**1. Introduction**

156 Salmonella – A Dangerous Foodborne Pathogen

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During the last decades, it has become increasingly clear that bacteria, including foodborne pathogens such as *Salmonella enterica*, grow predominantly as biofilms in most of their natural habitats, rather than in planktonic mode. A biofilm can be broadly defined as a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription (Donlan & Costerton, 2002; Kuchma & O'Toole, 2000; Lazazzera, 2005; Shemesh et al., 2007). Interestingly, it has been observed that the resistance of biofilm cells to antimicrobials is significantly increased compared with what is normally seen with the same cells being planktonic (Gilbert et al., 2002; Mah & O'Toole, 2001). Thus, it is believed that biofilm formation enhances the capacity of pathogenic *Salmonella* bacteria to survive stresses that are commonly encountered both within food processing, as well as during host infection.

In food industry, biofilms may create a persistent source of product contamination, leading to serious hygienic problems and also economic losses due to food spoilage (Brooks & Flint, 2008; Carpentier & Cerf, 1993; Ganesh Kumar & Anand, 1998; Lindsay & von Holy, 2006; Zottola & Sasahara, 1994). Improperly cleaned surfaces promote soil build-up, and, in the presence of water, contribute to the development of bacterial biofilms which may contain pathogenic microorganisms, such as *Salmonella*. Cross contamination occurs when cells detach from biofilm structure once food passes over contaminated surfaces or through aerosols originating from contaminated equipment. Till now, there is only limited information on the presence of *Salmonella* in biofilms in real food processing environments. However, numerous studies have shown that *Salmonella* can easily attach to various food-contact surfaces (such as stainless steel, plastic and cement) and form biofilms under laboratory conditions (Chia et al., 2009; Giaouris et al., 2005; Giaouris & Nychas, 2006; Hood & Zottola, 1997a,b; Marin et al., 2009; Oliveira et al., 2006; Rodrigues et al., 2011; Vestby et al., 2009a,b).

The natural environments that most bacteria inhabit are typically complex and dynamic. Unfortunately, this complexity is not fully appreciated when growing microorganisms in monocultures under laboratory conditions. Thus, in real environments, biofilm communities

Attachment and Biofilm Formation by Salmonella in Food Processing Environments 159

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 &

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.,

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,

Stoodley, 2002, 2005; Klausen et al., 2006).

2002; Lewis, 2001; Mah & O'Toole, 2001).

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

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