**3. Biofilm formation in food processing environments and implications**

The ability of bacteria to attach to abiotic surfaces and form biofilms is a cause of concern for many industries, including the food ones (Chmielewski & Frank, 2003). Poor sanitation of food-contact surfaces is believed to be an essential contributing factor in foodborne disease outbreaks, especially those involving *Listeria monocytogenes* and *Salmonella*. This is because the attachment of bacterial cells to such surfaces is the first step of a process which can ultimately lead to the contamination of food products. Thus, biofilms formed in food processing environments are of special importance since they may act as a persistent source of microbial contamination which may lead to food spoilage or/and transmission of diseases (Brooks & Flint, 2008; Zottola & Sasahara, 1994). While food spoilage and deterioration may result in huge economic losses, food safety is a major priority in today's globalizing market with worldwide transportation and consumption of raw, fresh and minimally processed foods (Shi & Zhu, 2009).

Besides food spoilage and safety issues, in the dairy industry, bacterial attachment in heat exchangers (a process commonly known as "biofouling") greatly reduces the heat transfer and operating efficiency of the processing equipment, while it can also causes corrosion problems (Austin & Bergeron, 1995). Additionally, in the various filtration systems, biofilm formation reduces significantly the permeability of the membranes (Tang et al., 2009). However, it should be noted that in the industry of fermented food products (sausages, cheeses etc), biofilm formation by some useful and technological bacteria (e.g. staphylococci, lactococci, lactobacilli) can be desirable, as a mean of the enhancement of food fermentation process, and more importantly as a mean of protection against the establishment of pathogenic biofilms (Chorianopoulos et al., 2008; Zhao et al., 2006).

Adhesion of *Salmonella* to food surfaces was the first published report on foodborne bacterial biofilm (Duguid et al., 1966). Since that time, many documents have described the ability of foodborne pathogens to attach to various surfaces and form biofilms, including *L. monocytogenes* (Blackman & Frank, 1996; Chorianopoulos et al., 2011; di Bonaventura et al., 2008; Poimenidou et al., 2009), *Salmonella enterica* (Chia et al., 2009; Giaouris et al., 2005; Giaouris & Nychas, 2006; Habimana et al., 2010b; Joseph et al., 2001; Kim & Wei, 2007, 2009; Oliveira et al., 2006; Marin et al., 2009; Rodrigues et al., 2011; Solomon et al., 2005; Stepanović et al., 2003, 2004), *Yersinia enterocolitica* (Kim et al., 2008*), Campylobacter jejuni* (Joshua et al., 2006) and *Escherichia coli* O157:H7 (Habimana et al., 2010a; Skandamis et al., 2009).

Modern food processing supports and selects for biofilm forming bacteria on food-contact surfaces due to mass production of products, lengthy production cycles and vast surface areas for biofilm development (Lindsay & von Holy, 2006). *In situ* biofilms have been recognised in various food processing industries, such as processors of cheese and other milk products, raw and cooked/fermented meats, raw and smoked fish etc (Austin & Bergeron, 1995; Bagge-Ravn

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

**3. Biofilm formation in food processing environments and implications** 

The ability of bacteria to attach to abiotic surfaces and form biofilms is a cause of concern for many industries, including the food ones (Chmielewski & Frank, 2003). Poor sanitation of food-contact surfaces is believed to be an essential contributing factor in foodborne disease outbreaks, especially those involving *Listeria monocytogenes* and *Salmonella*. This is because the attachment of bacterial cells to such surfaces is the first step of a process which can ultimately lead to the contamination of food products. Thus, biofilms formed in food processing environments are of special importance since they may act as a persistent source of microbial contamination which may lead to food spoilage or/and transmission of diseases (Brooks & Flint, 2008; Zottola & Sasahara, 1994). While food spoilage and deterioration may result in huge economic losses, food safety is a major priority in today's globalizing market with worldwide transportation and consumption of raw, fresh and

Besides food spoilage and safety issues, in the dairy industry, bacterial attachment in heat exchangers (a process commonly known as "biofouling") greatly reduces the heat transfer and operating efficiency of the processing equipment, while it can also causes corrosion problems (Austin & Bergeron, 1995). Additionally, in the various filtration systems, biofilm formation reduces significantly the permeability of the membranes (Tang et al., 2009). However, it should be noted that in the industry of fermented food products (sausages, cheeses etc), biofilm formation by some useful and technological bacteria (e.g. staphylococci, lactococci, lactobacilli) can be desirable, as a mean of the enhancement of food fermentation process, and more importantly as a mean of protection against the establishment of

Adhesion of *Salmonella* to food surfaces was the first published report on foodborne bacterial biofilm (Duguid et al., 1966). Since that time, many documents have described the ability of foodborne pathogens to attach to various surfaces and form biofilms, including *L. monocytogenes* (Blackman & Frank, 1996; Chorianopoulos et al., 2011; di Bonaventura et al., 2008; Poimenidou et al., 2009), *Salmonella enterica* (Chia et al., 2009; Giaouris et al., 2005; Giaouris & Nychas, 2006; Habimana et al., 2010b; Joseph et al., 2001; Kim & Wei, 2007, 2009; Oliveira et al., 2006; Marin et al., 2009; Rodrigues et al., 2011; Solomon et al., 2005; Stepanović et al., 2003, 2004), *Yersinia enterocolitica* (Kim et al., 2008*), Campylobacter jejuni* (Joshua et al.,

Modern food processing supports and selects for biofilm forming bacteria on food-contact surfaces due to mass production of products, lengthy production cycles and vast surface areas for biofilm development (Lindsay & von Holy, 2006). *In situ* biofilms have been recognised in various food processing industries, such as processors of cheese and other milk products, raw and cooked/fermented meats, raw and smoked fish etc (Austin & Bergeron, 1995; Bagge-Ravn

2006) and *Escherichia coli* O157:H7 (Habimana et al., 2010a; Skandamis et al., 2009).

the switch to the biofilm mode of growth.

minimally processed foods (Shi & Zhu, 2009).

pathogenic biofilms (Chorianopoulos et al., 2008; Zhao et al., 2006).

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

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

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 material (Chorianopoulos et al., 2010).

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

Attachment and Biofilm Formation by Salmonella in Food Processing Environments 163

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

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

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

with low productivity (Iibuchi et al., 2010).

origin of different serotypes (Marin et al., 2009).

ability and bacterial physicochemical surface characteristics.

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.
