Bacteriophages as Potential Tools for Detection and Control of Salmonella spp. in Food Systems

The global problem of antibiotic resistance in bacteria is quickly developing in most antibiotics used in hospitals and livestock. Recently, the infections with multi-drug resistant (MDR) bacteria become a major cause of death worldwide. Current antibiotics are not very effective in treating MDR Salmonella infections, which have become a public health threat. Therefore, novel approaches are needed to rapidly detect and effectively control antibiotic-resistant pathogens. Bacteriophages (phages) have seen renewed attention for satisfying those requirements due to their host-specific properties. Therefore, this review aims to discuss the possibility of using phages as a detection tool for recognizing bacterial cell surface receptors and an alternative approach for controlling antibiotic-resistant pathogens in food systems.


Introduction
Salmonella, a Gram-negative rod-shaped bacilli bacterium, is the most common foodborne pathogen and is known as one of the four key global causes of diarrheal diseases, according to the World Health Organization (WHO). As estimated by the Centers for Disease Control and Prevention, 23,000 hospitalizations and 450 deaths have been caused by Salmonella every year in the United States. Among the 1.2 million illnesses caused by Salmonella, contaminated food caused 1 million illnesses [1,2]. Contaminated meat, poultry, eggs, and milk are the main reservoirs for Salmonella infections [3]. Some direct contact with infected animals, blood, urine, and feces may also cause the problem to human health [4]. Antibiotic use has been increased for controlling the bacteria in animals or increasing food production, which has accelerated the emergence of antimicrobial-resistant bacteria [5][6][7]. The antibiotic-resistant bacteria are likely to contaminate in food products throughout the handling or other production stages [8][9][10]. Fruit and vegetable products are susceptible to cross-contamination during harvest and post-harvest periods [11][12][13]. Salmonellosis caused by Salmonella is one of the serious infections that is difficult to treat due to the reduced antibiotic activities that become less effective [14]. Thus, novel antibiotics or alternative methods are required to control the antibiotic-resistant bacteria.
Bacteriophages (phages) are predominant in nature, defined as viruses that can infect bacteria alone [15,16]. In the 1920s, Frederick Twort first observed the glassy and transparent colonies in micrococci cultures, which could cause an acute infectious disease in 1915, but the term "phage" was neither correctly defined nor clearly understood at this time [17]. Two years later, Félix d'Herelle isolated a microbe that showed an antagonistic effect against Shiga bacillus and first described this microbe as an obligate bacteriophage with a high specificity to the host and no pathogenic effects [18]. However, the interest in phages decreased because of the lack of proper quality controls and reproducible results in the western countries [21][22][23]. Another important reason was the discovery of antibiotics, which were used as the most powerful reagents for controlling bacterial infections. Ironically, the frequent use of antibiotics has resulted in the development of the multidrug-resistance or superbugs [24][25][26]. Figure 1 shows the advantages and disadvantages of bacteriophage applications. A phage as a biorecognition agent provides many advantages for rapid bacterial sensing [27], including target specificity [28], release of high titer phages, tolerance to environmental stresses [29], safe handling, and effectiveness against live bacteria [30]. The phage-based biosensors include the combination of whole phages or phage-constituents, which can be converted to electrical, colorimetric, fluorescent, or luminescent signals. Phages are thus shown as cheap, fast, sensitive, selective, and specific tools for detecting bacteria [31]. From a therapeutic viewpoint, phage therapy provides many benefits over chemotherapy, since phages are active against antibiotic-resistant bacteria and no side effect occurs during phage treatment [32]. Bacteriophages (phages) are predominant in nature, defined as viruses that can infect bacteria alone [15,16]. In the 1920s, Frederick Twort first observed the glassy and transparent colonies in micrococci cultures, which could cause an acute infectious disease in 1915, but the term "phage" was neither correctly defined nor clearly understood at this time [17]. Two years later, Félix d'Herelle isolated a microbe that showed an antagonistic effect against Shiga bacillus and first described this microbe as an obligate bacteriophage with a high specificity to the host and no pathogenic effects [18].   With more scientifically rigorous approaches in the recent years, more researchers have paid attention toward the bacteriophages as a promising tool to treat the pathogens [33]. Bacteriophages also show additional advantages, including a high specificity to the host, an ability to differentiate alive and dead cells, and the most abundant biological entity in nature, which draws renewed attention to the detection and control of antibiotic-resistant pathogens [34][35][36]. The effectiveness of phage applications in treating pathogenic bacteria is influenced by several factors, such as the multiplicity of infection (MOI), treatment mode, environmental conditions, phage neutralization, and target bacteria. The bacteriophage survival an adverse environment is also a desired characteristic for therapeutic use. Currently, the commercial products based on bacteriophages targeting Escherichia coli O157:H7 [37][38][39], Listeria monocytogenes [40][41][42], Salmonella spp. [43,44], and Shigella spp. [45][46][47] are available in the market. Furthermore, application trials in foods are also performed, which would help enhance the food safety. The lytic activities of specific bacteriophages against Salmonella and other pathogenic bacteria are compared in Tables 1 and 2. In this review, the detection methods and biocontrol applications based on bacteriophages targeting Salmonella are summarized and discussed in detail.

Phage Plaque Assay as A Traditional Method for Salmonella Detection
The traditional agar overlay method is a very useful technique for studying bacteriophage, including identification, enumeration, purification, and isolation of phage mutants. This method is based on lysis plaques, which are formed on lawns of the host bacteria, immobilized in the top soft agar. After incubation, a series of reaction events, such as phage infection, multiplication, and liberation, occur in the host [49,50]. The soft agar overlay method is a common titer assay, which was introduced by Mark Adams [51]. The plaque assay has been improved based on several modifications, such as the addition of effective supplements to modify growth media and agar composition and the plate spreading method, which can enhance the visibility of the plaques [49,52,53]. However, a long time (more than 24 h) is required for obtaining detection results and it is also labor intensive, which may not meet the demands of rapid pathogen detection. Thus, many researchers devote their efforts to developing reliable and rapid detection methods. Figure 2 shows two different life cycles of bacteriophages and lytic and lysogenic cycles. The active virulence phages can produce progeny phages that burst out of the host cell through the lytic cycle, while the lysogenic cycle involves the integration of the temperate phage genome into the host chromosome, which can remain in an inactive state, known as prophage [54]. The distinct life cycles of bacteriophages are used for designing novel diagnostic tools, including reporter bacteriophages [55] and phage display technologies [56].

Rapid Detection Methods Associated with Bacteriophage for Salmonella
With the growing need of food safety, several detection methods for targeting Salmonella were developed by combining bacteriophages, including molecular based real-time PCR [59,60], immunomagnetic separation based on fluorescence immunoassays [61,62], enzyme-linked

qPCR-Based Detection
Bacteriophages have been used for the detection of foodborne pathogens based on their specificity towards bacteria and amplification ability, which is like the "enrichment" procedure and shortens the detection time [36]. In this approach, qPCR is used to directly amplify and detect the nucleic acids of progeny bacteriophage after propagation. The bacteriophages coupled with qPCR were used for the detection of Ralstonia solanacearum, Escherichia coli O157:H7, Mycobacterium avium, and Acinetobacter baumannii [60,[65][66][67]. The amplification of Salmonella phage B_SenS_PVP-SE2 combined with qPCR was then developed for the detection of viable Salmonella Enteritidis in chicken samples [68]. The proposed method detected a low concentration level of viable S. Enteritidis (8 colony-forming unit (CFU)/g) in chicken samples within 10 h, which saved much time when compared to the culture-based method and also enhanced sensitivity, specificity, and accuracy. Thus, this protocol can be used in the food industry for self-monitoring, which successfully completed a "same-day" detection within 10 h.

Immunomagnetic-Based Detection
A novel assay composed of immunomagnetic separation (IMS) and amplification of Salmonella bacteriophage SJ2 was developed and optimized for the detection of Salmonella enterica serovar Enteritidis [62]. In the IMS procedure, Dynabeads ® anti-Salmonella was used for capturing and concentrating Salmonella. Bacteriophage SJ2 was then added, and the mixture was incubated for attachment and amplification. The final sample was detected using fluorescence or optical density measurements. This assay showed a detection limit of less than 10 4 CFU/mL with a short time (4.0-4.5 h). However, the pre-enrichment process was required in food samples. When this technique was applied to contaminated food samples, including skimmed milk, chicken, and beef, at an average of 3 CFU/25 g, S. Enteritidis could be detected within 20 h, including a pre-enrichment time of 16 h [62].

Enzyme-Linked Immunosorbent Assay
The commercial ELISA kits have been applied for detecting Salmonella in poultry, seafood, milk, and meat [69][70][71][72]. ELISA detects the protein in a liquid form using antibodies against the target samples. Instead of using antibodies, bacteriophages can be used in ELISA for detecting bacteria [73]. The ELISA procedure works by replacing antibodies with bacteriophages and was applied for the detection of S. enterica and E. coli [59]. The modified ELISA showed a detection limit up to 10 6 cells/mL, which is comparable with other ELISA methods. Thus, bacteriophages in ELISA can be an alternative way to detect pathogenic bacteria without specific antibodies. Since phages are highly abundant in nature, this assay becomes cheap compared with using specific antibodies.

Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry
MALDI-TOF MS is now becoming a most common method for bacterial identification, differentiating from the advantages of high throughput and rapidity and excepting a high cost of the initial installment [74,75]. MALDI-TOF MS has been applied for screening, identification, and detection of foodborne bacteria, which can enhance food safety. The MALDI-TOF MS-based detection method depends on progeny bacteriophage proteins or peptides. In this method, progeny bacteriophage proteins are applied to the test plate with a UV-absorbing matrix and then ablated by a laser. After 60-120 min of bacteriophage amplification, samples were used for analysis by MALDI-TOF MS. The parameters of MALDI-TOF MS, including matrix preparation, sample preparation, acid added to the matrix, growth medium, and setting parameters, were optimized and a standard protocol was set for the identification of Salmonella subspecies, and consequently the classification results were comparable to DNA sequence-based methods [76]. A whole-cell MALDI-TOF MS for rapid prescreening of S. enterica subspecies Enterica, isolates based on specific biomarker ions, rather than antigenic determinants that could reduce the sample numbers for subsequent serotyping analysis [77]. The MALDI-TOF MS combined with selective enrichment broth was developed for the identification of Salmonella sp. in clinical stool samples. The discrimination of bacteria species was mainly based on the comparison of peaks of peptides and small proteins with the reference database [78]. Strains of Salmonella and E. coli were simultaneously detected based on the characteristics of proteins by using two phages, MS2 and MPSS-1, respectively, [63]. The simultaneous detection of two bacteria, using MALDI-TOF MS coupled with bacteriophage amplification, provides the possibility of three or more target detections, which may require the specific bacteriophage biomarkers.

Genetically-Engineered Phages
Recent advances in genetically-engineered bacteriophages have been created as a powerful tool for the monitoring and detecting of bacterial pathogens. This novel technique is useful for detecting bacteria from contaminated foods with high selectivity and sensitivity [64,79]. Reporter bacteriophages are genetically modified bacteriophages that have a reporter gene inserted into their genomes, such as lux, gpf, and lacZ, which are activated by the interaction between bacteriophages and target bacteria [64,80]. The expression of the reporter gene upon infection emits a detectable signal, indicating the presence of target bacteria. The main advantage of using a reporter bacteriophage is the higher specificity for detecting viable host bacteria. To date, most commonly used reporter bacteriophages are associated with the formation of bioluminescence luciferase protein, which emits light in the presence of aldehyde substrates [81]. Several bioluminescent reporter bacteriophage systems have been designed using the lux operon, luxCDABE. The LuxCDE proteins encode a fatty acid reductase complex, including reductase, synthetase, and transferase, responsible for providing the aldehyde as a substrate, and the LuxAB encodes luciferase αand β-subunits, which feed the bioluminescent reaction [82,83]. Reporter bacteriophages containing the luxAB gene were the first bioluminescent for the detection of Salmonella strains [84]. The luxAB (P22 luxAB) reporter gene without the luxCDE was used to detect S. Enteritidis up to 63 CFU/g in whole eggs [84]. The advantage of the luxAB reporter system is that it can avoid the toxicity and noise signals by emitting a physical signal. However, this system needs the specific substrates for bacterial detections [82]. To avoid this inconvenience, a complete set of luxABCDE operons was constructed for Salmonella Typhimurium detection in different food matrices [83]. The reporter bacteriophage system could detect Salmonella up to 37 CFU/g in sliced pork, 22 CFU/g in iceberg lettuce, and 20 CFU/mL in pure culture. These reporter bacteriophages could be useful for diagnostics and rapid detection of Salmonella spp. in different food samples with no substrates and with the reporter host required.
Apart from the luciferase-based reporter bacteriophage systems, green fluorescent protein (GFP) and β-galactosidase have also been used for the detection of foodborne pathogens. The GFP gene is originated from Aequorea victoria and has many advantages, including high stability and low toxicity [85,86]. The GFP-labeled PP01 bacteriophage (PP01-GFP) system was applied on the surface of E. coli O157:H7, which can emit a fluorescent signal at an MOI of 1000 at 4 • C [87]. Although the specificity and host range are well defined, this system is not widely used in food because of the interference of food components. The lacZ gene encoding β-galactosidase can catalyze the hydrolysis of β-galactosides. The lacZ-based reporter bacteriophage needs various substrates, such as colorimetric, fluorescent, or luminescent substrates, which emit signals to detect bacteria [36,88]. The detection limits of this system were up to 10 3 CFU/100 cm 2 for the colorimetric method and 10 CFU/100 cm 2 for luminescence in beef slice samples [88]. The use of additional substrates can be a drawback of this system as it only allows single time point measurements. Although this reporter bacteriophage system can detect viable Salmonella at high specificity and a low detection limit, the construction of new reporter bacteriophages is still difficult. Therefore, further studies are needed for the development of reporter bacteriophage systems, which can detect Salmonella spp. in various food matrices.

Bacteriophage-Based Biosensors for Detecting Salmonella
Over the last few decades, biosensors have been developed as a novel analytical platform for pathogen detection [89,90]. A classical biosensor can be defined as an analytical device that measures biological responses by incorporating bioreceptors (antibodies, enzymes, cells, aptamers, bacteriophage, and organelle) with physical transducers and electrochemical (amperometric, impedimetric, and potentiometric), optical (surface plasmon resonance, surface-enhanced resonance spectroscopy, and fluorescence), and mass-based receptors (magnetoelastic and piezoelectric). The continuous efforts have been successfully developed on bacteriophage-based biosensors for the detection of Salmonella in food samples. The immobilization of bacteriophage receptors on the sensor surface is crucial to develop bacteriophage-based biosensors. The immobilization steps include physical adsorption, covalent attachment, and genetic modification of receptors [91,92].
Appendix A summarizes different bacteriophage-based biosensors for the detection of Salmonella. The first transducers of bacteriophage biosensors for Salmonella detection are mass-based transducers and magnetoelastic assays (ME), and Figure 3 shows a schematic illustration for the principle of ME biosensors for detection of target analytes [93]. Over the last few decades, biosensors have been developed as a novel analytical platform for pathogen detection [89,90]. A classical biosensor can be defined as an analytical device that measures biological responses by incorporating bioreceptors (antibodies, enzymes, cells, aptamers, bacteriophage, and organelle) with physical transducers and electrochemical (amperometric, impedimetric, and potentiometric), optical (surface plasmon resonance, surface-enhanced resonance spectroscopy, and fluorescence), and mass-based receptors (magnetoelastic and piezoelectric). The continuous efforts have been successfully developed on bacteriophage-based biosensors for the detection of Salmonella in food samples. The immobilization of bacteriophage receptors on the sensor surface is crucial to develop bacteriophage-based biosensors. The immobilization steps include physical adsorption, covalent attachment, and genetic modification of receptors [91,92]. Table S1. summarizes different bacteriophage-based biosensors for the detection of Salmonella. The first transducers of bacteriophage biosensors for Salmonella detection are mass-based transducers and magnetoelastic assays (ME), and Figure 3 shows a schematic illustration for the principle of ME biosensors for detection of target analytes [93]. ME-based detection methods are the most prominent type of biosensors due to their easy and cheap fabrication, composing of amorphous ferromagnetic ribbon that contracts and expands when exposed to the external magnetic field and generates magnetic fluxes by binding targets to the sensor surface. The ME biosensor was employed as a transduction platform in bacteriophage biosensors for the detection of Salmonella (Table S1). ME biosensors were developed by using filamentous bacteriophage specific for S. Typhimurium. The bacteriophages were immobilized by a physical adsorption method for S. Typhimurium detection by the changes in the resonance frequency of the sensor [93]. The numbers of S. Typhimurium in fresh tomato surfaces were quantified using the ME biosensor detection method by the immobilization of E2 bacteriophages on the sensor surface [94]. The tomato surfaces contaminated with S. Typhimurium were measured using a resonance frequency with a detection limit of 10 3 CFU/mL. The results show that E2-bacteriophage-based biosensors could detect Salmonella directly on the surface of tomatoes. The same principle for detection of S. Typhimurium in spinach leaves showed a similar detection limit as low as 10 2 CFU/mL [94].
Other bacteriophage-based biosensor detection systems have been developed using an acoustic wave piezoelectric biosensor combined with filamentous bacteriophages [95]. The detection limit was 10 2 CFU/mL for S. Typhimurium by measuring the changes in resonance frequency as a consequence of binding bacteria to the bacteriophage. In addition, the recombinant prophage coupled with a flow cytometer and specific fluorescence filter was used for sensitive and specific detection of Salmonella ME-based detection methods are the most prominent type of biosensors due to their easy and cheap fabrication, composing of amorphous ferromagnetic ribbon that contracts and expands when exposed to the external magnetic field and generates magnetic fluxes by binding targets to the sensor surface. The ME biosensor was employed as a transduction platform in bacteriophage biosensors for the detection of Salmonella (Table A1). ME biosensors were developed by using filamentous bacteriophage specific for S. Typhimurium. The bacteriophages were immobilized by a physical adsorption method for S. Typhimurium detection by the changes in the resonance frequency of the sensor [93]. The numbers of S. Typhimurium in fresh tomato surfaces were quantified using the ME biosensor detection method by the immobilization of E2 bacteriophages on the sensor surface [94]. The tomato surfaces contaminated with S. Typhimurium were measured using a resonance frequency with a detection limit of 10 3 CFU/mL. The results show that E2-bacteriophage-based biosensors could detect Salmonella directly on the surface of tomatoes. The same principle for detection of S. Typhimurium in spinach leaves showed a similar detection limit as low as 10 2 CFU/mL [94].
Other bacteriophage-based biosensor detection systems have been developed using an acoustic wave piezoelectric biosensor combined with filamentous bacteriophages [95]. The detection limit was 10 2 CFU/mL for S. Typhimurium by measuring the changes in resonance frequency as a consequence of binding bacteria to the bacteriophage. In addition, the recombinant prophage coupled with a flow cytometer and specific fluorescence filter was used for sensitive and specific detection of Salmonella with a detection limit of 10 CFU/mL [86]. A surface enhanced Raman scattering (SERS) by conjugating bacteriophage tail spike proteins to silica-encapsulated Raman reporter-embedded nanoprobes could detect single Salmonella cells [96]. Therefore, the use of bacteriophage as a bioreceptor in biosensors can contribute to the development of desirable detection tools for Salmonella in food samples. The stability, low cost, environment-friendly production, and genetic modification provide benefits for biosensor development. For the successful development of biosensors, the immobilization of phage onto the biosensor surface plays an important role. The genetically modified phages provide effective immobilization by introducing the functional ligands on their heads. In addition, the ability to manipulate the genetic material provides the possibility of creating novel recognition systems for biosensor applications, such as expanding the host range of phages by manipulating the receptor-binding protein [27]. However, further work should focus on detecting Salmonella in complex food matrices for extending the range of application of bacteriophage-based diagnostic tools from the laboratory to clinical diagnosis, environmental monitoring, and further food analysis in the near future.

Bacteriophage-Based Tool for Salmonella Control
Bacteriophages have been used for controlling bacterial infections based on their specificity to the host bacteria [35,[97][98][99][100][101][102][103]. Bacteriophages kept stable in thermal conditions from 30 to 60 • C and pH ranges from 3 to 13 can suggest the possibility of using bacteriophages in variable conditions. Recently, bacteriophages as a biocontrol tool have gained great attention and are recognized as an alternative for antibiotics [104][105][106]. Listeria bacteriophages were approved by the Food and Drug Administration (FDA) and the United States Department of Agriculture (USDA) in all food products, which were granted as generally recognized as safe (GRAS) [107]. Appendix B summarizes the applications of bacteriophage or bacteriophage-based treatments as biocontrol tools for Salmonella.
Bacteriophage control technique has been applied for Salmonella in vivo and food samples. When lytic bacteriophages was applied to the chicken skin contaminated with S. enterica serovar Enteritidis, less than one log reduction was obtained at the MOI of 1 and no viable bacteria were observed at the MOI of 10 5 [108]. A new virulent bacteriophage, F01-E2, was isolated for controlling S. Typhimurium [109]. F01 belongs to Myoviridae with a double-stranded deoxyribonucleic acid dsDNA genome of 86.2 kb and a broad host range [110,111].
Five log reductions were obtained for turkey deli meat and chocolate milk at 15 • C, and three log reductions were observed for hot dogs and seafood, implying that bacteriophage immobilized on the food surfaces were affected by the structure and chemical composition of the foods [112]. Salmonella Enteritidis bacteriophage SE07 showed a potential against S. Enteritidis in both solid and liquid food [113]. The isolated SE07 belongs to Podoviridae and is stable from 28 • C to 65 • C and pH 4 to 11. As shown in Table 3, two log reductions were obtained for the different food matrices after 48 h incubation at 4 • C. Additionally, the bacteriophage ΦCJ07 was applied for controlling S. Enteritidis in chicken [114].
Because Salmonella in contaminated, chickens can survive under the acidic conditions in the digestion system. Bacteriophage ΦCJ07 was added as a feed additive, which was effective against Salmonella by protection from other ingested feed constituents [115]. The bacteriophage ΦCJ07 isolated from the sewage effluent showed a lytic activity against most Salmonella spp., including S. Enteritidis, S. Typhimurium, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Choleraesuis, and Salmonella Derby. Further evaluations in vivo also demonstrated the good performance of phage ΦCJ07 in reducing both S. Enteritidis colonization and environment contamination levels. This provides a promising alternative of bacteriophage for preventing and controlling S. Enteritidis infections and reducing the incidence of Salmonella food poisoning.

Phage Cocktails
Despite the advantages of bacteriophages, bacteria can become resistance to bacteriophages through surface modification, superinfection exclusion, restriction modification, abortive infection, and clustered regularly interspaced short palindromic repeatsCRISPR-associated 9 (Cas9) systems [117,118]. Therefore, phage cocktails with different host specificities have been of great interest and are more practical for expanding the bacteriophage application, since the combined bacteriophage cocktails can reduce the development of bacteriophage-resistant mutants.
A mixture of two bacteriophages was used for controlling Salmonella in sprout seeds [119]. Bacteriophage A belongs to the Myoviridae family, while bacteriophage B is a member of the Siphoviridae family. The reductions of S. Typhimurium, S. Enteritidis, and Salmonella Montevideo were noticeable at the bacteriophage mixture of A and B in broccoli seeds compared to single bacteriophage treatment. The isolated bacteriophages effectively reduced the numbers of S. Typhimurium and S. Enteritidis in chickens [120]. The mixture of three phages, UAB_Phi20, Phi78, and Phi87, showed higher lytic activity than that obtained by any of the three phages alone, while the phage cocktail lysed Salmonella Virchow, Salmonella Hadar, Salmonella Infantis, S. Typhimurium, and S. Enteritidis, showing a broad spectrum lytic capability. The bacteriophage cocktail was applied in different food systems [121]. Significant reductions of S. Typhimurium and S. Enteritidis were observed for different food matrices, including pig skin, chicken breasts, and lettuce. Recently, a phage cocktail (BSPM4, BSP101, and BSP22A) based on targeting different cell surface receptors, including flagella, O-antigen, and BtuB, has been developed for the inhibition of Salmonella Typhimurium from fresh produce foods [122]. The multiple receptor-targeting bacteriophage cocktail can reduce Salmonella by up to 4.7-5.5 log CFU/cm 2 in iceberg lettuce and 4.8-5.8 log CFU/cm 2 in cucumber after 12 h incubation at 25 • C, without the development of bacteriophage resistance [122]. At present, the commercial bacteriophage cocktail has been applied for controlling Salmonella in poultry products [123].

Phage Endolysins
Bacteriophage endolysins have been used as a novel biocontrol agent and natural food preservatives over the past decades. The endolysins are peptidoglycan hydrolases that can lyse host cells after phage replication and propagation. The endolysins are mainly active against Gram-positive bacteria, which do not contain an outer membrane [36,124]. The outer membrane of Gram-negative bacteria can prevent contact between free endolysins and peptidoglycan. However, some Salmonella bacteriophage endolysins can bypass the outer membrane barriers when combined with different outer membrane permeabilizers, such as ethylene diamine tetra-acetic acid (EDTA), citrate, and malate [114,125,126]. The bactericidal activity of a Salmonella phage endolysin (Lys68) combined with organic acids was increased against Gram-negative bacteria [127]. A Salmonella bacteriophage endolysin, Gp110, has currently proved to show enzymatic activity [128]. In addition, bacteriophage endolysins have also been engineered to increase the bactericidal effect against Gram-negative bacteria. The modified endolysin combined with lipopolysaccharide (LPS)-destabilizing peptides showed promising results against Pseudomonas aeruginosa, showing more than 5 log CFU/mL reduction. However, there are still some limited effects against Salmonella Typhimurium (<1 log CFU/mL reduction) [129]. Although many bacteriophage endolysins have been introduced and characterized, further optimization is still needed to increase the host specificity and lytic activity. The genetic engineering endolysin can be one of the useful approaches for satisfying these requirements.

Phage Control Combined with Other Preservatives
The hurdle concept (or barrier technology) is applied to foods to enhance the microbiological safety and quality. Many preservative methods are employed, together with other barriers to effectively control microbial contamination in food [130]. Many studies have demonstrated bacteriophages as alternative antimicrobials to control bacteria. Several Salmonella bacteriophages, such as Salmonelex™, SalmoFresh TM , and SalmoPro TM , have been approved as GRAS by the United States Food and Drug Administration (US FDA) and the US Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) [131,132]. The combinations of bacteriophages and antimicrobials or sequential applications showed an effective biocontrol ability against the target bacteria [38,133]. SalmoFresh TM bacteriophages combined with cetylpyridinium chloride (CPC) or lauric arginate (LAE) showed more than 5 log reductions against Salmonella spp. in chicken products [133][134][135][136]. However, an in vivo test on chicken breast fillets showed that a lower number of Salmonella (0.5 to 1.3 log CFU/g) was reduced by the combinations of bacteriophages with CPC or LAE, which may be attributed to the complex matrix of the meat components [137]. Sequential treatment of chlorine, CPC, LAE, or peracetic acid (PAA) with concentrations of 50 and 400 ppm, respectively, followed by phage spray, were carried out to evaluate the hurdle effect of Salmonella on chicken skin. The high reductions of 1.7 to 2.2 and 2.2 to 2.5 log CFU/cm 2 were obtained with an immersion in 50 and 400 ppm of PAA, followed by phage spray, which may be used in industries for the reduction of Salmonella contamination in cut meat. With growing interest of the combinations of bacteriophages and antimicrobials, further studies are needed to evaluate the inhibitory effect of antimicrobials combined with bacteriophages, the potential synergistic effect of the combination, and the mode of phage application, such as immersion and spraying [38,[138][139][140].

Conclusions
Notably, research related to bacteriophages and their promising applications has increased in recent decades due to frequent outbreaks and the emergence of antibiotic-resistant bacteria. The effective detection and biocontrol of Salmonella, based on the potential bacteriophages, are of importance to reduce the incidence of Salmonella and ensure the food safety. Since many studies have been performed in the laboratory with well-controlled conditions, bacteriophages showed a significant effect on the inhibition of bacteria both in vivo and in vitro. Novel hurdle technology-coupled phages with antimicrobials, UV, or antagonistic bacteria are of interest to find synergistic effects against pathogens, which provide potential effective ways to be used in industries for control pathogens and alleviate the risk of pathogen contaminations in foods.

Conflicts of Interest:
The authors declare no conflict of interest. Pig skin, chicken breasts, fresh eggs, and packaged lettuce 10 10 pfu/mL for pig skin and fresh eggs, 10 9 pfu/mL for chicken breasts and lettuce

Appendix A
Spraying for pig skin and fresh eggs, while agitation 5 min and 60 min for chicken breasts, and lettuce, respectively In pig skin, >4 and 2 log/cm 2 for S. Typhimurium and S. Enteritidis were reduced for 6 h, respectively; in chicken breasts, 2.2 and 0.9 log cfu/g for S. Typhimurium and S. Enteritidis were reduced for 7 days, respectively; in lettuce, 9 and 2.2 log cfu/g, respectively; in fresh eggs, a reduction of 0.9 log cfu/cm 2 for 2h [120,121] SalmoFresh TM Commercial product S. Newport Whole and fresh-cut cucumbers 10 10 pfu/mL Spraying S. Newport was significantly lower when treated by phage at 10°C on day 1 and 4. [159] A phage cocktail of S16 and FO1a Both belong to the order Caudovirales and Myoviridae family S. enterica, S. Heidelberg, S. Newport, and S. Enteritidis C, Se 13 Ground meat including beef and pork trim, and poultry including chicken and turkey thighs 10 7 or 10 8 pfu/mL for samples, and 10 9 pfu/mL for vitro study Tumbling for 2 min at 4 rpm In vitro study, 99% were reduced for all strains; in vivo test, bacteria reductions of 1, 0.8, 1.1 and 0.9 log cfu/g were obtained in beef, pork, chicken, and turkey, respectively. [160] A phage cocktail of vB_SnwM_CGG4-1, 4-2, 3-1, and 3-2 vB_SnwM_CGG4-1, and 4-2 belong to Myoviridae family and vB_SnwM_CGG3-1, and 3-2 belong to Siphoviridae family S. Newport Cherry tomato 10 6 and 10 8 pfu/mL In vitro study, 3 log reduction was obtained after up to 7 h incubation; in vivo test, 2 log reduction with a MOI of 10 3 and about 4.4 log reduction was observed after 2, 3, and 4 days with a MOI of 10 5 [161] SalmoFresh TM Myoviridae family S. Newport, S. Braenderup, S. Typhimurium, S. Kentucky, and S. Heidelberg Lettuce, mung bean sprouts and seeds 10 8 pfu/mL Spraying, immersion, Reductions of 0.76 and 0.83 log10 CFU/g were obtained on lettuce and sprouts by spraying, respectively, while 2.43 and 2.16 log10 CFU/g by immersion.
[162] In vivo, reduction of 5.7 to 6.4 log CFU/mL were obtained. In sprouting mung bean sprouts, an additive effect was observed with the combination resulted in a detectable Salmonella only after enrichment. For sprouting alfalfa seeds, no Salmonella was recovered even with enrichment [163] A phage cocktail of S16 and FO1a combined with UV Both belong to the order Caudovirales and Myoviridae family S. Infantis, S. Heidelberg, S. Newport, and S. Enteritidis C, Se 13 Ground beef 10 9 pfu/mL Tumbling for 2 min at 4 rpm Approximately 1 log CFU/g reduction for bacteriophage and UV, separately, while 2 log CFU/g for combination [160,164] oFresh TM combined with chlorinated water Myoviridae family S. Newport, S. Braenderup, S. Typhimurium, S. Kentucky, and S. Heidelberg Lettuce, mung bean sprouts and seeds 10 8 pfu/mL Immersion 15 min for lettuce and sprouts, 1 h for mung seeds Reductions of 3.8, and 2.7, 1.28 log CFU/g were obtained by hurdle treatment on lettuce, sprouts, and mung seeds, respectively. [162]