Engineering bacteriophages for gut health: precision antimicrobials and beyond.

Introduction
In recent years, the global burden of gastrointestinal diseases has continued to increase, with approximately 8 million deaths annually attributed to disorders of the digestive system, among which inflammatory bowel disease (IBD), colorectal cancer (CRC), and infectious enteritis have drawn particular attention [1]. Epidemiological studies have shown that the incidence of IBD continues to rise in newly industrialized countries and, although stabilized in Western nations, still affects more than 0.3% of the population [2]. CRC remains a leading cause of cancer-related incidence and mortality worldwide, accounting for approximately 7% of newly diagnosed cancers and 11% of cancer deaths [3]. More concerningly, Clostridioides difficile infection (CDI), as a representative of pathogen-associated enteritis, has emerged as one of the top five most urgent antibiotic resistance threats in the United States due to its increasing resistance to antibiotics [4]. Despite differences in pathogenesis, these diseases are all closely associated with gut microbiota dysbiosis, typically marked by diminished microbial diversity, increased relative abundance of pathogenic bacteria, and chronic low-grade inflammation [5]. These features impose considerable challenges to conventional antimicrobial and anti-inflammatory treatments. In clinical practice, antibiotics may exacerbate microbiota imbalance (“therapeutic paradox”), immunosuppressants increase the risk of opportunistic infections, and chemotherapeutic agents may cause irreversible mucosal injury [6, 7]. Therefore, there is an urgent need for novel therapeutic strategies that can selectively eliminate pathogens while preserving or restoring microbial homeostasis.
Bacteriophages are viruses that infect and lyse bacteria. Owing to their high host specificity and ability to self-replicate, they represent the most abundant biological entities in nature and offer unique advantages for controlling bacterial infections. Phage therapy, initially introduced by Félix d’Hérelle in the early 1900 s, has shown notable antibacterial potential in both preliminary clinical trials and laboratory studies [8]. However, with the widespread use of antibiotics, phage research was largely marginalized until its recent resurgence, prompted by the growing crisis of antibiotic resistance [9]. In contrast to the broad-spectrum and non-specific nature of antibiotics, phages target specific bacteria while largely sparing beneficial microbiota [10]. Nonetheless, naturally occurring phages often exhibit a narrow host range and remain undercharacterized; only a small proportion of known phages have been fully sequenced and evaluated for host specificity, and their representation in public genomic and proteomic databases remains limited [11]. Moreover, in the complex gastrointestinal environment, phages are often rapidly cleared by the host immune system, which further restricts their clinical application [12, 13].
To overcome these limitations, researchers have increasingly turned to genetic engineering, chemical modification, and nanotechnology to create engineered phages with enhanced clinical utility. Advances in synthetic biology have enabled the creation of phages with broadened host spectra, reduced immunogenicity, and customizable functions such as drug delivery [14]. For example, He et al.. developed an aggregation-induced emission (AIE)-modified phage conjugate (AIE–PAP) by combining phage PAP with photodynamic therapy agents, which conferred enhanced bacterial imaging and synergistic bactericidal capabilities [15]. In addition, nanoplatform-assisted systems—such as DNA nanopatch-phages (DNPs@P)—represent a promising direction for targeted bacterial elimination and immune microenvironment modulation in gastrointestinal diseases [16]. These modifications not only improve the ability of phages to lyse pathogens, disrupt biofilms, and regulate the gut microbial composition, but also enhance their physicochemical stability and mucosal targeting within the gastrointestinal tract when integrated with nanomaterials and surface functionalization strategies [17–19]. Moreover, the emergence of chimeric phages and phage–antibiotic combination therapies has further expanded the scope of phage applications in gastrointestinal diseases [20]. The advancement of engineered phage platforms is therefore essential not only for therapeutic efficacy but also for driving innovation at the interface of microbiology, synthetic biology, and material science.
This review aims to systematically explore the therapeutic applications and potential of engineered bacteriophages in gastrointestinal diseases (Fig. 1). We begin by summarizing the basic principles of phage therapy and outlining current engineering strategies, including genetic and chemical modifications that have significantly improved phage efficacy and expanded their functional repertoire. We then focus on the applications of engineered phages in IBD, CRC, and infectious enteritis, highlighting recent preclinical advances and underlying mechanisms. Ultimately, we evaluate the major advantages and translational challenges of engineered phages—such as the trade-off between specificity and breadth, colonization efficiency in the complex gut environment, production scalability and stability, and bacterial resistance evolution—and offer a perspective on their future integration into precision and personalized medicine. This review intends to support future research and clinical translation of engineered phage-based therapies for gastrointestinal health and disease.

Mechanisms of phage therapy
Most bacteriophages consist of a protein-based tail and a head that encapsulates the viral genome. The tail structure enables the precise recognition of receptors on the surface of host bacteria [21]. Phages are typically classified into two categories according to their replication strategies postinfection: lytic and temperate (lysogenic) phages [22]. As viruses, phages modulate bacterial communities either through direct lysis or via lysogenic cycles (Fig. 2). Lytic phages directly destroy pathogenic bacteria, while temperate phages may influence host metabolism and virulence through horizontal gene transfer [23]. Phage therapy harnesses these naturally occurring “bacterial killers” to specifically infect and lyse target pathogens, providing a therapeutic strategy for bacterial infections. Beyond direct bactericidal activity, phages can modulate the composition of the gut microbiota and host immune responses, contributing more broadly to intestinal homeostasis [24].

Direct bactericidal activity
The antibacterial activity of phages primarily stems from their ability to recognize and lyse specific bacterial hosts. Through the use of tail fiber proteins, phages bind to bacterial surface receptors such as lipopolysaccharides, flagella, or pili, inject their genome, and initiate the lytic cycle. Once inside the host, the phage hijacks bacterial biosynthetic systems to replicate its genome and produce structural proteins, eventually assembling them into progeny virions. Upon reaching a critical number, phages express holins to perforate the bacterial inner membrane, allowing lysins to degrade the peptidoglycan layer, culminating in cell lysis and liberation of progeny phages for infection propagation [25–27]. For example, the ΦCD27 phage specifically lyses toxin-producing Clostridioides difficile strains, significantly reducing toxin release and mitigating the severity of pseudomembranous colitis [28].

Indirect modulation
In addition to direct lysis, phages can influence host physiology by reshaping the gut microbiome and modulating immune system. Their host specificity allows selective removal of pathogens and restoration of microbial balance, reducing inflammation and improving barrier function [29, 30]. Moreover, phage-mediated lysis reduces the bacterial release of pro-inflammatory components like lipopolysaccharides (LPS), indirectly modulating host immune responses. This can suppress the overproduction of pro-inflammatory cytokines and mitigate tissue damage [31]. Phages can also modulate immune cells, especially neutrophils, to promote the resolution of inflammation. This effect has been demonstrated in models of multidrug-resistant Klebsiella pneumoniae infection [32].
Phages also possess a self-replicating nature, allowing them to accumulate at infection sites and maintain high local concentrations, thereby dynamically suppressing emerging resistant strains [33]. In the ongoing co-evolutionary arms race, bacteria develop defenses such as CRISPR-Cas and restriction-modification systems, while phages counter these through anti-CRISPR proteins and receptor-binding adaptations [34–37].
In summary, phages exert their therapeutic effects not only through direct bacterial lysis but also via indirect mechanisms, including microbiota modulation, immunomodulation, and suppression of resistance development. With advances in genetic engineering and high-throughput screening, future phage optimization is expected to enhance these indirect functions, thereby accelerating the clinical application of phage therapy in microbiota-associated gastrointestinal disorders.

Engineering strategies of phages
Advances in molecular and synthetic biology have diversified engineering strategies for bacteriophages, addressing limitations such as narrow host range, high immunogenicity, and bacterial resistance. Synthetic biology enables rational phage design through genome editing and chemical modification, allowing functional modules such as enzybiotic genes for targeting phage-resistant bystander cells or diagnostic payloads for pathogen detection to be incorporated into phage genomes [38, 39]. Chemical conjugation with nanomaterials enhances phage stability in harsh gastrointestinal environments. Together, these approaches eliminate unwanted genes and enable safer, more stable, and versatile engineered phages.
Synthetic biology enables rational phage design through genome editing and chemical modification, allowing functional modules such as enzybiotic genes for targeting phage-resistant bystander cells or diagnostic payloads for pathogen detection to be incorporated into phage genomes.

Genome editing
A suite of genome editing tools has been developed to modify phage genomes with varying precision and complexity (Fig. 3). Early methods such as homologous recombination enable gene insertion or replacement but suffer from low efficiency. Subsequent techniques like Bacteriophage Recombineering of Electroporated DNA (BRED) significantly improved editing efficiency using short homology arms and the λ-Red system. For large and complex genomes such as T4, yeast-based platforms offer robust assembly capabilities, while in vitro strategies including Gibson assembly allow for full genome synthesis. More recently, CRISPR–Cas systems have enabled precise multi-site editing, and cell-free transcription–translation (TXTL) platforms support rapid in vitro phage production and screening.

Although these strategies differ in mechanism, they are complementary in application: homologous recombination and BRED are well suited for targeted gene modification, yeast-based and Gibson approaches facilitate large-scale genome assembly, CRISPR–Cas systems provide high-precision control, and TXTL enables rapid prototyping in vitro. Together, these tools offer a balanced framework for tailoring phage infectivity, safety, and therapeutic performance across diverse experimental and clinical contexts. The main features of these genome editing platforms are summarized in Table 1.

Homologous recombination
Homologous recombination is a conserved intracellular DNA repair mechanism that utilizes similar or identical DNA sequences as templates to achieve precise exchange of DNA fragments (Fig. 3A). By leveraging this natural process, researchers can recombine plasmids containing desired modification sequences with phage genomes, enabling targeted insertion, deletion, or replacement of key functional genes [40].
For example, in phage genome editing, designed DNA fragments with homologous arms can induce recombination with the target region in the phage genome, allowing fine-tuned control of phage functionality [41]. Using this approach, Zhang et al.. replaced the long tail fiber protein (pb1) of the narrow-host-range T5-like phage vB STyj5–1 with the corresponding region from the broad-host-range phage vB BD13. This modification successfully expanded the host range from 20 to 30 strains and improved adsorption rates from 0.28 to 28.84% to 28.10–99.49% [42]. Although homologous recombination is relatively straightforward to perform, its efficiency is generally low and depends on the endogenous recombination systems of the host bacteria.

Bacteriophage recombineering of electroporated DNA (BRED)
BRED is a recombination engineering strategy specifically designed for phage genomes (Fig. 3B). It involves electroporating editing templates directly into phage-infected bacterial cells, where high-efficiency homologous recombination enables precise modification of large genomic segments [43]. This technique allows for the insertion, deletion, and mutation of large DNA fragments with high recombination efficiency and flexibility. First reported by Marinelli et al.. in 2008, BRED has been widely used in the construction of mutant phages [43]. It facilitates genome modification during natural phage replication cycles and is particularly effective for generating customized phage libraries and rapidly screening engineered phages with expanded host ranges. With reported recombination efficiencies of 3.4% to 22.2%, BRED overcomes the limitations of traditional low-efficiency recombination techniques and is considered an important tool in phage engineering [44].

Yeast-based platforms
Yeast platforms exploit the robust homologous recombination machinery of Saccharomyces cerevisiae to assemble and manipulate large or complex phage genomes in vitro (Fig. 3C). Yeast artificial chromosomes (YACs) have a large DNA carrying capacity and are insensitive to potentially toxic phage-encoded proteins such as endolysins and bacteriocins. Co-transformation of yeast cells is performed using multiple overlapping PCR fragments encompassing the complete phage genome and linearized YAC vectors [45]. Homologous recombination at the terminal regions facilitates integration of the phage genome into the YAC, which is subsequently isolated and transferred into Escherichia coli (E. coli) for recombinant phage rescue [45]. This strategy circumvents the handling difficulties posed by large or toxic phage genes in bacterial systems and enables multiplexed, high-fidelity editing across entire genomes.

Gibson assembly
Gibson assembly utilizes a combination of enzymatic reactions—specifically exonuclease, DNA polymerase, and DNA ligase—in a single isothermal reaction to seamlessly join multiple DNA fragments with overlapping ends into a complete DNA molecule [46]. This method is simple, efficient, and does not require restriction enzyme recognition sites, making it particularly suitable for the synthesis and editing of entire phage genomes (Fig. 3D). Using Gibson assembly, researchers can construct phage genomes ranging from several hundred to thousands of kilobases in vitro, and subsequently “rescue” and validate phage function via transformation or TXTL systems [38].

CRISPR-Cas technology
CRISPR–Cas systems allow for precise, site-specific editing of phage genomes by introducing double-strand breaks (DSBs) at target loci via programmable guide RNAs (Fig. 3E). These breaks can then be repaired through homologous recombination pathways, allowing for the precise insertion or deletion of target sequences [47]. This approach enables high-precision genome editing and is especially useful for deleting immunogenic genes from phages or inserting anti-CRISPR modules to enhance phage stability in vivo [48, 49].
CRISPR-Cas9 has been successfully used to edit the genome of virulent Klebsiella phages, enabling point mutations, gene deletions, and gene exchanges using short homology arms [50]. Recent studies further demonstrate that combining CRISPR-Cas systems with homologous recombination significantly improves both the efficiency and biosafety of phage engineering. For instance, Guan et al.. integrated homologous recombination with RNA-targeting CRISPR-Cas13a, using the anti-CRISPR gene acrVIA1 as a selectable marker, to insert foreign genes, delete genes, and fluorescently label genes in the genome of jumbo phage Pseudomonas ФKZ [51].

Cell-free transcription–translation (TXTL) systems
Cell-free TXTL systems reconstruct phage assembly in vitro by transcribing and translating synthetic genomes outside living cells [52]. This method bypasses the low transformation efficiency often encountered in bacteria and allows rapid functional testing of phage genomes (Fig. 3F). Rustad et al.. have successfully synthesized various phages—such as MS2, ΦX174, T7, and T4—using TXTL, demonstrating the feasibility of constructing complex biological entities entirely in test tubes [53]. Leveraging T7 phages and an E. coli-based TXTL system, Levrier et al. developed PHEIGES (Phage Engineering via In vitro Gene Expression and Selection), achieving up to 10^11 PFU/ml within one day and supporting high-throughput phage construction [54]. These cell-free systems open new avenues for studying fundamental biological processes and developing innovative biomedical applications.
Each genome-editing approach offers distinct advantages and application contexts. Homologous recombination and BRED are straightforward and well established, making them suitable for small-scale or targeted gene modifications, though they suffer from relatively low efficiency and dependence on host recombination systems. Yeast-based platforms enable manipulation of large and complex genomes independent of bacterial toxicity, offering flexibility for multiplexed editing but requiring additional transfer steps for phage rescue. Gibson assembly provides rapid, scarless in vitro construction and is ideal for synthetic or modular genome design, though it depends on the quality and length of DNA fragments. CRISPR–Cas systems introduce high-precision, site-specific modifications and can be combined with homologous recombination to enhance editing accuracy, yet their use may be limited by host defense mechanisms and off-target effects. Finally, TXTL systems facilitate high-throughput, cell-free synthesis and testing of phages, representing a powerful tool for rapid prototyping but still face challenges in scalability and cost [47]. Overall, integrating these complementary methods allows researchers to balance precision, efficiency, and scalability in engineered phage development.

Chemical modification
Chemical modification represents an additional strategy to enhance the stability and targeting ability of phages in the complex gastrointestinal environment. Surface functionalization of phages, including PEGylation, glycosylation, and nanoparticle conjugation, can protect them from degradation in gastric acid and from immune clearance. These modifications also enhance their retention and infection efficiency at target sites. For example, the encapsulation or surface modification of phages with nanomaterials results in the formation of “nano-phages,” which exhibit superior stability and specificity in simulated gastrointestinal conditions, offering a novel approach for the development of oral phage therapy (Fig. 4).

Polyethylene glycol (PEG) modification
PEGylation, one of the earliest surface modification techniques applied to viruses and drugs, entails the covalent linkage of PEG molecules to the phage surface proteins or capsid structures (Fig. 4A). This significantly enhances phage resistance to acidic conditions and protease degradation while reducing recognition by the host immune system [55]. Kim et al.. conjugate non-immunogenic methoxy-PEG (mPEG) to viral proteins, successfully prolonging phage circulation time in the bloodstream and reducing the release of T-helper 1-related cytokines such as IFN-γ and IL-6. In vivo, PEGylated A511 phages retained 83.7% of initial infectivity at 1.5 h and 20.9% at 6 h post injection, compared with only 3.3% and 0.016% for unmodified phages. Similarly, PEGylated Felix-O1 phages maintained 59.6% and 9.0% viability at 1.5 and 6 h, respectively, while native phages declined to 0.25% and 0.03% over the same period. Moreover, PEGylation reduced neutralization in mouse serum, with over 80% infectivity preserved for 4 h, whereas unmodified A511 phages dropped to 63.3% viability after 4 h of incubation [56]. These results suggest that PEGylation increases the survival of infectious phages by delaying immune responses, thereby improving therapeutic efficacy [56]. Compared to non-PEGylated solid lipid nanoparticles (SLNs), PEGylated SLNs (pSLNs) exhibit enhanced mucus penetration, improved stability in simulated intestinal fluid, and increased absorption efficiency [57].

Biodegradable polymer encapsulation
The encapsulation of phages using biodegradable materials, such as chitosan and sodium alginate, is another important strategy for improving in vivo stability and targeted delivery. This approach not only protects phages from gastric acid but also facilitates sustained release and targeted action in the intestinal tract (Fig. 4B).
Oral phage therapy for intestinal bacterial infections is often challenged by the harsh acidity of the stomach, which compromises phage viability during gastrointestinal transit. Ma et al.. develop a microencapsulation system using chitosan–alginate–CaCl₂ for the Salmonella-specific phage Felix O1, demonstrating remarkable protection under acidic and bile conditions. In simulated gastric fluid (SGF), free Felix O1 rapidly lost viability, becoming undetectable after a 5-min exposure at pH below 3.7, whereas microencapsulated phages showed only a 0.67-log reduction at pH 2.4 and 2.58-log loss after 1 h incubation with pepsin. In bile solutions (1%–2%), the free phage count decreased by 1.29–1.67 logs after 3 h, while encapsulated phages maintained full viability. Furthermore, encapsulated phages were completely released in simulated intestinal fluid (pH 6.8) within 6 h, retaining full activity for 6 weeks at 4 °C, and achieving 12.6% survival in dried form with trehalose as a stabilizer. These findings indicate that microencapsulation significantly improves phage survival through the gastrointestinal tract and facilitates targeted delivery of therapeutic phages to the gut [58]. Recent advancements employ pH-responsive polymers combined with compounds such as trehalose or sodium alginate to encapsulate Felix O1. These microcapsules provide superior acid protection and significantly inhibit bacterial growth, highlighting their potential for gastrointestinal-targeted delivery [59, 60].

Nanomaterial-based modification and integration
Nanoparticles serve as effective drug delivery carriers, enabling the phage to overcome multiple physiological barriers and enhance delivery efficiency [61]. Since the physical size of most phages falls within the classical nanomaterial definition (1–100 nm), they exhibit good compatibility with inorganic nanomaterials, thereby facilitating high conjugation efficiency [62]. The integration of phages with nanoparticles significantly prolongs their detectable presence in the bloodstream—up to 24 h longer than unmodified controls [63]. Gold nanoparticles are most commonly employed to stabilize T4-like phages for the detection of E. coli cells [64]. Furthermore, gold nanoparticle-modified phage probes can specifically target the DNA of Bacillus anthracis [65]. For cost-effective applications, silica nanoparticles have also been widely used due to their ability to conjugate efficiently with phages [66]. A one-step synthesis strategy has been developed to generate biofunctionalized silica nanoparticles using engineered phages for optical biosensing applications [67].
Combining phages with nanomaterials can endow them with additional functionalities (Fig. 4C). Li et al.. immobilized polyvalent PEL1 phages (Podoviridae family) on chitosan-coated Fe₃O₄-based magnetic carbon nanotubes (CNCs), enabling the phages to penetrate biofilms under a relatively low magnetic field and achieve effective bacterial inhibition [68]. Temsaah et al.. encapsulated phage HK6 in chitosan nanoparticles (CS-NPs), achieving a high encapsulation efficiency of 97%. This formulation significantly improved phage stability, therapeutic efficacy, and delivery to Enterobacter cloacae, retaining infectivity at alkaline pH values (11–12) and thermal stability up to 80 °C [69]. In a study by Park et al.., phages were assembled with cationic polymers to form phage-polymer nanostructures (PPNs), which were capable of penetrating bacterial biofilms and killing bacteria. In a mouse model, PPNs significantly reduced the bacterial load within methicillin-resistant Staphylococcus aureus (MRSA) biofilms [70].

Other chemical modification strategies
In addition to the approaches mentioned above, researchers have chemically conjugated phages with functional molecules such as antibody fragments and antimicrobial peptides (AMPs) (Fig. 4D). This strategy enables more precise and targeted modification. Vaks and Benhar tethered antibiotics to M13 phage particles via ester linkages and further conjugated them with Staphylococcus-specific antibody fragments. This dual-targeting strategy enabled the phages to aggregate near pathogenic bacteria and release high local concentrations of antibiotics, effectively killing even resistant strains [71]. Through genetic engineering, AMPs have been incorporated into phage capsid proteins, allowing the therapeutic payload to cross the blood–brain barrier and be released upon infection, thereby enhancing the antimicrobial activity against resistant bacteria [72].
In addition to antibody fragments and AMPs, phages can also be conjugated with imaging agents such as fluorescent molecules. These modifications not only provide phages with novel functionalities but also expand their utility in biomedical diagnostics and therapeutics. For instance, PAP phages conjugated with aggregation-induced emission luminogens (AIEgens) possessing photodynamic inactivation (PDI) activity have been developed into AIE–PAP bioconjugates. These constructs exhibit both bacterial imaging capabilities and synergistic bactericidal effects against specific pathogens [15]. In vitro and in vivo experiments confirm their selective targeting and killing of both antibiotic-sensitive and multidrug-resistant bacteria, demonstrating excellent biocompatibility.
In conclusion, chemical modification provides an essential route for the functional enhancement of engineered phages. These strategies not only improve phage stability and bioavailability in vivo but also introduce novel functionalities such as targeted delivery, imaging, and combined photothermal therapy. Such multifunctional modifications lay a robust foundation for the application of engineered phages in gastrointestinal disease treatment and open promising avenues for future precision medicine.

Applications of engineered phages in gastrointestinal diseases
The human gastrointestinal tract harbors a vast and complex microbial ecosystem, primarily composed of bacteria, archaea, viruses, fungi, and protozoa. These microorganisms play indispensable roles in nutrient metabolism, immune regulation, maintenance of the intestinal barrier, and protection against pathogenic invasion [73]. Recent studies have shown that dysbiosis, referring to an imbalance in the gut microbiota, is closely associated with gastrointestinal disorders including IBD, CRC, irritable bowel syndrome (IBS), and infectious enteritis [74]. Advances in metagenomics and synthetic biology have further unveiled the complexity of the human gut virome, particularly bacteriophages. In healthy individuals, the gut phageome is dominated by members of the order Caudovirales, whereas in patients with IBD or CRC, significant changes have been observed in phage diversity, the ratio of virulent to temperate phages, and host specificity [5]. These findings offer dual therapeutic opportunities: on the one hand, engineered phages can directly eliminate specific pathogenic strains, contributing to microbial homeostasis and attenuation of inflammation [75]; on the other hand, phages can modulate the microbiota–immune axis to promote the repair of intestinal barrier function. Promising therapeutic outcomes have been demonstrated in preclinical models of CDI, IBD, and CRC [76, 77]. Table 2 summarizes the applications of various engineered phages in the treatment of gastrointestinal diseases.

Inflammatory bowel disease (IBD)
IBD is a multifactorial disorder driven by an abnormal immune response to intestinal microbiota in genetically susceptible hosts. Extensive research has indicated that individuals with IBD, including Crohn’s disease (CD) and ulcerative colitis (UC), exhibit pronounced dysbiosis of the gut microbiota, marked by a reduction in commensal bacteria and a relative increase in potentially pathogenic microorganisms [95, 96]. This microbial imbalance undermines barrier integrity by impairing tight junctions, thinning the mucus layer, and promoting epithelial permeability, which allows microbial products to infiltrate the lamina propria and trigger local inflammation via pattern recognition receptors such as Toll-like and NOD-like receptors [95, 97]. This leads to excessive production of proinflammatory cytokines such as TNF-α, IL-6, and IL-17 and the recruitment of immune cells, thereby mediating chronic inflammation and tissue damage [97]. At the same time, reduced production of short-chain fatty acids (SCFAs), particularly butyrate, weakens immunoregulatory signaling, impairs epithelial renewal, and decreases the expression of anti-inflammatory mediators [98, 99]. The combined effect of barrier disruption, immune overactivation, and metabolic dysregulation contributes to initiation and progression of IBD. In Fig. 5, these processes are illustrated in parallel with the roles of different engineered bacteriophages that target gut microbiota imbalance, inflammatory signaling, and epithelial barrier restoration, which are discussed in detail in the following paragraphs.

Beneficial bacteria such as Faecalibacterium prausnitzii are typically abundant in healthy individuals, but their levels are significantly reduced in IBD patients, which is believed to be associated with their anti-inflammatory properties [100]. F. prausnitzii is a prevalent commensal species and is considered a key biomarker of intestinal health [101]. This depletion is especially evident in CD patients with ileal involvement [102]. The key anti-inflammatory mechanism of F. prausnitzii involves butyrate generation, which mediates essential functions in colonic mucosal homeostasis maintenance [103]. Its depletion in IBD and other gastrointestinal disorders has prompted researchers to consider it a potential probiotic candidate for therapeutic intervention [103]. However, its oxygen sensitivity presents a major challenge for formulation as a probiotic.
An increase in opportunistic pathogens, particularly adherent-invasive Escherichia coli (AIEC), is strongly linked to inflammation and mucosal injury in IBD, especially in Crohn’s disease [104]. AIEC colonizes the intestinal mucosa of CD patients, adheres to epithelial cells, and invades macrophages [76]. Recent studies have explored phage therapy as a promising strategy to target AIEC in IBD. Galtier et al.. demonstrated that a cocktail of three phages effectively reduced AIEC levels in both mouse models and human ileal biopsy samples (Fig. 5A). It significantly decreased AIEC abundance in fecal and mucosa-associated microbiota of both transgenic and wild-type mice, concurrently ameliorating colitis symptoms induced by dextran sulfate sodium (DSS). Preventive administration decreased fecal AIEC levels by nearly four orders of magnitude and significantly lowered the disease activity index within ten days, while curative treatment achieved a similar but milder effect, maintaining reduced inflammation for over two weeks. Furthermore, the phage cocktail actively replicated in ileal biopsies from Crohn’s disease patients, increasing by more than ten thousand-fold within 24 h. This result confirms its ability to effectively target AIEC in human tissues [76]. Notably, Titécat et al.. reported that the seven-phage cocktail (EcoActive™) effectively targeted 95% of clinical AIEC strains in vitro and, when administered twice daily over 15 days, markedly attenuated inflammation in a murine colitis model [78]. These findings suggest that phage therapy may be a safe and effective strategy to reduce AIEC colonization in IBD patients, potentially offering a new avenue for treatment.
Other gut pathogens, such as Klebsiella pneumoniae (Kp), are also thought to induce uncontrolled intestinal inflammation in IBD patients [105]. However, the feasibility of selectively targeting IBD-associated pathogens without disrupting the gut microbiota requires further investigation. Federici and colleagues developed a lytic five-phage cocktail tailored to clonal members of Kp associated with IBD, which effectively suppressed Kp in a murine colitis model and alleviated inflammation and disease severity (Fig. 5B). Proof-of-concept assessments conducted in an artificial human gut model and healthy volunteers demonstrated that these Kp-targeting phages were acid-resistant, safe, and capable of surviving in the lower gastrointestinal tract [79].
Persistent inflammation in IBD has been shown to increase colonization by Streptococcus gallolyticus (Sg), which in turn promotes colorectal cancer progression via activation of the cyclooxygenase-2 (COX-2) pathway and upregulation of β-catenin. Zhao et al.. developed a DNA nanoplatform–phage system (DNPs@P) targeting Sg, aiming to treat IBD and prevent colorectal cancer [16]. The core component consists of Sg-specific bacteriophages (P-Sg) that are chemically conjugated to DNA origami nanostructures (DNPs). The DNPs, functioning as robust reactive oxygen species (ROS) scavengers, protect the phages from the oxidative microenvironment of the inflamed gut, thereby preserving their lytic activity. Simultaneously, the phages execute precise targeting and lysis of Sg, directly reducing the bacterial load and interrupting the associated pro-tumorigenic signaling (Fig. 5C). To ensure site-specific delivery, the construct is encapsulated within an enteric polymer (Eudragit L100-55), which shields it from degradation in the stomach and facilitates pH-dependent release in the intestinal tract. This coordinated strategy yielded compelling therapeutic outcomes. In murine models of IBD, treatment with DNPs@P significantly restored colon length, suppressed pro-inflammatory cytokines (IL-6, TNF-α, IL-1β), and enhanced the diversity of the gut microbiota, outperforming conventional treatments like 5-aminosalicylate. Moreover, in mouse models, this system effectively prevented colonic tumor formation, demonstrating its potential for reshaping the gut microenvironment and offering a novel strategy for treating gastrointestinal diseases.
Genetically engineered temperate phages can be used to reprogram host bacteria to express and release therapeutic proteins. Baker et al.. integrated genes encoding target proteins into the phage genome, constructing engineered T4 phages that, upon infecting E. coli, were able to express and release heterologous proteins. This approach was validated in two mouse models [80]. For example, a T4 phage was engineered to express the serine protease inhibitor serpin B1a, which inhibits neutrophil elastase (NE), an enzyme upregulated in UC. In a DSS-induced colitis model, the T4::serpin phage significantly reduced NE activity and ameliorated colitis symptoms (Fig. 5D). Another T4 phage was engineered to express ClpB, a protein with melanocyte-stimulating hormone (α-MSH)-like appetite-suppressing activity. Application of the T4::clpB engineered phage in mice fed a high-fat diet resulted in notable reductions in both body weight gain and caloric intake.
Gut dysbiosis plays a pivotal role in the onset and progression of IBD. By precisely targeting disease-associated bacteria and mitigating inflammation, engineered bacteriophages provide a promising and mechanistically grounded strategy for restoring gut homeostasis and improving intestinal healt.

Colorectal cancer (CRC)
Bacteria play a dual role in tumor initiation and progression. Based on their mechanistic roles, they can be categorized into pro-carcinogenic, anti-carcinogenic, and bacteria with currently unclear functions. Bacteria and their metabolites can promote tumor progression either directly, by damaging host DNA or interfering with cellular signaling pathways, or indirectly, by disrupting host immune regulation and metabolic homeostasis. Helicobacter pylori, a classical pro-carcinogenic bacterium, induces chronic inflammation and aberrant activation of signaling pathways, leading to gastric mucosal lesions and gastric cancer [106]. Recent studies have shown that Clostridium difficile is abnormally enriched in CRC tissues, and its sustained secretion of TcdB toxin significantly promotes CRC development [107]. Notably, different strains within the same bacterial genus may exert opposite effects on tumorigenesis. A typical example is the “double-edged sword” effect of E. coli: strains harboring the PKS genomic island produce the genotoxin colibactin, which induces DNA double-strand breaks and local inflammation to promote tumorigenesis [108, 109]; in contrast, Butler et al.. demonstrated that uropathogenic E. coli could suppress tumor growth and extend survival in mouse models of bladder and colorectal cancer by downregulating MYC protein expression [110].
The development of CRC is closely associated with changes in the gut microbiota (Fig. 6A). For example, Fusobacterium nucleatum (Fn) is frequently enriched in CRC patients and may promote cancer cell proliferation and metastasis through pro-inflammatory mechanisms and modulation of the tumor immune microenvironment [111, 112]. Fn selectively increases immunosuppressive myeloid-derived suppressor cells (MDSCs), hindering host anti-tumor immune responses [77]. Enterotoxigenic Bacteroides fragilis (ETBF), a toxin-producing strain, is also associated with CRC. It produces Bacteroides fragilis toxin (BFT), which activates inflammatory pathways, thereby inducing cellular proliferation and gene mutations [113]. Compared with control groups, the bft gene is more prevalent in the colonic mucosa of CRC patients, indicating that mucosal exposure to BFT may be a risk factor for CRC [114]. Recombinant BFT-1 promotes CRC cell proliferation and tumor growth by upregulating CCL3, CCR5, NF-κB, and TRAF-6 [115]. BFT also initiates a pro-carcinogenic inflammatory cascade in colonic epithelial cells involving IL-17R, NF-κB, and Stat3 signaling pathways. This cascade recruits CXCR2-expressing myeloid cells, contributing to distal colon tumor formation [116]. These findings underscore the complex mechanisms by which ETBF and BFT promote CRC through inflammation and altered cell signaling.

Modulating the gut microbiota offers promising therapeutic opportunities for CRC. Both therapeutic bacteria and bacteriophages have demonstrated potential in targeting CRC-associated taxa and remodeling the tumor immune microenvironment. These strategies can enhance intestinal immunity, improve epithelial barrier function, and regulate anti-tumor immune responses [117, 118]. Moreover, specific microbial features can influence the host’s sensitivity to chemotherapy/radiotherapy and impact CRC prognosis [117]. Combining microbiota-based therapies with immune checkpoint inhibitors or chemotherapeutics has shown great promise in prolonging overall survival in CRC models [77]. Recent evidence has highlighted the role of Bacteroides fragilis in promoting chemoresistance in CRC [119]. Mechanistically, the surface protein SusD/RagB of B. fragilis binds to the Notch1 receptor on CRC cells, activating Notch1 signaling and inducing epithelial–mesenchymal transition (EMT) and cancer stemness, thereby attenuating chemotherapy-induced apoptosis [119]. Targeting B. fragilis with a specific lytic phage VA7 selectively eliminated the bacterium and restored chemosensitivity in multiple CRC models, offering a promising microbiome-based strategy to overcome chemoresistance. Studies have revealed a significant association between phages and CRC progression, potentially through modulation of the host microbial structure. Geoffrey D. Hannigan’s team applied an integrative multi-omics analysis strategy, including 16 S rRNA sequencing, fecal whole-genome shotgun metagenomics, and purified viral metagenomic sequencing, to systematically characterize the gut phage community and its corresponding bacterial dynamics. In-depth analyses revealed that temperate phages dominate the gut virome and that specific phage community features are significantly associated with CRC. Mechanistic insights suggest that phages may influence tumor progression by reshaping the microbial microenvironment of the host [120].
Phages can specifically lyse such pro-carcinogenic bacteria, serving as indirect indicators of microbial dysbiosis and aiding early CRC screening. Shen et al.. constructed a predictive model and identified five gut phages, including Peptacetobacter hiranonis phage, three Fusobacterium nucleatum variants, and Parvimonas micra phage, as candidate biomarkers. These findings expand the repertoire of CRC-related biomarkers and provide new perspectives for phage-based therapy in colorectal cancer [81]. Phage display technology has become a powerful tool for developing diagnostic and imaging agents for cancer (Fig. 6B). Che et al.. used this technique in combination with an integrated microfluidic system to screen phage-displayed peptides specific to CRC cells and cancer stem cells, successfully identifying three highly specific peptides [82]. These peptides exhibited high affinity and specificity toward their target cancer cells, making them promising candidates for early detection and targeted therapy [121]. Using phage display, Dong et al.. selected an M13 phage that specifically binds Fn and electrostatically assembled silver nanoparticles (AgNPs) on its surface proteins (M13@Ag). This engineered phage effectively eliminated Fn and reprogrammed the tumor immune microenvironment by enhancing antigen-presenting cell activation and promoting M1 macrophage polarization, which increased about two- to threefold compared with controls. In orthotopic colorectal cancer models, M13@Ag treatment markedly inhibited tumor growth and extended mouse survival from 23 to 35 days when combined with immune checkpoint blockade or chemotherapy. These findings demonstrate that M13@Ag not only targets Fn-associated chemoresistance but also remodels the tumor microenvironment to enhance antitumor immunity and therapeutic efficacy [77]. In addition, engineered phages carrying fluorescent or radioactive labels that target tumor-associated bacteria or directly bind receptors on cancer cell surfaces can enable tumor localization imaging. Jin et al.. genetically modified M13 phages by fusing collagen-mimetic peptides and streptavidin-binding peptides, allowing them to target and image degraded and denatured collagen in tumor tissues. Upon binding with streptavidin-conjugated fluorescent reagents, the engineered phages could specifically label abnormal collagen expressed on A549 human lung adenocarcinoma cells [122].
Due to their precise bacterial targeting and low toxicity, phages have emerged as important carriers for cytokines, tumoricidal agents, and chemotherapeutics, gaining attention in both immunotherapy and gene therapy. The team led by Paladd Asavarut innovatively utilized tumor-targeting phage capsid proteins to construct an interparticle delivery system, successfully enabling targeted delivery of tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), and interleukin-15 (IL-15) and their encoding genes. In xenograft models, this system efficiently enriched anti-tumor cytokines at the tumor site and significantly suppressed tumor cell proliferation [83]. Notably, this phage delivery system avoids the risk of a “cytokine storm” and is more cost-effective than monoclonal antibodies, highlighting its immense potential in tumor immunotherapy. In the field of nanodelivery system development, Wang’s team assembled PEG-phosphatidylethanolamine (PEG-PE) conjugates with phage proteins (P38 and L1) specific to pancreatic cancer cells (PANC-1) to form mixed micelles, which were loaded with the poorly soluble anticancer drug paclitaxel (PCT) [123]. This phage-targeted micelle system significantly enhanced targeting ability toward PANC-1 cells and greatly increased cytotoxicity. Moreover, irinotecan-loaded nanoparticles guided by phages demonstrated unique advantages in spontaneous CRC mouse models, enhancing first-line chemotherapy efficacy while simultaneously suppressing the growth of the pro-carcinogenic bacterium Fusobacterium nucleatum [84]. These innovative delivery systems provide new approaches to enhance the tumoricidal capacity of chemotherapeutic agents.
In situ vaccines (ISVs) can leverage locally delivered chemotherapy or radiotherapy to stimulate the release of endogenous tumor antigens, thereby inducing systemic and sustained immune activation [124, 125]. Engineered phages, as natural nanomaterials with inherent immunogenicity, genetic plasticity, and scalability, hold great promise as platforms for cancer vaccine development (Fig. 6C). They offer an ideal solution for personalized and in situ immunotherapy strategies. Dong et al.. assembled natural filamentous phage M13 with cationic polymer PEI to create a hybrid phage (M13@PEI) and loaded it with various tumor-specific antigens to construct a phage vaccine (HMP@Ag). This vaccine activated antigen-presenting cells and enhanced innate and adaptive immune responses through the TLR9 signaling pathway [85]. The vaccine effectively prevented and delayed tumor growth while synergizing with immune checkpoint inhibitors. It eradicated both primary and metastatic tumors, significantly suppressed postoperative recurrence in clinically relevant surgical models, and induced durable immune memory. Lei et al. developed an M13 phage-based ISV targeting CD40 (M13CD40), termed H-GM-M13CD40, to stimulate dendritic cells (DCs). This system locally delivered (S)−10-hydroxycamptothecin (HCPT) to induce immunogenic cell death and was combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) to enhance tumor antigen release. Together, these actions promoted dendritic cell recruitment and activation, reversing the immunosuppressive tumor microenvironment [86]. Furthermore, H-GM-M13CD40 synergized with PD-1 blockade to induce abscopal effects in “cold tumor” models, validating its potential as an immune-enhancing agent for in situ vaccination.
In summary, engineered bacteriophages hold significant promise across multiple stages of CRC management, including diagnosis, therapy, and prevention. Their strong targeting capabilities and versatility as delivery vectors have demonstrated great potential in cancer immunotherapy, imaging, gene therapy, drug delivery, and vaccine development.

Infectious enteritis
Infectious enteritis is typically caused by a variety of pathogenic bacteria, notably Clostridium difficile (C. difficile) and certain pathotypes of E. coli. These pathogens often colonize the intestine by forming biofilms and releasing proinflammatory factors, which presents dual challenges to conventional antibiotic therapy: antimicrobial resistance and recurrent infections. Moreover, these bacteria frequently proliferate excessively following antibiotic-induced dysbiosis, resulting in severe diarrhea and pseudomembranous colitis [126]. Engineered phages have recently gained traction as an innovative therapeutic approach for infectious enteritis, owing to their high specificity, flexible engineering capabilities, and capacity to deliver customized functional molecules.
CDI has become one of the most critical nosocomial infections and a leading cause of hospital-acquired diarrhea. Recent investigations have evaluated the effectiveness of phage therapy and phage-derived enzymes to combat CDI. Engineered phages, designed through modifications of tail fiber proteins or by incorporating antibiotic-sensitizing modules, have shown potent bactericidal activity in vitro and in animal models. The phage ΦCD27 demonstrated the ability to reduce C. difficile counts and toxin production in a human colonic in vitro model without adversely affecting commensal bacteria [28]. Engineered phages carrying the CRISPR-Cas3 system exhibited high specificity and efficacy against C. difficile both in vitro and in vivo. The engineered phages expressing genome-targeting CRISPR components showed significantly enhanced killing efficiency compared to their wild-type parental strains [90]. Additionally, endolysins derived from phages, such as CD27L_EAD, have been studied as potential antibiotic alternatives that selectively eliminate C. difficile [127]. Another study characterized a C. difficile phage and its endolysin, which exhibited activity against 30 distinct C. difficile strains, including the epidemic ribotype 027, while preserving commensal gut flora [128]. These findings highlight the potential of phage-based therapies as targeted treatments for CDI.
Other pathogens, such as Salmonella and Shigella, are also commonly associated with acute enteritis. Among them, E. coli O157:H7 is one of the most clinically relevant Shiga toxin-producing strains. Wang et al.. compared the efficacy of a phage cocktail and the antibiotic enrofloxacin in a murine model of E. coli O157:H7 infection. Compared to enrofloxacin, phage therapy resulted in more rapid pathogen clearance, lower expression of inflammatory markers, and more effective recovery in infected mice [91].

Irritable bowel syndrome (IBS)
Beyond infectious enteritis, inflammatory bowel disease, and colorectal cancer, the application of engineered phages has recently gained attention in other gastrointestinal disorders, especially functional conditions such as IBS. While the exact cause of IBS is still under investigation, current evidence implicates gut microbiota dysbiosis, low-grade inflammation, and gut-brain axis dysfunction as key contributors to its pathogenesis. Notably, altered ratios of bacterial genera such as Bacteroides and Prevotella in IBS patients have been associated with typical symptoms including abdominal pain and bloating [129, 130].
Preliminary studies suggest that targeted regulation of the gut microbiota may represent a promising strategy for the treatment of IBS. Engineered phages, through rational design, can specifically target and eliminate abnormally proliferating bacterial strains, thereby restoring microbial balance and potentially alleviating IBS-related symptoms.
Although direct applications of engineered bacteriophages in IBS remain unreported, recent investigations into the gut virome and microbiome provide indirect support for phage-based strategies. For example, a metagenomic study of virus-like particles in IBS patients found altered bacteriophage populations including elevated Myoviridae and Podoviridae families compared with healthy controls [131]. Another multi-kingdom analysis revealed that viral taxa and phage-bacterial interactions significantly contributed to gut microbial network dysregulation in IBS patients with depression and anxiety [132]. These findings suggest that phage modulation of the gut virome may represent a feasible therapeutic approach in IBS, and we emphasise the urgent need for engineered-phage research specifically addressing this functional disorder.

Comparative insights into engineered phage applications across gastrointestinal diseases
Engineering bacteriophage therapies across different gastrointestinal disorders reveals both shared principles and disease-specific considerations. In IBD, engineered phages are designed not only to eliminate pathobionts like AIEC or Kp, but also to recalibrate host–microbiota–immune interactions. In CRC, the focus shifts toward targeting tumor-promoting bacteria like Fn and ETBF, and employing phage constructs that can assist in in situ imaging or biomarker detection. For infectious enteritis, the strategy is more direct: phages must survive harsh GI transit and potently lyse invading pathogens such as C. difficile or STEC. In IBS, where overt pathogens are rarely implicated, engineered phages are envisioned to modulate dysbiotic microbial networks rather than eradicate a single species.
These differing objectives create distinct engineering and clinical requirements. Therapeutic approaches for IBD must balance targeted antimicrobial action with preservation of commensal microbes and mitigation of inflammation. CRC applications demand integration of diagnostic and delivery functions under the constraint of the tumor microenvironment. Treatments for enteritis require robust formulation stability and rapid bacteriolysis. IBS-directed interventions must effect subtle microbiota modulation in the context of a multifactorial disorder. The maturity of evidence also varies: engineered phage applications in enteritis have advanced to animal models and some human settings, IBD and CRC remain primarily in preclinical or early-phase trial stages, and IBS approaches are largely hypothetical. Recognising these differences will guide tailored phage design, delivery strategies and clinical trial structure according to the particular biology and therapeutic context of each gastrointestinal disease.

Discussion
The gut microbiota plays a critical role in maintaining intestinal health, while its dysregulation is closely associated with the onset and progression of various gastrointestinal diseases. Current approaches to modulating the microbiota, such as probiotics, prebiotics, and fecal microbiota transplantation, have demonstrated potential in ameliorating intestinal inflammation and restoring barrier function [133]. Although conventional therapies remain central to disease management, their limitations have become increasingly apparent. Broad-spectrum antibiotics indiscriminately kill both pathogenic and commensal bacteria, exacerbating microbiota imbalance [134]. Prolonged use of immunosuppressants amplifies risks for both opportunistic infections and malignancies [135]. In addition, the global emergence of multidrug-resistant organisms, particularly extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Clostridium difficile, further complicates clinical treatment [136]. Against this backdrop, phage therapy, a century-old strategy once sidelined by antibiotics, has reemerged as a promising focus in translational medicine due to its precise targeting ability and potential for microbiota modulation.

Optimization of phage therapy and its combination with other treatments
Broad-spectrum antibiotics severely disrupt the gut microbiota, leading to reduced microbial diversity and altered microbial equilibrium [134, 137]. Additionally, antibiotic treatment fosters the emergence of resistant bacteria and enhances the likelihood of horizontal gene transfer, promoting the development of antibiotic-resistant (MDR) pathogens [138]. Combining phages with antibiotics has emerged as a promising strategy to combat MDR bacterial infections. Compared to monotherapies, this approach enhances bacterial suppression, improves biofilm penetration, and reduces the likelihood of resistance development [139]. Mechanistically, antibiotic treatment may weaken bacterial defenses, increase cell wall permeability or filamentation, and thereby enhance phage adsorption, replication and lysis of pathogens [140]. As pathogens are lysed by phages, the release of endotoxin fragments, lipopolysaccharides (LPS) or bacterial debris is altered, lowering the burden of pro-inflammatory stimuli upon the mucosa [23]. Concurrent antibiotic action accelerates reduction of pathogen load, thereby shortening the duration of innate immune activation and decreasing neutrophil and monocyte recruitment. In this way, the combined therapy facilitates restoration of epithelial barrier function, suppression of cytokines such as TNF-α and IL-6, and re-establishment of a tolerogenic microbial community [141, 142]. Phage-antibiotic synergy (PAS) has been observed in several bacterial species, including Pseudomonas aeruginosa, E. coli, and Acinetobacter baumannii [20]. In a preclinical study, the combination of phage øFG02 and ceftazidime showed superior efficacy against A. baumannii compared to either agent alone [143]. Furthermore, phage-induced bacterial evolution toward antibiotic-susceptible phenotypes underscores the potential of this combined strategy [143]. Compared to broad-spectrum antibiotics, combining phage therapy with narrow-spectrum antibiotics is more likely to become a frontline clinical approach [144]. Nonetheless, careful selection of phages, antibiotics, and their dosages is critical to optimizing therapeutic outcomes [145]. Comprehensive understanding of these interactions remains essential to developing effective treatments that eliminate pathogens while preserving the beneficial gut microbiota.
Due to the host specificity of phages and their limited activity against diverse pathogenic genotypes, particularly MDR or “superbugs”, phage cocktails have been proposed to overcome these limitations [146]. A study in mice showed that single phage therapy had limited efficacy in reducing intestinal colonization of ESBL-producing E. coli [147]. In contrast, phage cocktail therapy has demonstrated superior effectiveness. Rahimzadeh et al.. evaluated a chitosan-encapsulated phage cocktail in rats infected with E. coli, Salmonella, and Shigella, reporting significant therapeutic efficacy [148]. The seven-phage cocktail EcoActive™ also proved effective against clinical AIEC strains in vitro [78]. However, phage cocktails still face challenges such as low killing stability, inconsistent efficacy, and the lack of imaging guidance tools, which hinder their clinical translation [149].
Wu et al.. introduced a novel strategy combining phages with aggregation-induced emission photosensitizer (AIE-PS), specifically TBTCP-PMB, which retains the phage’s host specificity while enabling fluorescence imaging and reactive oxygen species (ROS) generation. This synergistic strategy combining phage cocktail therapy and photodynamic therapy (PDT) demonstrated potent antibacterial activity and good biocompatibility in a murine sepsis model [149].
Phage therapy also shows great promise when combined with other treatment modalities. For example, mixtures of phages and probiotic strains have shown synergistic effects in reducing colonization of MDR E. coli ST131-H30R in mice [150]. Additionally, phage-delivered CRISPR-Cas systems have been proposed as novel tools to target and eliminate specific MDR pathogens within the gut microbiome, potentially resensitizing them to antibiotics [151]. Lysogenic phages such as λ phage can dynamically regulate microbiota through genome integration, but may carry virulence genes such as the stx gene encoding Shiga toxin, which can enhance pathogenicity. Safe application can be achieved by removing non-therapeutic elements via genetic hybridization or CRISPR-Cas-based genome editing [152, 153].
These combinatorial strategies illustrate the modular potential of engineered bacteriophages for gastrointestinal therapy. Phage–antibiotic pairings combine selective microbial lysis with restored antibiotic sensitivity, overcoming multidrug resistance and reducing collateral damage to the microbiota. Nanomaterial-assisted phages enhance delivery, imaging and targeted activation within the gut microenvironment. Phage–probiotic combinations aim to rebalance microbial ecosystems and fortify mucosal defences through complementary actions. Finally, phage-delivered CRISPR systems offer genome-level precision by eliminating resistant strains without disrupting beneficial taxa. Additionally, surface modification with metallic nanoparticles, magnetic nanomaterials, or quantum dots not only enhances phage stability in complex environments like the gastrointestinal tract but also improves biofilm disruption via physical or chemical interactions, thereby increasing antibacterial potency [77]. Together, these modalities present a coherent framework in which engineered phages serve as adaptable platforms merging antimicrobial, immunomodulatory and precision-targeting functions within the complex gut ecosystem.

Key scientific challenges and translational bottlenecks
Several challenges remain in the clinical application of engineered phages. A major impediment to rational design is our limited understanding of phage behavior within the complex intestinal ecosystem. Critical parameters such as phage adsorption and replication kinetics in the mucus layer, spatial distribution and persistence in different gut niches, and the dynamics of phage-bacteria-immune cell tripartite interactions in vivo are poorly characterized. Without this basic knowledge, it is difficult to predict whether an engineered phage will successfully colonize, amplify at the target site, and exert a sustained therapeutic effect.
The rapid emergence of phage-resistant mutants through receptor alteration, restriction–modification, or CRISPR-based defense mechanisms can compromise long-term therapeutic efficacy. Although engineered phages can target new receptors or include anti-CRISPR modules, the co-evolutionary “arms race” demands constant adaptation. Through ongoing co-evolution with bacteriophages, bacteria can acquire resistance via multiple strategies, including CRISPR-Cas systems, restriction-modification systems, and adsorption inhibition [47]. While engineering can broaden host range, designing phages that preempt or outpace these resistance strategies—for example, by creating phage cocktails that target multiple independent receptors or that carry anti-CRISPR genes—requires a dynamic, adaptive approach that is currently more reactive than proactive.
Although high specificity is a key advantage, it limits the applicability of individual phages, necessitating the use of phage cocktails to achieve broad-spectrum antibacterial coverage. Despite enthusiasm around phage cocktails, uncertainty remains over predicting synergistic versus antagonistic interactions among phages or with antibiotics, and aligning cocktail composition with clinical realities is an ongoing technical challenge.
Immunogenicity within the host remains a concern. While phages can modulate immune responses beneficially, they are also subject to rapid clearance by neutralizing antibodies and phagocytic cells Host immune responses to bacterial infection may indirectly suppress phage activity, shortening their half-life and affecting therapeutic continuity [154, 155]. Host immune responses constitute another barrier: neutralizing antibodies can severely limit phage circulation with repeated dosing, raising concerns over long-term use. More subtly, the immunomodulatory effects of phage therapy, including how phage or bacterial components influence immune cells and tolerance, remain poorly understood. Engineering phages to achieve “immune stealth” while preserving or enhancing desirable immunomodulation is a delicate balancing act.
Phage production and storage demand strict control of environmental factors such as temperature and pH. These requirements affect phage viability and stability, creating challenges for large-scale manufacturing and clinical use. Key unanswered questions include: what constitutes definitive proof of efficacy, how to design adequate and controlled clinical trials for personalized phage cocktails, and what the long-term safety monitoring requirements should be. A collaborative dialogue between researchers, industry, and regulatory agencies is essential to create clear and feasible pathways to market.

Future perspectives
In recent years, clinical evidence has gradually demonstrated the feasibility and safety of engineered phage therapy in humans. In 2019, a landmark case reported the first successful use of genetically engineered bacteriophages for treating Mycobacterium abscessus infection. A three-phage cocktail containing two engineered temperate phages was administered intravenously and produced marked clinical improvement. The treatment resulted in sternal wound closure, recovery of hepatic function, and regression of cutaneous lesions, with no serious adverse events reported [156]. Subsequently, in 2022, Jerry et al. described another successful application of engineered phages for M. abscessus-associated advanced pulmonary disease [157]. Beyond these examples, several human gastrointestinal trials are now underway. The Phase 1/2a clinical trial (NCT03808103) is evaluating the safety and efficacy of oral EcoActive™, a phage cocktail that specifically targets adherent-invasive Escherichia coli (AIEC), in patients with inactive Crohn’s disease. The trial aims to determine whether selective phage-mediated eradication of AIEC can restore gut microbial balance and reduce inflammation without disturbing commensal flora. Another ongoing Phase 1/2a trial (NCT05715619) investigates VRELysin, a phage preparation targeting vancomycin-resistant Enterococcus (VRE), in healthy and VRE-colonized adults. This study focuses on assessing safety, phage persistence, and the potential of reducing intestinal VRE burden to prevent subsequent infection and nosocomial spread. Collectively, these clinical cases and trials underscore the growing translational momentum of bacteriophage-based therapeutics, especially engineered phages. While large-scale randomized controlled trials remain limited, the accumulating clinical data affirm both the safety and therapeutic promise of phage therapy for refractory bacterial infections, including those of gastrointestinal origin.
To advance phage therapy toward precision and personalized medicine, future research must overcome technical limitations in genome engineering, delivery, and clinical translation. In the genetic engineering of phages, combining CRISPR-Cas systems with homologous recombination or BRED techniques can enable precise editing of tail fiber proteins, thereby expanding host range and overcoming bacterial resistance [158, 159]. Meanwhile, introducing anti-CRISPR modules or immune evasion strategies may enhance phage persistence in vivo, reducing immune clearance and improving therapeutic outcomes [160]. The integration of nanotechnology and chemical modification presents new possibilities for functionalizing engineered phages. Strategies such as polymer coating, nanoparticle conjugation, and quantum dot labeling can enhance phage stability and targeting in the gastrointestinal tract while enabling real-time imaging and therapeutic delivery [161]. Furthermore, incorporating bioresponsive or pH-sensitive materials can facilitate site-specific release, improving treatment efficiency under physiological conditions [161]. Receptor-binding proteins (RBPs) can be rationally redesigned or evolved to broaden phage host range and circumvent receptor mutation–mediated escape. Incorporation of anti-CRISPR (Acr) genes into phage genomes helps neutralize bacterial CRISPR–Cas defenses, while surface PEGylation, glycosylation, or encapsulation within nanocarriers reduces recognition by host antibodies, thereby extending systemic circulation. In parallel, adaptive phage cocktails integrating these engineered features provide multilayered protection against bacterial resistance and immune inactivation, ensuring durable antibacterial efficacy in vivo.
Personalized phage cocktails tailored to individual microbiota profiles represent a promising therapeutic strategy. By leveraging high-throughput screening platforms and integrating multi-omics data with artificial intelligence, it becomes feasible to rationally design phages that restore microbial balance and selectively target pathogens [162, 163]. Such individualized approaches could enhance efficacy while minimizing microbiota disruption, laying the groundwork for precision microbiome therapy. Furthermore, upon infection, engineered phages can be programmed to express specific immunomodulators, such as cytokines or tolerogenic peptides, thereby actively steering the local immune environment toward a therapeutic state.
To ensure successful clinical translation, it is essential to establish standardized systems for phage manufacturing, purification, storage, and quality control. The production of engineered phages requires adherence to Good Manufacturing Practice (GMP) conditions, with validated host strains, genomic integrity checks, and removal of endotoxins and bacterial debris. Formulation stability remains a major technical challenge. Because phages are sensitive to temperature, pH, and ionic strength, optimized lyophilization or encapsulation methods are needed to preserve viability during storage and transportation. Furthermore, robust batch-to-batch consistency and potency assays must be implemented to meet regulatory expectations. Currently, the absence of harmonized international guidelines for phage-based biologics impedes large-scale clinical deployment. Collaborative frameworks among regulatory agencies, academic institutions, and industry are therefore essential to develop standardized evaluation criteria, ensuring both efficacy and safety for clinical translation.
In summary, integrating genome editing, nanotechnology, and systems biology will accelerate the development of multifunctional, precise, and efficient engineered phages. These next-generation biotherapeutics hold tremendous promise for combating antibiotic resistance and microbiota-associated gastrointestinal diseases.

Conclusion
Engineered bacteriophages exhibit unique and promising potential in the treatment of gastrointestinal diseases. Through genome editing, chemical conjugation, integration with nanomaterials, and synthetic biology tools, phages can be tailored to expand their host range, enhance antibacterial activity, and acquire additional functions such as biofilm disruption, microbiota modulation, and therapeutic delivery. Despite challenges such as host specificity, immunogenicity, and manufacturing constraints, technological advances and interdisciplinary collaboration are expected to address these hurdles. In the future, personalized phage therapy based on individual gut microbiota profiles, together with multimodal strategies that integrate nanotechnology and drug delivery systems, is expected to open new avenues for precision treatment. As a novel and sustainable class of biotherapeutics, engineered phages offer valuable insights from their success in anti-infective and antitumor applications and warrant further exploration and clinical translation for gastrointestinal disease management.