000; Molin & Tolker-Nielsen, 2003).

Bacteria experience a certain degree of shelter and homeostasis when residing within a biofilm and one of the key components of this microniche is the surrounding extrapolymeric substance (EPS) matrix (Flemming & Wingender, 2010). This matrix is composed of a mixture of components, such as exopolysaccharides, proteins, nucleic acids, and other substances (Branda et al., 2005). The nature of biofilm matrix and the physiological attributes of biofilm microorganisms confer an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants or germicides. Thus, established biofilms can tolerate antimicrobial agents at concentrations of 10-1000 times that need to kill genetically equivalent planktonic bacteria, and are also extraordinary resistant to phagocytosis, making rather difficult to eradicate biofilms from living hosts (Cos et al., 2010). Mechanisms responsible for resistance may be one or more of the following: (i) delayed penetration of the antimicrobial agent through the biofilm matrix, (ii) altered growth rate of biofilm microorganisms, and (iii) other physiological changes due to the biofilm mode of growth, e.g. existence of subpopulations of resistant phenotypes in the biofilm, which have been referred to as "persisters" (Donlan & Costerton, 2002; Gilbert et al., 2002; Lewis, 2001; Mah & O'Toole, 2001).

Scientific interest in the process of bacterial biofilm formation has erupted in recent years and studies on the molecular genetics of biofilm formation have begun to shed light on the driving forces behind the transition to the biofilm mode of existence. Evidence is mounting that up- and down-regulation of a number of genes occurs in the attaching cells upon initial interaction with the substratum (Donlan, 2002; Sauer, 2003). Thus, high-throughput DNA microarray studies have been conducted to study biofilm formation in many model microorganisms and have identified a large number of genes showing differential expression under biofilm conditions (Beloin et al., 2004; Hamilton et al., 2009; Lazazzera,

Attachment and Biofilm Formation by Salmonella in Food Processing Environments 161

et al., 2003; Gounadaki et al., 2008; Gunduz & Tuncel, 2006; Sharma & Anand, 2002). Several studies were also focused on the attachment of bacterial pathogens to food surfaces such as *Escherichia coli* to beef muscle and adipose tissue (Rivas et al., 2006) and *S.* Typhimurium,

Biofilm formation depends on an interaction between three main components: the bacterial cells, the attachment surface and the surrounding medium (Van Houdt & Michiels, 2010). Adhesion of bacterial cells, the first phase of biofilm formation, is influenced by the physicochemical properties of the cells' surface, which in turn are influenced by factors such as microbial growth phase, culture conditions and strain's variability (Briandet et al., 1999; Giaouris et al., 2009). The surfaces of most bacterial cells are negatively charged, and this net negative charge of the cell surface is adverse to bacterial adhesion, due to electrostatic repulsive force. However, the bacterial cell-surface possesses hydrophobicity due to fimbriae, flagella and lipopolysaccharide (LPS) (Ukuku & Fett, 2006). Hydrophobic interactions between the cell surface and the substratum may enable the cell to overcome repulsive forces and attach irreversibly (Donlan, 2002). The properties of the attachment surface (e.g. roughness, cleanability, disinfectability, wettability, vulnerability to wear) are important factors that also affect the biofilm formation potential and thus determine the hygienic status of the material. Stainless steel type 304, commonly used in the food processing industry, is an ideal material for fabricating equipment due to its physico-chemical stability and high resistance to corrosion. Teflon and other plastics are often used for gaskets and accessories of instruments. These surfaces become rough or crevice with continuous reuse and

Environmental factors such as pH, temperature, osmolarity, O2 levels, nutrient composition and the presence of other bacteria play important roles in the process of biofilm formation (Giaouris et al., 2005; Hood & Zottola, 1997a; Stepanovic et al., 2003). The integration of these influences ultimately determines the pattern of behavior of a given bacterium with respect to biofilm development (Goller & Romeo, 2008). In food processing environments, bacterial attachment is additionally affected by food matrix constituents, which can be adsorbed onto a substratum and create conditioning films (Bernbom et al., 2009). For example, skim milk was found to reduce adhesion of *Staphylococcus aureus*, *L. monocytogenes*, and *Serratia marcescens* to stainless steel coupons (Barnes et al., 1999). Additionally, in real environments, the presence of mixed bacterial communities adds additional complexity to attachment and biofilm formation procedure. For instance, the presence of *Staphylococcus xylosus* and *Pseudomonas fragi* affected the numbers of *L. monocytogenes* biofilm cells on stainless steel (Norwood & Gilmour, 2001), while compounds present in *Hafnia alvei* cell-free culture supernatant inhibited the early stage of *S*. Enteritidis biofilm formation on the same

Once biofilms have formed in the factory environment, they are difficult to be removed often resulting in persistent and endemic populations (Vestby et al., 2009b). Interestingly, persistent *L. monocytogenes* strains had the added ability of enhanced adhesion within shorter times to stainless steel surfaces compared to non-persistent strains (Lundén et al., 2000). It has been suggested that such persistence is likely due to physical adaptation of cells in biofilms, particularly resistance to cleaning and sanitizing regimes, since it is generally accepted and well documented that cells within a biofilm are more resistant to biocides than their planktonic counterparts (Carpentier & Cerf, 1993). For example, nine disinfectants commonly used in the feed industry and efficient against planktonic *Salmonella* cells, showed a bactericidal effect that varied considerably for biofilm-grown cells with products containing

*Yersinia enterocolitica* and *L. monocytogenes* to pork skin (Morild et al., 2011).

form a harbourage to protect bacteria from shear forces in the food fluid.

material (Chorianopoulos et al., 2010).

2005; Shemesh et al., 2007; Whiteley et al., 2001). In *S*. Typhimurium, 10% of its genome (i.e. 433 genes) showed a 2-fold or more change in the biofilm, using a silicone rubber tubing as a substratum for growth, compared with planktonic cells (Hamilton et al., 2009). The genes that were significantly up-regulated implicated certain cellular processes in biofilm development, including amino acid metabolism, cell motility, global regulation and tolerance to stress. Obviously, the more we learn about the genetic regulation of biofilm formation, the more we understand about the relative roles of benefits and forces that drive the switch to the biofilm mode of growth.
