1.1. Salmonella as an Enteric Pathogen

S. enterica is a Gram-negative, rod-shaped, facultative anaerobe that belongs to the Enterobacteriaceae family. More than 2600 serovars or serotypes of this bacterium have been identified [7]. Based on the major serotypes of S. enterica, it can be divided into typhoidal S. enterica and nontyphoidal S. enterica [8]. Typhoidal S. enterica includes S. serotype Typhi (S. Typhi) and S. enterica serotype Paratyphi (S. Paratyphi), which cause typhoid fever and paratyphoid fever in humans, respectively [9, 10]. In contrast, common nontyphoidal S. enterica strains include various serovars such as S. enterica Typhimurium (S. Typhimurium), S. enterica serovar Enteritidis (S. Enteritidis), S. enterica serovar Newport (S. Newport), and S. enterica serovar Heidelberg (S. Heidelberg). These serovars are associated with foodborne gastroenteritis, leading to symptoms like fever, diarrhea, vomiting, abdominal pain, nausea, and bacteremia. While their impact is generally nonfatal, infections can be lethal for young children, the elderly, immunocompromised individuals, or those with chronic conditions [4, 11].

Most nontyphoidal S. enterica serovars colonize many other warm-blooded animals, particularly chickens [12]. As a result, outbreaks involving these pathogens are commonly linked to poultry or poultry products. The predominant serotypes involved in salmonellosis outbreaks vary from year to year. According to a recent report, S. Enteritidis, S. Typhimurium, S. Heidelberg, and S. 1,4,[5],12:i:- are commonly linked to foodborne salmonellosis outbreaks in the United States [13] and S. Enteritidis, S. Typhimurium, S. 1,4,[5],12:i:-, and S. Infantis in the EU/EEA [5]. A recent study conducted in the Maryland-DC region of the United States revealed that the prevalence of S. enterica was 7.80% at the preharvest level and 1.91% in organically grown postharvested food products [14].

Transmission of nontyphoidal S. enterica, such as S. Enteritidis, S. Heidelberg, and S. Typhimurium occurs through several routes, including the consumption of contaminated foods or drinks, close contact with contaminated environments or animals, and horizontal or vertical transmission among animals [12]. In particular, partially or uncooked eggs, chickens, chicken/egg dishes, and chicken/egg products are significant contributors to S. enterica infection [15]. S. Enteritidis also can penetrate the eggshell and lead to the contamination of egg contents through infection of the reproductive organs [16]. Additionally, S. Enteritidis can spread among chickens, pigs, and other animals through contaminated feed or free-range environments [12].

Furthermore, the increasing trend of antimicrobial resistance (AMR) bacterial pathogens poses a significant concern [14, 17, 18]. Over the past few decades, the overuse and misuse of antimicrobials in both humans and animals have resulted in AMR in enteric pathogens, particularly S. enterica [19]. AMR bacteria can be transferred from animals to humans through the consumption of animal products or contact with animals containing AMR pathogens [19]. In 2019, it was estimated that approximately 1.27 million deaths occurred globally each year due to AMR bacteria [20]. AMR strains of S. enterica now pose a substantial threat to both animal and human health worldwide, contributing to an annual economic burden of $35 billion in the United States [21]. It is estimated that AMR strains of nontyphoidal S. enterica are responsible for approximately 212,500 infections and 70 deaths annually in the United States [22].

Many countries, including the United States and the EU, have prohibited the use of antibiotics as feed additives in animal agriculture to address the AMR issue [21, 23, 24]. To satisfy the rising demand from consumers for products free of antibiotics, this restriction made it easier to expand the production of organic and free-range poultry [25]. However, these alternative farming methods are now more susceptible to foodborne microbial contamination, particularly AMR bacteria, because of the decreased management of outside surroundings in free-range poultry production [26]. In addition, some pathogens for chickens, such as Salmonella pullorum and Salmonella gallinarum, are commonly present in preharvest and postharvest [27]. Investigating alternate tactics like bacteriophage therapy or bacteriophage-encoding enzymes becomes more and more critical in these changing difficulties.

1.2. Potential Strategies to Control S. enterica

In addition to the prohibition of using antibiotics as feed additives in many countries, many conventional poultry productions have shifted toward alternative antimicrobials to prevent colonization of zoonotic bacterial pathogens in livestock including poultry [28]. Among these alternative antimicrobials, such as prebiotics, probiotics, synbiotics, plant-derived antimicrobial compounds, essential oils, vaccination, and bacteriophage therapies, are gaining popularity for controlling colonization of S. enterica in poultry [11, 28]. Probiotics are a group of nonpathogenic microorganisms, such as Lactobacillus and Bifidobacterium, that provide health benefits to their host when administered in sufficient quantities [11, 29, 30]. Probiotics must meet safety and efficacy for their host, possess immunomodulatory properties, effectively colonize the intestinal epithelium, withstand bile salts and low pH, and maintain both phenotypic and genetic stability [29]. Prebiotics are nondigestible components that undergo selective fermentation, leading to beneficial changes in the composition and activity of the gastrointestinal microbiota [31]. Prebiotics, such as mannan-oligosaccharides (MOSs) and fructo-oligosaccharides (FOSs), have been shown to enhance beneficial gut bacteria and prevent S. Enteritidis infection by altering gut microbial diversity in poultry [32, 33]. Synbiotics, which combine probiotics and prebiotics, further enhance the survival of beneficial bacteria in the gastrointestinal tract, improving immune responses and overall health in poultry [34]. However, the effectiveness of synbiotics depends on factors like the specific strains used and the conditions of the gastrointestinal environment [35].

Plant-derived natural antimicrobial compounds, also known as phytobiotics, such as garlic, oregano, and cinnamon, have been shown to improve nutrient absorption, feed intake, and immune function in poultry [36]. These plant-derived metabolites, specifically flavonoids/phenolics have, demonstrated antimicrobial activity against Salmonella [37, 38]. Polyphenolic components, such as berry pomace extracts (BPEs), a byproduct with shell and seeds after juicing of blueberry or blackberry fruits, are shown to significantly inhibit the growth of S. Typhimurium and its virulent functions [39–41]. Other polyphenolic components, such as gallic acid, protocatechuic acid, and vanillic acid, are identified as properties that inhibit the growth of S. Typhimurium in vegetables and are potential candidates as alternative antimicrobial agents [42–44]. Essential oils (volatile oils) are aromatic compounds with distinct flavors and fragrances. They are extracted from various parts of plants and contain various compounds such as alcohols, acetones, phenolic acids, terpenes, aldehydes, and esters, all of which can act as critical antimicrobial agents or nutritional supplements [31]. One common type of essential oil, citrus oil, effectively prevents the contamination/colonization of Salmonella in poultry and poultry products [45].

Vaccinations are widely used to boost immune responses and prevent Salmonella shedding [46]. However, each vaccination has its limits regarding safety and effectiveness. The most common challenges are improving animal vaccine administration and costs for animal industries [47]. Each vaccination has its own set of limits in terms of safety and effectiveness. Bacteriophage (phage) therapy is gaining significant attention as a promising approach to combat bacterial infections. This review is aimed at assessing the progress of phage therapy and the potential of phage-encoded endolysins in controlling S. enterica infection. These advancements present new opportunities to overcome the limitations of conventional treatments and enhance food safety. Table 1 lists the benefits and challenges of these alternative strategies for controlling Salmonella.

2.1. Bacteriophage

Bacteriophages, or phages, are viruses that specifically target and infect bacterial cells rather than animal cells. They were first discovered in 1915 by William Twort, and 2 years later, in 1917, Felix d’Herelle identified their potential to combat bacteria, establishing them as a promising therapeutic agent for treating and preventing bacterial infections in both humans and animals [48]. Due to the development of synthetic antibiotics during World War II, phages lost prominence in the pharmaceutical industry. However, with the emerging AMR bacterial pathogens, phage research has become a promising alternative to replace antibiotics [49].

Phages are the most abundant microbes on Earth, estimated at 10³¹ phage particles, and they can be found ubiquitously in various natural environments where bacteria exist [50]. Phages are highly specific to their hosts/bacteria, typically infecting only a single bacterial species or specific strains within a species, regardless of their AMR pattern. Phages display remarkable diversity in size, appearance, and genetic organization [51]. Their size ranges from 24 to 200 nm, containing either single or double-stranded DNA or RNA [52]. Notably, there are certain phages referred to as “giant phages” or “jumbo phages” due to their exceptionally large genome sizes, ranging from 200 to 500 kbp [53]. These giant phages include Salmonella phage SPN3US, Proteus Phage 7, Enterobacteria phage SEGD1, and Salmonella bacteriophage-1252 [54]. Many of the gene functions within these jumbo phages remain unknown, and their essential roles require further investigation [52]. Therefore, agricultural animal producers, specifically poultry farmers, are searching for bacteriophage as an alternative strategy to prevent zoonotic pathogens in livestock gut and control contamination of animal food products/environment to limit foodborne bacterial illness [28].

2.2. Mechanisms of Phage Infection

Lytic phages, such as the Coliphage T4, initiate infection with the attachment of the phage tails to receptor proteins located at the surface of the host/bacterial cell. This interaction occurs between the tip on the phage tail fiber and various receptor proteins on the bacterial surface, such as outer membrane proteins, peptidoglycan, lipopolysaccharide, teichoic acids, oligosaccharides, flagellum, capsule, Type IV fimbriae, and sex pili, and this interaction allows the phage to inject its genome into the bacterial cell [55]. In general, most narrow host-range phages interact with specific receptor-binding proteins on the surface of a particular bacterial species, allowing them to infect only the target bacterial species [56].

Phages replicate within their host cell and then proceed through two main lifecycles: the lytic and the lysogenic (temperate) lifecycles [57]. During a lytic cycle, a phage genome utilizes the host metabolism and ribosomes for phage DNA replication and protein synthesis. Only lysogenic phages can be converted to the lytic cycle, whereas lytic phages can replicate only through the lytic cycle. At the end of the lytic process, most phages produce two major proteins: holins and endolysins. In addition, other phages may produce proteins such as pinholins, signal-anchor-release (SAR) endolysins, antiholins, and spanins [58], depending on the phage type. These proteins facilitate host cell lysis and the release of progeny phages.

In the lysogenic or temperate cycle, some phages undergo a latent infection, integrating their DNA into the chromosome of the host as a prophage. Instead of lysing the bacterium, the prophage postpones virion production and undergoes replication and multiplication during the bacterial cell replication and binary fission processes. Subsequently, the phage genome is transmitted to daughter bacterial cells without causing their death [59]. During the lysogenic cycle, prophages encode membrane-anchored proteins through “superinfection exclusion (Sie) systems” in the host cell, which block the entry of phage genomes from the same strain or specific phages [60, 61]. Well-characterized Sie systems, such as those in coliphage T4, utilize the Immunity protein (Imm) [62] and Spackle protein (Sp) [63]. Imm alters the phage injection site, while Sp inhibits T4 lysozyme activity, preventing host cell wall degradation and subsequent phage DNA entry [60, 61].

The lysogenic state can be maintained for long periods and converted to a lytic lifecycle, causing lysis of the host cell. This switch is often triggered by exposure to environmental stressors [64], such as the bacterial Son of Sevenless response [65]. It is believed to be a survival tactic employed by phages to escape from host cells that are in danger of dying [66] or to maintain their populations during harsh conditions [67]. However, lysogenic phages may lose their capability over time to be induced or to exit from the bacterial chromosome [68]. Holins are small transmembrane proteins that accumulate in the host cell membrane unit and serve as a lysis “clock” on the length of the lytic cycle. Their accumulation leads to an oligomeric complex that disrupts the bacterial cell inner membrane [69]. At a specific moment, holin triggers the host cell membrane to suddenly become permeable toward endolysin, and it also plays a significant role in the length of the lytic cycle [70]. Another similar functional protein is pinholins, which form small pores (approximately 2 nm in diameter) and lead to membrane depolarization, triggering SAR endolysin activation and degradation of the host cell wall [58].

Endolysins are enzymes phages produce and accumulate in the cytosol during the lysis cycle [71]. Endolysins can be categorized into canonical endolysins and SAR endolysins. SAR endolysins are preanchored to the periplasmic side of the cell membrane. They are released and degrade the cell wall due to membrane depolarization induced by pinholins [58, 72]. In Gram-negative bacteria, spanins are essential for lysing bacteria in inner membrane and outer membrane after the cell wall has been degraded by endolysin or SAR endolysin [58]. Spanins traverse both the inner membrane and outer membrane with two forms: a single molecule as an inner membrane-spanin and a heterodimeric structure as an outer membrane-spanin [73]. Lysis is triggered when spanin complexes are released and accumulate on the inner side of the peptidoglycan layer, followed by fusion of the inner membrane and outer membrane, causing disruption of the cell membranes [74]. Disruption of the cell membranes and cell wall will release newly formed phages from the host cell and start another round of infection in neighboring host cells [59].

Certain phage-derived enzymes, such as integrase and transposase, lead to genetic mutations and bacterial evolution by integrating phage DNA into host chromosomes [64]. Integrases enable site-specific recombination between phage and bacterial attachment sites (attP and attB), making them useful tools for genetic manipulation, particularly in organisms with large genomes like mammals and plants [75]. Transposase is a key contributor to the field of mobile DNA from phage Mu and promotes both replication and integration during the lytic cycle [76]. Although these enzymes provide potential for genetic engineering, their use in medical applications carries associated risks of AMR gene transfer.

2.3. Phage Therapy

Phage therapies have been applied successfully in the current application, specifically lytic phages, which can lyse targeted foodborne bacterial pathogens in foods or in livestock gut while leaving the beneficial microflora unaffected. This alternative strategy not only efficiently inhibits pathogenic bacteria and extends the shelf life of foods but also offers advantages such as the absence of harmful antimicrobial residues [77]. “Listex P100,” the first commercial phage product for L. monocytogenes, was approved by the FDA in 2006 and is used in ready-to-eat foods and poultry products in the United States [78, 79]. This FDA approval recognized the benefit of phages in producing safer food and established them as generally recognized as safe (GRAS) material [80]. As a result, numerous phage preparations for food applications have since been granted approval in the United States [79–81]. Table 2 presents FDA-approved phage products to control foodborne pathogens from 2006 to 2024. These products have a wide range of applications in meat, poultry, fruits, vegetables, dairy products, and seafood. Of the 17 products, eight specifically target Salmonella, while the remaining address E. coli, Shigella, Listeria, and Campylobacter. These demonstrate significant potential requirements in combating Salmonella contamination in food safety and the urgent need for effective solutions to reduce foodborne illnesses associated with Salmonella. In contrast, there is a lack of approved phage products for human or animal use in EU [82]. Only Stafal is available in Slovakia and the Czech Republic against Staphylococcus aureus [83]. However, Stafal has not been evaluated under EU regulations because it entered the market before both countries joined the EU [82]. Overall, phage therapy offers several advantages, including its noninterference with mammalian cells/hosts, making it safe for humans and animals, and technological advancements lowering the costs of phage production compared to traditional antibiotic production [84, 85].

3.1. Challenges in Phage Therapy for Host Cell

While the therapeutic applications of phages show immense promise, there are still several challenges, especially when the targeted host is S. enterica. The major obstacles include the emergence of phage resistance [84], gene transduction [86], and serotype or serovar variation of S. enterica. Phage cocktails, a combination of phages with different complementary properties, such as different host ranges, have been developed to combat the issue of variation in S. enterica [87]. Compared to single-phage treatments, which cause phage-resistant colonies, phage cocktails can eliminate bacteria while preventing the emergence of resistant strains [84]. However, phage cocktails may contain phages that target not only pathogenic bacteria but also beneficial bacteria in the microbiota. This could lead to unintended consequences, including microbiome disruption [88], and it can serve as a crucial mediator of genetic exchange between pathogenic and nonpathogenic bacteria [89, 90].

Lytic phages are generally preferred for therapeutic purposes. In contrast, lysogenic phages tend to be avoided due to their innate ability to promote gene transduction and potentially enhance bacterial pathogenicity by acquiring additional genes [84]. Although lytic phages have the potential to kill a significant portion of bacterial cells effectively, it is rare for them to completely eradicate the whole population of their host cells, implying that a small amount of the bacterial population employs multiple strategies to counteract invading phages [84]. These strategies include adsorption inhibition, restriction-modification (R/E) systems, utilization of plasmids, temperate genes, CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems [91], mobile genetic islands (capable of carrying genes encoding AMR) [61, 92], and abortive infection (Abi) [93]. These interventions enable bacteria to avoid phage invasion during various periods of the phage lifecycle. Table 3 lists the obstacles related to using phage therapy to manage S. enterica, as well as suggested solutions and their drawbacks.

3.2. Immune Response of Animal/Human Organisms to Phages

Phages have been considered safe for humans and animals because they only infect prokaryotic cells rather than eukaryotic cells [48]. However, recent research has revealed that phages can elicit immune responses in eukaryotic cells and influence the effectiveness of phage therapy [94]. Repeated phage exposure can induce phage-specific neutralizing antibodies, which may accelerate the clearance of phages from tissues and reduce their effectiveness [95]. For instance, myophages, a phage type, induce a neutralizing antibody response compared to siphophages. This antibody response limits the ability to reuse the same phage treatment in an individual in the long term [95]. In addition to the humoral immune response, phages may interact directly with eukaryotic cells and induce cellular responses through receptors, such as TLR3 (for RNA phages) or TLR9 (for DNA phages) [96]. These interactions produce interferons and a process of innate immune response [97].

Phages can also shift microbiome patterns and increase gut permeability, causing endotoxemia and inflammation [98]. Phages may decrease the population of beneficial bacteria, such as Lactobacillus and Faecalibacterium, and increase potentially harmful species, such as Butyrivibrio and Ruminococcus [98]. Additionally, overdoses of phages could overburden the immune system of humans or animals, leading to possible unintended side effects [98]. Other limitations for phage therapy include restricted narrow specificity, stringent quality and safety standards, stability of preparations, effective assays in screening, activity limitations within biofilms, short lifespan within the animal body [99, 100], selection of suitable candidates, delivery to infection sites, and navigating regulatory approval [101]. Based on these challenges, scientists are exploring alternative approaches to address bacterial infections, such as phage-encoded endolysin (Table 3).

4.1. Endolysin as a Promising Alternative

Endolysins are muralytic enzymes produced by phages that hydrolyze bacterial cell walls, facilitating the release of progeny phages at the end of a lytic lifecycle [71]. In recent years, the potential of endolysins is due to their broad lytic effects on both Gram-positive and Gram-negative bacterial pathogens [102]. Endolysins are divided into five groups based on their distinct muralytic activities involving covalent bonds: (I) N-acetylmuramidase (lysozyme), (II) N-acetyl-β-d-glucosaminidase (glycosylase), (III) N-acetylmuramoyl-l-alanine amidase (amidases), (IV) l-alanoyl-d-glutamate endopeptidase (endopeptidase), and (V) interpeptide bridge-specific endopeptidase [103]. These enzymes target specific bonds within the peptidoglycan polymer of the bacterial cell wall, including glycosidic bonds (glycan portion of peptidoglycan), amide bonds (between the glycan moiety and the peptide moiety), and peptide bonds (within the stem peptide) [103]. While endolysins were initially used primarily on Gram-positive bacteria, recent studies have shown that utilizing endolysins as outer membrane permeabilizers (OMPs) has proven effective in eliminating Gram-negative bacteria [102].

Endolysins, autolysins, and exolysins are members of a small family of mammalian peptidoglycan recognition proteins [71, 104]. Autolysins primarily play a key role in remodeling peptidoglycan during cell growth and division, while exolysins are produced to eliminate competitive strains or species. Endolysin generally consists of two domains, including a cell wall-binding domain and an N-terminal enzymatic activity domain, enabling it to bind to a particular host cell wall and lyse the host cell wall [105]. The cell wall-binding domain identifies and specifically binds to the receptor of bacterial cell walls, and the enzymatic activity domain contributes to the cleavage of different peptidoglycan linkages [71]. The structure of phage endolysins differs between Gram-positive and Gram-negative specific phages. Most endolysins from Gram-positive specific phages consist of two domains, an enzymatic activity domain and a cell wall-binding domain, which are connected through a short flexible linker [106].

Unlike endolysins from Gram-positive specific phages, most endolysins from Gram-negative phages are small, globular, and single-domain proteins (range from 15 to 20 kDa) that usually lack a specific cell wall-binding domain [71]. Only a few endolysins from Gram-negative specific phages possess both a wall-binding domain and an enzymatic activity domain, which enable them to present a broad binding spectrum [71].

4.2. Effect of Endolysins Against Salmonella and Other Enteric Bacterial Pathogens

Endolysin has been demonstrated as a promising alternative against various AMR bacterial pathogens [102]. Since the first identification of endolysin from the L. monocytogenes phage in 1995, these enzymes have generated interest as a novel class of natural food preservatives [107]. Other studies have shown that endolysins are effective against many other Gram-positive bacteria, such as Enterococcus faecalis [108], Streptococcus pneumoniae [109], Clostridium perfringens [110], S. aureus [111], Bacillus cereus [112], and L. monocytogenes [113].

Endolysins perform specific, targeted bactericidal activity without disrupting beneficial or normal microbiota in ecosystems, including in the human and animal gut [114]. In addition, they have other benefits, such as quick lysis of bacterial cells, a low risk of resistance, effectiveness of synergistic function with other antibacterial agents, and the capacity to effectively act against biofilms and mucosal surfaces [114]. Due to these advantages, many purified or recombinant endolysins have been used to combat AMR bacteria, making them a promising alternative treatment [71].

Applications of exogenous endolysins against Gram-negative bacteria are limited due to the structure of their cell wall, which consists of a peptidoglycan layer, an internal cytoplasmic cell membrane, and an outer membrane with a lipopolysaccharide layer that prevents endolysins from accessing and lysing the peptidoglycan layer [115]. However, several recent reports have shown that endolysins can act against Gram-negative bacteria, including S. enterica [116–119], Pseudomonas aeruginosa [120], Shewanella putrefaciens [121], and Vibrio parahaemolyticus [122]. To facilitate the effectiveness against Gram-negative bacteria, these studies utilized OMPs, such as ethylenediaminetetraacetic acid (EDTA) [123], Triton X-100 [124], trichloromethane [124], organic acids [71], or the edible ε-poly-L-lysine (EPL) [125], to pretreat or cotreat the bacteria when applying exogenous endolysins. A recent study has shown that combining the fusion endolysin with KL-L9P, a sensitizer peptide known to enhance the effectiveness of antibiotics by remodeling the outer membrane of Gram-negative bacteria [126], could serve as a potential biocontrol agent against Gram-negative bacteria and S. enterica [127].

On the other hand, limited studies have revealed the effectiveness of endolysins using animal models [71]. Therefore, during the preclinical development of endolysins, it is imperative to address several key factors such as safety, toxicity, and immunogenicity. Despite numerous published animal trials, few endolysins have been tested in humans. Nevertheless, several studies have indicated no adverse health effects, elevated proinflammatory cytokine levels, or complement activation in mice, providing reassurance about the favorable safety and toxicity profiles of these endolysins [71]. So far, several clinical trials for phage-derived endolysins have reached various clinical trial phases, with some showing promising results (Table 4). However, all these trials have focused on the treatment of S. aureus rather than Salmonella.

Due to inherent limitations, there are not commercially available endolysin products targeting Gram-negative bacteria, including Salmonella. Table 5 demonstrates research on phage-derived endolysin against Salmonella from 2014 to 2024. Despite the progress in developing these endolysins, they require a combination with OMPs or the use of fusion proteins to enhance their lytic ability and effectively penetrate the outer membrane of Salmonella.