Phage and enzyme therapies in wound infections: From lab to bedside

Abstract

Antibiotic-resistant (AR) bacterial wound infections (WIs) impose major burdens on healthcare systems, exacerbated by ineffective therapies and stalled antibiotic development. Phage therapy and phage-derived enzymes have gained traction as potent alternatives, leveraging targeted bactericidal mechanisms to combat AR pathogens. In this review, we summarised the antimicrobial mechanisms of both phage therapy and phage-derived enzymes as antimicrobial therapy, and outlined recent advances in their use for in vitro, in vivo and clinical applications for WI management. In addition, we also highlights recent advancements in their development, driven by genetic engineering, chemical modifications, and artificial intelligence. Finally, we identified the potential barriers and challenges they may encounter in clinical practice and the corresponding strategies to address these issues. The entire review gives us a comprehensive understanding of the latest advances in phages and their derivative enzyme therapies for treating WIs, in the hope that research in this field will continue to improve and innovate, accelerating the transition from the laboratory to application at the bedside and ultimately improving the efficacy of treatment for AR bacterial WIs.

Introduction

Wound infections (WIs) significantly impair patient health and burden healthcare systems by prolonging inflammatory responses, delaying healing, and increasing risks of sepsis and mortality.^[1] Globally, burn infections occur in 26.93% of cases with 18.27% mortality.^[2] In China, surgical site infections (2.91%) increase death risk, prolong hospitalization, and raise costs.^[3] Diabetic foot ulcers (DFUs) occur in 7% (UK) and 9% (USA) of patients, amplifying amputation and reducing quality of life.^[4]

Conventional management of treating WIs includes debridement, covering dressings, and antimicrobial treatment. Debridement involves the removal of the necrotic infected tissue and apoptotic cells from the wound, which reduces the bacterial population at the wound site. Wound dressings are commonly used to cover wounds and prevent or treat WIs. To date, many clinical wound dressings from various compatible materials have been developed and used, including sponges, hydrofibers, hydrocolloids, fucoidan, collagen, hydrogels, and films.^[5] Antibiotics are the preferred first-line antimicrobials for treating WIs. Currently, the extensive use and overuse of topical or systemic antibiotics has resulted in the emergence of bacterial strains with antibiotic resistance (AR).^[5] AR is estimated to occur in 70% of wound-infecting bacteria.^[6] Biofilms are also present in most chronic wounds. Biofilms increase the difficulty of treating WIs by preventing the penetration of antimicrobial agents into the wound and protecting the bacterials at the wound site.^[7] Therefore, it is urgent to identify new antimicrobial treatment strategies for difficult-to-treat WIs because of the presence of wound biofilms and the emergence of bacterial strains with AR.

In this context, phage and phage-derived enzyme therapies are emerging as viable alternatives for AR-refractory WIs, such as dermatoses, burns and chronic wound/ulcer infections.^[8] Despite the excellent antimicrobial efficacy of phages, phage therapy was discontinued in most Western countries once the broad-spectrum antibiotic penicillin was discovered. Unexpectedly, phage therapy alone or in combination with antibiotics continued in Eastern European countries, particularly in Russia, Poland, and Georgia. Following the global emergence of “invincible drug-resistant bacteria” and the rapid development of genetic engineering, phage therapy for treating WIs caused by AR bacteria has shown promising clinical results.^[9–11] Moreover, phage-derived enzymes do not replicate or evolve independently; therefore, they are more compatible with the international regulatory framework for drugs. The USA Food and Drug Administration (FDA) has successively approved the phage-derived enzymes: N-Rephasin^® SAL200 (iNtRON Biotechnology, Gyeonggi-do, Korea), CF-301(ContraFect, New York, USA), and Staphefekt SA.100 (Micreos Pharmaceuticals AG, Zug, Switzerland). Taken together, based on favorable regulatory approval, phage-derived enzymes can be applied clinically for treating WIs caused by AR bacteria.

This review was summarized as follow: (1) Antimicrobial mechanisms of phage-derived therapy; (2) Progress in phage/enzyme applications for in vitro and in vivo wound models; (3) Advances in the clinical development of phage/enzyme therapies for treating WIs; (4) Current challenges in broad implementation. Additionally, this review explored innovative approaches enhancing phage/enzyme efficacy through genetic engineering, chemical modifications, and artificial intelligence (AI).

Mechanisms of Phage-derived Therapy

Phage-derived therapy has been around more than 100 years since d’Herelle discovered and characterized phages and used them to treat dysentery in 1917.^[12] Phage-derived therapy is designed to kill pathogenic bacteria and eliminate biofilms through multiple mechanisms involving various phage components. Phages are viruses with specialized capability to target and destroy bacteria and can act as natural bactericides. Phages typically undergo two types of life cycles: lytic and lysogenic cycle. The mechanism of action of the lysis cycle consists of five steps as follow: (1) Adsorption: phage achieves tight adsorption to the host bacteria through specific binding of proteins on its tail fiber to the surface receptor of the host bacterium. (2) Invasion: the irreversible attachment phase is followed by tail sheath constriction, tail pipe penetration, and genomic injection. (3) Proliferation: phage synthesizes progeny phage proteins and nucleic acids using structural components of the host bacterium. (4) Assembly: synthesized phage components are assembled together according to a certain procedure. (5) Release: after the assembly is completed, phage is released into the extracellular space of the host bacterium by lysis and secretion. In contrast, in the lysogenic cycle, the phage genome is integrated into the bacterial host chromosome [Figure 1A]. Thus, horizontal gene transfer or the lytic cycle depends on the external condition. Through repeated lytic cycles involving the abovementioned steps, many phages proliferate within the host bacterial cell and are released outside the cell, subsequently causing a large number of bacteria to undergo lysis and cell death, thereby exerting a strong bactericidal effect.

Figure 1.

Antimicrobial schematic of phage and enzymes. (A) Life cycles of phages infecting host bacteria; (B) antibacterial mechanism of PGHs during the lysis cycle; (C) phages-derived therapy for WIs (created by BioRender.com 2025). AI: Artificial intelligence; PEG: Polyethylene glycol; PGHs: Peptidoglycan hydrolases; VAPGHs: Virion-associated peptidoglycan hydrolases; WIs: Wound infections.

The antibacterial activity of phages is mediated by two phage-derived enzymes: peptidoglycan hydrolases (PGHs) and polysaccharide depolymerase, which degrade bacterial cellular peptidoglycan and extracellular or surface polysaccharides, respectively. PGHs are localized either inside the virion or in the tail of the virion and cleave peptidoglycan in the bacterial cell wall from inside and outside of the cell, respectively.^[13] PGHs are classified into two groups: ectolysins and endolysins. Ectolysins are used by phages during the lytic cycle to locally degrade bacterial peptidoglycan prior to insertion of the genomic material; they allow viruses to inject their genomes into the host bacterial cell [Figure 1B]. Because virion-associated peptidoglycan hydrolases do not completely and thoroughly affect the stability of the host bacterial cell structure, their relevance has been investigated to a limited extent. In contrast, endolysins induce cell wall disruption during phage-mediated internal lysis, which occurs after phage assembly during the lytic cycle. After phage assembly during the lytic cycle, most lysins cannot directly penetrate the cell membrane. Holins are small-molecule polar transmembrane proteins that form “transmembrane pores” in the cell membrane; these pores facilitate the entry of lysins into the bacterial cell wall and the subsequent degradation of the bacterial peptidoglycan layer, leading to lysis of the host bacterial cell [Figure 1B].^[14]

In contrast, most depolymerases are localized inside or adjacent to the structural gene regions (i.e. tail fibers and substrates) of the phage genome; they frequently occur as part of tail fibers, tail spikes, baseplates, or neck proteins. Depolymerases can be classified into two groups according to their different mechanisms of action: hydrolases and lyases. Hydrolases catalyze the hydrolysis of glycosidic bonds. Lyases cleave (1,4)-glycosidic bonds through a β-elimination mechanism. These enzymes can degrade several forms of polysaccharides, including gram-negative (G^–) bacterial podoplanar capsular polysaccharides (CPS), extracellular polysaccharides (EPS) (major components of biofilms), and lipopolysaccharides (LPS).^[15] Degradation of these polysaccharide structures on the cell surface or in the biofilm promotes invasion of the bacterial cell not only by phages but also by other antimicrobial agents.

Phages and their derived enzymes can be developed through genetic engineering, chemical modification, or AI. In general, phage-derived therapy for bacterial growth inhibition and biofilm removal is mediated by the following three mechanisms: (1) intra- to extra- cellular degradation of the bacterial structure; (2) extra- to intra-cellular degradation of the bacterial structure; and (3) chemical disruption of the biofilm matrix, notably the structures of extracellular polymeric substances [Figure 1C].^[16]

In Vitro and In Vivo Studies for WIs

Phage therapy

Monophage therapy

Recently, several animal experiments have shown that monophage therapy is useful for WIs [Supplementary Table 1, http://links.lww.com/CM9/C448]. The results of animal experiments revealed a significant decrease in the number of pathogens in infected wounds, substantial reduction of the wound area, and regeneration of high-quality skin in the phage therapy group as compared to that in the antibiotic-treated and noninfected groups.^[17–20] In addition, phage therapy significantly reduced the level of wound tissue inflammation.^[18]

Phage therapy can also effectively eliminate biofilms in WIs. In an in vitro study, phage therapy of extracutaneous infected wounds in pigs significantly reduced the number of Staphylococcus aureus (S. aureus) cells in the biofilm.^[21] The combination of phage therapy with surgical debridement substantially improved wound healing and significantly reduced biofilm cell count.^[22]

Although monophages have apparent advantages as antimicrobial agents, they also have some limitations. First, because phages exhibit high specificity in targeting bacterial cells, a phage can kill only the bacterial strain that corresponds to its inhibitory effect. Second, bacteria can also develop resistance to phages.

Phage cocktails therapy

Phage cocktails, a mixture of two or more phages with different modes of division, can be a good solution to address the shortcomings of monophage therapy, which is currently one of the hot topics of research in phage therapy [Supplementary Table 1, http://links.lww.com/CM9/C448].

Cocktail phages display synergistic bactericidal activity in treating WIs and have a positive effect on wound healing. Cocktail phages appear to function synergistically, where one phage targets the encapsulated bacterial cell, selectively causes loss of the bacterial cell receptor, transforms the bacterial cell into an unencapsulated state, and increases its susceptibility to other phages in the cocktail.^[23,24] As reported previously, although the application of monophage therapy to treat Streptococcus pneumoniae-infected burn wounds showed some efficacy in eliminating infection, the phage cocktail therapy was more effective in reducing bacterial load and improving wound tissue healing.^[25,26] Moreover, as compared to monophage therapy, the phage cocktail therapy significantly inhibited the emergence of drug-resistant mutants.^[24,25]

Combination therapy

The antimicrobial effects and the underlying mechanisms of the combination of phages and antimicrobial agents have been increasingly studied [Supplementary Table 1, http://links.lww.com/CM9/C448].^[27–29] The combination therapy exerts its effects through the following mechanisms. The phage and antimicrobial agents exhibit a synergistic inhibitory effect. The combination of phages and antibacterial agents inhibits bacterial growth through a “seesaw effect”.^[30] The mechanism of this “seesaw effect” was elucidated by the study of Ho et al^[31]; the authors found that mutations in the bacterial gene epaR decreased the adsorption of phages by enterococci. However, alteration in this gene simultaneously increased bacterial susceptibility to daptomycin and vice versa. Moreover, the induction of phenotypic changes in bacteria by antimicrobial drugs enhanced the ability of the corresponding phage to invade the bacterial cells. In vitro and in vivo experimental studies further confirmed that the combination of phage and antibiotics shows better antimicrobial efficacy, prevents the development of drug resistance, and promotes tissue healing.^[32,33] The combination of phage with honey also displayed better antimicrobial effects.^[34,35]

The combination therapy also facilitates the elimination of biofilms from the infected wounds. Phages can disrupt the extracellular matrix in the biofilm, thereby exposing the bacterial cells to antimicrobial drugs.^[36] Compared to monotherapy, the combination of the PEV20 bacteriophage and ciprofloxacin enhanced biofilm removal in cystic fibrosis patients and those with wound biofilms.^[37] The sequential combination of antimicrobial agents and phages should also be considered for biofilm removal. For example, the sequential addition of gentamicin or ciprofloxacin after 6 hours of phage treatment significantly reduced biofilm formation.^[38] Similarly, the phage-honey combination therapy facilitated the eradication of biofilms from infected wounds.^[34]

Phage-derived enzyme therapy

Lysin therapy

Lysin therapy is usually the most effective option against gram-positive (G⁺) bacteria as it causes direct exposure of the peptidoglycan cell wall to enzymatic degradation [Supplementary Table 2, http://links.lww.com/CM9/C448]. Yang et al^[39] found that ClyF is effective against all clinical isolates of S. aureus tested under planktonic and biofilm conditions. In mouse models of bacteremia and burn WI, a single intraperitoneal or local injection of ClyF yielded good clearance of methicillin-resistant S. aureus (MRSA). Fusion proteins consisting of cell-penetrating peptides (CPPs) derived from trans-activating transcription (Tat) factor fused to lysin JDlys (CPPTat-JDlys) inhibited MRSA growth in intracellular infections, effectively reduced inflammatory responses and cellular damage, and accelerated the healing of skin abscesses in mice.^[40]

Lysin therapy against G^– bacteria is, however, not simple. Phage lysins cannot easily damage the peptidoglycan layer in the cell wall of G^– bacteria because of the presence of a protective outer membrane (OM). Improving the permeability of the OM is a proven solution to this issue. OM permeants are usually categorized into two groups. The first group comprises multivalent cationic compounds such as polymyxins and their derivatives, aminoglycosides, and lysin polymers. They can compete with divalent cations to displace them from interactions with anionic LPS molecules, leading to the disintegration of the OM.^[41] The second group includes chelating agents. Chelation of divalent cations is a well-established method for penetrating the cell wall of G^– bacteria. Ethylenediaminetetraacetic acid (EDTA) is the most commonly used chelating agent. EDTA removes divalent cations that stabilize the OM from the binding site, thereby disrupting the cell membrane and making it more susceptible to direct attack by lysins.^[42] Lysin L-KPP10 exhibited significant activity only in the presence of 0.5 mmol/L EDTA.^[43] Alternatively, the use of positively charged peptide fusions developed by recombinant engineering could be a more convenient approach to ensure efficient OM permeabilization.^[44] The addition of a positively charged sheep myeloid antimicrobial peptide (SMAP-29) to the C-terminus of endolysin L-KPP10 (to generate AL-KPP10) eliminates the need for EDTA to increase cell permeability.^[43] Under certain conditions, all three phage lysins (LysAm24, LysECD7, and LysSi3) show the natural ability to penetrate the bacterial OM without the need for chemical permeabilizing agents; this is because the positively charged fragments can disrupt or perturb the OM.^[45] In addition, enhanced penetration against G^– bacterial OM was observed for cationic liposomes encapsulating lysin. In previous studies, the endolysin Lysqdvp001 was encapsulated in cationic guar gum containing liposomes; this approach achieved more than 5-log reduction in the number of Vibrio parahaemolyticus cells.^[46]

Although WIs can be treated with lysins alone, combination therapy can more efficiently clear biofilms from infected wounds through synergistic effects. LysGH15 and apigenin (api) were added to Aquaphor to yield a LysGH15-api-aquaphor (LAA) ointment. In a mouse model of MRSA-infected skin wounds treated with LAA, bacterial colony counts and the levels of proinflammatory cytokines were significantly reduced, and skin wound healing was promoted.^[47] Duarte et al^[48] found that the combination of the phage-derived lysogenic protein CHAPSH3b and the virulent phage phiIPLA-RODI synergistically inhibited S. aureus biofilm formation. CHAPSH3b also prevented the development of phage resistance. In an in vitro model of porcine skin WIs, the combination of lysin and phiIPLA-RODI promoted wound healing.^[48]

Depolymerase therapy

Depolymerases are used in two forms: (1) tail spike proteins (TSPs) with depolymerase domains as a virion component and (2) free enzymes. During phage therapy, phages multiply in the host cell and produce more viruses with TSPs. Subsequently, while treating biofilm infections, phages can deliver these enzymes to the target site more efficiently.^[49] In contrast, free depolymerase is a protein obtained through gene expression. It is active even under harsh environmental conditions and inhibits biofilm formation. Following phage-induced lysis, depolymerases could be released as free enzymes from the cell interior.^[50] This free enzyme originates either from the excess production of proteins that are not incorporated into the virus assembly during phage infection or from the use of alternative start codons during translation, which results in a soluble form of the virus-associated enzyme.^[15,50] Both these forms of depolymerases can freely diffuse and physically separate from the phage following bacterial polysaccharide degradation.^[50] Free depolymerases obtained by recombinant gene technology are unlikely to develop bacterial resistance.^[51] In addition, free depolymerases diffuse more rapidly and efficiently than virus-associated depolymerases.^[49]

Although depolymerases degrade CPS/EPS/LPS in biofilms and bacterial surface barriers, they do not destroy bacterial cells. Therefore, depolymerases are frequently used as anti-biofilm agents and antibacterial adjuvants. Given the complexity of polysaccharides and the diversity of bacterial species in biofilms, the use of phage-encoded depolymerases alone to disrupt bacterial growth and remove biofilms is inadequate. A recent review by Wang et al^[52] suggests the use of combination therapy to treat bacteria and biofilms. Combination therapy involves phage-derived depolymerase in combination with different therapeutic agents, including antibiotics, phages, or other antimicrobial agents.^[52] Borzilov et al^[53] showed that the number of Acinetobacter baumannii (A. baumannii) cells on the surface and deeper layers of the wound was significantly reduced after treating burn-infected wounds in mice with the combination of a K9-specific phage and recombinant depolymerase. Depolymerases can also be genetically engineered to enhance their effectiveness.^[52,54] Currently, in vivo efficacy analyses of phage depolymerases have been limited to animal studies, few clinical studies have focused on treating WIs with phage-derived depolymerases. Hence, it is critical to conduct in-depth studies on this topic.

In summary, the in vitro and in vivo experimental studies have confirmed that treatment with phages and their derived enzymes is effective in inhibiting wound-infecting bacteria and eliminating biofilms, which can reduce the level of wound tissue inflammation and subsequently promote wound tissue healing. However, these studies also have some limitations. First, the formulation of phages and their derived enzymes are often delivered using sterile buffer solutions such as phosphate-buffered saline or Tris-buffered saline-magnesium buffer. Although liquid phage formulations are generally considered stable when refrigerated, there are no relevant studies on the stability of phages and their lytic enzymes during long-term storage (>1 or 2 years). However, this is a crucial aspect of developing commercially viable phages and their derived enzymes. Second, in most of the in vivo experimental studies, WI models were generated by infection with a single strain of bacteria; however, this is in contrast to real-world clinical WIs that are frequently caused by multiple strains of bacteria. In addition, the wound tissue characteristics of patients with WIs and the associated bacterial strains show dynamic changes over time, which are not replicated in experimentally infected wounds. Another aspect that cannot be ignored is that patients with WIs often have accompanying underlying diseases such as hypertension and diabetes mellitus, and their WIs show a tendency to become increasingly complex; experimental wound models cannot closely reflect such complex, real-world clinical scenarios. Finally, it is worth noting that most of these in vitro studies were evaluated in an in vivo model of mouse skin infection.^[55] The skin structure of a mouse, however, differs from that of a human. The epidermis and dermis are thinner in mice than in humans. In addition, hairs on mouse skin are renewed once every 3 weeks, whereas hairs on human skin remain intact for several years. The mechanism of wound closure also differs between mouse skin and human skin, which limits the translational potential of these findings. Porcine skin is structurally similar to human skin, thus making it the most suitable option for skin and wound studies. Currently, however, only a few studies have been conducted using isolated porcine skin as a skin model for research.^[55,56] Economic factors may be one of the main reasons for choosing mice as in vivo wound models. Therefore, phages isolated and screened from natural environments and recombinant phage-derived enzymes generated through bacterial or yeast expression systems are not fully adequate for widespread clinical use.

Clinical Studies for WIs

Phage therapy

Case study

Phage therapy has demonstrated significant efficacy in treating chronic wounds and ulcers, particularly in AR cases.^[57,58] Studies report complete healing of DFUs in five patients using phage preparation Sb-1 (the Eliava Institute of Bacteriophages, Microbiology, and Virology, Tbilisi, Georgia), preserving tissue integrity after failed antibiotics.^[59] Similarly, 20 patients with WIs unresponsive to standard therapies achieved full recovery after topical phage treatment, with no residual infection detected.^[60] Two MRSA-colonized DFUs patients also showed ulcer resolution and MRSA eradication after Pyo phage therapy (National manufacturer of immunobiological products in Russia, Moscow, Russia).^[61]

Combining phages with antibiotics enhances outcomes in the difficult-to-treat bone and soft tissue infection. A 21-year-old patient with recurrent multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) osteomyelitis suppressed bacterial growth and biofilm formation when treated with phage cocktail BFC 1.10 (the Queen Astrid Military Hospital, Brussels, Belgium) and ceftazidime-avibactam.^[62] Similarly, Netherton syndrome patients with persistent soft tissue infections and antibiotic-refractory wounds experienced marked clinical improvement and enhanced quality of life following phage-antibiotic combination therapy.^[63] These cases underscore the synergistic potential of phage-antibiotic regimens in tackling biofilm-associated and complex infections.

Clinical trials

Due to the superiority of phage therapy, there are more and more phage-related clinical trials [Table 1]. Phagoburn was a randomized phase I/II clinical trial (NCT02116010) that recruited 26 patients with burn WIs and one patient without infection.^[64] The efficacy of the phage cocktail (PP1131, Pherecydes Pharma SA, Nantes, France) treatment did not meet expectations, mainly due to a rapid decrease in the total titer of cocktail phages to 10⁴–10⁵ PFU/mL within 6 months. The instability of PP1131 might be due to electrostatic interactions, adsorption of phages on the surface of the storage container, oxidation, phage-phage interactions, or hydrochemical interference.^[64,65]

Table 1.

Summary of clinical trials of phage therapy and lysin therapy for wound infections.

Clinical trial registration number	Registration date	Phases	Study status	Results
NCT00663091	April 22, 2008	Phase Ⅰ	Completed.	This study found no safety concerns with the bacteriophage treatment.
NCT02116010	December 11, 2012	Phase Ⅰ/Ⅱ	Forced to end early, and related research published.	The efficacy of phage therapy is not as expected. The only positive outcome of the trial was that it resulted in fewer adverse reactions compared to the control group.
NCT02664740	January 27, 2016	Phase Ⅰ/Ⅱ	Not yet recruiting.	Not provided.
NCT04323475	March 20, 2020	Phase Ⅰ	Recruiting.	Not provided.
NCT04803708	March 2, 2021	Phase Ⅰ/Ⅱ	Completed.	Not Provided.
NCT04815798	March 19, 2021	Phase Ⅰ/Ⅱ	Recruiting.	Not provided.
NCT06319235	March 13, 2024	Phase Ⅰ/Ⅱ	Recruiting.	Not provided.
ChiCTR2400084025	May 9, 2024	Phase Ⅰ/Ⅱ	Recruiting.	Not provided.
NCT01855048	May 16, 2013	Phase Ⅰ	Completed, and related research published.	SAL200 was well tolerated, and no serious adverse events were observed in this clinical study.
NCT02439359	May 8, 2015	Phase Ⅰ	Completed, but Not yet published.	A single dose of CF-301 has a low propensity to induce an inflammatory response.
NCT02840955	July 21, 2016	Phase Ⅰ/Ⅱ	Completed, but Not yet published.	Not provided.
NCT03089697	March 24, 2017	Phase Ⅱ	Terminated	Not provided.
NCT03163446	May 23, 2017	Phase Ⅱ	Completed, and related research published.	Compared to the control group, CF-301 treatment had a similar rate of adverse events, but was statistically significant in terms of 30-day mortality, and median length of stay.
NCT04160468	November 13, 2019	Phase Ⅲ	Completed, and related research published.	Rates of adverse events were similar across groups, and CF-301 in combination with antibiotics failed to improve clinical response at day 14 in patients with Methicillin-resistant Staphylococcus aureus bacteremia/endocarditis.

CF-301: One of phage-derived lysins N-Rephasin; SAL200: One of phage-derived lysins N-Rephasin.

The University Hospital Centre of Nîmes, France, is conducting a clinical trial (NCT02664740) to evaluate the efficacy of standard treatment versus topical application of the phage cocktail (PhagoPied) therapy for treating MRSA-infected DFUs. A phase Ⅰ/Ⅱ clinical trial showed that the phage therapy using P. aeruginosa, S. aureus, and Escherichia coli (E. coli) phages was safe and effective in treating 42 patients with chronic venous leg ulcers.^[66] To the best of our knowledge, the results of the phase Ⅰ/Ⅱ clinical trials (NCT02664740) have not yet been reported. Another phase Ⅰ/Ⅱ trial (NCT04803708) of phage preparations TP-102 (TechnoPhage SA, Lisboa, Portugal) has been completed, but the results of the study have not yet been published.

Four other phage therapy trials for treating wounds with secondary infections (e.g., second-degree burn wounds, pressure ulcers and surgical site infections) are ready to be initiated or are currently in progress, as shown in Table 1.

These case studies inform us that patients with WIs were treated with phage therapy or a combination of phage and antibiotics after a long period of ineffective antibiotic treatment. Surprisingly, these chronic wounds healed rapidly, with a significant improvement in patients’ quality of life. There are, however, only a few cases of successful phage therapy. Hence, there is a critical need to conduct high-quality controlled clinical trials to further validate the efficacy of phage therapy for treating chronic WIs. Although the results of Phagoburn’s program have not been as promising as expected, it should be noted that several trials are currently in progress or have not yet reported their results.

Phage-derived enzyme therapy

Lysins, as an antimicrobial agent, have received increasing attention from researchers because of their strong bactericidal effect, specificity, stability, and safety. Currently, all clinical trials are primarily targeting S. aureus. In this section, we describe the current progress of clinical trials of lysin products as summarized in Table 1.

Rephasin^® SAL200 (SAL200) is a recombinant SAL-1 endolysin. SAL200 is active against both planktonic and biofilm-embedded strains of S. aureus, including MRSA and vancomycin-resistant S. aureus (VRSA).^[67,68] The study (NCT01855048) done by Jun et al^[68] revealed that SAL200 was well tolerated with satisfactory pharmacokinetics (PKs), with no severe clinical adverse events.

CF-301, also known as PlySs2 or exebacase, is a lysin produced by Streptococcus suis phage and has a broad-spectrum of lytic activity against G⁺ pathogens.^[69] CF-301 also showed anti-biofilm, anti-herpetic cell, and anti-small colony mutation activities against S. aureus.^[70] In 2015, Jandourek et al^[71] found that CF-301 was well tolerated with no severe adverse clinical events in a phase I clinical trial. Fowler et al^[72] found that the combination of CF-301 and antibiotics was significantly better than the antibiotic-alone group in terms of length of hospital stay/re-admission rate/mortality rate of patients in a phase II clinical trial (NCT03163446). However, a subsequent study of a superiorly designed phase III clinical trial (NCT03163446) revealed that the incidence of adverse events was similar in all groups and that the combination of CF-301 with antibiotics failed to improve the clinical response at day 14 in patients with MRSA bacteraemia/endocarditis.^[73] This may be related to the small sample size and increased heterogeneity among the groups.

Staphefekt SA.100 is an engineered lysin specifically designed to inhibit MDR S. aureus strains associated with superficial skin infections, with no suppressive effects on other cutaneous commensals. A double-blind, drug-controlled superiority trial found that S. aureus lysin (Staphefekt SA.100) treatment significantly reduced the incidence of recurrent AD during the intervention period compare to topical corticosteroids (TCS).^[74] Long-term targeted lysin therapy against S. aureus was well tolerated in AD patients but did not reduce TCS use. Because patient compliance with therapy is excellent, concurrent application of TCS, emollients, or both may mask the clinical benefits of lysins. Hence, phase III clinical trials are required to provide further strong evidence of the efficacy of topical lysin versus antibiotics for treating S. aureus superficial skin infections.

Micreos is also developing a recombinant chimeric lysin against drug-resistant S. aureus, designated as XZ.700 (Micreos Pharmaceuticals AG, Zug, Switzerland). In vivo animal studies demonstrated that XZ.700 significantly reduced the number of drug-resistant S. aureus cells, restored the microbial diversity of wounds, and promoted wound repair.^[75,76] A phase I/IIa clinical trial of XZ.700 for treating 48 patients with S. aureus-infected AD was initiated in 2020. The results of this trial are yet to be reported.

Medolysin^® (Lysando AG, Regensburg, Germany) developed by Lysando, is a wound care spray for use on chronic and infected wounds. Unfortunately, the company has yet to disclose the exact ingredients and specific research data for its products.

Although lysin products are still in the early development stage, in the near future, we expect that several commercially promising lysin products will be available in the market for patients with WIs caused by AR bacterial strains.

Engineering, chemical modifications, and AI

Conventional phage therapy has several limitations, including narrow host range, phage resistance, and potential eukaryotic immune response. Phage engineering has attracted considerable interest from researchers and has a high potential for modifying the biological properties of phages. Phage engineering involves gene manipulation techniques such as the targeted nucleases (including clustered regularly interspaced short palindromic repeats/Cas9 [CRISPR/Cas9] and zinc-finger nucleases and transcription activator-like effector nucleases) and the application of molecular biology methods (e.g., synthetic biology approaches, homologous recombination, CRISPR/Cas9-bacteriophage recombineering with electroporated DNA [CRISPY-BRED] and CRISPR/Cas9-bacteriophage recombineering with infectious particles [CRISPY-BRIP] recombination, and restarting phage-based engineering). Nucleic acid manipulation of the phage genome through genetic manipulation techniques is used to enhance the antimicrobial activity of the phage against pathogens. The CRISPR/Cas9 method is currently the main phage genome engineering approach because of its ability to precisely address the abovementioned limitations of wild-type phages. All genetic engineering manipulation techniques have advantages and potential disadvantages, as mentioned in a recently published review.^[77] Modified phages may exclude undesirable properties, which could potentially alter their specificity or improve their therapeutic potential. Immunomodulation and genetic manipulation of phages through deletion of genes associated with the holin-endolysin system can yield noncellular phage variants that effectively eliminate bacterial hosts, reduce endotoxin production, and attenuate adverse inflammatory responses.^[78,79] Peng et al^[80] replaced the receptor-binding structural domain of phage M13 (the N-terminal structural domain of g3p) with the corresponding structural domain of phage Pf1; this substitution allowed chimeric M13-g3p (Pf1) to attach to P. aeruginosa through type IV epilator. Prokopczuk et al^[81] found that, in a burn-infected wound animal model, treatment of P. aeruginosa with the genetically engineered superinfectious variant Pf (eSI-Pf) completely eliminated the ability of the bacteria to spread from the burn site to internal organs.

Lysins contain a modular assembly of two functional structural domains: the N-terminal enzymatically active domain (EAD) and the C-terminal cell wall-binding domain (CBD), which are linked with a short peptide. This modular structure is the key factor for preparing novel second-generation lysins by modifying natural lysins through protein engineering techniques. Many natural lysins show low bactericidal activity and other shortcomings. To overcome these limitations, second-generation lysins have been designed through various approaches to broaden their cleavage spectrum and improve their intrinsic antimicrobial and biochemical properties (e.g., bactericidal activity, specificity, and half-life); these approaches include targeted mutagenesis, deletion and unfolding of structural domains, synthesis of truncated proteins, and production of chimeric or engineered lysins by combining structural domains of different lysins or by fusing peptides with different properties (polycationic, hydrophobic, or amphiphilic). Mutation in the 88th amino acid residue (glutamate) of lysin LysF1 yielded three mutants, namely Glu88Leu, Glu88Phe, and Glu88Met.^[82] These mutants showed improved thermal stability and bactericidal activity as compared to natural lysin LysF1. The C-terminal truncated protein of lysin CD27L with retained EAD showed higher lysis activity against Clostridium difficile and an expanded lysis spectrum; in contrast, the N-terminal truncated protein lacked lysis activity.^[83] The fusion of LysK with the albumin-binding domain improved its serum circulating half-life and reduced its renal deposition in vivo.^[84] Wang et al^[40] combined lysin JD007 from an S. aureus phage with a CPP; this modification promoted the passage of the lysin across the bacterial cell membrane to inhibit intracellular S. aureus infection. Yang et al^[85] constructed the chimeric lysin ClyR by fusing the CHAP structural domain of PlyC lysin with the CBD of lysin PlySs2. This combination yielded a new chimeric lysin with potent bactericidal activity and broad-spectrum streptococcal host range, including a wide variety of Streptococcus species, as well as representative Enterococcus and Staphylococcus species (including MRSA and VRSA). Briers et al^[86,87] examined the binding of lysins to peptides with polycationic and hydrophobic/amphiphilic characteristics. They constructed a library of 49 Artilysins and found that the screened Artilysins exhibited significant in vitro antimicrobial activity against P. aeruginosa, A. baumannii, E. coli, and Enterobacteriaceae members. However, the engineering of these proteins often involves cumbersome restriction-linkage cloning methods, which limits the number of engineered variants that can be studied within the rational design framework. Protein engineering also presents other difficulties such as undesirable effects due to the fusion of peptides with enzymes and discrepancies between in vitro and in vivo results. It is therefore difficult to predict the final outcome of the fusion, partly because the precise mechanism through which the phage lysin penetrates the eukaryotic cell membrane barrier is not yet fully understood. In addition, the production of enzymes requires large-scale fermentation, purification, and activity testing, which can increase the production cost. All these factors restrict the large-scale production and clinical application of phage lysins.

Phages and phage-derived lysins can also be chemically modified to alter their properties. Phage particles can be modified by conjugating with polyethylene glycol (PEG) to render them less immunogenic and prolong their blood circulation time.^[88] Wang et al^[89] synthesized phage-Ce6 conjugates (PCs), which were modified through carbodiimide chemistry. Subsequently, by performing mild biomineralization, KMnO₄ was reduced to MnO₂ and deposited on the PC surface to yield PCM. PCM shows the potent activity of targeting the host bacterial cell and efficiently transports Ce6 to penetrate the wound biofilm. Wang et al^[90] successfully coupled cadmium-based photocatalytic quantum dots to phage particles through avidin-biotin bioconjugation; this approach enabled the phage to precisely localize on the surface of P. aeruginosa cells expressing the green fluorescent protein. Thus, the phage functioned as a “shell-delivering targeting device” in a mouse wound intervention model. In summary, chemical modifications of phages provide new strategies for their use in antimicrobial therapy. To avoid the risk of triggering a hypersensitivity reaction or to avoid inhibition by antibodies, third-generation lysins are developed through biochemical “masking” strategies, for example, PEGylation.^[55] PEG molecules are covalently linked to certain amino acid residues such as cysteine or lysine. Lysostaphin is the first PEGylated lytic enzyme; its binding affinity for anti-lysostaphin antibodies was reduced by 10-fold.^[91] However, the PEGylation process also decreased the in vitro antimicrobial activity of lysostaphin.^[91] Nevertheless, the longer half-life of lysostaphin compensates for its reduced antimicrobial activity in vivo.

The use of AI to develop and discover phages and their derived enzymes could provide new breakthroughs. With advancements in AI technology, AI is expected to play an increasingly important role in the development of phage therapy, including identification of phages from macro-genomic samples, annotation of phage viral proteins from phage genome sequences, prediction of phage hosts, and determination of phage lifecycle.^[92] Phage genetic engineering technology is combined with AI to optimize phage genome design, prevent the emergence of resistance to phages, customize the composition of personalized phage cocktails, and individualize patient treatment.^[93,94] Typically, the network of phage-bacterial infections is massive, making it difficult to manually study all possible phage cocktails. Diaz-Galiana et al^[94] developed an R package containing four methods for designing phage cocktails; this package can automate the designing of efficient phage cocktails based on the phage-bacterial infection network. This tool saves considerable efforts of scientists or other users to design high-quality phage cocktails. Users can choose between the speed and accuracy of the results based on their requirements. Thus, the design, synthesis, preparation, and application of artificial phages with high efficiency and low cost can be achieved; this strategy provides numerous possibilities to accelerate the large-scale clinical application of phages.

Although novel phage lysins have emerged as potential antimicrobial agents, experimental screening methods for these lysins pose major challenges because of the enormous workload. Fu et al^[95] proposed an AI framework (deep mining of phage lysins from human microbiome [DeepMineLys]) based on the convolutional neural network (CNN) to search for novel phage lysins from three human microbiome datasets and found 11 active novel lysins. Zhang et al^[96] used AI to mine seven novel phage lysins with excellent in vitro antimicrobial activity from a huge genome library; of these lysins, LLysSA9 outperformed the best-in-class alternatives. The efficacy of LLysSA9 was further demonstrated in mouse bloodstream and WI models.^[96] Screening nonredundant phage lysins through AI lays a new methodological foundation for discovering potent antimicrobial protein drugs, as shown in Figure 2. Despite the highly promising prospects of AI, several challenges need to be overcome in predicting protein structures in the following areas: intrinsically disordered proteins and their regions, protein-ligand interactions and post-translational modifications, protein dynamic conformations, protein structural defects caused by mutations, and untrained absolute unknown structures.^[97]

Figure 2.

Diagram of the AI framework for the discovery of novel lysins. (A) Datasets inputs: Finding phage/bacterial macrogenomes from databases, which in turn collects phage-related protein sequences. (B) Predictions: Deleting redundant phage-related protein sequences by AI, inputting organized protein sequences into AI neural networks, mining putative lysins, and then predicting the MIC of putative lysins in AI models. (C) Experimental validations: Obtaining the novel lysins by putative protein gene transcription and expression, assessing the antimicrobial activity against pathogenic bacteria in the in vitro experiments, then comparing the therapeutic efficacy of the novel lysins, phage-derived lysins, antibiotics, and phages for the treatment of infected wounds in a mouse model (created by BioRender.com 2024). AAC: Amino acid chain; AI: Artificial intelligence; MIC: Minimum inhibitory concentration.

Prospects and Challenges of Phage-derived Therapy for WIs

Conventional antibiotics are frequently ineffective in treating WIs because of the presence of AR bacteria in wounds and biofilm formation. In the post-antibiotic era, phages and their derived enzymes are receiving increasing attention from researchers as a potential alternative therapy to antibiotics. Results of in vitro and in vivo experiments suggest that phage-derived therapy may play an important role in reducing the bacterial load of wounds, eliminating wound biofilms, and promoting wound healing. Clinical studies have demonstrated encouraging results of phage therapy in the treatment of patients with WIs such as trauma, surgical and burn WIs, and diabetic foot infections.

Despite the promising prospects of phage therapy, some issues and challenges remain to be addressed to promote its widespread clinical application. First, chronically infected wounds harbor a diverse range of bacterial strains, which change dynamically over time.^[98] Therefore, the high specificity of phages for pathogenic bacteria greatly limits the scope of their clinical application. In addition, WI treatment is often administered through traditional routes such as sterile buffer solutions, creams, or powders, which provide a single burst of drug release, thus making it difficult to exert antimicrobial effects on the infected wounds at different stages of the treatment process.

Phage engineering and novel drug delivery systems may provide solutions to the abovementioned issues. Phage structure is relatively simple and comprises a core and a protein shell. The shell generally consists of a head and a tail. Adhesion proteins in the tail fibers of the phage are critical for their ability to invade the host bacteria. Modification of adhesion proteins and structural domains of the phage through genetic engineering can change the host bacterial spectrum of the phage, thereby increasing its application range. Future studies should also focus on optimizing the other properties of phages through genetic engineering to improve their antimicrobial effects. In addition, several researchers worldwide have shown increasing interest in developing novel delivery systems for phages and phage-derived lysins (e.g., hydrogels, liposomes, nanoparticles, and nanoemulsions) for treating WIs.^[99,100] Future studies should also evaluate the use of these novel drug delivery systems to release phages and lysins into the infected wound sites precisely and systematically to achieve robust antimicrobial effects at different stages of wound healing.

Second, the use of phages alone to inhibit bacteria often results in poor outcomes. In this scenario, a combination of phages with other antimicrobial drugs is required to enhance the bactericidal effect on wound-infecting bacteria. Previous experiments have shown that phages and antibiotics function synergistically through the “see-saw effect” to efficiently eliminate wound-infecting bacteria and biofilms.^[11,30] The use of phage cocktail therapy also appears to be equally effective in reducing bacterial load and promoting wound healing. The establishment of protocols for the selection and proportion of phages in these cocktails, possibly through AI, can facilitate the rapid development of effective scientific approaches to achieve the optimal combination of cocktail phages. The current approach of optimizing cocktail phage combinations through AI should be further validated through more research.

Third, the PK and pharmacodynamic (PD) properties of phages and their derived enzymes require improvement. Phage PK properties are complicated by phage self-replication and the interaction of the body’s immune system with the phage. In humans, phage levels were detectable at 5 minutes after injection of 8.5 × 10⁷ PFU in 4 mL of lactated Ringer’s solution through a peripherally inserted central catheter.^[101] However, phages could not be detected in blood samples after 50 minutes. Based on the PD properties of phages, most current applications of phage therapy are achieved using very high titers of phage, phage cocktails, or combined antibiotics. The specific mechanisms of phage resistance and phage-host interactions also require in-depth studies. Phage engineering and novel delivery systems can also be used to optimize the pharmacological properties of phages. The half-life of phage-derived lysins CF-301 and SAL200 is more comparable to that of standard drugs than to the half-life of phages. However, the application of phage-derived lysins has some limitations: (1) immune response to lysin during administration, (2) limited half-life, and (3) protein hydrolysis by inflammatory proteases at the infection site. To improve the PK and/or PD properties of phage-derived enzymes and increase their relevance under clinical conditions, third-generation lysins are currently being developed through protein engineering and biochemical modifications such as increased hydrodynamic volume, dimerization of lysin, fusion to the albumin-binding domain, PEGylation, glycosylation, and T-cell epitope depletion.^[55]

Fourth, the application of phages and their derived enzymes requires strict regulatory guidelines. Globally, official authorities have defined AR as a serious threat to human health. Various countries are developing strategic action plans to find alternatives to antibiotics. Phage or phage-derived enzymes have not yet been used as the primary clinical treatment approach for drug-resistant infections. The main reason for this hesitation is the lack of high-quality, large-sample-size clinical trials with favorable results. Phages have been used as a standard medical treatment in the healthcare institutions of Georgia and Russia. An encouraging observation is that in recent years, several countries, such as Australia, Belgium, the USA, and France, have officially allowed the use of phage or phage derivatives (lysins) in clinical therapy only for special and urgent situations. Phage-derived enzymes do not self-replicate or evolve; therefore, they are more compatible with the regulatory framework for drugs. To date, the FDA has granted consecutive approvals for the phage cocktail Sb-1 and the phage-derived lysins N-Rephasin^® SAL200 and CF-301 for application in clinical emergencies and special situations. Staphefekt SA.100 is designated as a Class 1 medical device in Europe for treating S. aureus-infected superficial lesions. In summary, the receipt of regulatory approvals for phages and their derived enzymes has facilitated their application for treating WIs with AR bacteria in clinical emergencies.

Lastly, it is critical to ensure that the produced phages and their derived enzyme formulations have reliable quality. The results of the European multicenter Phagoburn project (NCT02116010) and the CF301 phase III clinical trial (NCT03163446) did not meet the expected definitive efficacy. This prompted us to redefine the quality of phages and their derived enzyme preparations. Despite the negative outcome, these previous studies have some important lessons for future study design. The production of safe phages and their derived enzyme preparations, for both small-scale production for personalized therapy and large-scale production, requires effective quality control measures. The sterility, stability, and absence of endotoxins, exotoxins, or other harmful impurities should be monitored for all therapeutic preparations containing phages or phage-derived enzymes. In addition, pharmaceutical preparations should be formulated using appropriate measures that ensure adequate stability and compatibility with the administration route. Therefore, the process of preparing these formulations must also be considered during industrial scale-up. All these steps should be implemented while adhering to good manufacturing practice standards. Notably, efforts should be undertaken to reduce the total cost of production so that the final products of phage and its derived enzymes are economically viable.

Although few in vitro, in vivo, and clinical application research studies have been conducted on phage therapy and phage-derived enzyme therapy for treating WIs, there is a need to carefully develop phage and its derived enzyme formulations and to design more rigorous clinical trials for assessing their efficacy and safety as a novel antimicrobial therapeutic approach. This will facilitate the acquisition of the required administrative regulatory approvals to accelerate drug development and provide a solid foundation for the widespread clinical application of phages and their derived enzymes. Despite the abovementioned challenges, we are optimistic that phage therapy and phage-derived enzyme therapy have a bright future for application in treating WIs caused by AR bacteria.

Conclusions

AR bacterial WIs increasingly threaten global health and strain healthcare systems. Phage therapy and phage-derived enzymes emerge as viable alternatives to antibiotics, with preclinical and clinical studies confirming their efficacy in reducing bacterial load, eradicating biofilms, and accelerating wound healing. To optimize these therapies, future research can enhance antimicrobial properties and broaden their scope via: (1) phage-antimicrobial combination therapies; (2) genetically engineered or chemically modified phages; (3) engineered proteins like artilysins and chimeric lysins; and (4) AI-driven phage/lysin development or discovery. Comprehensive in vitro, in vivo, and clinical trials across diverse phage/enzyme formulations are crucial to ensure safe, effective clinical translation. Continued innovation in phage and enzyme engineering, coupled with rigorous validation, will unlock their full potential in combating AR bacterial WIs. By refining these strategies, phage-derived therapy could revolutionize wound care, offering tailored solutions to improve healing outcomes and patient prognoses in the face of rising AR.

Funding

This work was supported by grants from the National Key Research Programme Project (No. 2023YFE0204500) and the Natural Science Foundation Project of Chongqing Science and Technology Commission (No. CSTB2024NSCQ-MSX0770).

Conflicts of interest

None.

Supplementary Material

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