Recent Development and Application of Phage-Mediated Biocontrol Strategies and Detection against Salmonella

Abstract

Salmonella has emerged as one of the most important indicators of contamination with foodborne pathogens. Thus, it is necessary to develop sensitive and stable methods for rapid detection and control of Salmonella to ensure food safety. Typically, bacteriophages, shortened to phages, can not only specifically recognize host bacteria but also lyse the pathogens to produce many progeny phages. When phages were applied to detect Salmonella, they could not only recognize live bacteria but also achieve signal amplification to improve detection sensitivity further. Meanwhile, phages can also be applied as antibacterial agents against Salmonella in raw materials in agriculture, processing environments, and extend shelf-time for food products. In this chapter, we reviewed the up-to-date research development to use phages as recognition elements and antibacterial agents for rapid detection and biocontrol of Salmonella in foodstuff and agriculture-related matrices, dissected the unavoidable challenges, and debated the upcoming prospects.

1. Introduction

1.1 Salmonella

Salmonella is an important pathogenic factor for foodborne diseases that has attracted widespread attention. It was first isolated from the intestines of pigs infected with classical swine fever by American bacteriologists Salmon and Smith in 1885 during the cholera pandemic [1]. Due to its low requirements for nutrients and living environment, Salmonella is widely distributed in nature and has numerous serotypes. Currently, over 2700 Salmonella serovars have been discovered worldwide according to the Kauffmann-White scheme [2]. Traditionally, phenotypic and genotypic typing methods are important for surveillance and outbreak detection of Salmonella infections. Among them, phenotypic typing includes serotyping and phage typing. The phenotype-based approach relied on the surface antigens detected by agglutination reactions between the flagella, capsule, or mucus layer on the surface of their bacterial cells, purified proteins, and specific anti-serum. Specifically, the O (lipopolysaccharide), H (flagellum), and capsule (Vi) antigens are the main types and are often used for characterization. Typically, the O-antigen is related to lipopolysaccharides (LPS) in the cell wall of Salmonella, with high stability and high-temperature resistance. The flagella proteins (H-antigens) have poor stability and are not resistant to high temperatures [3]. Genomic methods are comprehensive and high-resolution approaches that aid in identifying the species, subtypes, and potential virulence factors of Salmonella. Common genomic techniques include whole genome sequencing (WGS) technology, which sequences the entire genome of Salmonella, providing more comprehensive genetic information. This includes all genotypes, SNPs, insertions, deletions, and other variations, which can be used for more accurate strain identification, subtype analysis, and source tracking. Due to its excellent discriminatory ability and relatively low cost, it is often used as a routine first- or second-line typing method for national surveillance of foodborne pathogens [4]. Multilocus sequence analysis (MLST) is one of the important bacterial genome typing methods based on the sequences of five to seven housekeeping genes. It is used for bacterial typing by analyzing sequence variations of multiple core genes. In Salmonella MLST analysis, a scheme of seven housekeeping genes is commonly used [5]. Gene chip technology, with each chip capable of accommodating thousands of oligonucleotide probes, allows for the simultaneous detection of expression levels of thousands of Salmonella genes or specific sequence variations. This greatly reduces false positives caused by cross-contamination between pathogens and enables efficient and straightforward identification of gene expression, virulence factor expression, and host interactions of Salmonella [6]. Transcriptomics, through high-throughput RNA sequencing (RNA-Seq) and other methods, studies the transcriptional structure of genes, revealing gene expression patterns of bacteria under different conditions. It can be employed for the identification and classification of Salmonella, offering advantages of high sensitivity, high throughput, and ease of operation [7]. In addition to the above techniques, there are also epigenomics, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) methods [8, 9]. These techniques accurately determine the genetic relationships between strains and are of great value in reducing outbreaks of Salmonella-related diseases, thus widely applied in investigations and research on Salmonella.

Different serovars of Salmonella have different host spectrums and virulence factors. The serotypes related to human diseases mainly include SalmonellaTyphi, SalmonellaParatyphi A, Salmonella Paratyphi B,Salmonella Paratyphi C,Salmonella Anatum, Salmonella Choleraesuis in swine, Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Newport. All Salmonella infections are considered zoonoses, except for Salmonella Typhi, Salmonella Paratyphi A,Salmonella Paratyphi B,Salmonella Paratyphi C, and Salmonella Anatum which are human-specific diseases. Salmonella Choleraesuis in swine belongs to Salmonella partial tropism, which is adapted to specific animals to a certain extent and can cause sepsis. Salmonella is a Gram-negative bacteria found in a wide range of hosts and can be associated with disease not only in humans but also in animals.

1.2 Hazardous risks of Salmonella

Salmonella is the most common foodborne pathogen and was listed by the World Health Organization (WHO) as one of the most dangerous foodborne pathogens. It is also one of the four major causes of global diarrheal diseases [10, 11]. Most cases of salmonellosis originate from the consumption of contaminated food, including beef, milk, chicken, eggs, or vegetables. Symptoms of salmonellosis include diarrhea, fever, and abdominal spasms that occur 12 to 72 hours after eating contaminated food, and generally last for 4 to 7 days. Sometimes, individuals infected with Salmonella may also develop Reiter syndrome in the later stages, characterized by frequent joint pain, eye irritation, and painful urination. Furthermore, patients may even experience bacteremia and sepsis, which can lead to death.

Salmonella is naturally distributed widely and mainly inhabits the intestines of animals, such as birds, livestock, rodents, reptiles, and humans [12]. Food transmission is the main route of human infection with Salmonella [13]. While all serotypes of Salmonella can be pathogenic to humans, Salmonella mainly originates from one or several animals and is transmitted to food-related supply chains and consumer populations through wastewater and sewage in the environment, resulting in infection [14]. Salmonellosis is generally caused by contaminated food of animal origin (mainly meat products, egg products, seafood, poultry, and other fresh products, as well as quick frozen products and dairy products) [15, 16, 17]. These foods are rich in nutrients, which are conducive to the proliferation of Salmonella and exacerbate pollution levels. In addition, the above raw materials or products may cause cross-contamination with other products during breeding, food transportation, storage, and processing operations, increasing the risk of pathogen spread and infection. In recent years, foodborne outbreaks of salmonellosis have been associated with nonanimal-based food contaminated with feces, including fresh vegetables, fruits, grains, and ready-to-eat foods [18]. Salmonella infection can be acquired through contaminated foods regularly found in meat products (accounting for 9.0 and 12.5%, respectively), dairy products (accounting for 26.3 and 19.6%, respectively), fruits and vegetables and their products (accounting for 3.7 and 23.5%, respectively), indicating that Salmonella can easily adapt to various environments and survive.

1.3 Strategies to monitor Salmonella

In recent years, governments worldwide have established multiple warning prevention and control systems for foodborne pathogens. The World Health Organization (WHO) has listed Salmonella as a foodborne pathogen with severe hazards and moderate hazards (WHO-GSS, http://www.who.ch/salmsurv) and has established a global Salmonella monitoring network to strengthen monitoring of Salmonella pollution [19]. The United States has established three monitoring network systems to dynamically monitor the occurrence and trend of foodborne diseases in the United States, including the Foodborne Diseases Active Surveillance Network (Food Net), the National Molecular Subtyping Network (Pulse Net) and the National Antimicrobial Resistance monitoring system (NARMS) [20]. In Europe, it has established the Enter-Net international surveillance network (Enter-Net). In China, Salmonella is an important monitoring indicator for foodborne pathogens in the national food pollutant monitoring system initially established by the Nutrition and Food Safety Institute of the China Center for Disease Control and Prevention.

According to the data from the Chinese national food safety risk surveillance system, 43.0% of food safety issues are attributed to foodborne microorganisms. Salmonella is a primary contributor to foodborne illnesses worldwide. In China, 70–80% of foodborne disease outbreaks are attributable to Salmonella. Among them, Salmonella Enteritis and Salmonella Typhimurium are the most common. In 2019, out of the total 139 Salmonella samples collected in Xiamen City, Salmonella Enteritis and Salmonella Typhimurium predominated, constituting 40.3 and 19.4%, respectively [21]. In China, meat and meat products are highly susceptible to contamination by Salmonella spp. In the past decade (2008–2019), Salmonella has continued to be the leading cause of large foodborne outbreaks in the United States, according to the Foodborne Diseases Active Surveillance Network (FoodNet) reports [22, 23, 24]. Based on the data from the Foodborne Diseases Active Surveillance Network (FoodNet) of the Centre for Disease Control (CDC) reported that there are nearly 150,000 cases of foodborne diseases caused by Salmonella infection in the United States each year, leading to nearly 40,000 hospitalizations and 688 deaths [25]. It ranks first among the foodborne pathogens that cause foodborne diseases. Salmonella is the leading cause of food poisoning in European countries. Because of varying dietary habits, vegetables as well as meat products are important sources of Salmonella infection [10, 26]. Currently, food safety issues caused by foodborne pathogens are still frequent. For example, in 2020, the US Centers for Disease Control and Prevention reported a Salmonella infection incident caused by onions contaminated with Salmonella, resulting in a total of 640 infections with Salmonella incidents occurring in 43 states in the USA [27].

1.4 Rapid detection methods against Salmonella

The typical standard methods for the identification of Salmonella bacteria include conventional culture techniques, immunoassays, nucleic acid-based approaches, biochemical analysis, and biosensing methods [28]. For instance, traditional culture-based methods used for Salmonella identification are defined by the international standard ISO6579:2002 [29], and the standard method of China [30]. The steps of traditional methods involve preenrichment, selective enrichment, selective isolation, biochemical screening, and serological confirmation using methods including immunoassays and molecular methods, such as polymerase chain reaction (PCR). The entire process takes 4–7 days to complete, and the results are accurate and reliable, which can intuitively reflect the actual situation of the sample [30]. Typically, conventional cultivation methods are accurate, sensitive, and reproducible, but they are time-consuming, cumbersome, and rely on subjective judgment [31].

To avoid complex cultivation processes to reduce workload, rapid detection strategies are increasingly being studied and applied, such as immunoassays and molecular-based technologies. Immunoassays are based on antigen-antibody reactions, which have the advantages of good specificity and sensitivity but still suffer from long detection times and antibodies with high costs [32]. Molecular techniques are based on the detection of specific nucleic acid sequences, including polymerase chain reaction (PCR) [33], loop-mediated isothermal amplification (LAMP) [34], gene chip technology [35]. These methods have significant advantages in specificity, sensitivity, and rapidity but require high requirements for instrument operators and detection conditions, and they cannot distinguish live and dead bacteria [36]. Furthermore, novel technologies such as biosensing have also been developed and utilized, such as microfluidic technology [37]. It is based on the manipulation of trace fluid systems and is particularly suitable for the analysis of small-sized targets. Biosensors are analytical devices that employ specific biomolecules to recognize the target entity. They could integrate the biorecognition element with signal amplification methods to achieve extremely high sensitivity and effective transducers for signal readouts. They have the advantages of high sensitivity, fast detection, and good reproducibility [38]. Biosensors have been developed for rapid and quantitative detection of Salmonella in foods. For example, Liu et al. developed a microfluidic-based biosensor that can detect Salmonella within 1 hour, with a detection limit of 300 cells/mL [39]. The sensitivity and specificity of biosensors largely depend on the interference of background microorganisms, sample matrices, and inhibitory substances [40]. Nanotechnology has the advantages of being simple, low-cost, fast, sensitive, and easy to operate, and therefore could be effectively integrated with biosensing methods [41]. Different analysis technologies are compared by their advantages and disadvantages to provide novel strategies for more efficient and practical analysis methods [42, 43]. Up to now, the existing detection methods still could not meet the requirements for on-site and point-of-care testing for viable Salmonella, and further improvement is still needed to achieve higher sensitivity and specificity.

2. Application of phages for Salmonella biocontrol and detection

2.1 Bacteriophages

Bacteriophages, abbreviated as phages, are viruses that specifically infect bacteria and consist of proteins and nucleic acids. The first reported use of bacteriophages can be traced back to the early twentieth century in 1915. British microbiologist Frederick W. Twort first observed phages when culturing Staphylococcus aureus but failed to follow up on his original observations [44]. In 1917, microbiologist Felix d’Herelle first isolated phages and applied them to treat bacterial infections. The word “phage” literally means bacteria eaters and comes from the Greek words “bacteria” and “φαγει˜ν (phagein)” [45]. From then on, phages began advocating for the development of a new research field in natural science. Phages play an important role in establishing the central principles of molecular biology, providing support for the development of modern biological technologies and reagents, as well as the discovery of the CRISPR-Cas phage resistance system [46, 47].

Phages can be found in the environment in a wide variety and large quantities that are widespread on Earth. Researchers have isolated phages in various environments [48], such as soil, water, animal intestines, and saliva. Even in some extreme environments, small amounts of phages are present, such as in areas with high temperatures (geothermal hot springs and volcanic vents), cold temperatures, and high osmotic pressures (polar lakes and seawater). Previous studies have found that phages can be isolated almost anywhere bacteria live. The total number of natural phages can reach 10³⁰–10³², which is more than ten times the number of bacteria [49]. It is believed that there are 4.7 × 10³⁰ virions in the ocean (including seawater and marine underground), approximately distributed as 2.6 x 10³⁰ virions in soils [50]. Therefore, phages are highly abundant in nature and could be used as low-cost antimicrobials.

In 1928, antibiotics such as penicillin were discovered and widely applied in clinical trials. Under that condition, phage therapy was abandoned to combat bacteria [51]. Russia approved the introduction of phages to the official pharmacopeia in 2016 [52]. In Europe, phage therapy has been officially permitted to be applied in patients with cystic fibrosis by Eastern European health institutions. However, it is still not able to solve ethical concerns in patients with cystic fibrosis using phage therapy in Western European international regulatory bodies [53]. Phages are viruses that specifically infect bacteria. According to their life cycles of reproduction, they can be divided into lytic or lysogenic phages [54, 55]. To infect the host bacteria, the phage first adsorbs to the receptors on the host cell, then injects the genetic material, and then follows a series of processes, such as lytic or chronic phage pathways. After infecting their host bacteria, lytic phages could effectively use the machinery in the host bacteria to replicate their nucleic acid before finally lysing the bacterial cell and releasing many viral particles to start further bacterial infection. In the lysogenic cycle, phages integrate their genetic elements into the bacterial genome, forming a prophage [56]. Its genome, called a prophage, exists in a free plasmid-like state or is integrated into the bacterial chromosome under pressure conditions such as ultraviolet light and radiation. Under stress conditions, prophages could transfer from the lysogenic state to the lytic cycle before producing the phage progeny. The release of lysogenic phage progeny will cause different effects on the host bacteria, such as the progeny particles of filamentous phages, which are “secreted” and released through the outer membrane of the bacteria, which will not cause bacterial lysis but will slow down the growth of the host bacteria. The reproduction cycle of bacteriophages is shown in Figure 1.

Figure 1.

Phage life cycle [57]. To infect the host bacteria, the phage first adsorbs to the receptors on the host cell, then injects the genetic material, and then follows a series of processes, such as lytic or chronic phage pathways. After infecting their host bacteria, lytic phages could effectively use the machinery in the host bacteria to replicate their nucleic acid before finally lysing the bacterial cell and releasing many viral particles to start further bacterial infection. In the lysogenic cycle, the phage would integrate its genetic elements into the bacterial genome, forming a prophage.

Phages have many different forms of existence in nature, such as filamentous, rod-shaped, spherical, and quasi-spherical. The majority of phages are composed of icosahedral head structures (capsids) and tail structures. They can also be classified according to the morphological characteristics of viruses and are currently divided into six categories: Myoviridae, Siphoviridae, Podoviridae, Microviridae, Leviviridae, and Inoviridae [47, 58], with the specific morphological characteristics shown in Figure 1. Currently, most of the phages discovered and studied belong to tailed phages, including contractile tails, long and noncontractile tails, and short tails, accounting for 96% of the total number of studies [59]. In recent years, phages have been recognized as one of the effective alternatives or additions to antibiotics to overcome the challenge of the emergence of antibiotic resistance [60].

2.2 Phage-mediated biocontrol strategies against Salmonella

2.2.1 Potential methods for prevention and control of Salmonella

Physical, chemical, antibiotic, and phage methods have been applied to prevent the contamination of Salmonella (Figure 2). As most of the Salmonella strains are sensitive to heat, physical techniques such as ultraviolet radiation and heat treatment have been used to effectively kill or inhibit the growth of Salmonella in liquid foods. However, heat treatment can lead to the degradation of nutrients and may also produce advanced glycation end products that are harmful to health [61]. Besides, it may also affect the taste, odor, and texture of foods. For example, Gouma et al. found that when combined heat treatment at 60°C with ultraviolet radiation, the time used for Salmonella inactivation in chicken soup can be reduced two times lower than using ultraviolet radiation only. However, the quality of nutrition and sensory texture was reduced as well [62].

Figure 2.

Phages application in the food industry.

Chemical disinfectants have also been widely used for the prevention and control of Salmonella [63]. The efficacy of disinfectants was evaluated at a room temperature of 25°C for 5–10 minutes [64]. Results have shown that the disinfection process will require a longer processing time when the temperature decreases [64]. However, none of the tested disinfectants have shown an antibacterial effect on Salmonella under dry conditions [64]. Disinfectants are non-selective and can rapidly kill a broad spectrum of pathogens. Therefore, it is easy to produce drug-resistant Salmonella strains after being exposed to a high concentration of disinfectants frequently. The residues of disinfectants will cause serious issues in the food industry and finally threaten the health of human beings seriously [65]. Previous studies have shown that commonly used disinfectants in poultry processing may confer tolerance to disinfectants and pose a risk of entering the food chain [66].

Antibiotics are a class of chemical substances synthesized by microorganisms that can effectively inhibit or kill bacteria. Antibiotic therapy has been the main strategy to control Salmonella infection, which has allowed to improve the growth and health of food-producing animals. Currently, antibiotics used to control Salmonella in animal production and human infections mainly include ciprofloxacin, meropenem, and cefepime. They exhibit broad-spectrum antibacterial properties and can eliminate the growth of most pathogens in foods [67]. Antibiotics can inhibit bacterial growth by cross-linking with glycopeptides, binding with phospholipids in cell membranes, or inhibiting bacterial DNA, RNA, and protein synthesis. In recent years, antibiotics have been widely used as feed additives in the field of animal disease prevention during the breeding process. The residues of antibiotics can stably exist in the environment for a long time. The abuse of antibiotics to overcome bacterial infections has caused the emergence of multidrug-resistant bacteria, which pose a great hazard to public health. The mechanisms of antibiotic resistance involve (i) enzyme inactivation, (ii) reduction of intracellular drug accumulation, and (iii) modification of target sites. From 2009 to 2012, studies have reported that 99% of Salmonella was found to be antibiotic-resistant in Shandong Province, suggesting that the resistance rate of Salmonella has been on the rise for years [68]. The abuse of antibiotics that triggered antibiotic resistance is currently one of the main problems when it comes to Salmonella infection. Developing novel strategies to reduce Salmonella contamination and prevent biofilm formation in different environments becomes urgently needed.

2.2.2 Phage-mediated biocontrol and prevention strategies against Salmonella

Phages are a type of virus that can infect bacterial cells and replicate and proliferate within bacteria. Phages could effectively kill bacteria with a mechanism that is different from that of antibiotics. Therefore, antibiotic resistance mechanisms do not affect phage efficacy. Phages tend to be natural antibacterial agents with higher safety [69]. Among them, lytic phages can ultimately remove their host bacteria and release progeny phages at the same time. Phages have specific lysis ability on bacteria without affecting the eukaryotic cells. In the presence of sensitive target bacteria, phages can quickly replicate themselves in a short period. Phages are equipped with unique advantages such as high specificity to their hosts, rapid lysis of target bacteria, and the ability to self-proliferate in large quantities [70]. Meanwhile, since there are a large number of phages present in the environment, phage application in the food system can be considered a natural process. More than 10⁸ viable bacteriophages per gram may be present in fresh meats and meat products [71]. Phages are considered safe and promising as potential antibacterial agents. However, it is necessary to identify the types of bacteria before utilizing the phages [72].

Phages have been applied in the food industry. Typically, Salmonella and other foodborne pathogens that have presented in the process of collection, production, processing, and storage of food raw materials could cause food contamination, leading to food spoilage and foodborne diseases. Phages could be applied as an effective antibacterial agents that can control Salmonella in food raw materials, equipment, packaging, and sales to ensure food safety, which includes the following aspects:

1. Disinfection of animal breeding and raw materials in foods: During the process of raw materials collection such as animal slaughtering, pathogens can easily contaminate the slaughtered material through blood and feces. As a natural antibacterial agent, phages can reduce the colonization of pathogens on cultured animals. During the time of animal breeding, the pathogens in animals can be killed by oral administration of phages. The inherent characteristics of phages reduce the occurrence of antibiotic resistance and can be used as a disinfectant after slaughter. Therefore, it is possible to meet the demand for raw food materials that are free from pathogens and synthetic chemicals.

2. Disinfection of food processing and equipment in the food industry: As for nutrient-rich raw materials, the disinfectant residues in the equipment will accelerate the accumulation of pathogenic bacteria on food processing equipment and biofilms formation, leading to serious public health hazards and economic losses caused by food spoilage. The contamination of pathogens in food processing facilities is often closely related to the formation of biofilms. The resistance of biofilms to chemical/physical cleaning and sanitizing procedures poses a severe threat to human health. It is worth noting that there may be sources of pollution on hands, operating platforms, soaking tanks, industrial facilities, and any surfaces in contact with food during the food processing steps. Under these conditions, phages can disinfect contaminated surfaces, such as working surfaces, floors, walls, and processing equipment [73].

3. Food additives to extend the shelf life: Phages can also be directly used in ready-to-eat foods such as vegetables to reduce food contamination. Phages and phage cocktails provide a promising strategy not only for antibiotic-sensitive Salmonella but also for antibiotic-resistant Salmonella strains. The standard form of phage preparation is phage suspension, which can be directly used or sprayed on the surface of foods after dilution. Spraying and immersion techniques are the two coldest methods for disinfecting phages. Phages can also be prepared into freeze-dried powder or on packaging paper for food preservation. Phage products can free foods from pathogen contamination, as well as extend their shelf life.

2.2.3 Commercialized phage products

Phages are widely distributed with high specific lysis of host bacteria. They have small genome size, and are easy to modify, making them suitable as antibacterial agents for sanitizing food processing environments and detecting specific bacteria in food products. There are commercialized phage products that can be applied in raw food materials, cleaning production environments, and processing equipment to extend food storage time and disinfect fresh fruits and vegetables (Figure 3) [74]. The phage preparation named ListShield™ was approved by the US Food and Drug Administration (FDA) for ready-to-eat meat products in 2006 antibacterial food additives. This approval was extended so that phages could also be applied in the fresh/processed fruits and vegetables, and dairy products in 2015 [75]. Moreover, commercial phage products have also been approved for use against Salmonella. For instance, FDA has approved the phage product SalmoFresh™ to be directly used in meat and poultry, as well as fresh vegetables and fruits [76]. Another phage product Salmonelex™ was approved in 2013 as a processing aid for meat and poultry product production [77]. They have also been applied to fruits and vegetables. For example, Bao et al. [78] mixed two different phages to prepare phage cocktails and sprayed them on the surface of cabbage to inhibit the proliferation of Salmonella [78]. The results showed that phage cocktails can effectively inhibit the growth of Salmonella at 4°C and 25°C. The effect of phages and chemical disinfectants on Salmonella on fruit slices was compared. The antibacterial effect of phages was significantly better than that of chemical disinfectants without affecting the sensory characteristics of fruit quality [79].

Figure 3.

Approved phage products in the food industry.

2.3 Phage-mediated detection strategies against Salmonella

Except for acting as natural antibacterial agents to control foodborne pathogens, phages could also use as recognition elements for the detection of Salmonella. Phage receptor binding proteins on its tail could act as receptors for binding to target bacteria hosts. In the presence of target bacteria, the interaction between phage and bacteria could be transferred into measurable detection signals such as optic and electrical signals. Phages were easy to culture and rapidly amplify, thus making their production economical. As a recognition element, phages mainly have unique advantages when it comes to Salmonella detection. Firstly, phages are considered highly monodispersed with clear shape and size in a buffer with nanometer range [54]. Secondly, phages can be genetically engineered to display exogenous peptides [80, 81]. Phage particles could also equipped with novel characteristics such as ability to specifically bind to desired target analytes through chemical modification. Thirdly, phages with different characteristics and types were chosen to be used in the biosensors to broaden their detection range. Finally, phages only recognize and infect live bacteria. After characterizing the host range of phages, they can be used for the recognition and detection of target cells [82]. Conversely, phages often have high specificity which could also be problematic, since it would narrow their potential detection spectrum. Multiplexed detection has been developed using different broad-spectrum phages as the probes [83]. Meanwhile, phages can also be used to amplify signals or develop detection methods based on biosensor principles, such as electrochemical and optical biosensors [84]. At present, the main methods for detecting specific pathogens using phages include (i) Traditional phage detection methods (such as phage typing and phage amplification), (ii) phage-labeled bioluminescence and chemiluminescence, (iii) reporter phages construction with molecular biology techniques, (iv) phage-derived components and enzymes, and (v) phages-mediated biosensors [85].

2.3.1 Conventional culture-based detection methods based on phage plaque assay

Lytic phages can specifically recognize and lyse host bacteria to produce progeny phages. The host bacteria were confirmed based on a visual examination of phage plaques on the plates. Traditional phage detection methods determine the quantity of host bacteria based on the number and size of phage plaques, including phage typing and phage amplification strategies. Phage typing is an important bacterial typing method, which is simple, easy to operate, low cost, and rapid. At present, phage typing methods have been developed for the detection of foodborne pathogens such as Campylobacter, Escherichia coli, Salmonella, and Listeria monocytogenes [86].

2.3.2 Phage-mediated bioluminescence and chemiluminescence

The bacterial intracellular components, such as adenosine triphosphate (ATP), adenylate kinase (AK), and beta-glucosidase, have been released upon phage infection and could be used as the signal probes for Salmonella detection. Firefly luciferase emits light in the presence of ATP and AK. The intensity of light emitted correlates with the amount of ATP, which enables luciferase to catalyze the luminescent reaction. When it integrated with phage amplification assay, it was possible to detect Staphylococcus aureus. However, it is insensitive due to the influence of ATP background [87]. In addition, reducing coenzyme amide adenine dinucleotide (NADH) can also be used as a marker to determine the target bacteria under the action of FMN-NADH oxidoreductase and luciferase [88]. The ATP/NADH bioluminescence method is simple and easy to operate. It had a short detection time, high sensitivity, low cost, and no need for cultivation. These methods have been used for the detection of foodborne pathogens such as Salmonella, Escherichia coli, and Bacillus under various conditions. However, this method suffers from the drawbacks of poor stability and requirements of specialized instruments.

Fluorescent labeling detection techniques were developed based on the fluorescent dye for detecting pathogens. The quantum dots (QDs) were prepared as fluorescent labels for rapid and sensitive detection of pathogenic bacteria in food products. A previous study has combined immunomagnetic separation with a quantum dot-labeled phage for the detection of Salmonella in milk [89]. The quantum dots (QDs) were prepared as fluorescent labels for rapid and sensitive detection of Salmonella in food products, providing a low detection limit (2 CFU/mL) [90]. Fluorescent labeling detection technology has the advantages of high throughput, accuracy, and rapidity. However, it still suffers from high cost and the inability to distinguish between live and dead bacteria.

2.3.3 Phage-mediated biosensing methods

Phage-mediated biosensors have been developed to combine phages with biosensors (Figure 4). They are ideal bio-receptors for the construction of highly specific and sensitive probes to construct biosensors. By monitoring the signal changes of certain intracellular substances in bacteria through biosensors, pathogenic bacteria can be detected. For instance, phages were integrated with portable magnetic relaxation switching biosensors to detect Salmonella in orange juice [83]. The method, following a 4-hour preenrichment step, enables the detection of low concentrations of Salmonella in orange juice (<10 CFU / 25 mL), with the detected Salmonella counts consistent with those obtained using the plate counting method. To improve the sensitivity, the sensing of phage-triggered amplification technology was developed, which utilizes phages to lyse target host bacteria, release intracellular ions, and alter potential potentials [92]. This method has been successfully applied to specifically quantify viable Salmonella in lettuce and milk, with a LOD of 1 CFU/mL and the ability to only detect live bacteria. After being coupled with real-time PCR (qPCR), PAA-qPCR further reduced the detection time and achieved an LOD of 10 CFU/mL in 3.5 h without DNA extraction or purification process. The phage-mediated biosensors have the advantages of simple operation and fast speed, but they have shown poor anti-interference ability and low reliability.

Figure 4.

Phage-mediated detection strategies against Salmonella [52, 83, 89, 91]. Various phage-mediated methods have been reported in recent years. These methods include culture-based methods, visualized methods using reporter phages, bioluminescence and chemiluminescence, and phage-mediated biosensing platforms.

2.3.4 Salmonella detection using reporter phages

Reporter phages have been defined as genetically modified phages that carry the reporter genes (Figure 4). After infection, the reporter gene could be transferred and expressed in the host bacteria upon infection. Expression of reporter genes inside host cells yields a detectable signal, in the presence of target bacteria. Reporter phages have been applied to target the major foodborne pathogens, such as Salmonella spp., Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Reporter phages containing the luxAB gene were the first bioluminescent for the detection of Salmonella strains and were able to detect Salmonella in 16 h [93]. In addition, reporter phages P22: inaW, which incorporated the inaW gene of ice nucleation bacteria, were constructed and applied for the detection of Salmonella with a detection limit of 10 CFU/mL [94]. This method can detect not only live bacteria but also detect noncultivable bacteria in various environments. Reporter phages-mediated technology was found to have a short time, high sensitivity, and low energy consumption. However, due to the specificity of phages, it is not possible to simultaneously detect all serotypes of the target bacteria.

3. Conclusion

Phages are known to have many advantages, including being able to specifically recognize host bacteria and effectively remove the bacteria. However, there is still uncertainty, especially due to their continuous evolution with host bacteria. Phages may be involved in the transfer of genes encoding toxins. For instance, it has been reported that the first mechanism of phage-mediated gene transfer was identified in Salmonella phage P22. High-frequency exchanges of phage genetic materials have enhanced the occurrence of horizontal gene transfer [95]. Naturally, phages and bacteria have been found to transfer an average of approximately 10²⁹ genes per day [96]. Therefore, it is necessary to select potent phages through whole genome sequencing instead of temperate phages to control pathogenic bacteria.

Due to the emergence of multidrug-resistant strains, numerous studies have shown that naturally occurring phages can quickly adapt to the bacterial population in their environment. In turn, bacteria can develop resistance to phages [97]. Consequently, phages can combat antibiotic-resistant bacteria, but it is still essential to explore novel strategies to improve phage efficacy, which might be limited by resistance evolution.

Acknowledgments

We thank the Natural Science Foundation of Hubei Province of China (2023AFB330) and the China Postdoctoral Science Foundation (2022 M721275) for financial support.

Conflict of interest

The authors declare no competing financial interest.