**2.** *Salmonella* **biofilms**

#### **2.1 Basic concepts on biofilms**

Costerton et al. [14] were the first researchers in stablish the term biofilm in paper published in *Scientific American* in 1978. They propose that most bacteria in *An Overview of* Salmonella *Biofilms and the Use of Bacteriocins and Bacteriophages… DOI: http://dx.doi.org/10.5772/intechopen.98208*

aquatic ecosystems growth attached to surfaces in a closed self-produced matrix. Researchers also postulates that sessile cells (biofilm) differ from the planktonic cells (floating). It is important to note that the authors include the reference to aquatic environment because it was the first place where bacterial biofilms were observed. But, at present it is known that biofilms are the predominant style of life of bacterial in environment and its related with 80% of bacterial infections. Actually, biofilm is defined as a community of bacterial cells enclosed in a selfproduced polymeric matrix and adhered to biotic (plant surfaces, epithelial cells, gallstones) or abiotic surfaces (plastic, rubber, glass, stainless steel). Biofilms have a great importance in the food production chain and human health because cells enclosed in this matrix are extremely difficult to eradicate because are more resistant to environmental stressors as antibiotics, disinfectants, host immune system [15–18].

There are four different steps of biofilm formation: 1) bacterial attachment, 2) microcolony formation, 3) bacterial maturation and 4) dispersion (**Figure 1**). The initial adhesion of bacterial cells is highly influenced by surface properties (roughness, hydrophobic interactions), environmental changes and bacterial regulation. Biofilm maturation and architecture is regulated by the signals of bacteria cells that compose biofilm and its stability depends on the accumulation of specific proteins, eDNA and polysaccharides. The presence of disruptive factors as proteases and nucleases and other enzymes activates biofilm dispersion. Factors as quorum sensing play an important role in this last step which function is the colonization of new niches [19].

#### **2.2 Biofilm formation steps**

#### *2.2.1 Adhesion*

*Salmonella* cells adhesion can be active or passive according the motility of bacteria or gravitational transport of planktonic cells. Both surfaces of bacterial cells and substrate surface highly influence the initial cell attachment. At this point bacterial cells have small quantities of extracellular polymeric substance (EPS) and maintain independent movement from other bacterial cells. Adhesion is reversible during this phase and cells do not present the morphological changes associated with biofilm cells and they can return to its planktonic state [16].

#### **Figure 1.**

*Steps involved in* Salmonella *biofilm formation. Created with biorender.com.*

#### *2.2.2 Irreversible adhesion*

The change from a weak interaction to a strong interaction between surface and bacterial cells is responsible to the switch from a reversible adhesion to an irreversible adhesion step. This change can happen in minutes and the production of EPS is key. The secretion of this polymeric substance by bacterial cells enhances the cellsurface interaction being necessary shear forces or chemical substances to break the adhesion [16, 20].

#### *2.2.3 Microcolony formation*

The formation of biofilm microcolony results from the accumulation of bacteria growth and the production and association with EPS. As a result, the bond between bacteria and substrate increases and protect bacteria from different environmental stressors. The cell-to-cell communication mechanism play an important role in this step of biofilm formation by regulating the expression of biofilm related genes. This results in an increased EPS production and caption of planktonic cells [21].

#### *2.2.4 Maturation*

The small microcolonies formed join to form the mature biofilm and its characteristic three-dimensional structure. The production of EPS and union between cells permits that mechanical pressure do not detach the biofilm from the surface. There are three different parts in mature biofilm. The bottom layer is a biofilm that forms a network structure that did not completely covers the surface that supports the biofilm. The intermediate layer is composed by a compact basement membrane. Finally, in the outer layer are located the planktonic cells [16].

#### *2.2.5 Dispersion*

The last step of biofilm formation is dispersion. In this phase the biofilm cells revert to their planktonic form. There are different factors that influences biofilm dispersion including external disturbance, starvation, endogenous enzymes, the release of EPS or surface binding proteins. This is an important step for the colonization of new niches by bacterial cells [22].

#### **2.3 Structural components of** *Salmonella* **biofilms**

*Salmonella* biofilm matrix is composed by proteins and exopolysaccharides among other things. There are two main proteins related with biofilms. Curli, an amyloid fimbria, and BapA protein. In the other hand, cellulose and colonic acid are the main exopolysaccharides of biofilm matrix. Also the type I fimbriae, Lpf and Pef are important in the initial steps of biofilm formation. Other components as fatty acids and lipopolysaccharides have also a role in biofilm formation.

Curli fimbriae is the most important protein involved in biofilm formation. Also is related to other processes as colonization, persistence, motility and invasion. This is a highly aggregative, unbranched, amyloid-like protein that promote cell-to-cell interactions through surfaces interactions and forms a complex with cellulose and O-capsule antigen. Other protein involved in biofilm formation is fimbriae type I. This protein is necessary for adhesion and biofilm formation in enterocytes. The protein BapA has an important role in bacterial aggregation and biofilm formation in air-liquid interface through homophilic interaction between bacterial cells [23–26].

*An Overview of* Salmonella *Biofilms and the Use of Bacteriocins and Bacteriophages… DOI: http://dx.doi.org/10.5772/intechopen.98208*

Cellulose is the main polysaccharide involved in *Salmonella* biofilm formation. It is necessary for biofilm maturation phase in different surfaces, and it is inversely correlated with virulence as its production is suppressed in *Salmonella* enterocyte colonization phase. Another exopolysaccharide is the lipid bound O-antigenic capsule, with importance in resistance to desiccation and environmental persistence. This exopolysaccharide has demonstrated a role in biofilm formation in gallstones and plants but lower importance in adhesion to abiotic surfaces as glass or plastic. In other hand, cholinic acid is important for three-dimensional structure formation in enterocytes but not in abiotic surfaces, gallstones or alfalfa seeds. Therefore, some polysaccharides are only important for some types of biofilm formation [27–33].

Flagella, which are basic for cell movement and swarming in *Salmonella* also play a role in biofilm formation. In the initial step of reversible and irreversible adhesion, motility is important. Also, motility is necessary for 3D biofilm structure and the dispersion phase. But in other steps of biofilm formation the expression of flagella is inhibited. There is switch mechanism system that causes a reduction of flagella function and increased the expression of cellulose, resulting in the inhibition of flagellar rotation. This demonstrates the ambivalent role of flagella in biofilm formation. Fatty acids have also a role in *Salmonella* biofilm formation, especially in hydrophilic surface such as glass but not in hydrophobic surfaces as gallstones [34–36].

#### **2.4 Genetic control of** *Salmonella* **biofilms**

The change from a planktonic to a biofilm cell lifestyle needs some physiological changes. This switch is controlled by a complex genetic machinery that regulates the production of substances that conform the biofilm extracellular matrix, bacterial metabolism and the response to environmental signals. The transition between planktonic to biofilm cells and the expression of specific biofilm matrix-associated components is the master regulator of biofilm formation CsgD. It forms part of the operon that control the synthesis of curli fimbriae and acts as a transcriptional activator of the quorum sensing LuxR family. CsgD expression respond to different environmental signals as nutrient concentration, temperature, growth phase, oxygen tension, osmolarity, membrane integrity, tryptophan, and indole. CsgD positively regulates cellulose biosynthesis in *Salmonella* through direct stimulation of adrA transcription. AdrA synthetize c-di-GMP, a signaling molecule, that also activates the cellulose synthase BcsA, resulting in increased production of cellulose. Although it is the most important, there are other enzymes involved in cellulose synthesis [37–40].

RpoS and Crl are other important regulators of *Salmonella* biofilm formation regulating the expression of several components. Gene *rpoS* encodes a sigma factor called σS that regulates genes involved in stress response and stationary phase. It has been observed that almost the 25% of genes regulated with this sigma factor are overexpressed in biofilm cells of *S.* Typhimurium. For example, RpoS increases the expression of *csgD* and biofilm formation in environments with limited iron availability and regulate the expression of *adrA* in some steps of biofilm formation and is involved in the expression of genes related with motility. In other hand, the transcriptional regulator Crl protein regulates the activity of σS. RpoS and Crl have an effect in each other and their concentration are negatively correlated. Crl is necessary for maximal expression of *csgB, csgD* or *bcsA* and increased the expression of other genes related to RpoS. It is also remarkable that its effect are higher at 28°C than at 37°C. This indicates that this transcriptional regulator acts as a temperature sensor of *Salmonella* biofilm formation [41–44].

The bacterial messenger molecule c-di-GMP regulates several biological functions as virulence, motility, cell cycle regulation, differentiation, and biofilm formation. This molecule promotes *Salmonella* biofilm formation by regulating the production of some important components of biofilm matrix as cellulose and curli fimbriae. The c-di-GMP has a positive feedback on *csgD* expression. Thus, high levels of c-di-GMP increased the levels of CsgD, this increased the levels of AdrA and therefore c-di-GMP and cellulose synthesis [45, 46].

Other regulatory system implicated in motility and biofilm formation is the twocomponent system BarA/SirA. This system is modulated by factors as external pH, metabolic end products (formate, acetate), short chain fatty acids or bile salts. SirA modulates the *Salmonella* Csr system, an important regulator of motility, virulence, carbon storage, secondary metabolism and biofilm formation. CsrA control the change between sessile cells and motility, mainly activating motility. SirA activate the transcription of small RNAs CsrB and CsrC that inhibits CsrA activity and motility related genes. This increases type I fimbriae production and therefore biofilm formation [47, 48].

#### **2.5 Quorum sensing**

Another mechanism implicated in biofilm formation is Quorum sensing (QS). This is a cell-to-cell communication mechanism used by bacteria to adapt to environmental changes and implant a common bacterial strategy to respond to environmental stressors. QS is implicated in responsive defense against eukaryotic host cells, nutrient access, growth restriction environments, survive in hostile environments as well as cell differentiation to other form of life as biofilm cells. This communication is based in small molecules called autoinducers and that diffuse through bacterial membranes. Autoinducers are secreted at a basal level during bacterial growth. The concentration of this molecules increases with the growth of bacterial population until reach a threshold level and modulate the expression of QS target genes (**Figure 2**) [49, 50].

Gram-negative bacteria QS is divide into three categories: (i) N-acyl homoserine lactones (AHLs) called AI-1; (ii) furanosyl borate diester derived from the recycling of S-adenosyl-homocysteine to homocysteine called Autoinducer II (AI-2) for interspecies and intraspecies communication; and (iii) Autoinducer (AI-3) related to the recognition of host catecholamines epinephrine and norepinephrine. In the

#### **Figure 2.**

*Schematic representation of quorum sensing mechanisms AI-1 and AI-2 in* Salmonella*. Created with biorender.com.*

*An Overview of* Salmonella *Biofilms and the Use of Bacteriocins and Bacteriophages… DOI: http://dx.doi.org/10.5772/intechopen.98208*

case of *Salmonella*, only encode the receptor for AHLs but do not produce AI-1 molecules. But *Salmonella* can recognize AHLs produced by other bacterial genera as *Pseudomonas aeruginosa* or *Yersinia enterocolitica*. QS is basic in the formation of healthy biofilms and have a role in every stage. Genes regulated by the AI-1 receptor SdiA promote *Salmonella* cell adhesion and the production of extracellular proteins that compose biofilm matrix. Thus, *Salmonella* can response to the presence of AHLs molecules produced by other bacteria and increase biofilm formation. In the same way, AI-2 LuxS also can increase the expression of motility and biofilm related genes. Therefore QS is key a component of biofilm formation regulation [51–53].

## **3. Biofilms in the food industry**

Nowadays, it is totally accepted that most bacteria grow in biofilm in the environment. Biofilms can have beneficial effects. For example, biofilm formation by *Lactobacillus* and *Lactococcus* results in more efficient fermentation processes and in the case of human health protect against the adhesion of pathogenic bacteria in the gut. But biofilm formation by undesirable bacteria, as food-borne pathogens, has a negative impact on food industry. Also, bacteria growing in biofilm can cause deterioration in the machinery as corrosion, efficiency reduction in heat transfer or clogging filters [54, 55].

Biofilms are a persistent source of contamination in the food industry. This cause hygiene and economic issues due to the spoilage of different food product batches with bacteria that persist in biofilms [56]. This is especially important in today's globalized world where food is globally distributed. Also, in the last years consumers demand fresh and minimally processed food products. Hygiene measures must therefore be strict to avoid contamination of food products. The presence of food-borne pathogen biofilms in the food processing environment can result in large number of food batches contaminated and outbreaks worldwide [57]. A good example was the salmonellosis outbreak caused by contamination of different batches of infant formula manufactured in a single facture and causing an outbreak that affected different countries around the world. Poor cleaning and disinfection procedures of food industry surfaces results in the presence of food residues that in the presence of humidity favors the development of bacterial biofilms as *Salmonella.* Cross-contamination occurs when food contact with surfaces with bacterial biofilms or also through aerosols from contaminated equipment. Until now, there is limited information of the real presence of *Salmonella* biofilms in the food processing environment. But *in vitro* studies have demonstrated that *Salmonella* can attach to different material commonly present in the food industry as plastic, glass, or stainless steel [57, 58].

Biofilm formation is influences but different factors as bacterial genus, species and even strains. But surface have a high influence on the ability of bacteria to adhere and form biofilm [59, 60]. Different type of material as stainless steel, glass, rubber, polystyrene and polyurethane, Teflon, nitrile and rarely wood are present in the food industry [61–63]. Physical properties have influence on biofilm formation, especially surface tension. Bacterial adhesion is favor by moist, energy free surfaces. Bacterial cells have better adherence to hydrophilic surfaces in comparison to hydrophobic surfaces. Surface roughness also influence cell adherence [57, 64]. In this sense, polished stainless steel showed less bacterial adherence than unpolished stainless steel [65]. Also, a study that compared stainless steel, glass and wood found that this latter surface favor biofilm formation because its porosity and ability to hold organic matter [66]. But also, surface influences biofilm formation in food industry. In this sense, welds, joints, corners or equipment design could enhance

initial bacterial cell adherence [67]. But the presence of organic molecules on food industry surfaces is one of the major factors that influences biofilm formation. The presence of a layer of molecules as milk or meat proteins, EPS produced by other bacteria, favor the initial adhesion of bacterial cells. Diverse studies have observed that the presence of chicken juice macromolecules in stainless steel surfaces favor the initial adhesion of *C. jejuni* or *S.* Typhimurium. However, in some occasion macromolecules have the opposite effect. In this sense, an study observed that milk proteins reduced the initial adhesion of *L. monocytogenes* [68–70]*.*

In the food production chain, there are different environmental conditions that can modulate *Salmonella* biofilm formation ability through modulation of initial adherence. Nutrient availability is one of these environmental conditions to which bacteria have to adapt. Under specific conditions, *Salmonella* has to persist under limited nutrient availability [71]. Biofilm formation is one strategy used for *Salmonella* cells to survive under this environmental stress conditions [72]. *In vitro* studies have demonstrated that *Salmonella* enhance a biofilm under limited nutrient conditions. These studies used common laboratory media as Tryptic Soy Broth or peptone water. These studies are a first approximation of the possible behavior of *Salmonella* under nutrient-limited conditions [71]. Temperature is another factor that changes through the food production chain. Several studies have demonstrated that *Salmonella* strains showed different biofilm formation amount under different temperatures tested. Interestingly, temperatures below 37°C and specially temperatures of 20°C favored *Salmonella* biofilm formation. The pH also influences *Salmonella* biofilm formation. A study that evaluated a total of 60 *S. enterica* strains under different pH, NaCl concentrations and temperature concluded that pH was the environmental factor that most influenced biofilm formation in *S. enterica* strains tested. This is probably due to the different ability of strains to adapt to acidic pH through an acid tolerance response mechanism [60, 73]. In the same way, another study found that weak acidic pHs (6) increased initial adhesion to stainless steel surfaces in comparison to neutral pHs. But curiously, acidic pHs reduced the number of cells present in mature biofilms due among other things to a lower presence of biofilm matrix components as polysaccharides and proteins [74]. Gene expression showed that acidic pH caused changes in the expression of virulence and biofilm related genes [75]. The environmental conditions under biofilms are formatted also influences its resistance to disinfectants. In this sense, biofilms formed under refrigeration temperatures showed higher sensitivity to disinfectants than those produced at 25°C under nutrient restriction as well as biofilm formed under acidic pH. In the other hand, mature biofilm are more resistant to substances such as quaternary ammonium compounds, peroxyacetic acid or organic acids. This is probably due a higher presence of matrix compounds as cellulose and curli fimbriae [76].

Although monospecies biofilm studies are interesting to understand the mechanism involved in biofilm formation under different environmental conditions of a specific bacteria, in nature biofilms are commonly composed by bacteria of different species and genera. These different bacteria communicate with each other through diverse mechanism as quorum sensing stablishing synergistic interactions that increase the resistance of biofilm to stressful environments. Also, genetic exchanges between different bacteria can occur in the biofilm environment [77]. This is specially interesting when resistance genes are transmitted. Dual biofilm studies are the first step to study multi-species biofilms. In this kind of studies, the biofilm formation ability of each bacterial group is studied individually, and then conjunct studies are carried out to determine the synergic mechanism stablished between the different groups [78]. In this sense, a study observed that *Salmonella* and *E. coli* mixed biofilms are more sensitive to disinfectants that biofilm of only

*An Overview of* Salmonella *Biofilms and the Use of Bacteriocins and Bacteriophages… DOI: http://dx.doi.org/10.5772/intechopen.98208*

one species [79]. In other hand, *S.* Enteritidis and *P. aeruginosa* mixed biofilms are more resistant to chlorine treatments [80]. In the same way, it was observed that mixed biofilms of *S.* Typhimurium and cultivable lettuce microorganism increased resistance to cold oxygen plasma treatments [81]. These studies provide a first clue of mixed biofilms. These studies are a first approach to multi-species studies. But undoubtedly the study of biofilms composed of hundreds of different bacterial genera will provide valuable information to fully understand how biofilms behave in nature. Such studies supported by genomics, metabolomics and high-resolution imaging will be the trend of the coming years in this field of microbiology.
