**5. Bacteriophages and derived protein endolysin**

#### **5.1 Briefly definition and characteristics**

Bacteriophages are viruses that infects bacterial cells with a high specificity. The life cycle of bacteriophages can be classified in two general categories: the lytic cycle (virulent) and the lysogenic cycle (temperate phage). In the lytic cycle the infection process starts with the irreversible attachment of the phage tail proteins to a receptor of the bacterial cell surface (protein or lipopolysaccharides). The ability of the bacteriophages to recognize and attach to molecules of the bacterial cell surface defines its host range. Once the phage DNA is in the host cell, specific enzymes are synthetized to drive host cell to the production of proteins necessary for the generation of new phage particles and cell lysis enzymes. At the end of the phage cycle, cell lysis, release of progeny phage and infection of neighboring susceptible cells occurs. Temperate phages combine its capacity to carried out the lytic cycle with the ability to persist as a prophage in the genome of the host cell and replicate with them. Diverse environmental signal can result in the prophage entering in the lytic cycle [96]. The use of temperate phages in medical and food applications is avoided because can cause transduction of genetic material between bacteria including virulence genes. In addition, due to its cycle, they do not kill all the bacteria that infect [97].

Lytic phages are those chosen for being used in phage therapy because they can replicate exponentially on bacterial culture and can eliminate multidrug resistant bacteria [86]. Based on their activity spectrum can be defined as monovalent phages when they are specific to one type of bacterial species and polyvalent phages when they are able to attack two or more bacterial species. But normally phages have a narrow host range, strains specific in most cases, and therefore cocktails composed by two or more phages are normally used to broaden the antimicrobial spectrum and reduce phage resistance [98].

Although bacteriophages have been known for over a century, the development of antibiotics resulted in their use not being explored in the Western world. However, the global problem of antimicrobial resistance and the need to seek alternatives has resulted in bacteriophages being brought back into the spotlight. Its applications in the food chain are very wide. They can be used for the treatment of bacterial diseases of production animals, for the disinfection of facilities and the elimination of biofilms or they can be added to food or packaging to inhibit the growth of food pathogens [86, 97]. In fact, there are different commercially available bacteriophage solutions to be applied to food or food processing facilities. Some examples are ListShield™, SalmoFresh™ and EcoShield PX™ commercialized by Intralytix or PhageGuard Listex and PhageGuard S commercialized by PhageGuard.

The bacteriophages synthetized at the end of the phage multiplication cycle peptidoglycan hydrolases commonly called endolysin. Its function is to lyse the host bacterial cell by directly target bonds in the bacterial cell wall peptidoglycan structure. This result in the degradation of the rigid murein layer and the release of newly assembled bacteriophage virions [99]. While endolysins can act as exolysins in the Gram-positive bacterial peptidoglycan layer, they cannot degrade the bacterial outer membrane of Gran-negative bacterial cells. Therefore, the outer membrane can prevent the access and the effect of endolysins [100]. For that reason, it is necessary to combine endolysins with other treatments for the lysis of Gram-negative bacteria. The combination of endolysin with outer membrane disruptors in one the main options for the application of enzymes in Gram-negative bacteria. Gram-positive phages endolysins have a modular structured formed by a cell-wall-binding domain that specifically recognizes the cell wall-associated

ligand molecules and an enzymatically active domain that cleaves the peptidoglycan structure. Although gram-negative bacteriophage endolysins may also have this structure, they usually have a globular structure that only possesses a an enzymatically active domain [101, 102].One of the main advantages of the use of endolysins is that a very small amount of purified enzyme is enough to lyse in minutes or even seconds a dense suspension of bacterial cells. This in combination with their substrate specificity makes them have great potential for application in food science [103]. Endolysins are considered to be safe and also have some advantages compared to the use of bacteriophages because do not create gene transduction issues and therefore not contribute to the emerging problem of antimicrobial resistant bacteria [104]. Its applications in the food industry are very wide. They can be added directly to food, can be part of bioactive packaging or can even be used to remove biofilms in the food industry environment Furthermore, due to their specificity, they can be applied directly to treat intestinal infections in farm animals without causing alterations in the intestinal microbiota [103].

### **5.2 Applied studies on** *Salmonella* **biofilms**

Tiwari et al. [105] tested a specific *S.* Enteritidis virulent phage called SE2 against planktonic and biofilm cells of an antimicrobial resistant *S.* Enteritidis strain. The phage showed a high bacteriolytic effect. This phage reduced in 4.2 log UFC/mL the count of *S.* Enteritidis after incubation of 4 h at 37°C and 2.5 log UFC/mL after incubation at 4°C. These results demonstrate that this phage can also be used effectively at refrigeration temperature. Also, biofilm studies showed that treatments with phage SE2 concentrations of 1011 PFU/mL reduced in 97% viable cells present in biofilms formed in glass. Also this phage showed that could maintain its activity at different ranges of pH and temperature. It has been also proposed that phage predation could increase biofilm formation by bacteria in some specific conditions. Hosseinidoust et al. [106] carried out and study to evaluate this theory in different pathogens including *S.* Typhimurium and to determinate if the increase of biofilm formation is due to the development of phage resistance or to non-evolutionary mechanism as spatial refuge. The results indicate that phage resistance was the mechanism implicated in increased biofilm formation in *P. aeruginosa.* However, in the case of *S.* Typhimurium it was due to non-evolutionary mechanisms [106]. Karaca et al. [107] evaluated the effect of phage P22 in *S.* Typhimurium biofilm formation in polystyrene and stainless-steel surfaces. The authors evaluated both the incubation of phage particles with *Salmonella* in biofilm studies and the treatment of preformed biofilms. *S.* Typhimurium biofilm formation was significantly reduced at high phage titer (≤106 PFU/mL). Also, all phage titers were effective against biofilm formation in 24 h incubation period but only higher phage titers were effective in 48–72 h incubation time. In addition, the ability to reduce biofilm formation was lower in polystyrene than in stainless steel. In the other hand, phage treatment was not effective in eradicating pre-formed *Salmonella* biofilms. This is probably due to the presence of extracellular matrix components that prevent bacteriophages from binding to specific receptors on the bacterial surface. In this sense, Yüksel et al. [108] combined phage P22 with EDTA and nisin to improve the antibiofilm activity of phage. The combination of the three inhibit in 93% *S.* Typhimurium biofilm formation at low phage titer concentrations but only reduced 70% mature biofilms. Therefore, the combination of phages with other antimicrobial substances could enhance antibiofilm activity. But it is still difficult to reduce biofilm in mature stages, when high quantities of extracellular matrix substances are present.

Garcia et al. [109] tested a cocktail of lytic bacteriophages biofilm to eradicate biofilms formed by different *Salmonella* serotypes in different surfaces (stainless

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

steel, glass, and polyvinyl chloride) at short and long incubation times. Preformed biofilms were treated with 108 PFU/mL during 3, 6 and 9 h. The results were not very promising and had a lot of variation between different surfaces and *salmonella* serotypes. In the same way, Gong et al. [110] tested different phage concentration (104 –108 PFU/mL) against hard *Salmonella* biofilms formed in microtiter plates. Phages were selected based in its range activity against the different *Salmonella* serotypes included in the study. The reduction of biofilm formation was of 90% when *Salmonella* was incubated in combination with phages and 66% in preformed biofilms. Milho et al. [111] tested the phage PVP-SE2 against *S.* Enteritidis biofilms formed in food contact surfaces polystyrene and stainless steel. This phage caused reductions of 2 to 5 log CFU cm2 at room temperature of 24 h and 48 h old *Salmonella* biofilms, showing its efficacy to control *S.* Enteritidis biofilms. Also, it was observed that this phage inhibited the growth of *S.* Enteritidis in poultry skin, even in freezing phage-pretreated poultry skin. The same research group evaluated the antibiofilm effect of phages in *E. coli* and *S. Enteritidis* dual-species biofilms [112]. The results of this study showed that phages were more effective to eradicate mono-species biofilms than dual-species biofilms. It is important to consider this when designing products that include phages to eradicate biofilms as biofilms in the food industry are often composed of various bacterial species. Kosznik-Kwasnicka et al. [113] evaluated three phages vB\_SenM-1, vB\_SenM-2, and vB\_SenS-3 with lytic activity against different *Salmonella* serotypes. The phages were able to reduce biofilm cells and biomass in different strains tested and under different temperatures. This is important as there are different temperatures in the food chain and this study would indicate that phage treatment could be used over a wide temperature range. In the same way, Esmael et al. [114] tested to *S.* Typhimurium lytic phages against 72 h-old biofilms formed in microtiter plates. Concentrations of 8 log10 PFU/mL reduced more than three times biofilm formation. However, most of the studies conducted so far focus on specific *Salmonella* serotypes. One of the main characteristics of phages is their specificity. Thus, phages usually show activity against specific species, serotypes or even strains. This leads to a number of studies evaluating phage cocktails. Even so, it is difficult to find a phage cocktail effective against all *Salmonella* serotypes. This is one of the main problems to be solved with the use of phages in the food industry.

Using a food model, Sadekuzzaman et al. [115] evaluated the efficacy of 2 h bacteriophage treatment against *Salmonella* biofilms formed in lettuce surface. Although effective, phage treatment only reduced 1.0 log CFU/cm the count of *Salmonella*. Another alternative is the use of the active parts of the phages, for example the phage-encoded proteins. Altought some of the functions performed by proteins can be also performed by the phage itself, the use of proteins can have advantages in consumer acceptance and in terms of regulation. In this sense. Zhang et al. [116] tested endolysin LysSTG2 against *S.* Typhimurium biofilms. One hour treatment with 100 μg/mL of this endolysin, reduces 72 h biofilm in 13%. However, the combination of this endolysin with slightly acidic hypochlorous water containing 40 mg/L available chlorine reduces *S.* Typhimurium biofilm cells in 99%. Therefore, the combination of endolysin with other antimicrobial substances is a potential alternative against *Salmonella* biofilms.

### **6. Conclusion**

*Salmonella* biofilm formation in the food production chain is a major public health problem. Mechanisms regulating biofilm formation in *Salmonella* are complex and is regulated by a wide range of environmental factors. The ability of *Salmonella* to form biofilm in a wide temperature or pH range as well as in other stressful situations poses a major problem for its eradication. Also of concern is the increase of *Salmonella* strains with resistance to multiple biocides. Both bacteriocins and bacteriophages are a potential alternative to eliminate *Salmonella* biofilms. In addition, they can be combined synergistically with traditional antimicrobials, thus reducing the amount of antimicrobials used. One of the main limiting factors in its application is its range of activity. Normally bacteriocins and bacteriophages present a narrow spectrum of activity. They are therefore very useful for use against a specific pathogen. But in order to have a broad spectrum of activity to prevent different bacterial groups in the food chain, formulations combining a cocktail of bacteriocins and phages are needed. Studies evaluating such products as an alternative to traditional biocides are still limited, but future research and the use of recombinant technologies will make it possible to obtain products with high efficacy against *Salmonella* biofilms.
