Recent insights on challenges encountered with phage therapy against gastrointestinal-associated infections

Pathogenic E. coli is classified into extraintestinal (ExPEC) and diarrheagenic (DEC) groups. Key DEC pathotypes include Enteropathogenic (EPEC), Enterohemorrhagic (EHEC), ETEC, Enteroaggregative (EAEC), Entero-invasive (EIEC), and Diffusely Adherent (DAEC) [9]. Gastrointestinal (GIT) infections, including those caused by E. coli are responsible for 443,832 deaths annually from diarrheal illnesses [10], making it the second leading cause of death in this age group in underdeveloped nations, after pneumonia [11]. Healthcare infrastructure, water quality, and hygiene are considered important factors that greatly influence the prevalence of E. coli-associated infections [12]. EHEC infections are particularly significant in both industrialized and developing nations. STEC causes over 265,000 infections annually in the US [13], and Studies in Upper and rural Egypt found DEC in 20 and 66% of diarrhea cases in children respectively [14, 15]. The rise of antimicrobial resistance (AMR) and diverse pathogenic strains has led to widespread antibiotic resistance, with threats from carbapenemase-producing isolates (KPC-2, NDM, OXA-48-like) and extended-spectrum β-lactamases (ESBLs), often resistant to aminoglycosides, quinolones, and cephalosporins [16]. High prevalence of multidrug-resistant (MDR) isolates resistant to three or more classes has also been reported [17]. This antimicrobial resistance is multifactorial driven by genetic and adaptive mechanisms. It is primarily propagated through horizontal gene transfer of mobile genetic elements encoding β-lactamases, including ESBLs, AmpC, and carbapenemases. Enzymatic inactivation and target site alterations—such as mutations in DNA gyrase, topoisomerase IV, and ribosomal components—confer resistance to fluoroquinolones, macrolides, and β-lactams. Efflux pumps and porin mutations reduce intracellular antibiotic accumulation, while biofilm formation and stress responses enhance survival under antimicrobial pressure. Chromosomal mutations further modulate resistance to trimethoprim-sulfamethoxazole and β-lactams. Additionally, colistin resistance arises from lipid A modifications mediated by mcr genes or chromosomal regulators. These mechanisms collectively contribute to the resilience of E. coli against a broad range of antibiotics [18].

S. enterica includes six subspecies, with enterica being the most common, causing 99% of infections in humans and animals [19]. Salmonella is divided into typhoidal (TS) and non-typhoidal (NTS) types, with TS affecting humans and NTS having a broader host range, including animals [20]. NTS is a main source of foodborne infections globally, with 1.3 billion cases and 155,500 deaths annually. In 2018, it was the second leading cause of foodborne illness in the EU and the USA, and the third leading cause of death worldwide [21, 22]. In China, infections represented 70–80% of foodborne diseases [23], with symptoms ranging from gastroenteritis to more severe complications like bacteremia and osteomyelitis, particularly in vulnerable groups like children, the elderly, and immunocompromised individuals [24]. NTS is primarily zoonotic and transmitted through contaminated food, especially poultry, and also through manure-contaminated vegetables [21, 25]. Antibiotic resistance, especially due to plasmids, poses a significant public health threat [26]. Resistance to ciprofloxacin and extended-spectrum β-lactamase producers (ESBLs) have become more common, with MDR strains emerging against most of the utilized antibiotics, including ciprofloxacin, amoxicillin, ceftriaxone, ampicillin, and trimethoprim-sulfamethoxazole, which are used to treat invasive Salmonella infections [26‐28].

TS includes S. Typhi, S. Sendai that cause typhoid fever, and S. Paratyphi A, B, and C that cause enteric fever, transmitted mainly through contaminated water and food, with food handlers playing a major role in its spread [29]. TS is endemic in areas with poor sanitation and affects travelers to these regions. MDR typhoidal strains appeared in the 1980s, and in the 2000s, extensively drug-resistant (XDR) typhoid emerged, resistant to cephalosporins, fluoroquinolones, and first-line antibiotics, with cases reported in Bangladesh and Pakistan [30, 31]. Since then, reports of XDR typhoid have emerged from every endemic nation, including the US [32, 33]. Despite the effectiveness of azithromycin and carbapenems, antibiotic resistance remains a challenge, with azithromycin-resistant S. Typhi strains also identified [34]. A recent case study of 6 years old child having Down syndrome reported pan drug resistant S. Typhi to all the tested antibiotics including Ampicillin, chloramphenicol, ceftriaxone, cefixime, trimethoprim-sulphamethoxazole, ciprofloxacin, meropenem, imipenem and azithromycin [35]. Salmonella exhibits a complex array of antimicrobial resistance mechanisms that include the production of β-lactamases and aminoglycoside-modifying enzymes, which enzymatically inactivate antibiotics. At the same time, the expression of multiple efflux pump systems (such as AcrAB–TolC and MacAB–TolC) actively expel antimicrobial agents from the cell. Additionally, mutations in target sites like DNA gyrase and topoisomerase IV contribute to fluoroquinolone resistance, while porin loss and outer membrane alterations reduce drug permeability. Biofilm formation further protects Salmonella populations by limiting antibiotic penetration and supporting persistent cell development. Moreover, plasmid-mediated gene transfer facilitates rapid dissemination of resistance traits across strains. Together, these mechanisms pose significant challenges to treatment and underscore the importance of novel therapeutic strategies and enhanced surveillance efforts [36].

Campylobacteriosis has been the most commonly stated foodborne gastroenteritis in humans, with 50–80% of reported cases due to the poultry reservoir [37]. In the US. 9.4 million cases of foodborne diseases are recorded each year, whereas 9% of those infections are caused by Campylobacter species, rendering them the third most common bacterial pathogen [38]. While in the European Union (EU) campylobacteriosis is the most common foodborne bacterial illness, with 127,840 registered cases in 2021 only [39]. The two most common reported species were Campylobacter (C.) jejuni and C. coli (88.4% and 10.1%, respectively, in 2021) [37]. It is important to point out that finding an alternative to antibiotic therapy is crucial, especially with the recorded substantial levels of resistance to antibiotics such as ciprofloxacin and erythromycin [40]. Campylobacter species deploy an extensive arsenal of antimicrobial resistance mechanisms that are mediated through enzymatic inactivation—such as β-lactamases (e.g., blaOXA variants), aminoglycoside-modifying enzymes (aadE, aphA-3), and erythromycin methylases (ermB)—as well as ribosomal protection (TetO). Mutations in DNA gyrase (gyrA) and 23 S rRNA underpin fluoroquinolone and macrolide resistance, respectively. Additional defense involves the overexpression of efflux pumps (e.g., cmeABC), coupled with porin alterations that reduce drug uptake. Plasmids and other mobile genetic elements drive the dissemination of resistance determinants, while biofilm formation and adaptive stress responses bolster drug tolerance. Widespread antibiotic use in both human and animal settings has favored the propagation of these mechanisms, resulting in multidrug-resistant Campylobacter strains that hinder successful treatment and raise the demand for vigilant monitoring and targeted control strategies [41].

Shigella causes shigellosis (bacillary dysentery) with S. flexneri being considered as one of the major causes of bacillary dysentery cases in the world especially in underdeveloped areas with poor sanitation. Shigella is responsible for about 5 to 15% of all diarrheal cases worldwide, including 1.1 million fatalities, especially in children under the age of five where it accounts for two-thirds of all morbidity and mortality [42].

Shigella isolates exhibited significant resistance to most antibiotics, with notable regional variations. In Asia, the highest resistance rates were observed for streptomycin (98.4%), trimethoprim (95.5%), and ticarcillin (90.5%), while the lowest resistance rates were for carbapenem antibiotics and nitrofurantoin, with rates close to 0% [43]. There is also a growing concern regarding the increasing resistance to third-generation fluoroquinolones, cephalosporins, and, most recently, to azithromycin, thus, this should be taken into consideration in treatment policies [44]. Shigella species have harnessed a sophisticated array of antimicrobial resistance mechanisms, fostering their persistent threat to public health, including the overexpression of (resistance-nodulation-cell division) RND-family efflux pumps (e.g., AcrABTolC) alongside loss or downregulation of porins, which together decrease intracellular antibiotic accumulation. They acquire target-site mutations—particularly within the quinolone-resistance–determining regions of gyrA, parC, and occasionally parE—conferring resistance to fluoroquinolones. Additionally, production of diverse β-lactamases, including classical and extended-spectrum types (TEM, CTX-M, SHV, OXA), degrades β-lactam antibiotics. Resistance is further exacerbated by plasmid-mediated mechanisms such as mobile elements that carry PMQR genes (qnr, aac(6’)-Ib-cr), macrolide resistance genes (e.g., mphA), integrons clustering multiple resistance determinants, and even colistin-resistance genes like mcr-1, all of which facilitate rapid horizontal gene transfer. Through the convergence of active efflux, target modification, enzymatic degradation, and plasmid-borne gene exchange, Shigella evolves into formidable multidrug- and extensively drug-resistant strains, underscoring the urgent need for novel treatment approaches and vigilant surveillance [45, 46].

C. difficile produces toxins, leading to gastrointestinal conditions ranging from mild diarrhea to life-threatening complications like pseudomembranous colitis, toxic megacolon, colon perforation, and septic shock. In the US, C. difficile infections (CDI) affects around approximately half a million individuals each year with approximately 80,000 recurrences and 30,000 deaths [47]. In the EU/ European Economic Area, an estimated 123,997 healthcare-associated C. difficile infections occur annually, resulting in around 3,700 deaths [48]. Both the incidence and severity of C. difficile infections have increased due to the emergence of hypervirulent strains, such as the BI/NAP1/027 strain [49]. Increasing resistance to metronidazole and vancomycin has also been reported worldwide. The long-term antibiotic utilization is considered the primary risk factor for CDI, especially with antibiotics, such as clindamycin, cephalosporins, and fluoroquinolones and certain penicillins like co-amoxiclav, which can promote CDIs [48]. Resistance to other drugs like rifampin and fidaxomicin is also increasing globally [50]. C. difficile is responsible for approximately 15–25% of antibiotic-associated diarrhea cases, 50–75% of antibiotic-related colitis cases, and nearly all instances (90–100%) of antibiotic-associated pseudomembranous colitis [51]. In two tertiary-care hospitals in Mexico, highly drug-resistant C. difficile ribotypes 027 and 001 were identified, with 40.3% and 3.2% of isolates showing reduced susceptibility to vancomycin and fidaxomicin, respectively. This is particularly concerning given that fidaxomicin is not currently accessible in Mexican healthcare facilities [52]. This resistance is a multifaceted resistance that includes intrinsic and acquired adaptations. Intrinsic adaptations include biofilm production and sporulation that confer phenotypic tolerance by physically shielding cells and reducing metabolic activity. Moreover, acquired adaptations alters drug targets through mutations in RNA polymerase (rpoB) for rifamycins—and modify peptidoglycan precursors via mutations in the vanG operon homolog (vanG_Cd), reducing vancomycin affinity. Resistance to metronidazole arises through chromosomal changes affecting oxidoreductive and iron-dependent metabolic pathways, and, in some cases, via plasmidencoded inactivation mechanisms. Active efflux systems also extrude drugs, decreasing intracellular antibiotic concentrations. This combination of target al.teration, enzymatic drug modification, efflux, and persistence strategies has fostered the emergence of multidrug-resistant C. difficile strains, reinforcing the need for novel therapeutic tactics and meticulous epidemiological tracking [50].

S. aureus is a significant foodborne pathogen responsible for a range of illnesses, including staphylococcal food poisoning caused by the ingestion of pre-formed enterotoxins, primarily enterotoxin A. Human carriers, particularly food handlers, are the main sources of contamination. Symptoms typically occur rapidly and include nausea, vomiting, and abdominal cramps, resolving within 24–48 h, though severe cases can affect vulnerable populations. Despite causing an estimated 241,000 cases annually in the U.S., underreporting is widespread due to the self-limiting nature of the illness and limited surveillance systems, especially in developing countries, indicating a far greater global burden than currently documented [53]. A key concern with S. aureus is its ability to acquire antibiotic resistance, particularly methicillin-resistant S. aureus (MRSA), which is driven by the mecA gene encoded on the SCCmec element. MRSA strains are typically resistant to most β-lactams, making treatment challenging and often reliant on vancomycin, although resistance to vancomycin is also emerging. MRSA has evolved beyond hospital settings to cause community-associated and livestock-associated infections, with increasing prevalence in humans and animals due to antimicrobial misuse [54]. S. aureus mechanisms of resistance to antimicrobials includes β-lactam resistance mediated by mecA-encoded PBP2a from SCCmec cassettes, alongside β-lactamases and occasional PBP mutations; glycopeptide resistance, featuring vanA acquisition or cell wall thickening in VISA strains that reduce vancomycin accessibility; oxazolidinone resistance, driven by Cfr- and Erm‐mediated 23 S rRNA methylation and ribosomal mutations; aminoglycoside resistance, through plasmid‐encoded modifying enzymes (acetyl-, phospho-, nucleotidyltransferases); fluoroquinolone, tetracycline and MLS‐B resistance via gyrA/gyrB/parC/parE mutations and overactive efflux pumps (NorA, MdeA, MepA from MATE (multidrug and toxic compound extrusion), MFS (major facilitator superfamily), SMR (small multidrug resistance) and ABC (ATP-binding cassette) families); and other mechanisms including resistance to rifampicin, chloramphenicol, fusidic acid, mupirocin, and daptomycin. This formidable genetic toolkit is compounded by active efflux systems that also support biofilm formation, vancomycin sequestration, and integration of resistance genes on mobile genetic elements—SCCmec, plasmids, integrons, transposons—facilitating horizontal transfer. Cumulatively, these mechanisms have given rise to multidrug- and extensively drug-resistant S. aureus, underscoring the imperative for novel therapeutic options and rigorous epidemiological surveillance.

Among VC strains, those belonging to serogroups O1 and O139 are linked to cholera outbreaks due to the production of cholera toxin and toxin-coregulated pilus, which are crucial for intestinal colonization and diarrhea [55]. Cholera remains endemic in regions like Asia, Africa, Central and South America, and Bangladesh, with outbreaks primarily linked to the El Tor (O1) biotype [56]. AMR in VC emerged in the 1970s, with resistance to polymyxin B and other antibiotics observed, particularly in the El Tor biotype. Resistance patterns vary globally, with selective antibiotic pressures influencing the spread of resistance genes [57]. In sub-Saharan Africa, VC isolates mostly belong to the O1 El Tor biotype, with common resistance to trimethoprim-sulfamethoxazole, ampicillin, chloramphenicol, and streptomycin [58, 59]. VC exhibits a wide array of AMR mechanisms that contribute to its persistence and treatment failure in clinical settings. Key strategies include active efflux of antibiotics via multiple transporter families—such as MATE, MFS, RND, SMR and ABC—many of which also support virulence and environmental adaptation. Efflux pumps like NorM, VcmA, and EmrD-3 are implicated in resistance to a broad range of drugs. Resistance is also mediated by chromosomal mutations, particularly in the quinolone resistance-determining regions (QRDRs) of gyrA and parC, leading to reduced susceptibility to fluoroquinolones. Additionally, VC acquires β-lactamase genes, including blaNDM-1, which confer resistance to β-lactams and carbapenems. Structural modifications, such as lipid A alteration by the almEFG operon, contribute to polymyxin resistance. Outer membrane impermeability further limits antibiotic entry, particularly for large molecules, while inducible stress responses and enzymatic degradation of drugs add to the problem of resistance. These combined mechanisms underscore the adaptive plasticity of VC under antimicrobial pressure and highlight the urgency of continuous surveillance and novel therapeutic strategies [57].

Y. enterocolitica primarily causes yersiniosis, leading to gastrointestinal illness, mesenteric lymphadenitis, and endocarditis, particularly in children [60]. Infections typically result from consuming contaminated food, especially raw or undercooked pork, as pigs are the primary reservoir [61]. The bacterium is biochemically and serologically diverse, with serotype O8 is the most virulent and can sometimes cause severe gastrointestinal ulceration or even death [60]. Yersiniosis is the third most reported zoonotic infection in the EU, though actual cases may be underreported [62]. Y. entericolitica develops antimicrobial resistance through multiple mechanisms, primarily via β-lactamase production (BlaA and BlaB), which hydrolyze β-lactam antibiotics [63]. Efflux pumps, notably the AcrAB-TolC system, actively expel diverse antibiotics such as tetracycline resistance typically results from plasmid-borne tet genes (e.g., tet(A), tet(B)) that encode efflux pumps and ribosomal protection proteins [63, 64]. Target site modifications, including mutations in gyrA and parC, reduce fluoroquinolone efficacy, while changes in membrane porins limit drug entry [63]. Furthermore, horizontal gene transfer via plasmids, integrons, and transposons (e.g., Tn2670) facilitates the acquisition and dissemination of resistance genes, enhancing the survival under antimicrobial pressure and increasing the public health risks. Aminoglycoside resistance is mediated by enzymatic inactivation via acetyltransferases, phosphotransferases, or nucleotidyltransferases [64]. Together, these mechanisms—enzymatic drug inactivation, decreased permeability, target modification, and gene mobility—underpin the adaptive resistance landscape of this pathogen.

Gram-negative H. pylori is a major cause of gastrointestinal infections, affecting around 50% of the global population [65]. Infections with H. pylori range from peptic ulcers and gastritis to more serious problems such as gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. In addition, H. pylori infection has been connected to biliary tract malignancies, which is quite intriguing [66]. Even though H. pylori only colonizes the stomach epithelium, the infection has also been linked to several extra-gastric illnesses, such as colorectal cancer (CRC). Direct causative and functional linkages between the chronic infection and CRC have only recently been discovered, despite epidemiological studies reporting a nearly two-fold greater risk for infected persons to acquire CRC [65]. H. pylori exhibits antimicrobial resistance primarily through chromosomal mutations and adaptive mechanisms that impair antibiotic efficacy. Resistance to clarithromycin is most commonly associated with point mutations in the 23 S rRNA gene (A2142G, A2142C, A2143G), which prevent effective ribosomal binding [67]. Fluoroquinolone resistance results from mutations in the gyrA and gyrB genes within the quinolone resistance-determining region (QRDR), while metronidazole resistance involves loss-of-function mutations in rdxA and frxA, which encode enzymes essential for drug activation [68]. Tetracycline resistance is less common but arises through alterations in the 16 S rRNA gene that interfere with ribosomal interaction [68]. Additionally, reduced membrane permeability and overexpression of efflux pumps contribute to decreased intracellular antibiotic concentrations, further enhancing resistance and contributing to treatment failure which poses a significant threat to public health [68].

Ensuring delivery of phages to the target site in sufficient quantities is a complex process due to several factors. For example, the acidic pH of the stomach is considered an important obstacle against successful phage therapy. It was reported that the co-administration of approved pharmacological agents that suppress gastric acid secretion, significantly improved phage survival and therapeutic efficacy within the gastrointestinal tract. In a single-blind, randomized, placebo-controlled clinical trial to evaluate the safety and feasibility of oral delivery of a two-phage cocktail in healthy adults, all participants were pretreated with oral esomeprazole to reduce gastric acidity to enhance phage survival through the gastrointestinal tract [130]. Another strategy includes encapsulation methods, like microencapsulation and liposome-based delivery, are being researched to protect phages from the harsh gastric environment. A study reported that liposome coating enhanced the retention of phages within the chicken intestinal tract. When both encapsulated and nonencapsulated phage cocktails were given to broilers, encapsulated phages were recovered from 38.1% of the birds after 72 h, compared to just 9.5% for nonencapsulated phages, indicating a significant difference in retention. Additionally, in vitro tests revealed that cecal contents from broilers facilitated the release of phages from the liposomes. In broilers infected with Salmonella, administering both phage types daily for six days provided comparable protection against colonization. However, the protective effect of nonencapsulated phages vanished once treatment ended, whereas the encapsulated phages continued to offer protection for at least a week, demonstrating their superior efficacy over time [131]. Another research study explored using alginate–carrageenan microcapsules with or without CaCO₃ to protect Salmonella bacteriophages from harsh gastrointestinal conditions. While unprotected phages were inactivated at low pH, encapsulation improved phage survival, especially with added CaCO₃, which further reduced phage loss. Phages were effectively released when the pH shifted to neutral, simulating the duodenum. The findings suggest that this encapsulation method could be a promising strategy for targeted phage delivery to the intestines [132].

Bacteria, like other genetic entities, can evolve in response to environmental changes, including exposure to chemotherapies such as antibiotics or phages [146]. Resistance to phages can be inherent in certain bacterial species or acquired over time. Acquired resistance can either emerge before exposure to the antagonist or as a result of selective pressures during treatment [140]. Resistance may be horizontal, arising through the transfer of mobile genetic elements, or mutational, involving changes to bacterial molecules that phages need to infect successfully [147]. Bacteria can also develop either phenotypic tolerance (temporary resistance) or genetic resistance (stable changes). Mutations that lead to phage resistance can cause antagonistic pleiotropies, meaning that while resistance provides benefits, it may also come with costs, such as slower growth, reduced virulence, or greater susceptibility to antibiotics [148]. These trade-offs may decrease the bacteria’s ability to cause infections [149].

Resistance to phages has been noted in research analyzing clinical samples from patients infected with V. cholerae, as well as in phage-treated chickens infected with C. jejuni and calves infected with E. coli [150‐152]. This requires continuous monitoring and development of phage cocktails. It is advisable to utilize a cocktail of phages with varying host ranges and receptor targets in all phage therapy studies to enhance effectiveness and minimize the development of phage resistance in targeted pathogens [153]. Phage cocktails are generally less toxic and can be safely used regularly compared to untested antibiotic combinations, which might cause unpredictable side effects [154].

Some bacterial strains can form biofilms, which are protective layers that phages find difficult to penetrate. Recent research has shown that bacteria have developed several defense mechanisms to survive phage infections, including surface modification, superinfection exclusion (Sie), restriction-modification (R-M) systems, CRISPR-Cas systems, and abortive infection (Abi) systems [155]. Surface modification prevents phages from attaching to bacterial cells, making it one of the safest defenses. For example, glycosylated type IV pili can block phage replication and protect P. aeruginosa from pilus-specific phages [156]. Sie prevents secondary infections by a phage when the bacterium is already infected [157], allowing bacteria to resist further attacks from similar or closely related phages [158]. The R-M and CRISPR-Cas systems are common and diverse bacterial defenses. R-M systems, which include restriction endonucleases and methyltransferases, cut phage DNA that is not properly modified, while protecting the bacterial genome through methylation [159]. Bacteria can also enhance their phage resistance by increasing the concentration of restriction enzymes [160]. CRISPR-Cas systems, an adaptive immune mechanism, target and cut foreign DNA from phages, preserving the bacterial genome’s integrity [161].

Abi systems represent a final defense where infected bacteria undergo “altruistic suicide,” halting phage spread and protecting the population [162]. These systems prevent phage replication by interfering with its lifecycle after adsorption and DNA injection, resulting in few or no infectious virions and the death of the infected cells [163]. In response to bacterial resistance to PT through surface modifications, mutations, and modified restriction enzymes, phage cocktails, which combine multiple phages, overcome this developing resistance, by broadening host range and reducing resistance [164]. Studies demonstrated that, they are more effective at eradicating bacterial biofilms compared to single-phage treatments [165, 166]. Also, combining phages with antibiotics has been shown to enhance bacterial killing and improve treatment efficacy [167]. Phages can weaken biofilm structures, making bacteria more susceptible to antibiotics when administered in the right sequence [168, 169].

Furthermore, genetically engineered phages are being developed to target a broader range of bacteria and enhance biofilm degradation [170]. These modified phages can improve biofilm removal and bacterial elimination [171‐173].

Last but not least, phage-derived enzymes, such as depolymerases, lysins, and DNases, are also effective in breaking down biofilms and facilitating bacterial elimination [174]. These enzymes can be used alone or as adjuvants for better biofilm eradication [175, 176]. Finally, phages can be combined with chemical disinfectants or nanomaterials to enhance biofilm removal and penetration [177, 178]. These strategies improve phage delivery to biofilm layers, increasing their effectiveness in treating infections.

Bacteriophages contribute to horizontal gene transfer primarily through transduction, a process by which bacterial DNA is inadvertently packaged into phage particles and delivered to new host cells. This occurs either randomly during the lytic cycle (generalized transduction) or specifically during excision of temperate phages from the host genome (specialized transduction). Although this mechanism raises concerns about the spread of antibiotic resistance and virulence factors, recent genomic analyses indicate that such genes are rarely found in therapeutic phages. Additionally, while functional assays have detected transduction events in laboratory settings, these may overestimate the actual clinical risk. Importantly, gene transfer potential depends more on the phage’s DNA packaging system than on its lifecycle, suggesting that not all temperate phages pose a high risk [179]. Therefore, risk assessments for phage therapy should be based on detailed genetic and mechanistic evaluations rather than generalized exclusions.

The regulatory pathway for phage therapy is still developing, with different standards across countries, making global approval challenging [180‐182]. PT involves single phages or “phage cocktails” and can be tailored to an individual patient’s bacterial infection, offering a personalized approach. However, only a few centers worldwide, including those in Georgia, Belgium, Poland, the USA, and Australia, continue to provide phage-based treatments and conduct clinical trials on their effectiveness [183‐185]. Phage therapy has been used experimentally for decades at institutions like the Eliava Institute in Georgia and the Ludwik Hirschweld Institute in Poland. It follows the principles of the Helsinki Declaration and is intended for patients for whom no proven medical treatments exist or have been ineffective [181, 186‐189]. Following Poland’s EU accession in 2005, its PT became a global model, supported by the establishment of the Phage Therapy Center (PTC), which operates in compliance with EU and US regulations [190]. Belgium introduced regulations for individual patient treatments with personalized phage preparations in 2018, making it one of the few Western countries to embrace PT [191]. That same year, the Center for Innovative Phage Applications and Therapeutics (IPATH) was established at the University of California, San Diego, focusing on PT for life-threatening infections and incorporating clinical trials [192]. Australia, with its favorable regulatory environment, is well-suited for conducting controlled phage trials, with the Therapeutic Goods Administration (TGA) aligning with European Medicines Agency (EMA) policies [193]. The therapeutic use of bacteriophages in the UK is regulated under the Human Medicines Regulations 2012, with oversight by the Medicines and Healthcare products Regulatory Agency (MHRA), which treats phages as biological medicinal products requiring compliance with quality, safety, and efficacy standards. While personalized phage therapies can be administered under the “specials” exemption for individual cases, they remain outside routine licensing pathways and must still adhere to Good Manufacturing Practice (GMP) standards. Key regulatory challenges include ensuring product consistency, sterility, and generating clinical evidence to support broader use. The current framework does not yet fully accommodate the adaptive and personalized nature of phage therapy, highlighting the need for further regulatory refinement [194].

Phage banks, which hold extensive collections of phages, play a crucial role in matching the appropriate phage to a bacterial infection. Notable collections include the American Type Culture Collection (ATCC) and the Public Health England (PHE). Creative Biolabs, a biopharmaceutical company, aids in developing PT by screening natural phages and engineering synthetic ones targeting specific bacteria [195]. The rise in MDR organisms has driven the need for expanding phage collections. The global market for phage therapy is projected to reach USD 1.4 billion by 2026 [195]. Advancements in the production and purification of phages are expected to further propel the development of PT in Western Europe and beyond [196].

PT holds significant therapeutic promise but faces safety concerns that have limited its clinical use. One primary concern is the presence of endotoxins in phage preparations. These endotoxins, LPS from Gram-negative bacteria, are released when bacteria divide or die and can provoke severe immune reactions, including fever, reduction in white blood cells, and even shock. To mitigate these risks, the U.S. FDA mandates that LPS levels in intravenously administered biologics be below 5 Endotoxin Units per kilogram of body weight, requiring improvements in endotoxin removal methods for PT to meet these safety standards [197]. Another concern is the potential production of antibodies against phages. Immunoglobulins like IgM and IgG can neutralize or diminish the effectiveness of phages by triggering their rapid clearance from the bloodstream [198]. Additionally, phages consist of proteins and nucleic acids that may induce immune responses, including allergic reactions. While these immune responses are typically mild, caution is still necessary [199].

Phages may also transfer virulence or antibiotic resistance genes to host bacteria, particularly during the lysogeny process, where phage genetic material is integrated into bacterial genomes [200]. This could increase the pathogenicity of bacteria within the normal human microbiota, creating potential health risks. Moreover, the use of broad-spectrum phages could disrupt the natural microbiota, which may have unintended consequences [201]. Phages can also evolve, potentially losing their ability to kill bacteria during manufacture or use, which poses further challenges to their effectiveness [202]. However, targeted manipulation of bacterial communities using engineered bacteriophages can be used to allow phage-delivered genome editing for microbiome modulation. In parallel to the prompt advancement in molecular biology, bioinformatics, and gene-editing technologies, engineered phages grant an effective strategy for targeting harmful genes in the gut microbiota without distracting the integrity of the microbiome, thus presenting potential antimicrobial therapy. Phage-based engineering of the gut microbiome aims to genetically modify bacterial pathogens and consequently eradicate them. This treatment strategy relies on the application of engineered phages to treat diarrhea caused by Escherichia Coli, Clostridium difficile infection, as well as colorectal cancer due to Fusobacterium nucleatum infection [203].

To ensure the safety and efficacy of PT, it is important to select phages that are highly effective against bacteria while being non-toxic to the patient [204]. Phage preparations must be carefully characterized, ideally through whole genome sequencing, to exclude any harmful genes. Purifying phages can also minimize the risks of allergic or toxic reactions [201]. Despite these advancements, the complex and costly process of producing phage preparations at scale remains a challenge, limiting their widespread use in clinical settings. However, technological progress may eventually make large-scale clinical applications of PT possible. Phages are specific to bacteria and do not replicate in mammalian cells. However, mammalian cells can internalize phages through processes like macropinocytosis and receptor-mediated endocytosis [205]. Some phages can bind to specific cell receptors, facilitating their entry into mammalian cells. While phages are generally considered safe for human use, studies have shown that they can stimulate immune responses and even enhance cell growth and metabolism [206]. Phages are also being explored for their potential as adjuvants or delivery systems for vaccines [207]. Moreover, Bacteriophage-derived endolysins are emerging as superior alternatives to whole bacteriophage therapy due to their immediate, targeted lytic action, absence of genetic material, and lower risk of horizontal gene transfer or unintended ecological impact [208]. Unlike whole phages that require bacterial replication and may trigger immune responses or carry resistance genes, lysins act directly on bacterial cell walls, offering precise and rapid antimicrobial effects, especially against Gram-positive pathogens [209]. Recent advances in encapsulation techniques—using polymers, liposomes, nanofibers, and stimuli-responsive materials—have significantly enhanced lysin stability, bioavailability, and targeted delivery in harsh physiological environments [210]. These systems allow lysins to maintain activity across various applications, including gut infections, chronic wounds, and foodborne pathogen control [208]. In contrast, while whole phages are advantageous in dynamic microbial ecosystems due to their self-replicating nature and biofilm penetration capabilities, their unpredictable pharmacokinetics and regulatory hurdles limit their broader use [209]. Therefore, lysins, especially when encapsulated, offer a safer, more tunable, and clinically viable antimicrobial strategy for precision-targeted infection control [210]. Although PT has shown promise, the interactions between phages and mammalian cells remain poorly understood, and further research is needed to fully assess the implications of these interactions. A better understanding of how phages interact with the human immune system and cells will be essential for optimizing their use in clinical treatments and ensuring their safety.