**4. Attachment to food-contact surfaces and biofilm forming ability of**  *Salmonella*

Salmonellae represent a group of Gram-negative bacteria that are recognized worldwide as major zoonotic pathogens for both humans and animals. In the EU, salmonellosis was the second most commonly reported zoonotic infection in 2009, with 108,614 human cases confirmed and a case fatality rate of 0.08%, which approximately corresponds to 90 human deaths (EFSA-ECDC, 2011). That year, *Salmonella* was most often found in fresh broiler, turkey and pig meat where proportions of positive samples, on average 5.4%, 8.7% and 0.7%, were detected respectively. The two most common *Salmonella* serotypes, implicated in the majority of outbreaks, are Typhimurium and Enteritidis (52.3% and 23.3% respectively of all known serovars in human cases). The native habitat of salmonellae is considered to be the intestinal tract of taxonomically diverse group of vertebrates, from which salmonellae can spread to other environments through released faeces (Litrup et al., 2010).

Interestingly, salmonellae have been shown to survive for extended periods of time in nonenteric habitats, including biofilms on abiotic surfaces (White et al., 2006). Thus, several reports have demonstrated the ability of *Salmonella* to form biofilms on abiotic surfaces outside the host, such as stainless steel (Austin et al., 1998; Chorianopoulos et al., 2010; Giaouris et al., 2005; Giaouris & Nychas, 2006; Hood & Zottola, 1997a,b; Joseph et al., 2001; Kim & Wei, 2007, 2009; Møretrø et al., 2009), plastic (Asséré et al., 2008; Iibuchi et al., 2010; Jain & Chen, 2007; Joseph et al., 2001; Ngwai et al., 2006; Stepanović et al., 2003, 2004; Vestby et at., 2009a,b), rubber (Arnold & Yates, 2009), glass (Kim & Wei, 2009; Korber et al., 1997; Prouty & Gunn, 2003; Solano et al., 1998), cement (Joseph et al., 2001), marble and granite (Rodrigues et al., 2011). Taken into account, that all these surfaces are commonly encountered in farms, slaughter houses, food industries and kitchens, it is obvious that the risk for public health is quite serious.

It is strongly believed that the ability of *Salmonella* to form biofilms on inanimate surfaces contributes to its survival and persistence in non-host environments and its transmission to

70% ethanol being most effective (Møretrø et al., 2009). Other studies similarly indicated that compared to planktonic cells, biofilm cells of *Salmonella* were more resistant to trisodium phosphate (Scher et al., 2005) and to chlorine and iodine (Joseph et al., 2001). In a comparative study of different *S.* Enteritidis phage type 4 isolates it was found that those isolates that survived better on surfaces also survived better in acidic conditions and in the presence of hydrogen peroxide and showed enhanced tolerance towards heat (Humphrey et al., 1995). The cellular mechanisms underlying microbial biofilm formation and behaviour are beginning to be understood and are targets for novel specific intervention strategies to control problems caused by biofilm formation in fields ranging from industrial processes like food processing, to health-related fields, like medicine and dentistry. In food industry, various preventive and control strategies, like hygienic plant lay-out and design of equipment, choice of materials, correct selection and use of detergents and disinfectants coupled with physical methods can be suitably applied for controlling biofilm formation. Right now, bacterial biofilms have not been specifically addressed in the HACCP system that has been employed in the food processing facilities. However, surveying of biofilms in food environments and developing an effective sanitation plan should be considered in the HACCP system (Sharma & Anand, 2002). An upgraded HACCP with biofilm assessment in food plants will provide clearer information of contamination, and assist the development of

biofilm-free processing systems in the food industry.

risk for public health is quite serious.

*Salmonella* 

**4. Attachment to food-contact surfaces and biofilm forming ability of** 

can spread to other environments through released faeces (Litrup et al., 2010).

Salmonellae represent a group of Gram-negative bacteria that are recognized worldwide as major zoonotic pathogens for both humans and animals. In the EU, salmonellosis was the second most commonly reported zoonotic infection in 2009, with 108,614 human cases confirmed and a case fatality rate of 0.08%, which approximately corresponds to 90 human deaths (EFSA-ECDC, 2011). That year, *Salmonella* was most often found in fresh broiler, turkey and pig meat where proportions of positive samples, on average 5.4%, 8.7% and 0.7%, were detected respectively. The two most common *Salmonella* serotypes, implicated in the majority of outbreaks, are Typhimurium and Enteritidis (52.3% and 23.3% respectively of all known serovars in human cases). The native habitat of salmonellae is considered to be the intestinal tract of taxonomically diverse group of vertebrates, from which salmonellae

Interestingly, salmonellae have been shown to survive for extended periods of time in nonenteric habitats, including biofilms on abiotic surfaces (White et al., 2006). Thus, several reports have demonstrated the ability of *Salmonella* to form biofilms on abiotic surfaces outside the host, such as stainless steel (Austin et al., 1998; Chorianopoulos et al., 2010; Giaouris et al., 2005; Giaouris & Nychas, 2006; Hood & Zottola, 1997a,b; Joseph et al., 2001; Kim & Wei, 2007, 2009; Møretrø et al., 2009), plastic (Asséré et al., 2008; Iibuchi et al., 2010; Jain & Chen, 2007; Joseph et al., 2001; Ngwai et al., 2006; Stepanović et al., 2003, 2004; Vestby et at., 2009a,b), rubber (Arnold & Yates, 2009), glass (Kim & Wei, 2009; Korber et al., 1997; Prouty & Gunn, 2003; Solano et al., 1998), cement (Joseph et al., 2001), marble and granite (Rodrigues et al., 2011). Taken into account, that all these surfaces are commonly encountered in farms, slaughter houses, food industries and kitchens, it is obvious that the

It is strongly believed that the ability of *Salmonella* to form biofilms on inanimate surfaces contributes to its survival and persistence in non-host environments and its transmission to new hosts. To this direction, Vestby et al. (2009b) found a correlation between the biofilm formation capacities of 111 *Salmonella* strains isolated from feed and fish meal factories and their persistence in the factory environment. Another study on colonization and persistence of *Salmonella* on egg conveyor belts indicated that the type of egg belt (i.e. vinyl, nylon, hemp or plastic) was the most important factor in colonization and persistence, while rdar morphotype, a physiological adaptation associated with aggregation and long-term survival which is conserved in *Salmonella* (White & Surette, 2006), surprisingly, was not essential for persistence (Stocki et al., 2007). Interestingly, inoculation onto fresh-cut produce surfaces, as well as onto inert surfaces, such as polyethersufone membranes, was found to significantly increase the survival of salmonellae during otherwise lethal acid challenge (pH 3.0 for 2 hours) (Gawande & Bhagwat, 2002). Similarly, *Salmonella* strains with high biofilm productivity survived longer on polypropylene surfaces under dry conditions than strains with low productivity (Iibuchi et al., 2010).

In the food processing environments, food-contact surfaces come in contact with fluids containing various levels of food components. Under such conditions, one of the first events to occur is the adsorption of food molecules to the surface (conditioning). Both growth media and surface conditioning were found to influence the adherence of *S*. Typhimurium cells to stainless steel (Hood & Zottola, 1997b). A study of 122 *Salmonella* strains indicated that all had the ability to adhere to plastic microwell plates and that, generally, more biofilm was produced in low nutrient conditions, as those found in specific food processing environments, compared to high nutrient conditions (Stepanovic et al., 2004). A study conducted in order to identify the risk factors for *Salmonella* contamination in poultry farms, showed that the most important factors were dust, surfaces and faeces, and nearly 50% of the strains isolated from poultry risk factors were able to produce biofilm, irrespective of the origin of different serotypes (Marin et al., 2009).

There are some studies which have investigated the influence of physicochemical and surface properties (e.g. charge, hydrophobicity, surface free energy, roughness) of *Salmonella* and surface materials on the attachment process. For instance, Sinde & Carballo (2000) found that surface free energies and hydrophobicity do not affect attachment of *Salmonella* to stainless steel, rubber and polytetrafluorethylene, while Ukuku & Fett (2002) found that there was a linear correlation between bacterial cell surface hydrophobicity and charge and the strength of attachment of *Salmonella*, *E. coli* and *L. monocytogenes* strains to cantaloupe surfaces. Korber et al. (1997) found that surface roughness influences susceptibility of *S*. Enteritidis biofilms, grown in glass flow cells (with or without artificial crevices) to trisodium phosphate. Chia et al. (2009) studied the attachment of 25 *Salmonella* strains to four different materials (Teflon®, stainless steel, rubber and polyurethane) commonly found in poultry industry and found out that materials more positive in interfacial free energies had the highest number of adhering bacteria. However, in that study, authors concluded that *Salmonella* adhesion is strain-dependent, and probably influenced by surface structures, such as cell wall and membrane proteins, fimbriae, flagella and polysaccharides. This was also the conclusion of another similar study which compared the adhesion ability of four *S*. Enteritidis isolates to three different materials (polyethylene, polypropylene and granite) used in kitchens (Oliveira et al., 2006). Ngwai et al. (2006) characterized the biofilm forming ability of eleven antibiotic-resistant *S*. Typhimurium DT104 clinical isolates from human and animal sources and concluded that there was a general lack of correlation between this ability and bacterial physicochemical surface characteristics.

Attachment and Biofilm Formation by Salmonella in Food Processing Environments 165

fimbriae occurs upon iron starvation at 37°C. Römling et al. (2003) showed that the majority (more than 90% of 800 strains) of human disease-associated *S*. Typhimurium and *S*. Enteritidis (isolated from patients, foods and animals) displayed the rdar morphotype at 28°C, but just rarely at 37°C. Interestingly, mutants in the *csgD* promoter have also been found expressing rdar morphotype independently of temperature (Römling et al., 1998b). Curli fimbriae are amyloid cell-surface proteins, and are involved in adhesion to surfaces, cell aggregation, environmental persistence and biofilm development (Austin et al., 1998; Collinson et al., 1991; White et al., 2006). The *csg* (curli subunit genes) genes (previously called *agf* genes) involved in curli biosynthesis are organized into two adjacent divergentlytranscribed operons, *csgBAC* and *csgDEFG* (Collinson et al., 1996; Römling et al., 1998a). Knocking out the gene encoding for the subunit of thin aggregative fimbriae, AgfA, results in pink colony formation, the pdar (pink, dry and rough) morphotype, which is characterised by production of cellulose without curli (Jain & Chen, 2007). Solano et al. (2002) stressed the importance of the applied biofilm system since they noticed that curli were not essential for biofilm mediated glass adherence under adherence test medium (ATM) conditions, while they were indispensable to form a tight pellicle under LB conditions. In addition to curli, the second component of the extracellular matrix of the *Salmonella* biofilms is cellulose, a *β*-1→4-D-glucose polymer, which is biosynthesized by the *bcsABZCbcsEFG* genes (bacterial cellulose synthesis) (Zogaj et al., 2001). Both operons are responsible for cellulose biosynthesis in both *S*. Enteritidis and *S*. Typhimurium (Jain & Chen, 2007; Solano et al., 2002). Cellulose production impaiment generates a bdar (brown, dry and rough) morphotype on congo red (CR) agar plates, characteristic of the expression of curli. Solano et al. (2002) showed that cellulose is a crucial biofilm determinant for *Salmonella*, under both LB and ATM conditions, without however affecting the virulence of the bacterium. Additionally, cellulose-deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of *Salmonella* in hostile environments. Prouty & Gunn (2003) identified its crucial importance for biofilm formation on glass coverslips. However, cellulose was not a major constituent of the biofilm matrix of *S*. Agona and *S*. Typhimurium strains isolated from the feed industry, but it contributed to the highly organized matrix structurization (Vestby et al., 2009a). Malcova et al. (2008) found that cellulose was not crucial for *S*.

Enteritidis adherence and biofilm formation on polystyrene.

Latasa et al. (2005) also reported another matrix component, BapA, a large cell-surface protein required for biofilm formation of *S*. Enteritidis. This protein was found to be loosely associated with the cell surface, while it is secreted through the BapBCD type I protein secretion system, encoded by the *bapABCD* operon. The expression of *bapA* was demonstrated to be coordinated with the expression of curli and cellulose through the action of *csgD* (Latasa et al., 2005). Also, these authors demonstrated that a *bapA* mutant strain showed a significant lower colonization rate at the intestinal cell barrier and consequently a

Motility was found to be important for *Salmonella* biofilm development on glass (Prouty & Gunn, 2003) and polyvinyl chloride (PVC) (Mireles et al., 2001). On the contrary, Teplitski et al. (2006) noticed that the presence of the flagellum on the surface of the cell, functional or not, is inhibitory to biofilm formation on polystyrene, as mutants lacking intact flagella, showed increased biofilm formation compared to the wild-type. Flagella were not found to be important for *S*. Typhimurium rdar expression on Congo Red (CR) agar plates (Römling & Rohde, 1999). Solano et al. (2002) noticed that flagella affect *S*. Enteritidis biofilm development

decreased efficiency for organ invasion compared with the wild-type strain.

The persistence of *Salmonella* within the food chain has become a major health concern, as biofilms of this pathogen formed in food processing environments can serve as a reservoir for the contamination of food products. The development of materials to be used for foodcontact surfaces with improved food safety profiles continues to be a challenge. One approach which has been developed to control microbial attachment is the manufacture of food-contact materials incorporating antimicrobial compounds. Triclosan-impregnated kitchen bench stones (silestones), although prone to bacterial colonization, were found to reduce *S*. Enteritidis biofilm development on them and also the viability of cells within the biofilm (Rodrigues et al., 2011).
