Efficiency of Bacteriophage-Based Detection Methods for Non-Typhoidal 
Non-typhoidal Salmonella (NTS) is among the most prevalent and prioritized bacterial pathogens and causes approximately 150 million gastroenteritis and 60,000 deaths in humans annually from food-borne, water-borne, and zoonotic infections worldwide . Approximately 2500 serovars of S. enterica have been identified to cause the infection in various vertebrates, but only ~100 of these serovars are linked to outbreaks in humans . The approximately 100 NTS serovars implicated in human infections are derived from the combinations of 46 O-antigens and 114 H-antigens. Among these, the serovars O:4, H:i:1,2 ( S . Typhimurium) and O:9, H:g,m:1,7 ( S . Enteritidis) are particularly significant due to their high prevalence and clinical importance . These antigens play a dual role, functioning as markers for serovar classification as well as key factors in interactions with external agents, including antibodies and bacteriophages (phages, viruses that specifically target and infect bacteria) . The diversity of NTS serovars and their niche dynamics highlight their public health importance, requiring strain-specific surveillance strategies and control measures. For this reason, highly sensitive and effective detection mechanisms of the pathogen in various sample sources is critical. Foods such as pork and poultry products remain the highest-risk sources of NTS infection . However, occasionally, Salmonella infection is also found to be associated with rarely reported food sources such as chocolate, which was linked to the largest recent outbreak in the EU/EEA, with 15.5 cases detected per 100,000 population in 2022 . Furthermore, Salmonella is recognized as the most commonly detected foodborne pathogen in imported foods in the EU, with the majority (64%) of the notifications being linked to sesame seeds outsourced from Africa . NTS is also a major cause of foodborne illnesses in Southeast Asian countries like Thailand, Vietnam, and Malaysia, with outbreaks frequently linked to undercooked poultry, eggs, seafood, and street food . These reports clearly indicate the diverse and widely variable sources of the infection, requiring effective detection strategies in all food processing systems and sources. Although NTS infections in healthy individuals often result in mild and self-limiting symptoms, they can lead to more complex and severe manifestations in vulnerable populations, such as infants, the elderly, and other immunocompromised individuals, than in healthy groups . The detection of NTS contamination in food supply chains is therefore extremely crucial for overall public health safety and to prevent disease outbreaks. Various methods for Salmonella detection exist, including immunological- and molecular-based techniques, mass spectrometry, spectroscopy, optical phenotyping, and biosensor methods . The electrochemical biosensor is particularly gaining more traction for Salmonella detection than the aforementioned methods due to its high sensitivity, speed, and portability. However, despite this advancement, specific bioreceptors like aptamers, antibodies, and phages, along with nanomaterial applications, are still required to enhance the effectiveness of these strategies . While phage-based detection systems may employ signaling mechanisms similar to those used in antibody- or aptamer-based methods, they exhibit the ability to distinguish between variable bacterial strains, offering greater specificity and versatility in detecting a broad range of bacterial pathogens . The extensive serovar diversity within Salmonella presents considerable challenges for antibody- or aptamer-based detection methods. These approaches are typically limited to recognizing only specific subsets of Salmonella serovars, which reduces the effectiveness of separation techniques in detecting the full genus. Also, these conventional methods for detecting Salmonella , as well as culture-based techniques, whilst reliable, are often time-consuming, labor-intensive, and unsuitable for throughput analyses . Consequently, there is a growing demand for more rapid, sensitive, and specific strategies for optimum Salmonella detection in all food sources and safety management systems to replace or supplement existing methods. Phages have emerged as a promising tool in the detection and control of bacterial pathogens, including Salmonella . Salmonella detection using innovative phage technology leverages the natural specificity and binding affinity of phages to their bacterial hosts, thus enabling the development of highly selective and sensitive detection assays. This method potentially offers several advantages over conventional Salmonella detection techniques, including shorter detection times, reduced need for sample preparation, and the capability for on-site testing . Crucially, phage-based methods precisely target live bacteria, effectively distinguishing viable cells from non-viable ones to ensure the detection of active infection. In contrast, molecular techniques (for example PCR) can detect very low levels of genetic material but usually necessitate additional steps to differentiate between live and dead cells. The use of phages for detecting Salmonella in food has been explored in various studies, employing different phage-based approaches such as targeting whole phage amplification , expression of phage-encoded reporter genes, and detection of phage-mediated bioluminescence . However, the efficiency and reliability of these methods can vary significantly depending on the type of food matrix, the specificity of phages used (whether narrow or broad host range phages), and the detection protocols applied . A recent review in this field focused on a limited selection of studies, narrowing down to just eleven studies that explored the use of phage-based sensors for detecting Salmonella spp. and their application in five different food samples . To further knowledge in the area and expand beyond the limited studies above, here, this systematic review was carefully designed to comprehensively evaluate the current state of phage-based detection methods for NTS serovars in various food sample types. To achieve this, we conducted three searches in PubMed/MEDLINE, Embase, and ScienceDirect of research studies in the area up until 4 November 2024 and screened the output against a list of inclusion and exclusion criteria. The resulting data was synthesized to present the overall efficiency, sensitivity, and specificity of the phage-based detection methods identified for each serovar and food type. Furthermore, factors which play key roles in the overall performance of the methods were also established, and mitigation steps to improve effectiveness were highlighted and discussed. Our work provides valuable insights into the potential of phage-based detection as a viable alternative to conventional methods in food safety diagnostics and management, using NTS as a model.   This systematic review was conducted to evaluate the effectiveness of phage-based detection methods for NTS serovars in food samples. The study followed the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) 2020 statement . The review was not registered due to time constraints and the extensive number of studies being conducted in this area. 2.1. Data Search Strategy and Extraction A comprehensive search was carried out in three databases, namely PubMed/MEDLINE, Embase, and ScienceDirect, to identify relevant literature from their inception dates up to 4 November 2024. The search strategy for each database and the number of studies searched were based on the terms provided in . Duplicate records were removed using both EndNote 21 tool  and manual screenings. Only studies written in English were considered for inclusion. Three reviewers independently screened the titles and abstracts of all retrieved records from the database search to determine eligible studies using Rayyan review manager . The reviewers retrieved and screened the full-text articles according to the pre-specified inclusion and exclusion criteria as highlighted below. All studies chosen to be excluded were discussed, and the reasons for the exclusion were recorded. All retrieval processes were performed manually and independently by three researchers. Any disagreement during the selection process was resolved by consulting a fourth reviewer. 2.2. Selection Criteria of Studies to Be Included in This Systematic Review Eligibility criteria were designed based on the PICO acronym  for the research question: Population: foods contaminated with NTS; Intervention: phage-based detection methods; Comparison: among all phage-based detection methods we identified; and Outcome: efficiency of detecting NTS in food samples. To ensure adherence to our research objectives, we implemented specific eligibility criteria for study inclusion. Only research articles published in English were considered for analysis. We gave precedence to studies investigating the detection of non-typhoidal S. enterica in food samples, encompassing research on both whole phage particles and phage component-based methods. Prioritizing study on NTS detection is critical for protecting public health. This is to ensure food safety and advance scientific expertise and technology in foodborne pathogen detection and management. Studies falling into categories such as reviews, mini-reviews, comments, or editorials were excluded. Additionally, studies involving environmental and clinical samples, pure non-typhoidal S. enterica cultures, typhoidal S. enterica , or strictly other food pathogens were excluded from our analysis. 2.3. Quality Assessment and Methodological Evaluation Following rigorous scientific protocols, the methodological quality of the included studies was assessed using the JBI Critical Appraisal Checklist for Systematic Reviews . Two independent reviewers (P.P. and C.M.) carefully evaluated each study based on four adopted critical questions. These items included: “Is the review question clearly and explicitly stated?”; “Were the inclusion criteria appropriate for addressing the review question?”; “Did the study provide fully available details of data?”; and “Was the measurement of the intervention conducted with validity and reliability?” Criteria indicating low methodological quality included one or more “no” answers or “unclear” responses to critical questions or three or more “no” answers to non-critical items. High methodological quality was determined when a study had a maximum of two “no” responses and one “unclear” response in non-critical items. Studies not meeting these criteria were considered to have moderate methodological quality . 2.4. Analysis of Data The systematic review was conducted using a pipeline and manual screening, following the PRISMA method. However, due to significant heterogeneity among the included studies, conducting a meta-analysis was deemed unfeasible. The studies and their results were grouped and analyzed based on the stage of phage implementation for NTS detection, either during bacterial enrichment or the final stage of bacterial detection, the efficiency of the detection methods based on the rapid phage detection platforms used, types of food samples, limits of bacterial load, and specificity and sensitivity of the assay.   A comprehensive search was carried out in three databases, namely PubMed/MEDLINE, Embase, and ScienceDirect, to identify relevant literature from their inception dates up to 4 November 2024. The search strategy for each database and the number of studies searched were based on the terms provided in . Duplicate records were removed using both EndNote 21 tool  and manual screenings. Only studies written in English were considered for inclusion. Three reviewers independently screened the titles and abstracts of all retrieved records from the database search to determine eligible studies using Rayyan review manager . The reviewers retrieved and screened the full-text articles according to the pre-specified inclusion and exclusion criteria as highlighted below. All studies chosen to be excluded were discussed, and the reasons for the exclusion were recorded. All retrieval processes were performed manually and independently by three researchers. Any disagreement during the selection process was resolved by consulting a fourth reviewer.   Eligibility criteria were designed based on the PICO acronym  for the research question: Population: foods contaminated with NTS; Intervention: phage-based detection methods; Comparison: among all phage-based detection methods we identified; and Outcome: efficiency of detecting NTS in food samples. To ensure adherence to our research objectives, we implemented specific eligibility criteria for study inclusion. Only research articles published in English were considered for analysis. We gave precedence to studies investigating the detection of non-typhoidal S. enterica in food samples, encompassing research on both whole phage particles and phage component-based methods. Prioritizing study on NTS detection is critical for protecting public health. This is to ensure food safety and advance scientific expertise and technology in foodborne pathogen detection and management. Studies falling into categories such as reviews, mini-reviews, comments, or editorials were excluded. Additionally, studies involving environmental and clinical samples, pure non-typhoidal S. enterica cultures, typhoidal S. enterica , or strictly other food pathogens were excluded from our analysis.   Following rigorous scientific protocols, the methodological quality of the included studies was assessed using the JBI Critical Appraisal Checklist for Systematic Reviews . Two independent reviewers (P.P. and C.M.) carefully evaluated each study based on four adopted critical questions. These items included: “Is the review question clearly and explicitly stated?”; “Were the inclusion criteria appropriate for addressing the review question?”; “Did the study provide fully available details of data?”; and “Was the measurement of the intervention conducted with validity and reliability?” Criteria indicating low methodological quality included one or more “no” answers or “unclear” responses to critical questions or three or more “no” answers to non-critical items. High methodological quality was determined when a study had a maximum of two “no” responses and one “unclear” response in non-critical items. Studies not meeting these criteria were considered to have moderate methodological quality .   The systematic review was conducted using a pipeline and manual screening, following the PRISMA method. However, due to significant heterogeneity among the included studies, conducting a meta-analysis was deemed unfeasible. The studies and their results were grouped and analyzed based on the stage of phage implementation for NTS detection, either during bacterial enrichment or the final stage of bacterial detection, the efficiency of the detection methods based on the rapid phage detection platforms used, types of food samples, limits of bacterial load, and specificity and sensitivity of the assay.   In this systematic review, we identified the pertinent literature on the application of phages to detect NTS in all food sample types. To accomplish this, we commenced with keyword searches across scientific research records and databases, as shown in . These searches yielded a comprehensive pool of 607 records sourced from the prominent platforms PubMed/MEDLINE, Embase, and ScienceDirect, with 197, 73, and 337 records, respectively. Subsequently, 35 English-language documents were meticulously handpicked for in-depth scrutiny by a panel of three independent reviewers based on our inclusion and exclusion criteria as stated above . To ensure rigor and consensus in the selection process, a fourth reviewer was engaged as an arbiter in cases of discordance. 3.1. Distribution of the Selected Studies The geographic distribution of studies on phage-based detection for NTS in foods is illustrated in . Approximately, half of the selected publications, approximately 51.43% (18 out of 35), originated from China. This is followed by 11.43% (four out of thirty-five) from the USA, 8.57% (three out of thirty-five) each from Canada and South Korea, 5.71% (two out of thirty-five) from Spain, and 2.86% (one out of thirty-five) each from Brazil, France, Portugal, Switzerland, and Turkey. This systematic review reveals that phage-based detection methods for NTS in foods have been in development since 1983, continuing to the present day. Notably, the highest number of research articles in a single year was published in 2023, accounting for eight out of the thirty-five articles. There has been a notable increase in research activity per year after 2020. 3.2. Concepts of Phage-Based NTS Detection in Food Safety Testing Phage-based bacterial detection methods, particularly when combined with enrichment steps, offer a promising and rapid alternative to traditional bacterial detection methods in food safety testing. Their high specificity and ability to rapidly detect live bacterial cells by infecting and replicating within their hosts make phage-based assays an attractive option for detecting active infection, ensuring food safety and preventing foodborne illnesses . However, whole phage particles have limitations as biorecognition elements. Their lytic activity can disrupt signal stability, and random immobilization often reduces sensitivity and bacterial capture efficiency . Additionally, their large size can impair signal sensitivity in distance-dependent systems . Phages recognize bacterial hosts through receptor-binding proteins (RBPs) such as tail fiber proteins (TFPs) and tail spike proteins (TSPs), which mediate adsorption and injection. RBPs are advantageous for their high sensitivity and specificity, stability without inducing bacterial lysis, compact structure, and ease of production via recombinant methods . Consequently, RBPs exhibit ideal molecular recognition properties, combining high specificity with strong resistance to interference from non-target bacteria. Generally, applications of phages as tools for bacterial detection in food can be categorized into two major purposes: enriching or capturing pathogens from food matrices and utilizing their specificity to detect pathogens . Based on these reasons, the selected studies here were categorized into two subgroups: Salmonella enrichment and Salmonella detection, as discussed below . The quality assessment of each study is summarized in . 3.2.1. Salmonella Enrichment Enrichment is a necessary step in the detection of Salmonella in food samples due to the low numbers of bacteria typically present, the potential for bacterial injury, competition from other microorganisms, and regulatory requirements . Scientific studies and regulatory standards support the use of enrichment to improve the sensitivity and reliability of Salmonella detection methods. This challenge can be addressed using high-affinity biorecognition reagents that bind to target pathogens . The combination of magnetic microbeads (MBs) and recognition elements, such as specific phages and aptamers, was used to recognize and separate pathogens . Here, eight out of the thirty-five included studies reported the efficiency of Salmonella captured from 12 types of food samples: pork, chicken meat, lettuce, skim milk, orange juice, eggs, milk, fresh milk, chocolate milk, infant formula, celery, and alfalfa sprouts. The recovery efficiency ranged from 65% to 102%, with an initial artificially inoculated bacterial concentration of approximately 10 5 CFU/mL , and each method did not discriminate between solid and liquid food samples. Magnetic microbeads (MBs-COOH) or magnetic nanoparticles (MNPs-COOH) were commonly used to separate the bacteria from the food matrix. The principle of separation entails immobilizing magnetic beads (MBs) with biorecognition molecules like antibodies, and phage proteins, which possess high selectivity for target analytes. Both intact phage particles and phage-derived bacterial binding proteins (PBPs) serve as effective biorecognition elements for the capture of bacterial pathogens. The remarkable binding specificity of phages and PBPs offers significant potential for applications in bacterial diagnostics and monitoring, spanning fields such as food safety, environmental surveillance, and healthcare . The phage-derived tail spike protein (TSP) from phage P22  demonstrated superior capture efficiency for S . Typhimurium, achieving rates exceeding 90% and outperforming antibodies, which achieved 80% capture efficiency . However, the mechanism of how the phage interacts with Salmonella binding protein is unclear. In addition, the streptavidin-coated MBs conjugated with the specific long tail fiber (LTF) protein of phage S16 can increase the capture efficiency to 98.00% ± 1.00, with an initial bacterial concentration range of 10 1–5 CFU/mL . To optimize the capturing efficiency of NTS, the immobilization efficacy of phages or their proteins on magnetic beads was tested. Phage P22, a member of the Podoviridae family, achieved a higher coupling efficiency with magnetic microparticles (92.40%) than with nanoparticles (83.80%) . When comparing the coupling efficiency of different magnetic nanoparticles of varying diameters (1000 nm, 250 nm, 180 nm, and 150 nm), the results indicated that larger magnetic nanoparticles allowed more phages such as LPST10, belonging to the Siphoviridae family, to immobilize. However, there was no significant difference in the capturing efficiency of the phage-magnetic conjugates for Salmonella . Additionally, various concentrations of phages or phage components were tested. In the P22-MBs covalent immobilization process, the number of phages per MBs increased significantly, plateauing at a phage/MB ratio of approximately 1650, with up to 2.0 × 10 11 phages immobilized on 7.0 × 10 7 MBs. To assess the impact of varying phage receptor- binding protein (RBP) 41 concentrations on the capture efficiency of the probe, different amounts of RBP 41 were coupled with MBs. Like the immobilization of whole phage particles with MBs, the capture efficiency of the RBP 41 for Salmonella increased with higher concentrations of the protein. A concentration of 115 µg/mL RBP 41 was found to be optimal, achieving a capture efficiency of approximately 83.43%. There was no significant difference in capture efficiency between 115 µg/mL and 1150 µg/mL of RBP 41 coupled MBs . The bacterial capture efficiency was influenced not only by the size of the MBs and the concentration of phages or phage proteins but also by several key parameters optimized to maximize the binding efficiency of Salmonella cells. These parameters included the single-reaction volume, the incubation time with Salmonella culture, the pH value of the reaction solution, and the NaCl concentration in the reaction solution. Further studies ascertained the optimal conditions for coupling phage proteins such as the long tail fiber proteins of STP4-a (LTF4-a) with MNPs to be 0.1 mg/mL protein solution per reaction, in a neutral to weakly alkaline environment (pH 7.5), with an incubation time of 30 min at room temperature. The T4-like phage, STP4-a, utilizes lipopolysaccharide (LPS) and outer membrane proteins as primary receptors, enabling high infectivity and a broad host range, successfully targeting 88 out of 91 Salmonella strains across 24 serovars . These conditions achieved optimal recovery efficiency, supporting rapid and effective Salmonella detection . Instead of solely relying on phages or phage components for capturing and enriching targeted NTS in food samples, they could also be employed as dual probes. To achieve this, composite probes were developed that consisted of deactivated phage-modified magnetic beads (MB-P) and PtNP-antimicrobial peptide (Pt-AMP) nanozyme probes. These probes form sandwich complexes with S . Typhimurium, emitting both bioluminescent and colorimetric signals for live and total Salmonella cells, respectively, and these can be simultaneously measured on a single microfluidic platform . The validated concepts for using specific phages or phage proteins as bio-probes to capture Salmonella from food matrices are summarized in A. Bacterial recovery or enrichment is the initial step in bacterial detection and must be combined with other methods to identify bacterial cells in samples. These methods include colony counting on selective agar, enzyme-linked immunoassay, and real-time PCR, as discussed below. 3.2.2. Salmonella Detection Phage-based methods for food bacterial detection utilize phage specificity and rapid replication, providing evidenced advantages. As indicated above, this strategy ensures accurate detection in food samples by targeting only viable bacteria. Virulent phages complete their infection cycle within approximately 1–2 h, and this speeds detection time compared to traditional methods . Moreover, phages are stable in a wide range of environmental conditions (temperature, pH, organic solvents), ensuring reliable detection across diverse food matrices and processing conditions . Based on these advantages, phages have shown great potential for Salmonella detection in food, through targeted phage-induced bacterial lysis, phage amplification, genetically modified reporter phages, and phage biosensors, as shown in B and discussed below. Phage-induced bacterial lysis The final step in the life cycle of lytic phages is the lysis from within of the host cell, leading to bacterial cell destruction. This process releases phage progeny and intracellular components, such as adenosine triphosphate (ATP), intracellular enzymes like adenylate kinase (AK) and caprylate esterase, and cytoplasmic components like β-D-galactosidase, which can serve as markers for detecting the lysis event . Caprylate esterase is an enzyme that catalyzes the hydrolysis of caprylate esters, resulting in products that can be easily detected through various assay techniques. In the context of phage-based detection, a recent study by Zhang et al. (2023) demonstrated the use of caprylate esterase as a detection marker for S . Enteritidis in milk. In this study, Salmonella cells lysed by phage LPSE1 released caprylate esterase, and the product of this reaction, salicylaldazine, was detected using a fluorescence assay . When combined with pre-enrichment of Salmonella cells using ampicillin-conjugated magnetic beads, the developed method offers high accuracy, with a broad detection range (10–10 6 CFU/mL), a low detection limit (6 CFU/mL), and a short detection time (within 2 h) for rapid S . Enteritidis detection in milk. Continued research and development in the area of utilizing caprylate esterase as a marker for bacterial pathogens is beneficial in four aspects: (1) Specificity: phages and bacterial cells are highly specific in their binding interactions. Phages typically recognize receptors on the bacterial cell surface with high precision, which is advantageous for targeting specific Salmonella serovars. The specificity of binding largely depends on the structural compatibility between the phage binding proteins and the target bacterial surface molecules. The application of phages to recognize caprylate esterase released from specific targeted bacterial cells can minimize false positives. (2) Sensitivity: phages generally provide a broader and more adaptable binding range, which enhances sensitivity especially in complex or challenging environments. Fluorescence assays can detect even small amounts of the reaction product, allowing for the identification of low levels of bacterial contamination. (3) Speed: due to their natural evolutionary design, phages bind quickly to their specific bacterial hosts. This rapid recognition often allows for a faster initial attachment to the target bacteria in a sample, particularly when high-affinity wild-type phages are used. (4) Simplicity: compared to culturing Salmonella , using phages is a lot easier, and the assay does not require sophisticated equipment, making it accessible for various laboratory settings and needs. Despite its advantages, the use of caprylate esterase as a marker also faces several challenges. These challenges include ensuring enzyme stability under various environmental conditions, maintaining phage specificity to minimize cross-reactivity with non-target bacteria, and optimizing the assay to ensure consistent performance . In addition to caprylate esterase, adenosine triphosphate (ATP) is also used as a marker for detecting NTS. Wang et al. (2024) developed a platinum nanoparticle- decorated phage 2-nanozyme (P2@PtNPs) for sensitive, on-site detection of live and dead S. Typhimurium. The detection limits for this are 30 CFU/mL for total bacteria and 40 CFU/mL for live bacteria. Recovery rates for spiked pork, fish, and milk ranged from 91% to 108% . To achieve a lower limit of detection of 20 CFU/mL, a phage-assisted DNA and ATP synergistically triggered Argonaute-mediated fluorescent (PDAAF) biosensor for DNA extraction- and amplification-free detection of viable S . Typhimurium with the phage LPST10. Spiked recoveries in chicken meat samples ranged from 87.72% to 113.97% . This rapid and versatile assay offers a reliable, cost-effective tool for assessing bacterial vitality and enhancing food safety diagnostics. 2. Phage amplification Phage amplification is the process of increasing phage populations through replication within bacterial hosts. This natural process involves phages infecting bacterial cells, replicating, and producing numerous progenies of phages. The phage amplification assay (PAA) is used for detecting specific bacterial pathogens by leveraging phages that specifically infect the target bacteria . The process starts by mixing the sample with phages, allowing them time to adsorb susceptible bacteria. Then, an antiviral agent (virucide) such as ferrous sulfate or tea extract is added to inactivate any extracellular phages, leaving only adsorbed particles, which subsequently engage in the infection process. To enhance detection, helper cells of a bacterial strain that supports phage growth are added to the sample after neutralizing the virucide  . This assay can detect low levels of bacterial contamination because of phage amplification . Capitalizing on the benefits of phage amplification assays, they have been combined with other methods such as spectrophotometry, quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP) to detect viable Salmonella cells . For example, Favrin et al. (2002) introduced an innovative immunomagnetic separation (IMS)-phage assay with four essential steps of capturing Salmonella cells using IMS, phage infection, recovery of phage progeny, and assessment of the phage progeny by evaluating their effects on a healthy population of host cells, referred to as signal amplifying cells (SACs) . The assay detected an average of 3 CFU of S . Enteritidis per 25 g of skim milk powder, chicken meat, and ground beef samples. Subsequently, phage amplification coupled with quantitative PCR (PAA-qPCR) enhanced the detection of S . Enteritidis in chicken meat samples . After selectively infecting Salmonella cells with phage vB_SenS_PVP-SE2, progeny phages were produced and released into the sample. Following this, qPCR was used to detect nucleic acids of progeny phages rather than bacterial DNA. The findings revealed the detection of 0.22 fg/μL of pure viral DNA (equivalent to 10 3 PFU/mL of phage particles). After a brief bacterial recovery phase, the introduction of phage into chicken samples spiked with 8 CFU/25 g resulted in detection within 10 h. In a similar experimental setup by another research group, PAA-qPCR was applied to detect S . Typhimurium in lettuce and skim milk . Furthermore, utilizing phage T156, this method achieves a detection limit of 10 CFU/mL within 3.5 h, offering high specificity for live bacteria and excluding detection of dead bacteria. Additionally, the assay exhibits specific detection capabilities for drug- resistant S . Typhimurium, reducing the detection time from 24 h to 6.5 h, offering a significant time-saving advantage compared to traditional culture-based methods. Incorporating a phage amplification assay with loop-mediated isothermal amplification (PA-LAMP), Lamas et al. (2023) detected viable S . Enteritidis via replicated phage DNA by using two strategies, a real-time fluorescence and naked-eye detection under UV light conditions . The approach reached detection limits as low as 0.2 fg/μL of pure phage DNA (equivalent to 2.2 log PFU/mL). Both real-time fluorescence and naked-eye detection methods showed a limit of detection with 95% of confidence (LOD 95 ) of 6.6 CFU/25 g and a limit of detection with 50% of confidence (LOD 50 ) of 1.5 CFU/25 g in spiked chicken breast samples, completed within an 8 h timeframe. The PAA reaction conditions, including the infection concentration and time for each phage, need to be optimized for the most efficient amplification. For instance, using phage T156 at 10 5 PFU/mL with a 30-min infection period yielded an optimal burst time for progeny phages within the same duration. Furthermore, ferrous ammonium sulfate (FAS) at a final concentration of 30 mM was stable and effectively neutralized all phages at 10 5 PFU/mL within 8 min . Under these optimized conditions, the PAA-qPCR method achieved a detection sensitivity of 1 CFU per 25 g sample of chicken meat or milk, with a total detection time of 3.5 h . Overall, phage amplification assays provide a robust, efficient, and cost-effective method for the specific detection of bacterial pathogens in various settings, including food safety, clinical diagnostics, and environmental monitoring. 3. Engineered reporter phages Reporter phages are modified to carry reporter genes, which allow for the detection of specific bacteria by producing easily measurable signals upon infection. Reporter genes can be chosen to produce various types of signals, such as luminescence (luciferase; lux/luc ), fluorescence ( gfp ), or color change (beta-galactosidase; lacZ ), thus making the detection process versatile and adaptable to different detection platforms . The reporter phages can be used to detect foodborne pathogens like NTS in food products . The rapid detection capability of the method can help in ensuring food safety and preventing outbreaks. The reporter luciferase gene is commonly used for detecting NTS in egg, iceberg lettuce, sliced pork, ground turkey, chicken breast, powdered infant formula, and milk . The first luciferase reporter phage for the purpose of detecting NTS in food was constructed by Chen and Griffiths in 1996 . Phage P22 was engineered to incorporate lux genes into its genome, enabling the detection of a luminescence signal using a photon-counting charge-coupled device (CCD) camera or X-ray film. This system was developed not only to detect Salmonella contamination in whole eggs but also to identify its precise location within the egg. It can detect 63 CFU/egg of S . Enteritidis contamination within 24 h. Regarding phage life cycles, lysogenic reporter phages are more efficient for host detection than lytic phages, as the latter quickly destroy the host cell’s signal production machinery . Temperate phage SPC32H was modified to include luxCDABE , generating a bioluminescent reporter signal . From this study with the modified phage SPC32H-CDABE, at least 20 CFU/mL of pure S . Typhimurium was detectable within 2 h, with signal intensity that increased proportionally to cell contamination levels in lettuce, sliced pork, and milk. Also, unnecessary genes from the phage SPC32H genome, including the int integrase gene, were deleted, and it was found that the int -defective phage produced similar levels of bioluminescence. The phage demonstrated significant bioluminescence production without causing immediate lysis of the host Salmonella . Further to the work on phage SPC32H, more luciferase reporter phages were engineered to express NanoLuc , an optimized luciferase derived from the deep-sea shrimp Oplophorus gracilirostris , which is 150 times brighter than traditional luciferase genes . This development produced a more detectable bioluminescence signal and minimized background noise than the luxCDABE engineered phage. More studies from the same research group introduced an enhanced method for detecting NTS in food matrices using NanoLuc -expressing phages . The combination of NanoLuc -expressing phages, SEA1 and TSP1, as a phage cocktail can accurately detect 1 CFU in either 25 g of ground turkey with a 7 h enrichment or 100 g of powdered infant formula with a 16 h enrichment . In another study , strains of S. enterica were detected within 9 h in all lettuce samples containing 10 CFU or more per 25 g. In summary, engineered reporter phages represent a promising and innovative approach for bacterial detection, offering high specificity, rapid results, and the ability to assess bacterial viability. Despite these advantages, their construction is labor-intensive and demands a thorough understanding of phage genetics. The limited volume of the phage capsid poses constraints on genetic material incorporation. Techniques such as direct cloning, transposition, and homologous recombination are employed for gene introduction, albeit applicable to only certain phages. Additionally, their use involves genetically modifying host cells, which raises concerns regarding consumer acceptance and regulatory approval. 4. Phages as bio-probes or phage biosensors Phages are increasingly utilized as bio-probes or biosensors due to their unique ability to specifically recognize and interact with bacterial targets. Phage biosensors, a notable advancement in phage-based bacterial detection, illustrate this trend. These biosensor platforms typically involve the use of either whole phages or partial phage particles. Upon infecting the host bacterium, they produce detectable signals such as colorimetric, electrical, fluorescent, or luminescent signals . The use of phages as bio-probes to detect NTS in milk was developed by Hirsh and Martin in 1983, who aimed to minimize cross-reactivity associated with immunological agents such as antibodies . The scheme employs the Salmonella -specific phage Felix-O1, which proliferates in the presence of NTS. The increased number of phages is detected using high-pressure liquid chromatography (HPLC), successfully identifying the presence of fewer than five Salmonella per milliliter of milk within 24 h. Defined as a simple but novel detection concept, a demonstration involved combining an affinity capture surface with an intracellular metabolic marker to visually detect the presence of bacteria on the affinity surface using colorimetry. This method can detect as little as 5 CFU/25 g of S . Typhimurium and S . Napoli, unaffected by the presence of food particles and the natural background microflora . Phage-based magnetoelastic (ME) biosensors were utilized to directly detect S. enterica in foods. The detection signal originates from ME materials, which are magnetostrictive alloys that change their magnetic properties in response to mechanical stress and vice versa. In this scenario, the binding of bacterial cells to the phage coated ME material increases the sensor’s mass. This mass loading alters the mechanical vibration characteristics of the ME material and the biosensor platform successfully detected S . Typhimurium at concentrations of 500 CFU/mL in spiked fresh tomato , 100 CFU/25 g on spinach leaves , and 1.31 to 1.85 log CFU/mL on Salmonella -inoculated chili peppers . The surface-scanning ME biosensor was tested on modified filamentous E2 phages, immobilized on the sensor, establishing distinct resonant frequencies and making them useful for Salmonella detection in food matrices . However, a study has shown that the wild tailed phage (TST) demonstrates greater sensitivity and binding affinity than the modified filamentous E2 phage, achieving a lower detection limit of detection (LOD) of 1.31 ± 0.27 log CFU/mL of S . Typhimurium tested on chili peppers, whereas the LOD value found in E2 phages was lower, at 1.85 ± 0.31 log CFU/mL . Phage biosensors for rapid detection of NTS (total detection time not exceeding 2 h) were implemented across various platforms. Five studies were developed to detect targeted bacteria within 30 min, without requiring an enrichment step. Firstly, a phage F5-4 (capture probe)-based lateral flow immunoassay (LFIA) coupled with surface- enhanced Raman spectroscopy (SERS) achieves the lowest LOD of 7 CFU/mL for S . Enteritidis in chicken meat and egg samples . Secondly, S. Wang et al. (2023) reported a phage/DNAzyme co-modified zeolitic imidazolate framework-encoded bio-probe that detected S . Typhimurium in spiked milk, pork, fish, and lettuce samples, achieving a limit of detection (LOD) of 8 CFU/mL . This strategy showcases the feasibility of S . Typhimurium, enabling on-site detection of contamination levels in foods. Thirdly, a novel electrochemical biosensor was introduced. The biosensor is constructed by electrostatically immobilizing phage vB_SenM_PA13076 onto MXene-nanostructured electrodes. MXene, known for its high surface area, biocompatibility, and conductivity, enables dense phage immobilization while maintaining phages’ viability and functionality for pathogen detection. The biosensor demonstrates enhanced sensitivity, with a low LOD of 5 CFU/mL, and excellent specificity, even in the presence of other foodborne bacteria . Fourthly, the ultrasensitive electrochemical detection of S . Typhimurium in food matrices was reported. The biosensor uses surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), functionalized with immobilized S . typhimurium-specific phage particles. The BC substrate’s fibrous and porous structure is enhanced by the oxidative polymerization of Ppy and RGO, creating a conductive and flexible bio-interface. It provided detection limits of 1 CFU/mL in PBS, 5 CFU/mL in milk, and 3 CFU/mL in chicken meat . Finally, in rapid detection within 20 min, the Det7 phage tail protein, used with a surface plasmon resonance (SPR) biosensor, enabled detection of S . Typhimurium at 5 × 10 4 CFU/mL in contaminated apple juice . A relatively low sensitivity appears to be a common limitation of using SPR biosensors for bacterial detection, as demonstrated in previous studies . Utilizing a detection time of approximately 2 h, a bioorthogonal reaction-mediated particle-counting sensing platform  was developed. Bioorthogonal reactions are high-yielding chemical reactions that can occur in organisms or simulated microenvironments using low-concentration, non-toxic reagents such as tetrazine ligation, the cycloaddition of s-tetrazine (Tz) with trans -cyclooctene dienophiles (TCO) . This platform was used for signal amplification in a phage LPST10-mediated sensor, enabling rapid detection of S . Typhimurium within 1 h in food matrices . Incorporating UV-spectrophotometry  and quantum dot fluorescence phage biosensors  achieved detection time frames of 80 min and 2 h, respectively. According to Anany et al. (2018), the first bioactive phage-based paper dipstick, when combined with qPCR, detects S . Newport in chicken homogenate samples with a detection limit of 50 CFU/mL . To achieve the lowest LOD of 1 CFU/mL, a phage-based electrochemical impedance spectroscopy biosensor was coupled with a 3.5-h pre-enrichment process in chicken breast samples . In contrast to other studies, Muldoon et al. (2007) proposed the idea that using specific phage cocktails to control non- Salmonella species in food metrices such as Citrobacter spp. and E. coli could reduce false-positive results . After removing non-target bacteria, serogroup-specific monoclonal antibodies on an immunochromatographic strip were used to detect NTS in raw ground beef, liquid egg, and sliced cooked turkey . When compared to the FSIS-USDA cultural reference method, the developed method demonstrated 100% sensitivity and specificity in detecting NTS in contaminated raw ground chicken . Based on the evidence provided, phage-based biosensors serve as a robust and adaptable tool for detecting bacterial contaminants in food, delivering rapid, sensitive, and specific results vital for safeguarding food safety and public health. Ongoing research endeavors seek to enhance these technologies by improving detection limits, accelerating assay times, and ensuring compatibility across diverse food matrices, thereby expanding their utility in food safety and beyond.   The geographic distribution of studies on phage-based detection for NTS in foods is illustrated in . Approximately, half of the selected publications, approximately 51.43% (18 out of 35), originated from China. This is followed by 11.43% (four out of thirty-five) from the USA, 8.57% (three out of thirty-five) each from Canada and South Korea, 5.71% (two out of thirty-five) from Spain, and 2.86% (one out of thirty-five) each from Brazil, France, Portugal, Switzerland, and Turkey. This systematic review reveals that phage-based detection methods for NTS in foods have been in development since 1983, continuing to the present day. Notably, the highest number of research articles in a single year was published in 2023, accounting for eight out of the thirty-five articles. There has been a notable increase in research activity per year after 2020.   Phage-based bacterial detection methods, particularly when combined with enrichment steps, offer a promising and rapid alternative to traditional bacterial detection methods in food safety testing. Their high specificity and ability to rapidly detect live bacterial cells by infecting and replicating within their hosts make phage-based assays an attractive option for detecting active infection, ensuring food safety and preventing foodborne illnesses . However, whole phage particles have limitations as biorecognition elements. Their lytic activity can disrupt signal stability, and random immobilization often reduces sensitivity and bacterial capture efficiency . Additionally, their large size can impair signal sensitivity in distance-dependent systems . Phages recognize bacterial hosts through receptor-binding proteins (RBPs) such as tail fiber proteins (TFPs) and tail spike proteins (TSPs), which mediate adsorption and injection. RBPs are advantageous for their high sensitivity and specificity, stability without inducing bacterial lysis, compact structure, and ease of production via recombinant methods . Consequently, RBPs exhibit ideal molecular recognition properties, combining high specificity with strong resistance to interference from non-target bacteria. Generally, applications of phages as tools for bacterial detection in food can be categorized into two major purposes: enriching or capturing pathogens from food matrices and utilizing their specificity to detect pathogens . Based on these reasons, the selected studies here were categorized into two subgroups: Salmonella enrichment and Salmonella detection, as discussed below . The quality assessment of each study is summarized in . 3.2.1. Salmonella Enrichment Enrichment is a necessary step in the detection of Salmonella in food samples due to the low numbers of bacteria typically present, the potential for bacterial injury, competition from other microorganisms, and regulatory requirements . Scientific studies and regulatory standards support the use of enrichment to improve the sensitivity and reliability of Salmonella detection methods. This challenge can be addressed using high-affinity biorecognition reagents that bind to target pathogens . The combination of magnetic microbeads (MBs) and recognition elements, such as specific phages and aptamers, was used to recognize and separate pathogens . Here, eight out of the thirty-five included studies reported the efficiency of Salmonella captured from 12 types of food samples: pork, chicken meat, lettuce, skim milk, orange juice, eggs, milk, fresh milk, chocolate milk, infant formula, celery, and alfalfa sprouts. The recovery efficiency ranged from 65% to 102%, with an initial artificially inoculated bacterial concentration of approximately 10 5 CFU/mL , and each method did not discriminate between solid and liquid food samples. Magnetic microbeads (MBs-COOH) or magnetic nanoparticles (MNPs-COOH) were commonly used to separate the bacteria from the food matrix. The principle of separation entails immobilizing magnetic beads (MBs) with biorecognition molecules like antibodies, and phage proteins, which possess high selectivity for target analytes. Both intact phage particles and phage-derived bacterial binding proteins (PBPs) serve as effective biorecognition elements for the capture of bacterial pathogens. The remarkable binding specificity of phages and PBPs offers significant potential for applications in bacterial diagnostics and monitoring, spanning fields such as food safety, environmental surveillance, and healthcare . The phage-derived tail spike protein (TSP) from phage P22  demonstrated superior capture efficiency for S . Typhimurium, achieving rates exceeding 90% and outperforming antibodies, which achieved 80% capture efficiency . However, the mechanism of how the phage interacts with Salmonella binding protein is unclear. In addition, the streptavidin-coated MBs conjugated with the specific long tail fiber (LTF) protein of phage S16 can increase the capture efficiency to 98.00% ± 1.00, with an initial bacterial concentration range of 10 1–5 CFU/mL . To optimize the capturing efficiency of NTS, the immobilization efficacy of phages or their proteins on magnetic beads was tested. Phage P22, a member of the Podoviridae family, achieved a higher coupling efficiency with magnetic microparticles (92.40%) than with nanoparticles (83.80%) . When comparing the coupling efficiency of different magnetic nanoparticles of varying diameters (1000 nm, 250 nm, 180 nm, and 150 nm), the results indicated that larger magnetic nanoparticles allowed more phages such as LPST10, belonging to the Siphoviridae family, to immobilize. However, there was no significant difference in the capturing efficiency of the phage-magnetic conjugates for Salmonella . Additionally, various concentrations of phages or phage components were tested. In the P22-MBs covalent immobilization process, the number of phages per MBs increased significantly, plateauing at a phage/MB ratio of approximately 1650, with up to 2.0 × 10 11 phages immobilized on 7.0 × 10 7 MBs. To assess the impact of varying phage receptor- binding protein (RBP) 41 concentrations on the capture efficiency of the probe, different amounts of RBP 41 were coupled with MBs. Like the immobilization of whole phage particles with MBs, the capture efficiency of the RBP 41 for Salmonella increased with higher concentrations of the protein. A concentration of 115 µg/mL RBP 41 was found to be optimal, achieving a capture efficiency of approximately 83.43%. There was no significant difference in capture efficiency between 115 µg/mL and 1150 µg/mL of RBP 41 coupled MBs . The bacterial capture efficiency was influenced not only by the size of the MBs and the concentration of phages or phage proteins but also by several key parameters optimized to maximize the binding efficiency of Salmonella cells. These parameters included the single-reaction volume, the incubation time with Salmonella culture, the pH value of the reaction solution, and the NaCl concentration in the reaction solution. Further studies ascertained the optimal conditions for coupling phage proteins such as the long tail fiber proteins of STP4-a (LTF4-a) with MNPs to be 0.1 mg/mL protein solution per reaction, in a neutral to weakly alkaline environment (pH 7.5), with an incubation time of 30 min at room temperature. The T4-like phage, STP4-a, utilizes lipopolysaccharide (LPS) and outer membrane proteins as primary receptors, enabling high infectivity and a broad host range, successfully targeting 88 out of 91 Salmonella strains across 24 serovars . These conditions achieved optimal recovery efficiency, supporting rapid and effective Salmonella detection . Instead of solely relying on phages or phage components for capturing and enriching targeted NTS in food samples, they could also be employed as dual probes. To achieve this, composite probes were developed that consisted of deactivated phage-modified magnetic beads (MB-P) and PtNP-antimicrobial peptide (Pt-AMP) nanozyme probes. These probes form sandwich complexes with S . Typhimurium, emitting both bioluminescent and colorimetric signals for live and total Salmonella cells, respectively, and these can be simultaneously measured on a single microfluidic platform . The validated concepts for using specific phages or phage proteins as bio-probes to capture Salmonella from food matrices are summarized in A. Bacterial recovery or enrichment is the initial step in bacterial detection and must be combined with other methods to identify bacterial cells in samples. These methods include colony counting on selective agar, enzyme-linked immunoassay, and real-time PCR, as discussed below. 3.2.2. Salmonella Detection Phage-based methods for food bacterial detection utilize phage specificity and rapid replication, providing evidenced advantages. As indicated above, this strategy ensures accurate detection in food samples by targeting only viable bacteria. Virulent phages complete their infection cycle within approximately 1–2 h, and this speeds detection time compared to traditional methods . Moreover, phages are stable in a wide range of environmental conditions (temperature, pH, organic solvents), ensuring reliable detection across diverse food matrices and processing conditions . Based on these advantages, phages have shown great potential for Salmonella detection in food, through targeted phage-induced bacterial lysis, phage amplification, genetically modified reporter phages, and phage biosensors, as shown in B and discussed below. Phage-induced bacterial lysis The final step in the life cycle of lytic phages is the lysis from within of the host cell, leading to bacterial cell destruction. This process releases phage progeny and intracellular components, such as adenosine triphosphate (ATP), intracellular enzymes like adenylate kinase (AK) and caprylate esterase, and cytoplasmic components like β-D-galactosidase, which can serve as markers for detecting the lysis event . Caprylate esterase is an enzyme that catalyzes the hydrolysis of caprylate esters, resulting in products that can be easily detected through various assay techniques. In the context of phage-based detection, a recent study by Zhang et al. (2023) demonstrated the use of caprylate esterase as a detection marker for S . Enteritidis in milk. In this study, Salmonella cells lysed by phage LPSE1 released caprylate esterase, and the product of this reaction, salicylaldazine, was detected using a fluorescence assay . When combined with pre-enrichment of Salmonella cells using ampicillin-conjugated magnetic beads, the developed method offers high accuracy, with a broad detection range (10–10 6 CFU/mL), a low detection limit (6 CFU/mL), and a short detection time (within 2 h) for rapid S . Enteritidis detection in milk. Continued research and development in the area of utilizing caprylate esterase as a marker for bacterial pathogens is beneficial in four aspects: (1) Specificity: phages and bacterial cells are highly specific in their binding interactions. Phages typically recognize receptors on the bacterial cell surface with high precision, which is advantageous for targeting specific Salmonella serovars. The specificity of binding largely depends on the structural compatibility between the phage binding proteins and the target bacterial surface molecules. The application of phages to recognize caprylate esterase released from specific targeted bacterial cells can minimize false positives. (2) Sensitivity: phages generally provide a broader and more adaptable binding range, which enhances sensitivity especially in complex or challenging environments. Fluorescence assays can detect even small amounts of the reaction product, allowing for the identification of low levels of bacterial contamination. (3) Speed: due to their natural evolutionary design, phages bind quickly to their specific bacterial hosts. This rapid recognition often allows for a faster initial attachment to the target bacteria in a sample, particularly when high-affinity wild-type phages are used. (4) Simplicity: compared to culturing Salmonella , using phages is a lot easier, and the assay does not require sophisticated equipment, making it accessible for various laboratory settings and needs. Despite its advantages, the use of caprylate esterase as a marker also faces several challenges. These challenges include ensuring enzyme stability under various environmental conditions, maintaining phage specificity to minimize cross-reactivity with non-target bacteria, and optimizing the assay to ensure consistent performance . In addition to caprylate esterase, adenosine triphosphate (ATP) is also used as a marker for detecting NTS. Wang et al. (2024) developed a platinum nanoparticle- decorated phage 2-nanozyme (P2@PtNPs) for sensitive, on-site detection of live and dead S. Typhimurium. The detection limits for this are 30 CFU/mL for total bacteria and 40 CFU/mL for live bacteria. Recovery rates for spiked pork, fish, and milk ranged from 91% to 108% . To achieve a lower limit of detection of 20 CFU/mL, a phage-assisted DNA and ATP synergistically triggered Argonaute-mediated fluorescent (PDAAF) biosensor for DNA extraction- and amplification-free detection of viable S . Typhimurium with the phage LPST10. Spiked recoveries in chicken meat samples ranged from 87.72% to 113.97% . This rapid and versatile assay offers a reliable, cost-effective tool for assessing bacterial vitality and enhancing food safety diagnostics. 2. Phage amplification Phage amplification is the process of increasing phage populations through replication within bacterial hosts. This natural process involves phages infecting bacterial cells, replicating, and producing numerous progenies of phages. The phage amplification assay (PAA) is used for detecting specific bacterial pathogens by leveraging phages that specifically infect the target bacteria . The process starts by mixing the sample with phages, allowing them time to adsorb susceptible bacteria. Then, an antiviral agent (virucide) such as ferrous sulfate or tea extract is added to inactivate any extracellular phages, leaving only adsorbed particles, which subsequently engage in the infection process. To enhance detection, helper cells of a bacterial strain that supports phage growth are added to the sample after neutralizing the virucide  . This assay can detect low levels of bacterial contamination because of phage amplification . Capitalizing on the benefits of phage amplification assays, they have been combined with other methods such as spectrophotometry, quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP) to detect viable Salmonella cells . For example, Favrin et al. (2002) introduced an innovative immunomagnetic separation (IMS)-phage assay with four essential steps of capturing Salmonella cells using IMS, phage infection, recovery of phage progeny, and assessment of the phage progeny by evaluating their effects on a healthy population of host cells, referred to as signal amplifying cells (SACs) . The assay detected an average of 3 CFU of S . Enteritidis per 25 g of skim milk powder, chicken meat, and ground beef samples. Subsequently, phage amplification coupled with quantitative PCR (PAA-qPCR) enhanced the detection of S . Enteritidis in chicken meat samples . After selectively infecting Salmonella cells with phage vB_SenS_PVP-SE2, progeny phages were produced and released into the sample. Following this, qPCR was used to detect nucleic acids of progeny phages rather than bacterial DNA. The findings revealed the detection of 0.22 fg/μL of pure viral DNA (equivalent to 10 3 PFU/mL of phage particles). After a brief bacterial recovery phase, the introduction of phage into chicken samples spiked with 8 CFU/25 g resulted in detection within 10 h. In a similar experimental setup by another research group, PAA-qPCR was applied to detect S . Typhimurium in lettuce and skim milk . Furthermore, utilizing phage T156, this method achieves a detection limit of 10 CFU/mL within 3.5 h, offering high specificity for live bacteria and excluding detection of dead bacteria. Additionally, the assay exhibits specific detection capabilities for drug- resistant S . Typhimurium, reducing the detection time from 24 h to 6.5 h, offering a significant time-saving advantage compared to traditional culture-based methods. Incorporating a phage amplification assay with loop-mediated isothermal amplification (PA-LAMP), Lamas et al. (2023) detected viable S . Enteritidis via replicated phage DNA by using two strategies, a real-time fluorescence and naked-eye detection under UV light conditions . The approach reached detection limits as low as 0.2 fg/μL of pure phage DNA (equivalent to 2.2 log PFU/mL). Both real-time fluorescence and naked-eye detection methods showed a limit of detection with 95% of confidence (LOD 95 ) of 6.6 CFU/25 g and a limit of detection with 50% of confidence (LOD 50 ) of 1.5 CFU/25 g in spiked chicken breast samples, completed within an 8 h timeframe. The PAA reaction conditions, including the infection concentration and time for each phage, need to be optimized for the most efficient amplification. For instance, using phage T156 at 10 5 PFU/mL with a 30-min infection period yielded an optimal burst time for progeny phages within the same duration. Furthermore, ferrous ammonium sulfate (FAS) at a final concentration of 30 mM was stable and effectively neutralized all phages at 10 5 PFU/mL within 8 min . Under these optimized conditions, the PAA-qPCR method achieved a detection sensitivity of 1 CFU per 25 g sample of chicken meat or milk, with a total detection time of 3.5 h . Overall, phage amplification assays provide a robust, efficient, and cost-effective method for the specific detection of bacterial pathogens in various settings, including food safety, clinical diagnostics, and environmental monitoring. 3. Engineered reporter phages Reporter phages are modified to carry reporter genes, which allow for the detection of specific bacteria by producing easily measurable signals upon infection. Reporter genes can be chosen to produce various types of signals, such as luminescence (luciferase; lux/luc ), fluorescence ( gfp ), or color change (beta-galactosidase; lacZ ), thus making the detection process versatile and adaptable to different detection platforms . The reporter phages can be used to detect foodborne pathogens like NTS in food products . The rapid detection capability of the method can help in ensuring food safety and preventing outbreaks. The reporter luciferase gene is commonly used for detecting NTS in egg, iceberg lettuce, sliced pork, ground turkey, chicken breast, powdered infant formula, and milk . The first luciferase reporter phage for the purpose of detecting NTS in food was constructed by Chen and Griffiths in 1996 . Phage P22 was engineered to incorporate lux genes into its genome, enabling the detection of a luminescence signal using a photon-counting charge-coupled device (CCD) camera or X-ray film. This system was developed not only to detect Salmonella contamination in whole eggs but also to identify its precise location within the egg. It can detect 63 CFU/egg of S . Enteritidis contamination within 24 h. Regarding phage life cycles, lysogenic reporter phages are more efficient for host detection than lytic phages, as the latter quickly destroy the host cell’s signal production machinery . Temperate phage SPC32H was modified to include luxCDABE , generating a bioluminescent reporter signal . From this study with the modified phage SPC32H-CDABE, at least 20 CFU/mL of pure S . Typhimurium was detectable within 2 h, with signal intensity that increased proportionally to cell contamination levels in lettuce, sliced pork, and milk. Also, unnecessary genes from the phage SPC32H genome, including the int integrase gene, were deleted, and it was found that the int -defective phage produced similar levels of bioluminescence. The phage demonstrated significant bioluminescence production without causing immediate lysis of the host Salmonella . Further to the work on phage SPC32H, more luciferase reporter phages were engineered to express NanoLuc , an optimized luciferase derived from the deep-sea shrimp Oplophorus gracilirostris , which is 150 times brighter than traditional luciferase genes . This development produced a more detectable bioluminescence signal and minimized background noise than the luxCDABE engineered phage. More studies from the same research group introduced an enhanced method for detecting NTS in food matrices using NanoLuc -expressing phages . The combination of NanoLuc -expressing phages, SEA1 and TSP1, as a phage cocktail can accurately detect 1 CFU in either 25 g of ground turkey with a 7 h enrichment or 100 g of powdered infant formula with a 16 h enrichment . In another study , strains of S. enterica were detected within 9 h in all lettuce samples containing 10 CFU or more per 25 g. In summary, engineered reporter phages represent a promising and innovative approach for bacterial detection, offering high specificity, rapid results, and the ability to assess bacterial viability. Despite these advantages, their construction is labor-intensive and demands a thorough understanding of phage genetics. The limited volume of the phage capsid poses constraints on genetic material incorporation. Techniques such as direct cloning, transposition, and homologous recombination are employed for gene introduction, albeit applicable to only certain phages. Additionally, their use involves genetically modifying host cells, which raises concerns regarding consumer acceptance and regulatory approval. 4. Phages as bio-probes or phage biosensors Phages are increasingly utilized as bio-probes or biosensors due to their unique ability to specifically recognize and interact with bacterial targets. Phage biosensors, a notable advancement in phage-based bacterial detection, illustrate this trend. These biosensor platforms typically involve the use of either whole phages or partial phage particles. Upon infecting the host bacterium, they produce detectable signals such as colorimetric, electrical, fluorescent, or luminescent signals . The use of phages as bio-probes to detect NTS in milk was developed by Hirsh and Martin in 1983, who aimed to minimize cross-reactivity associated with immunological agents such as antibodies . The scheme employs the Salmonella -specific phage Felix-O1, which proliferates in the presence of NTS. The increased number of phages is detected using high-pressure liquid chromatography (HPLC), successfully identifying the presence of fewer than five Salmonella per milliliter of milk within 24 h. Defined as a simple but novel detection concept, a demonstration involved combining an affinity capture surface with an intracellular metabolic marker to visually detect the presence of bacteria on the affinity surface using colorimetry. This method can detect as little as 5 CFU/25 g of S . Typhimurium and S . Napoli, unaffected by the presence of food particles and the natural background microflora . Phage-based magnetoelastic (ME) biosensors were utilized to directly detect S. enterica in foods. The detection signal originates from ME materials, which are magnetostrictive alloys that change their magnetic properties in response to mechanical stress and vice versa. In this scenario, the binding of bacterial cells to the phage coated ME material increases the sensor’s mass. This mass loading alters the mechanical vibration characteristics of the ME material and the biosensor platform successfully detected S . Typhimurium at concentrations of 500 CFU/mL in spiked fresh tomato , 100 CFU/25 g on spinach leaves , and 1.31 to 1.85 log CFU/mL on Salmonella -inoculated chili peppers . The surface-scanning ME biosensor was tested on modified filamentous E2 phages, immobilized on the sensor, establishing distinct resonant frequencies and making them useful for Salmonella detection in food matrices . However, a study has shown that the wild tailed phage (TST) demonstrates greater sensitivity and binding affinity than the modified filamentous E2 phage, achieving a lower detection limit of detection (LOD) of 1.31 ± 0.27 log CFU/mL of S . Typhimurium tested on chili peppers, whereas the LOD value found in E2 phages was lower, at 1.85 ± 0.31 log CFU/mL . Phage biosensors for rapid detection of NTS (total detection time not exceeding 2 h) were implemented across various platforms. Five studies were developed to detect targeted bacteria within 30 min, without requiring an enrichment step. Firstly, a phage F5-4 (capture probe)-based lateral flow immunoassay (LFIA) coupled with surface- enhanced Raman spectroscopy (SERS) achieves the lowest LOD of 7 CFU/mL for S . Enteritidis in chicken meat and egg samples . Secondly, S. Wang et al. (2023) reported a phage/DNAzyme co-modified zeolitic imidazolate framework-encoded bio-probe that detected S . Typhimurium in spiked milk, pork, fish, and lettuce samples, achieving a limit of detection (LOD) of 8 CFU/mL . This strategy showcases the feasibility of S . Typhimurium, enabling on-site detection of contamination levels in foods. Thirdly, a novel electrochemical biosensor was introduced. The biosensor is constructed by electrostatically immobilizing phage vB_SenM_PA13076 onto MXene-nanostructured electrodes. MXene, known for its high surface area, biocompatibility, and conductivity, enables dense phage immobilization while maintaining phages’ viability and functionality for pathogen detection. The biosensor demonstrates enhanced sensitivity, with a low LOD of 5 CFU/mL, and excellent specificity, even in the presence of other foodborne bacteria . Fourthly, the ultrasensitive electrochemical detection of S . Typhimurium in food matrices was reported. The biosensor uses surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), functionalized with immobilized S . typhimurium-specific phage particles. The BC substrate’s fibrous and porous structure is enhanced by the oxidative polymerization of Ppy and RGO, creating a conductive and flexible bio-interface. It provided detection limits of 1 CFU/mL in PBS, 5 CFU/mL in milk, and 3 CFU/mL in chicken meat . Finally, in rapid detection within 20 min, the Det7 phage tail protein, used with a surface plasmon resonance (SPR) biosensor, enabled detection of S . Typhimurium at 5 × 10 4 CFU/mL in contaminated apple juice . A relatively low sensitivity appears to be a common limitation of using SPR biosensors for bacterial detection, as demonstrated in previous studies . Utilizing a detection time of approximately 2 h, a bioorthogonal reaction-mediated particle-counting sensing platform  was developed. Bioorthogonal reactions are high-yielding chemical reactions that can occur in organisms or simulated microenvironments using low-concentration, non-toxic reagents such as tetrazine ligation, the cycloaddition of s-tetrazine (Tz) with trans -cyclooctene dienophiles (TCO) . This platform was used for signal amplification in a phage LPST10-mediated sensor, enabling rapid detection of S . Typhimurium within 1 h in food matrices . Incorporating UV-spectrophotometry  and quantum dot fluorescence phage biosensors  achieved detection time frames of 80 min and 2 h, respectively. According to Anany et al. (2018), the first bioactive phage-based paper dipstick, when combined with qPCR, detects S . Newport in chicken homogenate samples with a detection limit of 50 CFU/mL . To achieve the lowest LOD of 1 CFU/mL, a phage-based electrochemical impedance spectroscopy biosensor was coupled with a 3.5-h pre-enrichment process in chicken breast samples . In contrast to other studies, Muldoon et al. (2007) proposed the idea that using specific phage cocktails to control non- Salmonella species in food metrices such as Citrobacter spp. and E. coli could reduce false-positive results . After removing non-target bacteria, serogroup-specific monoclonal antibodies on an immunochromatographic strip were used to detect NTS in raw ground beef, liquid egg, and sliced cooked turkey . When compared to the FSIS-USDA cultural reference method, the developed method demonstrated 100% sensitivity and specificity in detecting NTS in contaminated raw ground chicken . Based on the evidence provided, phage-based biosensors serve as a robust and adaptable tool for detecting bacterial contaminants in food, delivering rapid, sensitive, and specific results vital for safeguarding food safety and public health. Ongoing research endeavors seek to enhance these technologies by improving detection limits, accelerating assay times, and ensuring compatibility across diverse food matrices, thereby expanding their utility in food safety and beyond.   Salmonella Enrichment Enrichment is a necessary step in the detection of Salmonella in food samples due to the low numbers of bacteria typically present, the potential for bacterial injury, competition from other microorganisms, and regulatory requirements . Scientific studies and regulatory standards support the use of enrichment to improve the sensitivity and reliability of Salmonella detection methods. This challenge can be addressed using high-affinity biorecognition reagents that bind to target pathogens . The combination of magnetic microbeads (MBs) and recognition elements, such as specific phages and aptamers, was used to recognize and separate pathogens . Here, eight out of the thirty-five included studies reported the efficiency of Salmonella captured from 12 types of food samples: pork, chicken meat, lettuce, skim milk, orange juice, eggs, milk, fresh milk, chocolate milk, infant formula, celery, and alfalfa sprouts. The recovery efficiency ranged from 65% to 102%, with an initial artificially inoculated bacterial concentration of approximately 10 5 CFU/mL , and each method did not discriminate between solid and liquid food samples. Magnetic microbeads (MBs-COOH) or magnetic nanoparticles (MNPs-COOH) were commonly used to separate the bacteria from the food matrix. The principle of separation entails immobilizing magnetic beads (MBs) with biorecognition molecules like antibodies, and phage proteins, which possess high selectivity for target analytes. Both intact phage particles and phage-derived bacterial binding proteins (PBPs) serve as effective biorecognition elements for the capture of bacterial pathogens. The remarkable binding specificity of phages and PBPs offers significant potential for applications in bacterial diagnostics and monitoring, spanning fields such as food safety, environmental surveillance, and healthcare . The phage-derived tail spike protein (TSP) from phage P22  demonstrated superior capture efficiency for S . Typhimurium, achieving rates exceeding 90% and outperforming antibodies, which achieved 80% capture efficiency . However, the mechanism of how the phage interacts with Salmonella binding protein is unclear. In addition, the streptavidin-coated MBs conjugated with the specific long tail fiber (LTF) protein of phage S16 can increase the capture efficiency to 98.00% ± 1.00, with an initial bacterial concentration range of 10 1–5 CFU/mL . To optimize the capturing efficiency of NTS, the immobilization efficacy of phages or their proteins on magnetic beads was tested. Phage P22, a member of the Podoviridae family, achieved a higher coupling efficiency with magnetic microparticles (92.40%) than with nanoparticles (83.80%) . When comparing the coupling efficiency of different magnetic nanoparticles of varying diameters (1000 nm, 250 nm, 180 nm, and 150 nm), the results indicated that larger magnetic nanoparticles allowed more phages such as LPST10, belonging to the Siphoviridae family, to immobilize. However, there was no significant difference in the capturing efficiency of the phage-magnetic conjugates for Salmonella . Additionally, various concentrations of phages or phage components were tested. In the P22-MBs covalent immobilization process, the number of phages per MBs increased significantly, plateauing at a phage/MB ratio of approximately 1650, with up to 2.0 × 10 11 phages immobilized on 7.0 × 10 7 MBs. To assess the impact of varying phage receptor- binding protein (RBP) 41 concentrations on the capture efficiency of the probe, different amounts of RBP 41 were coupled with MBs. Like the immobilization of whole phage particles with MBs, the capture efficiency of the RBP 41 for Salmonella increased with higher concentrations of the protein. A concentration of 115 µg/mL RBP 41 was found to be optimal, achieving a capture efficiency of approximately 83.43%. There was no significant difference in capture efficiency between 115 µg/mL and 1150 µg/mL of RBP 41 coupled MBs . The bacterial capture efficiency was influenced not only by the size of the MBs and the concentration of phages or phage proteins but also by several key parameters optimized to maximize the binding efficiency of Salmonella cells. These parameters included the single-reaction volume, the incubation time with Salmonella culture, the pH value of the reaction solution, and the NaCl concentration in the reaction solution. Further studies ascertained the optimal conditions for coupling phage proteins such as the long tail fiber proteins of STP4-a (LTF4-a) with MNPs to be 0.1 mg/mL protein solution per reaction, in a neutral to weakly alkaline environment (pH 7.5), with an incubation time of 30 min at room temperature. The T4-like phage, STP4-a, utilizes lipopolysaccharide (LPS) and outer membrane proteins as primary receptors, enabling high infectivity and a broad host range, successfully targeting 88 out of 91 Salmonella strains across 24 serovars . These conditions achieved optimal recovery efficiency, supporting rapid and effective Salmonella detection . Instead of solely relying on phages or phage components for capturing and enriching targeted NTS in food samples, they could also be employed as dual probes. To achieve this, composite probes were developed that consisted of deactivated phage-modified magnetic beads (MB-P) and PtNP-antimicrobial peptide (Pt-AMP) nanozyme probes. These probes form sandwich complexes with S . Typhimurium, emitting both bioluminescent and colorimetric signals for live and total Salmonella cells, respectively, and these can be simultaneously measured on a single microfluidic platform . The validated concepts for using specific phages or phage proteins as bio-probes to capture Salmonella from food matrices are summarized in A. Bacterial recovery or enrichment is the initial step in bacterial detection and must be combined with other methods to identify bacterial cells in samples. These methods include colony counting on selective agar, enzyme-linked immunoassay, and real-time PCR, as discussed below.   Salmonella Detection Phage-based methods for food bacterial detection utilize phage specificity and rapid replication, providing evidenced advantages. As indicated above, this strategy ensures accurate detection in food samples by targeting only viable bacteria. Virulent phages complete their infection cycle within approximately 1–2 h, and this speeds detection time compared to traditional methods . Moreover, phages are stable in a wide range of environmental conditions (temperature, pH, organic solvents), ensuring reliable detection across diverse food matrices and processing conditions . Based on these advantages, phages have shown great potential for Salmonella detection in food, through targeted phage-induced bacterial lysis, phage amplification, genetically modified reporter phages, and phage biosensors, as shown in B and discussed below. Phage-induced bacterial lysis The final step in the life cycle of lytic phages is the lysis from within of the host cell, leading to bacterial cell destruction. This process releases phage progeny and intracellular components, such as adenosine triphosphate (ATP), intracellular enzymes like adenylate kinase (AK) and caprylate esterase, and cytoplasmic components like β-D-galactosidase, which can serve as markers for detecting the lysis event . Caprylate esterase is an enzyme that catalyzes the hydrolysis of caprylate esters, resulting in products that can be easily detected through various assay techniques. In the context of phage-based detection, a recent study by Zhang et al. (2023) demonstrated the use of caprylate esterase as a detection marker for S . Enteritidis in milk. In this study, Salmonella cells lysed by phage LPSE1 released caprylate esterase, and the product of this reaction, salicylaldazine, was detected using a fluorescence assay . When combined with pre-enrichment of Salmonella cells using ampicillin-conjugated magnetic beads, the developed method offers high accuracy, with a broad detection range (10–10 6 CFU/mL), a low detection limit (6 CFU/mL), and a short detection time (within 2 h) for rapid S . Enteritidis detection in milk. Continued research and development in the area of utilizing caprylate esterase as a marker for bacterial pathogens is beneficial in four aspects: (1) Specificity: phages and bacterial cells are highly specific in their binding interactions. Phages typically recognize receptors on the bacterial cell surface with high precision, which is advantageous for targeting specific Salmonella serovars. The specificity of binding largely depends on the structural compatibility between the phage binding proteins and the target bacterial surface molecules. The application of phages to recognize caprylate esterase released from specific targeted bacterial cells can minimize false positives. (2) Sensitivity: phages generally provide a broader and more adaptable binding range, which enhances sensitivity especially in complex or challenging environments. Fluorescence assays can detect even small amounts of the reaction product, allowing for the identification of low levels of bacterial contamination. (3) Speed: due to their natural evolutionary design, phages bind quickly to their specific bacterial hosts. This rapid recognition often allows for a faster initial attachment to the target bacteria in a sample, particularly when high-affinity wild-type phages are used. (4) Simplicity: compared to culturing Salmonella , using phages is a lot easier, and the assay does not require sophisticated equipment, making it accessible for various laboratory settings and needs. Despite its advantages, the use of caprylate esterase as a marker also faces several challenges. These challenges include ensuring enzyme stability under various environmental conditions, maintaining phage specificity to minimize cross-reactivity with non-target bacteria, and optimizing the assay to ensure consistent performance . In addition to caprylate esterase, adenosine triphosphate (ATP) is also used as a marker for detecting NTS. Wang et al. (2024) developed a platinum nanoparticle- decorated phage 2-nanozyme (P2@PtNPs) for sensitive, on-site detection of live and dead S. Typhimurium. The detection limits for this are 30 CFU/mL for total bacteria and 40 CFU/mL for live bacteria. Recovery rates for spiked pork, fish, and milk ranged from 91% to 108% . To achieve a lower limit of detection of 20 CFU/mL, a phage-assisted DNA and ATP synergistically triggered Argonaute-mediated fluorescent (PDAAF) biosensor for DNA extraction- and amplification-free detection of viable S . Typhimurium with the phage LPST10. Spiked recoveries in chicken meat samples ranged from 87.72% to 113.97% . This rapid and versatile assay offers a reliable, cost-effective tool for assessing bacterial vitality and enhancing food safety diagnostics. 2. Phage amplification Phage amplification is the process of increasing phage populations through replication within bacterial hosts. This natural process involves phages infecting bacterial cells, replicating, and producing numerous progenies of phages. The phage amplification assay (PAA) is used for detecting specific bacterial pathogens by leveraging phages that specifically infect the target bacteria . The process starts by mixing the sample with phages, allowing them time to adsorb susceptible bacteria. Then, an antiviral agent (virucide) such as ferrous sulfate or tea extract is added to inactivate any extracellular phages, leaving only adsorbed particles, which subsequently engage in the infection process. To enhance detection, helper cells of a bacterial strain that supports phage growth are added to the sample after neutralizing the virucide  . This assay can detect low levels of bacterial contamination because of phage amplification . Capitalizing on the benefits of phage amplification assays, they have been combined with other methods such as spectrophotometry, quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP) to detect viable Salmonella cells . For example, Favrin et al. (2002) introduced an innovative immunomagnetic separation (IMS)-phage assay with four essential steps of capturing Salmonella cells using IMS, phage infection, recovery of phage progeny, and assessment of the phage progeny by evaluating their effects on a healthy population of host cells, referred to as signal amplifying cells (SACs) . The assay detected an average of 3 CFU of S . Enteritidis per 25 g of skim milk powder, chicken meat, and ground beef samples. Subsequently, phage amplification coupled with quantitative PCR (PAA-qPCR) enhanced the detection of S . Enteritidis in chicken meat samples . After selectively infecting Salmonella cells with phage vB_SenS_PVP-SE2, progeny phages were produced and released into the sample. Following this, qPCR was used to detect nucleic acids of progeny phages rather than bacterial DNA. The findings revealed the detection of 0.22 fg/μL of pure viral DNA (equivalent to 10 3 PFU/mL of phage particles). After a brief bacterial recovery phase, the introduction of phage into chicken samples spiked with 8 CFU/25 g resulted in detection within 10 h. In a similar experimental setup by another research group, PAA-qPCR was applied to detect S . Typhimurium in lettuce and skim milk . Furthermore, utilizing phage T156, this method achieves a detection limit of 10 CFU/mL within 3.5 h, offering high specificity for live bacteria and excluding detection of dead bacteria. Additionally, the assay exhibits specific detection capabilities for drug- resistant S . Typhimurium, reducing the detection time from 24 h to 6.5 h, offering a significant time-saving advantage compared to traditional culture-based methods. Incorporating a phage amplification assay with loop-mediated isothermal amplification (PA-LAMP), Lamas et al. (2023) detected viable S . Enteritidis via replicated phage DNA by using two strategies, a real-time fluorescence and naked-eye detection under UV light conditions . The approach reached detection limits as low as 0.2 fg/μL of pure phage DNA (equivalent to 2.2 log PFU/mL). Both real-time fluorescence and naked-eye detection methods showed a limit of detection with 95% of confidence (LOD 95 ) of 6.6 CFU/25 g and a limit of detection with 50% of confidence (LOD 50 ) of 1.5 CFU/25 g in spiked chicken breast samples, completed within an 8 h timeframe. The PAA reaction conditions, including the infection concentration and time for each phage, need to be optimized for the most efficient amplification. For instance, using phage T156 at 10 5 PFU/mL with a 30-min infection period yielded an optimal burst time for progeny phages within the same duration. Furthermore, ferrous ammonium sulfate (FAS) at a final concentration of 30 mM was stable and effectively neutralized all phages at 10 5 PFU/mL within 8 min . Under these optimized conditions, the PAA-qPCR method achieved a detection sensitivity of 1 CFU per 25 g sample of chicken meat or milk, with a total detection time of 3.5 h . Overall, phage amplification assays provide a robust, efficient, and cost-effective method for the specific detection of bacterial pathogens in various settings, including food safety, clinical diagnostics, and environmental monitoring. 3. Engineered reporter phages Reporter phages are modified to carry reporter genes, which allow for the detection of specific bacteria by producing easily measurable signals upon infection. Reporter genes can be chosen to produce various types of signals, such as luminescence (luciferase; lux/luc ), fluorescence ( gfp ), or color change (beta-galactosidase; lacZ ), thus making the detection process versatile and adaptable to different detection platforms . The reporter phages can be used to detect foodborne pathogens like NTS in food products . The rapid detection capability of the method can help in ensuring food safety and preventing outbreaks. The reporter luciferase gene is commonly used for detecting NTS in egg, iceberg lettuce, sliced pork, ground turkey, chicken breast, powdered infant formula, and milk . The first luciferase reporter phage for the purpose of detecting NTS in food was constructed by Chen and Griffiths in 1996 . Phage P22 was engineered to incorporate lux genes into its genome, enabling the detection of a luminescence signal using a photon-counting charge-coupled device (CCD) camera or X-ray film. This system was developed not only to detect Salmonella contamination in whole eggs but also to identify its precise location within the egg. It can detect 63 CFU/egg of S . Enteritidis contamination within 24 h. Regarding phage life cycles, lysogenic reporter phages are more efficient for host detection than lytic phages, as the latter quickly destroy the host cell’s signal production machinery . Temperate phage SPC32H was modified to include luxCDABE , generating a bioluminescent reporter signal . From this study with the modified phage SPC32H-CDABE, at least 20 CFU/mL of pure S . Typhimurium was detectable within 2 h, with signal intensity that increased proportionally to cell contamination levels in lettuce, sliced pork, and milk. Also, unnecessary genes from the phage SPC32H genome, including the int integrase gene, were deleted, and it was found that the int -defective phage produced similar levels of bioluminescence. The phage demonstrated significant bioluminescence production without causing immediate lysis of the host Salmonella . Further to the work on phage SPC32H, more luciferase reporter phages were engineered to express NanoLuc , an optimized luciferase derived from the deep-sea shrimp Oplophorus gracilirostris , which is 150 times brighter than traditional luciferase genes . This development produced a more detectable bioluminescence signal and minimized background noise than the luxCDABE engineered phage. More studies from the same research group introduced an enhanced method for detecting NTS in food matrices using NanoLuc -expressing phages . The combination of NanoLuc -expressing phages, SEA1 and TSP1, as a phage cocktail can accurately detect 1 CFU in either 25 g of ground turkey with a 7 h enrichment or 100 g of powdered infant formula with a 16 h enrichment . In another study , strains of S. enterica were detected within 9 h in all lettuce samples containing 10 CFU or more per 25 g. In summary, engineered reporter phages represent a promising and innovative approach for bacterial detection, offering high specificity, rapid results, and the ability to assess bacterial viability. Despite these advantages, their construction is labor-intensive and demands a thorough understanding of phage genetics. The limited volume of the phage capsid poses constraints on genetic material incorporation. Techniques such as direct cloning, transposition, and homologous recombination are employed for gene introduction, albeit applicable to only certain phages. Additionally, their use involves genetically modifying host cells, which raises concerns regarding consumer acceptance and regulatory approval. 4. Phages as bio-probes or phage biosensors Phages are increasingly utilized as bio-probes or biosensors due to their unique ability to specifically recognize and interact with bacterial targets. Phage biosensors, a notable advancement in phage-based bacterial detection, illustrate this trend. These biosensor platforms typically involve the use of either whole phages or partial phage particles. Upon infecting the host bacterium, they produce detectable signals such as colorimetric, electrical, fluorescent, or luminescent signals . The use of phages as bio-probes to detect NTS in milk was developed by Hirsh and Martin in 1983, who aimed to minimize cross-reactivity associated with immunological agents such as antibodies . The scheme employs the Salmonella -specific phage Felix-O1, which proliferates in the presence of NTS. The increased number of phages is detected using high-pressure liquid chromatography (HPLC), successfully identifying the presence of fewer than five Salmonella per milliliter of milk within 24 h. Defined as a simple but novel detection concept, a demonstration involved combining an affinity capture surface with an intracellular metabolic marker to visually detect the presence of bacteria on the affinity surface using colorimetry. This method can detect as little as 5 CFU/25 g of S . Typhimurium and S . Napoli, unaffected by the presence of food particles and the natural background microflora . Phage-based magnetoelastic (ME) biosensors were utilized to directly detect S. enterica in foods. The detection signal originates from ME materials, which are magnetostrictive alloys that change their magnetic properties in response to mechanical stress and vice versa. In this scenario, the binding of bacterial cells to the phage coated ME material increases the sensor’s mass. This mass loading alters the mechanical vibration characteristics of the ME material and the biosensor platform successfully detected S . Typhimurium at concentrations of 500 CFU/mL in spiked fresh tomato , 100 CFU/25 g on spinach leaves , and 1.31 to 1.85 log CFU/mL on Salmonella -inoculated chili peppers . The surface-scanning ME biosensor was tested on modified filamentous E2 phages, immobilized on the sensor, establishing distinct resonant frequencies and making them useful for Salmonella detection in food matrices . However, a study has shown that the wild tailed phage (TST) demonstrates greater sensitivity and binding affinity than the modified filamentous E2 phage, achieving a lower detection limit of detection (LOD) of 1.31 ± 0.27 log CFU/mL of S . Typhimurium tested on chili peppers, whereas the LOD value found in E2 phages was lower, at 1.85 ± 0.31 log CFU/mL . Phage biosensors for rapid detection of NTS (total detection time not exceeding 2 h) were implemented across various platforms. Five studies were developed to detect targeted bacteria within 30 min, without requiring an enrichment step. Firstly, a phage F5-4 (capture probe)-based lateral flow immunoassay (LFIA) coupled with surface- enhanced Raman spectroscopy (SERS) achieves the lowest LOD of 7 CFU/mL for S . Enteritidis in chicken meat and egg samples . Secondly, S. Wang et al. (2023) reported a phage/DNAzyme co-modified zeolitic imidazolate framework-encoded bio-probe that detected S . Typhimurium in spiked milk, pork, fish, and lettuce samples, achieving a limit of detection (LOD) of 8 CFU/mL . This strategy showcases the feasibility of S . Typhimurium, enabling on-site detection of contamination levels in foods. Thirdly, a novel electrochemical biosensor was introduced. The biosensor is constructed by electrostatically immobilizing phage vB_SenM_PA13076 onto MXene-nanostructured electrodes. MXene, known for its high surface area, biocompatibility, and conductivity, enables dense phage immobilization while maintaining phages’ viability and functionality for pathogen detection. The biosensor demonstrates enhanced sensitivity, with a low LOD of 5 CFU/mL, and excellent specificity, even in the presence of other foodborne bacteria . Fourthly, the ultrasensitive electrochemical detection of S . Typhimurium in food matrices was reported. The biosensor uses surface-modified bacterial cellulose (BC) integrated with polypyrrole (Ppy) and reduced graphene oxide (RGO), functionalized with immobilized S . typhimurium-specific phage particles. The BC substrate’s fibrous and porous structure is enhanced by the oxidative polymerization of Ppy and RGO, creating a conductive and flexible bio-interface. It provided detection limits of 1 CFU/mL in PBS, 5 CFU/mL in milk, and 3 CFU/mL in chicken meat . Finally, in rapid detection within 20 min, the Det7 phage tail protein, used with a surface plasmon resonance (SPR) biosensor, enabled detection of S . Typhimurium at 5 × 10 4 CFU/mL in contaminated apple juice . A relatively low sensitivity appears to be a common limitation of using SPR biosensors for bacterial detection, as demonstrated in previous studies . Utilizing a detection time of approximately 2 h, a bioorthogonal reaction-mediated particle-counting sensing platform  was developed. Bioorthogonal reactions are high-yielding chemical reactions that can occur in organisms or simulated microenvironments using low-concentration, non-toxic reagents such as tetrazine ligation, the cycloaddition of s-tetrazine (Tz) with trans -cyclooctene dienophiles (TCO) . This platform was used for signal amplification in a phage LPST10-mediated sensor, enabling rapid detection of S . Typhimurium within 1 h in food matrices . Incorporating UV-spectrophotometry  and quantum dot fluorescence phage biosensors  achieved detection time frames of 80 min and 2 h, respectively. According to Anany et al. (2018), the first bioactive phage-based paper dipstick, when combined with qPCR, detects S . Newport in chicken homogenate samples with a detection limit of 50 CFU/mL . To achieve the lowest LOD of 1 CFU/mL, a phage-based electrochemical impedance spectroscopy biosensor was coupled with a 3.5-h pre-enrichment process in chicken breast samples . In contrast to other studies, Muldoon et al. (2007) proposed the idea that using specific phage cocktails to control non- Salmonella species in food metrices such as Citrobacter spp. and E. coli could reduce false-positive results . After removing non-target bacteria, serogroup-specific monoclonal antibodies on an immunochromatographic strip were used to detect NTS in raw ground beef, liquid egg, and sliced cooked turkey . When compared to the FSIS-USDA cultural reference method, the developed method demonstrated 100% sensitivity and specificity in detecting NTS in contaminated raw ground chicken . Based on the evidence provided, phage-based biosensors serve as a robust and adaptable tool for detecting bacterial contaminants in food, delivering rapid, sensitive, and specific results vital for safeguarding food safety and public health. Ongoing research endeavors seek to enhance these technologies by improving detection limits, accelerating assay times, and ensuring compatibility across diverse food matrices, thereby expanding their utility in food safety and beyond.   Phage-based detection methods offer notable advantages over traditional approaches, particularly in their specificity and efficacy against pathogenic bacteria. Phages exhibit high selectivity for viable bacterial hosts or specific strains, ensuring that detection systems accurately target active Salmonella infection or contamination while minimizing cross-reactivity with other microorganisms . Whole phages are typically suitable for this purpose due to their innate ability to target and infect particular bacterial strains, but they can also be genetically engineered to enhance specificity. For example, the modifications of tail fiber proteins, which play a critical role in host recognition and to initiate infection, have been shown to improve phage target capacity and to differentiate between closely related bacterial strains, thereby increasing the accuracy of detection systems . Phage selectivity is essential for applications in complex food matrices and environments with diverse microbial communities, where the presence of non-target bacteria can potentially complicate detection efforts . However, dairy matrices such as milk and cheese are characterized by high concentrations of proteins and fats, which can significantly impede phage–host interactions . Similarly, the detection of foodborne pathogens in meat is also challenged by the presence of blood components (in addition to fats and proteins) and in fruit juices with a low pH, which are known to impact the activity of specific phages . Despite these limitations, phage detection methods for NTS have been successfully applied to various food types. These food types include dairy products such as milk, as well as meat products such as chicken, and fresh produce including fruits and vegetables . To perform this effectively and to ensure optimal phage activity in these complex food matrices, rigorous sample preparation protocols encompassing filtration, dilution, centrifugation, and enzymatic treatments are imperative to eliminate inhibitory substances and mitigate matrix-associated interferences . Further, to sample pre-treatment protocols, the initial phase of phage-based detection entails the recovery or enrichment of Salmonella from the food samples. The sensitivity of these methods was shown to be significantly improved by optimizing both the concentration of whole phages or phage-derived proteins and the conditions under which they interact with the bacterial cells . This combined strategy can enhance the effectiveness of traditional culture-based methods by reducing the time required and minimal Salmonella benchmark concentration for robust identification . For example, typical Salmonella detection techniques using qPCR produced results within fewer hours when combined with phage enrichment than when used alone, which is essential for timely intervention in food safety management . This strategy further supports the seamless integration of phage-based detection methods with other conventional techniques, such as colony counting on selective agar, enzyme-linked immunoassays (ELISA), and real-time PCR, to enhance detection. Additionally, LTF4-a conjugated with MNPs has been shown to enhance recovery efficiency . This integration allows for a more comprehensive and confirmatory approach to NTS detection . In addition to combined detection systems, parameters such as reaction volume, incubation time, pH, and NaCl concentration play critical roles in maximizing the binding efficiency and recovery of Salmonella cells. While the advantages of phage-based NTS detection methods are compelling, several practical considerations and challenges must be addressed before widespread implementation. One of the primary challenges is the regulatory landscape. Phage detection methods are relatively novel, and existing regulatory frameworks may not fully address their use. Gaining approval for these methods requires demonstrating their safety, efficacy, reliability and reproducibility, which can vary depending on the specific phage strains used . Each phage detection system may need individual evaluation for regulatory approval, adding complexity to the process, especially when different phage cocktails are involved. Another significant challenge is public understanding and acceptance of phage-based technologies in food safety. Misconceptions or lack of awareness about the safety and benefits of the detection could hinder or delay adoption . Although there is a growing interest in developing phage-based detection methods, most of these technologies remain at the experimental stage in laboratories, and their translation to commercial products has been grossly slow. Transparent communication and education efforts are necessary to build consumer trust. Another key limitation of phage-based bacterial detection is the narrow host range of phages, which can lead to missed detections, particularly with strain/serovar diversity in NTS . To address this, a cocktail of phages may be needed, but this can complicate the assay and increase the risk of phage interference . Rather than covering all serovars of NTS, priority should be given to the development of detection methods for the two most significant Salmonella serovars transmitted from animals to humans worldwide . Just as bacteria can develop resistance to phage-based therapies, there is a risk that resistance could also develop in the context of detection, potentially compromising the effectiveness of the system over time . This necessitates ongoing monitoring and possible adjustments to the phage selection process. Additionally, the widespread use of phages in detection systems raises concerns about their potential impact on natural microbial communities if released into the environment . Careful assessment and management of these ecological risks are essential to prevent unintended consequences. Addressing these challenges requires a collaborative approach involving scientists, regulatory bodies, industry stakeholders, and public health experts to develop, validate, and implement phage-based detection systems that are effective, safe, and economically viable.   Phage-based detection methods for NTS in foods demonstrate high specificity, sensitivity, and rapid detection capabilities. By optimizing recovery and enrichment steps and combining phage-based techniques with other detection methods, these approaches can enhance the overall efficiency of NTS detection in food safety applications. Further research and development are essential to address the challenges and fully realize the potential of phage-based detection systems for NTS to ensure food safety. The successful development of a highly effective phage-based NTS detection system can serve as a model for implementation in the detection of other foodborne pathogens.   