Bacteriophages as a modern diagnostic tool: innovations, applications and challenges

Abstract

Bacteriophages, viruses that specifically infect bacteria, have emerged as a valuable tool in diagnostics due to their unique specificity and adaptability. This review explores the diverse applications of bacteriophages in diagnostic methods, from traditional phage typing to advanced molecular techniques such as phage display and PCR-based diagnostics. It highlights their use in identifying bacterial strains, monitoring fermentation processes, and diagnosing critical conditions like tuberculosis, MRSA infections, and cancer. Innovations such as phage-based biosensors and reporter phages enhance the speed and precision of diagnostics, offering significant advantages over traditional methods. Challenges, including bacterial resistance and immune responses to phages, are also discussed alongside strategies for mitigation, such as phage cocktails and engineering. Integrating phage technology with modern bioscience holds promise for addressing antibiotic resistance and revolutionizing clinical and industrial diagnostics. This comprehensive analysis underscores the potential of bacteriophages to transform the diagnostic landscape while identifying areas requiring further research and development.

Introduction

Bacteriophages, or phages, are viruses that infect and replicate within bacteria with high specificity. While they have traditionally been explored as alternatives to antibiotics, especially in the context of rising multidrug resistance (MDR), their diagnostic potential stems from distinct characteristics. Specifically, their natural ability to recognize and bind to particular bacterial strains with remarkable precision enables their use in detecting and identifying bacterial pathogens. This specificity, combined with advances in molecular biology, bioengineering, and biosensor technologies, has fostered the development of innovative phage-based diagnostic platforms. Unlike conventional methods that often require time-consuming culturing steps, phage-based diagnostics offer rapid, sensitive, and specific detection options. These include phage amplification assays, reporter phages, and phage-based biosensors. This review focuses exclusively on the role of phages in diagnostics, excluding their therapeutic applications. It presents an overview of current technologies, recent developments, limitations, and future perspectives in this field [1–5].

Bacteriophages as diagnostic tools

Bacteriophages, viruses that specifically infect bacteria, are among the most numerous and genetically and evolutionarily diverse biological entities in the biosphere. They can be found in almost every environment, including even the most extreme conditions. Their exceptional specificity towards the bacterial host makes them ideal candidates for diagnostic applications. Unlike broad-spectrum antibiotics or general detection methods, phages can distinguish not only species, but often also specific strains of bacteria. This feature makes them extremely attractive diagnostic tools, especially in situations where rapid and precise detection of the pathogen is crucial for further clinical management.

In diagnostics, mainly lytic phages are used, which, after infecting the bacterial cell, multiply and lead to its lysis. Phage-based diagnostic technologies use their specificity in various ways. Lytic phages can be used in culture-based tests, integrated with biosensors, and also act as"reporters"by generating a signal (e.g., luminescence or fluorescence) after infection of the target bacteria. They can also be combined with molecular methods, such as quantitative PCR or CRISPR-based systems, which increases sensitivity and shortens the analysis time. Thanks to this, phages are becoming increasingly competitive with traditional diagnostic methods, offering not only specificity, but also the ability to detect living cells, which is not provided by, for example, classic molecular tests.

Depending on the clinical, environmental, or industrial application, phages are selected for properties such as stability, scope of infection, ease of genetic engineering, and compatibility with analytical systems [6–9].

Phage typing

Phage typing—origins

Bacteriophages were first discovered during World War I and have since become a frequent subject of research. Oskar Bail was one of the first to recognize the potential of using phages in diagnostics. He noted that specific bacteriophages destroy well-defined strains of bacteria. For him, phages were then considered fragments of bacterial chromatin that induced lysis or changes in bacterial cells. However, this research was crucial, as it enabled them to target specific bacteria. He used them as a tool to differentiate closely related pathogens, such as Salmonella and Shigella [10, 11].

Another, Curt Sonnenschein of the Institute of Hygiene in Cologne, significantly developed phage typing, focusing on practical applications in diagnosis and epidemiology. He discovered antiphage antibodies in patients’ serum, which enabled faster diagnosis of infections. He also isolated phages specific to a particular strain of bacteria, such as Shigella flexneri or Salmonella typhi. He stored them and made them available to other laboratories with instructions. Sonnenschein and his team became a major center for phage typing. Other laboratories, including those in Prague, Berlin, Zwickau, and Budapest, began adapting his techniques and developing their diagnostic phages. However, the lack of consensus on what bacteriophages are and their nature and character made it very difficult to standardize them, which stopped the development of phage typing. Additionally, scientists at the time struggled to maintain the stability of bacteriophages [11, 12]. However, they proved that bacteriophages can be an effective diagnostic tool and laid the groundwork for modern bacteriophage research Fig.1.

Fig. 1.

Historical timeline of bacteriophage research and applications

Phage typing—what it is based on

It is a microbiological technique used to identify and classify specific strains of bacteria by using bacteriophages that are specifically directed against them. When a phage infects a susceptible bacterium, it causes the bacterium to lyse. This can be observed on the plate as a zone of bacterial growth inhibition. This is visible as a distinct zone of translucence. This phenomenon is the basis of phage typing [13, 14]. The whole procedure involves several steps. First, a pure culture of the strain to be tested is prepared. Then, it is spread evenly on the plate. Later, various bacteriophages are applied to it, each explicitly targeting different bacterial strains. The plate is then incubated, after which a clear zone of translucence can be observed where the phage is applied. The results are then compared with phage typing patterns, and based on this, the corresponding strain can be identified [15, 16].

Phage typing—applications

Its use in epidemiology can be beneficial. It enables the identification of individual strains and the tracks of bacterial outbreaks. Phage typing has been used to type strains of Listeria monocytogenes and Escherichia coli [13, 17].

Currently, researchers are trying to improve the Phage typing method. A novel in silico phage typing method for Enterococcus faecium has been developed [12]. This bacterium is resistant to vancomycin and poses a significant threat, particularly to hospitalized, immunocompromised patients. Its rapid identification is essential to prevent the spread of nosocomial infections. The study tested whether phage analysis of the bacterial genome allows phage typing of VRE strains. E. faecium isolates were forfeited for their prophage content (this is an inactive form of phage that builds its genetic material into the bacteria’s DNA and does not lead to its immediate lysis). Their unique patterns for specific genetic lines were identified. The results showed that some prophages were present in multiple lines, while others were specific to single strains. Such analysis offers the possibility of differentiating strains, even if they have low genome variability. The study was verified based on 12 clinical isolates from three epidemiological outbreaks between 2015 and 2019. For each of these isolates, the prophages that were present in their genomes were identified. The profiles of these prophages were then compared with those from other epidemic outbreaks to determine whether they were related. It turned out that bacteria from the exact epidemic had very similar sets of bacteriophages. This made it possible to associate and identify them [12]. This study demonstrated the tremendous opportunities that phage typing offers [12]. It is possible to locate the source of a hospital-acquired infection quickly. However, further development and research on this method are needed. In conclusion, this method is a very valuable tool in microbiology, and its development and application can significantly contribute to public health safety.

Phage amplification and PCR-based diagnostics

Bacteriophages are increasingly being used as diagnostic tools in PCR-based applications due to their unique biological properties, including strict host specificity, environmental stability, and well-characterized genomes. Their integration into molecular diagnostic workflows enhances quality control, improves assay reliability, and opens new avenues for the development of targeted detection systems.

One of the primary uses of bacteriophages in molecular diagnostics is as internal process controls (IPCs) in PCR and RT-PCR assays. Their genetic material can be artificially introduced into clinical or environmental samples to monitor key stages such as nucleic acid extraction, reverse transcription, and amplification. For instance, Ninove et al. utilized DNA phage T4 and RNA phage MS2 as internal controls in clinical samples to detect the presence of PCR inhibitors that could otherwise lead to false-negative results. This approach is especially beneficial when working with complex biological matrices, such as heparinized blood or feces, where inhibition is a common issue. The inclusion of phage controls ensured that a successful amplification indicated a reliable diagnostic outcome, thus improving assay robustness and reproducibility [18].

In industrial contexts, while bacteriophages are indeed a threat to fermentation processes (particularly in the dairy industry), PCR-based detection of phages themselves can serve as a diagnostic tool to monitor and prevent contamination. In this sense, the phage genome becomes the diagnostic target, allowing early detection of contamination that might otherwise compromise product quality. Binetti et al. developed a PCR assay targeting a conserved nucleotide-binding gene in phages infecting Lactobacillus casei/paracasei. This tool enabled the rapid detection of phages in milk and fermented products at levels as low as 10⁴ PFU/mL, allowing for timely intervention in industrial fermentation lines [19].

A more advanced diagnostic approach was demonstrated by del Río et al., who developed a multiplex PCR method for the simultaneous detection of multiple phages infecting key starter culture species such as Streptococcus thermophilus, Lactobacillus delbrueckii, and Lactococcus lactis (including phage groups P335, 936, and c2). By targeting conserved genomic regions across these diverse phages, their method allowed sensitive, high-throughput detection (10³ PFU/ml) directly from milk samples. Though this application addresses phage contamination, it represents a diagnostic use of phage DNA as a target, with direct implications for quality assurance and production control in food biotechnology [20].

Bacteriophage detection also plays a diagnostic role in research settings, particularly in studies on phage diversity and epidemiology. Labrie and Moineau developed a multiplex PCR assay targeting structural genes, such as the major capsid protein gene (mcp), to monitor and classify phage populations in whey. This allowed not only detection but also genotyping of phages, which is crucial for developing informed phage management strategies, including the design of phage-resistant starter cultures or the application of engineered anti-phage systems [21].

In summary, although phages may pose a threat in some industrial environments, their genetic material is increasingly used in PCR methods—both as an internal standard for quality control of the reaction and as a detection target in environmental and industrial diagnostics. It is worth emphasizing, however, that the use of phage DNA as a control for PCR reactions (e.g., for monitoring extraction efficiency or the presence of inhibitors) is not equivalent to using phages as a direct diagnostic tool for detecting pathogens. In the former case, phages act as a technical indicator of the correctness of the reaction, while in the latter, their presence (or their genetic material) is the diagnosed phenomenon. Both strategies have high practical value: the first enhances the reliability of clinical results, and the second facilitates rapid monitoring of quality in biotechnological processes. Further research should focus on improving the sensitivity and specificity of these methods to meet the increasing demands of modern diagnostics and industry [22–24].

Phage display—origins

Phage display is a molecular biology technique that involves presenting proteins or peptides on the surface of a bacteriophage. In 1985, George Smith proved that it was possible to present proteins that were attached to the capsid of filamentous phage f1. More importantly, he could do this without losing the virus’s infectivity and preserving the protein’s function [25, 26]. This method has evolved over the years and is now utilized in various fields of molecular biology, medicine, and diagnostics. One of the first to utilize this method was the adalimumab antibody, which is used to treat rheumatoid arthritis, psoriasis, and inflammatory bowel diseases. Thanks to contributing to such tremendous development in many fields, the creator of this method was awarded the Nobel Prize in Chemistry in 2018 [27].

Principle of phage display

The phage display method is based on introducing phage genomes that encode relevant proteins or peptides. They are then presented on the surface of the phage. The entire process begins with the introduction of the sequences of interest (proteins or peptides) into the phage genome to be presented on the surface [28, 29]. It must be inserted into the gene encoding the capsid proteins to be presented. The most common genes are pIII or pVIII [29–31]. In the next step, the bacteriophage infects the corresponding bacterial cell. Replication of the gene along with the newly introduced gene occurs there. Proteins synthesized on the phage’s surface are deposited on it. A crucial step is establishing a library. Phages multiply to present a variety of proteins and peptides on their surface. These libraries are created to contain as many bacteriophages as possible, presenting up to hundreds of millions of variants. This enables the identification of individuals with the desired characteristics, such as the ability to bind to the appropriate receptor. Those best-fitting bacteriophages are incubated with a targeted protein or peptide to select from among so many variants. Selection is then performed on those with the strongest affinity [28, 30, 32].

Types of bacteriophages used

Bacteriophage M13—the prototypical Ff (filamentous) inovirus—remains the workhorse of phage display because of its non-lytic, chronic infection of E. coli, simple genetics, and flexible display formats. Structurally, it is a ~ 930 nm × ~ 6 nm filament that packages a circular ssDNA genome of ~ 6407 nt in a helical array of ~ 2700–3000 copies of the major coat protein pVIII, capped by minor proteins pIII/pVI and pVII/pIX (~ 5 copies each). Host recognition is mediated by pIII binding to the F pilus; assembly and secretion occur through the periplasm, which favors the display of disulfide-bonded and secretory proteins [33, 34]. In practice, pVIII libraries support multivalent display of short peptides (high avidity but stringent size limits), whereas pIII libraries tolerate larger inserts, including antibody fragments and folded domains; alternative fusions to pVI/pVII/pIX are also described to tune copy number and orientation [35, 36]. Library formats span phage or phagemid + helper phage, routinely reaching ~ 10⁵–10¹² diversity; ORF-enrichment strategies (e.g., pHORF/Hyperphage) improve in-frame pIII fusions and selection efficiency—useful for antigen libraries derived from complex transcriptomes [36, 37].

These features underpin M13’s central role across ligand and epitope discovery, antibody selection, diagnostics, and targeted delivery, including oncology applications where display-selected peptides/antibodies support immunodiagnostics and targeting strategies [38]. M13 is also a versatile biosensing scaffold: a recent engineered truncated M13 (tM13) enabled rapid, immunomagnetic capture and dark-field quantification of E. coli (limit of detection ~ 10 CFU/µL; ~ 30 min workflow), illustrating how M13’s biology and surface chemistry translate into fast diagnostics [39].

Bacteriophage T7 is widely used in biotechnology and synthetic biology due to its well-characterized genetics, high replication speed, and versatility as a vector system. It is particularly known for its ability to infect Escherichia coli, and for its structural properties—a double-stranded DNA genome encapsulated in a robust icosahedral capsid [40]. One of its most valuable features is its cloning capacity, enabling insertion of foreign DNA fragments up to approximately 3,000 base pairs, which makes it superior to commonly used filamentous phages like M13 in terms of library size and gene insert stability [41]. T7 exhibits high environmental stability, remaining active at low pH and elevated temperatures, and does not rely on the host’s secretion pathways, which avoids issues of steric hindrance when expressing foreign peptides or proteins. These traits have been successfully exploited in phage display applications, particularly in the fields of antigen discovery, vaccine development, and targeted cancer diagnostics [41].

Recently, T7 has been engineered for immunodiagnostic purposes. In a notable study, researchers constructed a nanobody phage display library using a T7-based system and identified several single-domain antibody (VHH) clones that bound specifically to chicken dendritic cells (DCs). Among these, phage-54 and phage-74 demonstrated strong affinity not only for chicken DCs, but also for those of duck and goose. These nanobody-decorated phages significantly enhanced antibody production in immunized chickens, suggesting their potential for avian DC-targeted vaccine strategies [42].

In another study, T7 was applied in the development of biosensors for the detection of Brucella abortus, showcasing its capability to serve as a reporter phage via the integration of luciferase genes. The resulting assay enabled rapid and specific detection of the pathogen, reinforcing T7’s diagnostic versatility [36].

Furthermore, modern approaches have explored the use of T7 in synthetic biology platforms to detect and kill bacterial pathogens through engineered gene circuits, fluorescent reporters, and antibiotic-sensitizing payloads, highlighting its role in both diagnostics and targeted bacteriophage therapy [43].

Compared to M13, T7 allows for faster plaque formation (within 3 h), supports direct fusion of exogenous genes with capsid protein gp10B, and tolerates a wider range of insert sizes. These advantages collectively make T7 an increasingly favored vector in both experimental and applied microbiology [41].

Bacteriophage T4, a lytic double-stranded DNA virus that infects Escherichia coli and is among the most thoroughly studied phages in molecular biology. With a large capsid (~ 120 × 86 nm) and a genome of approximately 169 kb, T4 is a structurally complex bacteriophage that encodes more than 50 different proteins [44]. Two non-essential but highly valuable capsid proteins—Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein)—serve as anchoring sites for foreign peptides and proteins, allowing high-density surface display without affecting phage infectivity or stability [45].

Compared to filamentous phages like M13, T4 enables the presentation of larger and more complex protein structures, including multimeric domains and conformational epitopes. Moreover, display using T4 can be achieved both genetically (in vivo) and post-purification (in vitro), by expressing antigens fused to Hoc or Soc in E. coli, purifying them, and assembling them onto Hoc⁻/Soc⁻ phage capsids. This modular system decouples protein expression from phage production, offering precise control over antigen density and configuration [45].

T4 has shown remarkable potential as a platform for next-generation vaccines. For instance, multivalent vaccines displaying Bacillus anthracis antigens—protective antigen (PA), lethal factor (LF), and edema factor (EF)—have been constructed on T4 capsids and demonstrated strong humoral and cellular immune responses in murine models [46]. More recent work has engineered T4 particles to co-display HIV antigens, such as p24, gp41, and Nef, thereby mimicking the natural epitope architecture and significantly enhancing antigenicity [47]. Beyond vaccine design, T4 phage has also been explored as a nanocarrier for diagnostics and targeted therapy. Its large structural capacity and surface versatility enable conjugation with DNA, proteins, or drugs, allowing for the delivery of complex payloads without the need for phage replication in the target cell [47].

Bacteriophage T4 is being engineered into a high-capacity, modular vector whose icosahedral head can be reloaded in vitro by the ATP-driven gp17 motor with ~ 171 kb DNA payloads, while its exterior is densely and programmably decorated via Hoc/Soc to add targeting ligands, cell‐penetrating peptides, or immune modulators—together enabling concurrent gene + protein (“progene”) co-delivery and robust transgene expression in vitro and in vivo [48]. Recent artificial viral vectors (AVVs) based on T4 add a lipid layer and achieve multiplex genome operations in human cells—editing, recombination, replacement, expression, and silencing—and can ferry very large plasmids, including full-length dystrophin, highlighting a programmable route to genome remodeling at sizes difficult for many eukaryotic viral vectors [49]. Reviews emphasize that T4’s payload capacity, surface modularity, and lack of mammalian replication provide a favorable safety and manufacturability profile compared with integrating vectors, while also outlining design levers for cell-specific targeting, endosomal escape, and nuclear delivery [50, 51]. Beyond nucleic acids, T4 nanoparticles can be co-functionalized to synergize with gene therapy—e.g., catalase/Chlorin-e6 formulations that relieve tumor hypoxia and potentiate photodynamic therapy—illustrating how the same chassis supports combination cargoes for oncology. Key challenges under active optimization include efficient entry and trafficking in primary human cells, control of biodistribution and immunity, and scalable, consistent production for clinical translation [50, 51].

Technically, working with T4 is more challenging than with M13 or T7. T4 follows a strictly lytic cycle, which complicates iterative selection rounds (biopanning), and construction of display libraries requires more advanced genetic engineering. Nevertheless, its high physical stability, large capsid surface area, and dense multivalent display capacity make T4 a powerful tool for synthetic biology, immunotherapy, and precision diagnostics.

Bacteriophage λ (lambda) is a temperate coliphage with a 48.5 kb double-stranded DNA genome packaged in an icosahedral head and delivered by a flexible tail. Its head is built from ~ 415 copies of gpE and ~ 405–420 copies of the decoration protein gpD; the tail comprises disks of gpV. After infection, λ can enter lysogeny or a lytic cycle that assembles capsids in the cytoplasm and releases progeny by lysis—features that remove the periplasmic-secretion constraint of filamentous systems and favor display of cytoplasmically folded proteins. In λ-display, foreign sequences are fused to gpD (high-density head display) or gpV (tail display); gpD is especially powerful, supporting very high decoration densities (reports up to ~ 90% of gpD copies) and enabling efficient selection of low-affinity binders [52]. Libraries of ~ 10⁷–10⁸ independent clones are readily built using λ packaging extracts, and have been applied to antigen/domain mapping, antigen discovery from complex cDNA or genomic repertoires, and functional genomics across pathogens (e.g., HCV, Toxoplasma gondii, Streptococcus pneumoniae) [53].

Head display on λ can exceed the effective density achievable on M13 and improves recovery from gene-fragment libraries: for multiple HIV-1 p24 fragments, gpD fusions on λ showed ≥ 100-fold stronger display than equivalent M13 constructs, with markedly less degradation and robust epitope enrichment[54]. Notably, λ can also present functional scFv antibodies as gpD fusions despite cytoplasmic assembly, retaining specific antigen binding and supporting selections, with both N- and C-terminal fusion formats validated [55].

Compared with M13 and T7: λ combines cytoplasmic assembly (like T7) with very high display valency via gpD, making it well-suited for complex ORF/cDNA libraries and for selecting low-to-moderate affinity interactions; M13 remains strong for periplasmic/disulfide-rich targets, while T7 often excels for very large inserts and rapid lytic workflows [53–55].

Across display platforms, four benchmark coliphages offer complementary strengths. M13 (filamentous, non-lytic) assembles through the periplasm and enables multivalent peptide display on pVIII or larger inserts on low-copy pIII, supporting very large libraries for ligand/epitope discovery, antibody selection, diagnostics, and biosensing. T7 (icosahedral, lytic) assembles in the cytoplasm, tolerates larger gp10B fusions, forms plaques within hours, and performs well for antigen discovery, reporter assays, and synthetic-biology kill/detect circuits. T4 (large, lytic) uses Hoc/Soc as high-density anchoring sites to present sizable or multimeric antigens, enabling modular in vivo or post-purification assembly for potent vaccine scaffolds and nanocarrier applications. λ (lambda) (temperate) combines cytoplasmic assembly with exceptionally high-density head display via gpD, making it effective for selections from complex gene-fragment or cDNA libraries and compatible with scFv display. In practice, choose M13 for disulfide-rich/secretory targets and massive libraries, T7 for rapid cycles and larger inserts, T4 for multimeric or structurally complex antigens, and λ when very high display density or robust recovery from gene-fragment libraries is needed.

Phage display—identification of biomarkers

This technique enables the identification of biomarkers for relevant diseases by matching peptides or proteins specific to them, thereby personalizing diagnostics for individual patient needs. One of the methods currently developed is the ORFeome technology. It involves studying entire proteomes to search for and identify immunogenic proteins. For this purpose, whole genomes are fragmented, multiplied, and then inserted into a phage vector. The genome fragments are then cloned into a unique system that allows the selection of ORFs (fragments of genetic material encoding functional proteins). When random cDNA or genomic fragments are cloned, most do not encode proteins. Statistically, only one in eighteen randomly selected DNA fragments forms a valid open reading frame (ORF) [56, 57]. The result is a library of proteins presented on the phage’s surface. Random non-functional DNA fragments are avoided by inserting only ORF sequences into the library [58]. The undeniable advantage of this technology is the reduction of the proportion of DNA fragments that do not encode the correct protein sequences, significantly increasing the chances of identifying specific protein interactions. This technology directly identifies immunogenic proteins using antibodies derived directly from patient serum. This enables the identification of only the immunogenic proteins present in the interaction between the host and the pathogen. So far, this method has been used, among other things, to identify immunogenic proteins of Salmonella Typhimurium, the pathogen that causes diarrhea. It has also been used to study the salivary proteins of ticks (Ixodes scapularis), which play a role in their feeding process [57, 59]. In addition, the technology was able to identify novel immunogenic proteins from Neisseria gonorrhoeae [60].

Phage display in HIV diagnosis

HIV has remained a global problem for many years despite numerous advances in the development of new treatments. The Phage display method has proven to be a valuable tool in the search for alternative options. It is used in two ways. The first step is to participate in identifying peptides that induce neutralizing antibodies, which could be potential components for vaccines. Libraries of peptides presented on bacteriophage surfaces are being created to map the epitopes of HIV-1 proteins, crucial to the virus’s proliferation. Studies have been conducted on monkeys that have been infected with these proteins, leading to a decrease in viral load and protection from AIDS after exposure to HIV-1. Thus, Phage display can contribute to the development of vaccines and a better understanding of the HIV-1 virus [61, 62].

The Phage display method has also discovered peptides that inhibit HIV-1 replication. They act on key steps in the replication cycle of this virus, disrupting their function [61]. Among other things, it was discovered that peptides involved in integrase binding (e.g., FHNHGKQ) can stop the integration of the virus’s genetic material into the host genome [63]. Through the discovery of these and other proteins that inhibit HIV-1 replication, Phage display may contribute to the discovery of drugs with higher efficacy than the treatments developed to date [61].

The discovery of peptides applicable to the search for alternative HIV treatments or the identification of biomarkers through Phage display is crucial for phage diagnostics. It demonstrates the significant potential of this technique for diagnosing and identifying specific molecules.

Use of bacteriophages in the diagnostics

Use in the diagnosis of tuberculosis

Tuberculosis is a bacterial disease caused by the bacterium Mycobacterium tuberculosis (mycobacteria) and is one of the most dangerous infectious diseases, leading to numerous deaths worldwide. In addition, the incidence of the disease is on the rise. According to the Global Tuberculosis Report 2022, for the first time in two decades, there has been a 4–5% increase in tuberculosis cases. In addition, the number of deaths caused by the disease increased from 1.5 million in 2020 to 1.6 million in 2021 [64]. The WHO launched the END-TB strategy in 2015, which aimed to reduce the number of TB cases through early detection, treatment, and prevention. The morbidity and mortality rates have remained unchanged due to the overall ineffectiveness of existing treatment and diagnostic methods [65]. It is particularly important to develop new diagnostic techniques, as the current ones have limitations in detecting early active tuberculosis or latent infection [66].

Bacteriophages have gained increasing popularity in the diagnosis of tuberculosis in recent years. FASTPlaqueTB was first developed as a test using them. It was used to detect Mycobacterium tuberculosis in sputum. It used bacteriophage D29 because of its ability to infect a wide range of mycobacteria. The test detects the proliferation of bacteriophages on host cells present in the sample. Its sensitivity was high, but similar to sputum microscopy, which limited its advantage over traditional methods [65, 67]. Later, the technique was further enriched by combining it with PCR tests to identify the pathogen more accurately. Samples containing bacteria are infected with phage. After the phage replicates and lysing the bacterial cells, the released DNA is detected by PCR [65, 68]. The most significant advantage of this method is that it detects only live bacterial cells, since bacteriophage multiplies only in live ones, and, in addition, it is susceptible [65, 69].

The newest and most streamlined method for using phages in TB diagnosis is the Actiphage method developed by PBD Biotech [70]. This method is beneficial when standard diagnostic methods are inadequate, such as in people with difficult-to-detect bacteria levels in blood samples. The technique uses phages that infect only live bacterial cells. Blood is drawn from the patient and subjected to preparation. Monocytes (PBMC) are then separated from the blood, removing interfering elements and concentrating the bacteria. The phages multiply inside the host cell, eventually leading to its breakdown and the release of the bacterial genetic material, among other things. The released bacterial DNA is then isolated and multiplied, enabling precise detection. The entire analysis takes 6–12 h, which allows extremely fast and more than precise diagnostics. In clinical studies using this method, the presence of Mtb in the blood was demonstrated in 73% of patients with active pulmonary tuberculosis, which indicates high sensitivity. In addition, the specificity was 100%, as the test did not detect Mtb in people with other conditions. Actiphage Rapid detects the presence of bacteria even in subclinical stages of the disease. The technology can also be used to monitor the risk of TBI progressing to active TB [65, 70–72].

Use of bacteriophages in the diagnosis of MRSA

Some of the most common nosocomial infections are those caused by methicillin-resistant Staphylococcus aureus (MRSA). It poses an exceptionally high risk due to its high resistance to many antibiotics, which makes treatment more difficult and increases the risk of complications. It is hazardous for hospitalized and immunocompromised patients [73, 74]. Therefore, it is necessary to develop more accurate and rapid diagnostic tests. Those currently in use have some limitations. For example, PCR or culture are costly, time-consuming, and require adaptation to appropriate MRSA strains [75, 76]. Developing new methods can help stop the spread of these pathogens and enable more effective treatment. A promising alternative to current diagnostic methods is bacteriophages that attack specific bacterial strains [76, 77].

One method under development includes a low-cost and rapid diagnostic test for MRSA, explicitly based on bacteriophages, using NanoLuc luciferase. This test is designed to detect MRSA in nasal samples [76]. This test uses bacteriophages ISP and MP115, which are widely used in phage therapy and diagnostic tests [76, 78]. Luciferase (NanoLuc) was inserted into the genome of the bacteriophage. It is supposed to generate a light signal to indicate that the phage has infected the bacteria [79]. The test also uses the antibiotic cefoxitin, which selectively limits the growth of methicillin-sensitive strains (MSSA). Finally, light is measured, and its signal indicates the presence of MRSA in the sample. Studies have shown that the test recognizes 97.7% of clinical MRSA strains, achieving 100% detection at higher bacterial concentrations (100–1000 CFU). It also showed high specificity, excluding MSSA or other strains. The method can provide results within 6 h, making it extremely practical for MRSA diagnosis. However, the low number of false positives requires further research to improve the method [76].

Use of bacteriophages in cancer diagnostics

Bacteriophages can also be an extremely valuable diagnostic tool in detecting bacterial diseases. Thanks to the previously discussed Phage display method, it can participate in cancer diagnostics. Bacteriophages can be used as molecular diagnostic probes. By presenting appropriate peptides on their surface, they can bind to cancer-specific markers, identify them, and distinguish them from healthy tissues [80, 81]. Additionally, presenting phages can be labeled with various substances, such as nanoparticles for precise imaging or fluorescent markers for microscopic analysis [82]. Such additional molecular imaging makes it possible not only to detect the tumors themselves but also their metastases. A fascinating example is the use of Phage typing in presenting peptides directed against the CEA antigen, a marker associated with colorectal cancer [83].

The CEA antigen is overexpressed in cancer cells but in minimal amounts in healthy tissues, making it an ideal diagnostic target. Presentation phages show high specificity in binding to CEA, allowing their use as diagnostic probes in various molecular imaging techniques [81, 83]. Such an application is very promising but has some limitations. These include the need for the precise selection of appropriate phages and diagnostic targets, which requires advanced methods of peptide selection from phage libraries. This process is time-consuming and requires accuracy to provide specific diagnostics for individual cancers.

Advantages and challenges

Bacteriophages, viruses that infect only bacteria, are a unique and promising tool in diagnosing and treating bacterial infections. Their use is becoming increasingly important in the face of the growing antibiotic resistance crisis, which is currently one of the most significant challenges to public health. In 2019, this resistance contributed to 1.27 million deaths worldwide, and forecasts indicate that by 2050, the number of deaths related to antibiotic-resistant bacteria could exceed the number of deaths caused by cancer, reaching more than 10 million per year. In this situation, bacteriophages, as a precise and effective tool, are becoming a real alternative to traditional therapies [84, 85].

Bacteriophages are characterized by their ability to selectively attack only specific bacterial strains, making them highly effective in combating infections caused by multi-resistant pathogens, such as those from the ESCAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). Their specific action allows for the avoidance of destroying the body’s natural microflora, a significant problem associated with broad-spectrum antibiotics. Bacteriophages, unlike antibiotics, can also evolve in response to bacterial defense mechanisms, which ensures their long-term effectiveness in combating infections [84–86].

One of the key advantages of bacteriophages is their ability to penetrate and break down bacterial biofilms, which pose a significant challenge in treating chronic infections, such as those associated with medical implants or catheters. Phage enzymes, such as endolysins, act directly on biofilm structures, breaking down the protective matrix of bacteria and enabling effective treatment. Moreover, bacteriophages can be used in combination with antibiotics, which in some cases leads to a synergistic therapeutic effect, increasing the effectiveness of therapy even in complex cases [87, 88].

The use of bacteriophages in diagnostics is based on their high specificity and ability to lyse bacteria quickly. Thanks to reporter phages, the presence of pathogens in biological or environmental samples can be identified in real time, using signals generated during the lysis process. This enables a significant reduction in the time required for diagnosis compared to traditional culture methods. An example of this is diagnostic systems based on phages functionalized with fluorescent markers, which enable precise detection of the pathogen within minutes or hours [5, 89, 90].

Despite the numerous advantages of bacteriophages, there are also limitations to their use. One of the key challenges is the potential for bacterial resistance to develop against phages. Mechanisms such as mutations of bacterial receptors, CRISPR-Cas systems or restriction-modification systems can limit the effectiveness of phage therapy. The use of phage cocktails, containing different types of phages, is one of the most effective strategies to counteract resistance, allowing for the maintenance of therapy effectiveness and reducing the risk of bacterial adaptation [85, 91].

Another challenge is the body’s immune response to repeated administration of phages, which can lead to their neutralization. Additionally, phage lysis is associated with the release of endotoxins, which can trigger inflammatory reactions in patients. In response to these challenges, new technologies are being developed, such as phage engineering or encapsulation, which aim to improve their stability, efficacy, and safety [85, 88].

Bacteriophages are increasingly utilized in personalized therapy, particularly in severe bacterial infections where traditional methods are ineffective. Examples include successful therapies for diseases caused by multi-resistant bacteria, such as Pseudomonas aeruginosa or Klebsiella pneumoniae. Innovative programs, such as IPATH (Innovative Phage Applications and Therapeutics) in the USA or national phage banks in Belgium, enable the rapid and efficient matching of phages to specific bacterial strains, thereby increasing the chances of successful therapy [85, 87, 92].

Bacteriophages are a tool with enormous potential that can revolutionize the treatment and diagnosis of bacterial infections. Their unique properties, such as specificity of action, the ability to eliminate biofilms, and adaptation to changing conditions, make them a promising alternative in the face of the growing problem of antibiotic resistance. Production technology development, standardization of procedures, and further clinical trials are crucial to exploit their potential in healthcare worldwide fully.

Author contribution

P.O, M.S, K.S, A.O and B.G wrote the main manuscript text and prepared Fig. 1. All authors reviewed the manuscript.

Funding

This research was funded by the National Centre for Research and Development project number LIDER/12/0069/L-12/20/NCBR/2021.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.