**2. Biofilm formation**

Biofilms are formed on all types of surfaces existing in food plants ranging from plastic, glass, metal, cement, to wood and food products [4]. Usually, biofilms form a monolayer or more often multilayers, in which bacteria may undergo a significant change in physiology with an increased tolerance to environmental stresses [5].

*L. monocytogenes*, the pathogen that proliferates at low temperatures, is able either to form pure culture biofilms or to grow in multispecies biofilms [6]. Prevalent strains in food processing environments have good adhesion ability due to the presence of flagella, pili, and membrane proteins [7]. Composition of biofilms produced by *L. monocytogenes* is different in comparison with that produced by other bacteria. For example, exopolysaccharides like alginate in Pseudomonas or poly-N-acetylglucosamine in *Staphylo-coccus* have not been put into evidence [8].

*Salmonella* spp. express proteinaceous extracellular fibers called curli that are involved in surface and cell-cell contacts and promotion of community behavior and host colonization [9]. Besides curli, different fimbrial adhesins have been identified to have implications in biofilm formation, dependent of serotype. The presence of cellulose in the biofilm matrix contributes to cells' resistance to mechanical forces and improved adhesion to abiotic surfaces [10]. Significant differences between serovars were put into evidence regarding biofilm formation the most persistent in food processing environments being the ones that are capable to form biofilms [11].

Flagella, pili, and membrane proteins are also used by *E. coli* to initiate attachment on inanimate surfaces. Flagella are lost after attachment and bacteria start producing an extracellular polymeric substance (EPS) that provides a better resistance of bacteria to disinfectants as hypochlorite [12]. Similarities in biofilm structure and composition as well as regulatory mechanisms with *Salmonella* spp. have been demonstrated for *E. coli*, mostly in terms of expression of small RNAs leading to a change in bacterial physiology regarding the cell motility and production of curli or EPS [13].

In general terms, different *E. coli* serotypes have been reported to enhance flexibility and adaptability in forming biofilms when exposed to different stresses. For example, *E. coli* seropathotype A isolates associated with human infection, O157:H7 and O157:NM, showed greater ability to form biofilms than those belonging to seropathotype B or C associated with outbreaks and hemolytic-uremic syndrome (HUS) or sporadic HUS cases but no epidemics, respectively [14]. In addition, synergistic interactions are taking place in a fresh-cut produce processing plant in which *E. coli* is interacting with *Burkholderia caryophylli* and *Ralstonia insidiosa* with the formation of mixed biofilms [15].

*C. jejuni*, which is known as an anaerobic bacterium, is able to develop biofilms both in microaerophilic conditions (5% O2 and 10% CO2) and in aerobic conditions (20% O2) [1]. The cells embedded in the biofilm matrix are better protected from oxygen and survive for days in food processing environments [1].

*Pseudomonas* spp. produce high amounts of EPS and have been shown to attach and form biofilms on stainless steel surfaces. They coexist within biofilms with *Listeria, Salmonella*, and other pathogens forming multispecies biofilms, more stable and resistant [6].

*B. cereus* is a cause of biofilm formation on many food contact surfaces such as conveyor belts, stainless steel pipes, and storage tanks [16], but it is also able to

**215**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

the bacterium to antimicrobials and cleaning procedures.

**3. Genes involved in the biofilm formation**

a specific genetic signal control.

just the bioinformatics analysis.

cells and surface.

form immersed or floating biofilms, and to secrete within the biofilm a vast array of metabolites, surfactants, bacteriocins, enzymes as lipases and proteases affecting the sensorial qualities of foods, and toxins. For floating biofilms, the production of kurstakin, a lipopeptide biosurfactant, that is regulated via quorum sensing (QS)

Within the biofilm, *B. cereus* exists either in vegetative or in sporal form, the spores being highly resistant and adhesive, properties that increase the resistance of

Four mechanisms based on the flagellar motility of *B. cereus* are described as being involved in biofilm formation. The first mechanism is used in static conditions when the bacterium must reach on its own suitable places for biofilm formation [18], at the air-liquid interface. The second one is represented by the creation of channels in the biofilm matrix to facilitate nutrients' access on one hand and penetration of toxic substances on the other hand [19]. The third mechanism refers to motile planktonic bacteria that penetrate the biofilm and increase its biomass [18, 19], while the fourth represents the extension of the biofilm based on the ability of motile bacteria located at the edge of the biofilm to colonize the surroundings [18]. It has been showed that, in its planktonic form, *S. aureus* does not appear resistant to disinfectants, compared to other bacteria, but it may be among the most resistant ones when attached to a surface [20]. It seems that different stress-adaptive responses may enhance biofilm formation, with certain differences in terms of their composition and architecture, especially for the wild-type biofilms colonizing the food and related processing environments. Examples include protein-based sources responsible for the structure of biofilms formed by *S. aureus* of food origin [21] similar to those put into evidence for the coagulase-negative ones. However, other studies demonstrated that simple carbohydrates, such as milk lactose, can modulate the biofilm formation especially by inducing the production of polysaccharide intercellular adhesins [22].

Over time, beside the conditions that favor the biofilm formation in food processing plants**,** the genetic background of biofilm forming microorganisms was also intensively studied. At each step of biofilm development and dispersal, there is

The *L. monocytogenes* pattern of the microarray gene expression was analyzed at different time intervals (4, 12, and 24 h) in order to depict genes' expression at different stages of biofilm formation. The results showed that more than 150 genes were upregulated after 4 h of biofilm formation and a total of 836 genes highlighted a slow increase in expression with time [23]. Although for many bacterial species the genome sequencing allowed the identification of genes that were involved in biofilm synthesis, for *L. monocytogenes*, these genes could not be identified using

In the biofilm formation, the attachment step is a prerequisite in which flagella and type I pili-mediated motilities are critical for the initial interaction between the

In order to find out the roles of the genes and regulatory pathway controlling the biofilm formation, researchers applied one or two genome-wide approaches, like transposon insertion mutagenesis or/and transcriptome analyses. With a transposon mutagenesis library, it was possible to identify 70 *L. monocytogenes* mutants, with Himar1 mariner transposon insertion, which produced less biofilms [24]. From a total of 38 genetic loci identified, 4 of them (**Table 1**) were found to be involved in bacterial motility (*fli*D, *fli*Q, *fla*A, and *mot*A), a required property for initial surface

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

signaling is important [17].

### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

associated with brand damage.

**2. Biofilm formation**

tion of curli or EPS [13].

stable and resistant [6].

the formation of mixed biofilms [15].

of processed foods [3]. When contamination of food products happens, recalls are necessary. These actions present large economic burden to industry and are also

Biofilms are formed on all types of surfaces existing in food plants ranging from plastic, glass, metal, cement, to wood and food products [4]. Usually, biofilms form a monolayer or more often multilayers, in which bacteria may undergo a significant change in physiology with an increased tolerance to environmental stresses [5]. *L. monocytogenes*, the pathogen that proliferates at low temperatures, is able either to form pure culture biofilms or to grow in multispecies biofilms [6].

Prevalent strains in food processing environments have good adhesion ability due to the presence of flagella, pili, and membrane proteins [7]. Composition of biofilms produced by *L. monocytogenes* is different in comparison with that produced by other bacteria. For example, exopolysaccharides like alginate in Pseudomonas or poly-N-acetylglucosamine in *Staphylo-coccus* have not been put into evidence [8]. *Salmonella* spp. express proteinaceous extracellular fibers called curli that are involved in surface and cell-cell contacts and promotion of community behavior and host colonization [9]. Besides curli, different fimbrial adhesins have been identified to have implications in biofilm formation, dependent of serotype. The presence of cellulose in the biofilm matrix contributes to cells' resistance to mechanical forces and improved adhesion to abiotic surfaces [10]. Significant differences between serovars were put into evidence regarding biofilm formation the most persistent in food processing environments being the ones that are capable to form biofilms [11]. Flagella, pili, and membrane proteins are also used by *E. coli* to initiate attachment on inanimate surfaces. Flagella are lost after attachment and bacteria start producing an extracellular polymeric substance (EPS) that provides a better resistance of bacteria to disinfectants as hypochlorite [12]. Similarities in biofilm structure and composition as well as regulatory mechanisms with *Salmonella* spp. have been demonstrated for *E. coli*, mostly in terms of expression of small RNAs leading to a change in bacterial physiology regarding the cell motility and produc-

In general terms, different *E. coli* serotypes have been reported to enhance flexibility and adaptability in forming biofilms when exposed to different stresses. For example, *E. coli* seropathotype A isolates associated with human infection, O157:H7 and O157:NM, showed greater ability to form biofilms than those belonging to seropathotype B or C associated with outbreaks and hemolytic-uremic syndrome (HUS) or sporadic HUS cases but no epidemics, respectively [14]. In addition, synergistic interactions are taking place in a fresh-cut produce processing plant in which *E. coli* is interacting with *Burkholderia caryophylli* and *Ralstonia insidiosa* with

*C. jejuni*, which is known as an anaerobic bacterium, is able to develop biofilms both in microaerophilic conditions (5% O2 and 10% CO2) and in aerobic conditions (20% O2) [1]. The cells embedded in the biofilm matrix are better protected from

*Pseudomonas* spp. produce high amounts of EPS and have been shown to attach and form biofilms on stainless steel surfaces. They coexist within biofilms with *Listeria, Salmonella*, and other pathogens forming multispecies biofilms, more

*B. cereus* is a cause of biofilm formation on many food contact surfaces such as conveyor belts, stainless steel pipes, and storage tanks [16], but it is also able to

oxygen and survive for days in food processing environments [1].

**214**

form immersed or floating biofilms, and to secrete within the biofilm a vast array of metabolites, surfactants, bacteriocins, enzymes as lipases and proteases affecting the sensorial qualities of foods, and toxins. For floating biofilms, the production of kurstakin, a lipopeptide biosurfactant, that is regulated via quorum sensing (QS) signaling is important [17].

Within the biofilm, *B. cereus* exists either in vegetative or in sporal form, the spores being highly resistant and adhesive, properties that increase the resistance of the bacterium to antimicrobials and cleaning procedures.

Four mechanisms based on the flagellar motility of *B. cereus* are described as being involved in biofilm formation. The first mechanism is used in static conditions when the bacterium must reach on its own suitable places for biofilm formation [18], at the air-liquid interface. The second one is represented by the creation of channels in the biofilm matrix to facilitate nutrients' access on one hand and penetration of toxic substances on the other hand [19]. The third mechanism refers to motile planktonic bacteria that penetrate the biofilm and increase its biomass [18, 19], while the fourth represents the extension of the biofilm based on the ability of motile bacteria located at the edge of the biofilm to colonize the surroundings [18].

It has been showed that, in its planktonic form, *S. aureus* does not appear resistant to disinfectants, compared to other bacteria, but it may be among the most resistant ones when attached to a surface [20]. It seems that different stress-adaptive responses may enhance biofilm formation, with certain differences in terms of their composition and architecture, especially for the wild-type biofilms colonizing the food and related processing environments. Examples include protein-based sources responsible for the structure of biofilms formed by *S. aureus* of food origin [21] similar to those put into evidence for the coagulase-negative ones. However, other studies demonstrated that simple carbohydrates, such as milk lactose, can modulate the biofilm formation especially by inducing the production of polysaccharide intercellular adhesins [22].
