Efficacy of phage therapy in controlling staphylococcal biofilms: a systematic review

Abstract

Background

Antibiotic-resistant bacteria pose an urgent health threat as mutations have led to resistant strains that evade treatment. These bacteria form biofilms, complicating infection management. Bacteriophages are being recognized for their potential in phage therapy due to their effectiveness in rapidly targeting and eliminating bacterial hosts.

Materials and methods

This systematic review examined the effectiveness of bacteriophages against biofilms created by antibiotic- and drug-resistant staphylococcal strains. A thorough search of the Embase, Web of Science, PubMed, and Scopus databases was conducted for studies published from 2012 to October 29, 2024, focusing on relevant research while excluding irrelevant studies.

Results

This systematic review assesses the effectiveness of phage-derived enzymes, including endolysins and depolymerases, as well as whole bacteriophages, in degrading biofilms and clearing bacteria. It also highlights how combining phages with antibiotics or other agents can improve biofilm removal. The review explores the potential applications of phage therapy in various contexts, such as infections related to milk, silicone surfaces, synovial fluid, and prosthetic joint materials. Overall, while phage therapy shows promise as an alternative to antibiotics, additional research is necessary to refine treatment methods and ensure safety.

Conclusions

Bacteriophages hold potential as a standalone treatment and a complementary option to traditional antibiotics for managing S. aureus biofilms, but further research is needed to understand their clinical potential. Additional studies on phage selection, dosing, and administration methods are necessary, along with exploration of phage–antibiotic synergy mechanisms and assessment of the safety and environmental impacts of phage therapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s40001-025-02781-3.

Introduction

Staphylococci are a group of bacteria classified under the genus Staphylococcus, which comprises several species, with Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) being the most clinically relevant. These Gram-positive cocci are round in shape and commonly aggregate in clusters that resemble grapes. S. aureus is well-known for causing a variety of infections, ranging from minor skin conditions to serious illnesses, such as pneumonia, sepsis, and endocarditis. It is particularly concerning due to its capacity for developing antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA), which presents significant difficulties in clinical environments [1]. Conversely, S. epidermidis is a member of the normal skin microbiota and is typically less pathogenic; however, it can become a threat, especially in patients with weakened immune systems or those with medical implants [2]. The capability of staphylococcal species to form biofilms—structured communities surrounded by a protective matrix they produce—further complicates treatment efforts, as this characteristic enhances their resistance to the immune system and various antimicrobial agents [3]. Biofilms are complex microbial communities that form in a self-produced extracellular matrix, which helps to bind the cells and enables communication and resource sharing among them. The structure of biofilms gives them unique properties compared to planktonic (free-floating) cells, including altered metabolism and increased resistance to environmental stressors. The clinical relevance of biofilms primarily arises from their significant resistance to antimicrobial agents, which creates substantial challenges in managing infections associated with biofilms. Cells within biofilms can exhibit antimicrobial resistance that is up to a thousand times greater than that of planktonic cells. Biofilms are linked to a wide range of chronic infections and contribute to the failure of many antimicrobial treatments, especially in cases involving medical devices and tissues [4, 5]. Current treatment approaches, which often depend on traditional antimicrobials, are increasingly ineffective in eliminating these robust microbial communities. Given the limitations of available therapies and the rising threat posed by multidrug-resistant organisms, there is an urgent need for the development of innovative anti-biofilm strategies [6]. At the same time, employing bacteriophages as prospective biological control agents is crucial for tackling issues associated with biofilms in both medical and industrial settings [7].

Phage therapy is gaining recognition as a safe and targeted alternative to antibiotics, especially for multidrug-resistant bacterial infections. Its safety profile is supported by preclinical and clinical studies that report minimal adverse effects, such as temporary flu-like symptoms or localized reactions at administration sites [8]. However, immunogenicity is a critical consideration, as phages can trigger both innate and adaptive immune responses. For example, intravenous or intralesional administration of phages may induce neutralizing antibodies, though this has not consistently affected therapeutic outcomes. In some cases, patients have shown clinical improvement despite immune activity. Moreover, phage-derived pathogen-associated molecular patterns (PAMPs) can influence host immune responses, potentially enhancing bacterial clearance while also requiring careful monitoring in immunocompromised individuals [9]. Another concern is horizontal gene transfer (HGT), particularly involving temperate phages or engineered variants. While lytic phages are preferred for therapy due to their non-integrative lifecycle, regulatory frameworks must address the risk of HGT, which could spread antibiotic resistance or virulence genes. Advances in phage genomics and stringent screening protocols—such as the exclusion of lysogenic or toxin-encoding phages—help mitigate these risks. Standardizing phage preparations further ensures safety and efficacy. For instance, Belgium’s magistral phage preparations comply with a monograph-based quality control system that includes genome sequencing, endotoxin testing, and potency assays. Such protocols are essential for scalable clinical adoption [10]. Real-world applications demonstrate the practicality of phage therapy. Clinical trials targeting Pseudomonas aeruginosa in cystic fibrosis patients and Staphylococcus aureus in chronic wound infections have yielded promising results, particularly when phages are used in conjunction with antibiotics [11]. Compassionate use cases, such as the treatment of a 100-patient cohort at the Queen Astrid Military Hospital, achieved a 77.2% clinical improvement rate, with bacterial eradication in 61.3% of cases. These outcomes highlight the synergy between phages and antibiotics, where phages can resensitize resistant bacteria to conventional drugs, for example, by targeting efflux pumps [11].

This systematic review aims to examine the efficacy of phage therapy in managing staphylococcal biofilms. A significant challenge in treating staphylococcal infections is their capacity to form biofilms. Biofilms are structured communities of bacteria encased in a self-produced extracellular matrix, enhancing their resistance to the immune system and antimicrobial agents. Cells within biofilms can exhibit antimicrobial resistance that is up to a thousand times greater than that of planktonic cells. These biofilms are linked to a wide range of chronic infections and contribute to the failure of many antimicrobial treatments, especially in cases involving medical devices and tissues. Given the limitations of available therapies and the rising threat posed by multidrug-resistant organisms, there is an urgent need for the development of innovative anti-biofilm strategies.

Materials and methods

Research questions

The primary aim of this research was to investigate the effectiveness of phage therapy in managing staphylococcal biofilms, with a focus on developments from the beginning of 2012 to October 29, 2024.

Search strategy

This systematic review conducted a thorough search across four electronic databases: Web of Science, Embase, Scopus, and PubMed. The focus was on original research articles published in English. To examine the effectiveness of phage therapy in managing staphylococcal biofilms, a range of keywords and medical subject headings (MeSH) were utilized, including “Phage therapy,” “Bacteriophage Therapy,” “Staphylococcus aureus biofilm,” “Staphylococcus biofilm,” and “biofilm” (supplementary file Table 1). Two researchers carried out the search independently, while a third researcher reviewed the results (Fig. 1).

Table 1.

Inclusion/exclusion criteria

Inclusion criteria	Exclusion criteria
Research studies	Review article
English language studies	Non-English language articles
Studies investigate Efficacy of phage therapy in biofilm	Observational studies

Fig. 1.

Prism of study selection

Data extraction and critical appraisal

The information gathered from the chosen articles (n = 93) included the following details: authorship, publication date, study location, study design, number of relevant reports, microbiological specifics of the condition, clinical status, and, when available, patient ages and pre-existing treatments. In addition, information about the phage therapy, such as the treatment schedule, routes of administration, and any simultaneous therapies (including antibiotics), was documented. Treatment outcomes were recorded, along with any relevant data or comments regarding safety and adverse effects. Data extraction for each eligible study was conducted independently by at least two of the three authors, with any inconsistencies resolved through discussion. The discussion section examines the potential influences of publication bias and selective reporting on the results.

Study selection criteria

The process of selecting studies included applying the inclusion and exclusion criteria specified in Table 1. Initially, the review targeted the titles and abstracts of the articles to identify studies that met the inclusion criteria. For those abstracts that qualified, a detailed evaluation of the full texts was performed.

Quality assessment

To evaluate the methodological quality of the included articles, the Joanna Briggs Institute (JBI) checklist for quasi-experimental studies was employed. The results revealed varying levels of methodological rigor across studies, with key strengths including clear inclusion criteria and appropriate statistical analyses in most articles. However, some studies had limitations, such as potential selection bias or insufficient detail on confounding factors. These findings were considered during data synthesis to ensure a balanced interpretation of the evidence. Two reviewers carried out independent evaluations of the studies, followed by a review of the final outcomes by a third researcher.

Results

Effect of phage lysin on biofilm

Bacteriophage endolysins are enzymes that degrade the cell wall of bacteria, leading to bacterial cell death [12, 13]. Endolysins have been shown to be effective against both planktonic S. aureus cells and biofilms [13]. They work by hydrolyzing peptidoglycans in the bacterial cell wall, causing the cell to lyse [14]. Bacteriophage endolysin LysSte134_1 has been shown to be effective against both planktonic and biofilm forms of S. aureus. It can hydrolyze peptidoglycans from the cell wall of S. aureus and reduce colony forming units by 50-fold [13]. The addition of zinc ions enhances the lytic activity of LysSte134_1 against S. aureus, indicating that it is a zinc-dependent enzyme. Studies have also shown that other bacteriophage endolysins, such as HY-133, LysK, and LysH5 are effective in killing S. aureus cells that are attached to surfaces or embedded in biofilms [12, 15–17]. The effectiveness of these endolysins, coupled with their low toxicity to mammalian cells and low potential for resistance development, make them a promising alternative to antibiotics for the treatment of S. aureus infections [14].

The molecular mechanisms of phage-mediated biofilm disruption involve a complex interaction between three key phage-derived components and bacterial extracellular polymeric substances (EPS). Central to this process are depolymerases, enzymes that specifically target and degrade the polysaccharide components of bacterial cell walls and biofilm matrices. These enzymes function through two main mechanisms: hydrolases cleave bonds using water molecules, while lyases break bonds via β-elimination reactions. Together, these actions enable phage particles to penetrate the dense EPS matrix effectively [18]. The degradation of extracellular polymeric substances (EPS) by depolymerases creates significant vulnerabilities in the biofilm structure. This allows both whole phages and lysins to reach previously protected bacterial cells. Whole phages take advantage of these openings by attaching to specific receptors on exposed bacterial surfaces, injecting their genetic material, and initiating processes that take over the cell [19]. At the same time, endolysins produced during phage infection work in conjunction with depolymerases to hydrolyze the peptidoglycan layers of bacterial cell walls. Associated proteins such as holin and spanin further compromise the cytoplasmic and outer membranes [20].

This multi-component approach to disrupting biofilm integrity unfolds in several distinct phases. First, depolymerases target and weaken the polysaccharide matrix, diminishing its protective barrier and allowing phages to penetrate. Once a successful infection occurs, endolysins promote the release of progeny phages through controlled cell lysis while also creating channels that facilitate further phage diffusion throughout the biofilm. The efficiency of this process is enhanced by the phages' capability to alternate between lytic and lysogenic cycles, allowing for strategic penetration and spread within the biofilm structure [21] (Fig. 2).

Fig. 2.

Diagrammatic representation of phage therapy for the breakdown of biofilms

The effectiveness of molecular interactions is greatly affected by the composition and organization of the extracellular polymeric substances (EPS), which can account for up to 90% of the total organic components in a biofilm. Although the EPS matrix forms a strong barrier against many antimicrobial agents, phage-derived enzymes exhibit remarkable specificity for their targets, particularly in breaking down capsular polysaccharides, lipopolysaccharides, and exopolysaccharides. This targeted approach allows for precise disruption of biofilm structure while reducing collateral damage to surrounding tissues, showcasing the intricate nature of these molecular interactions in achieving effective biofilm control [22]. A summary of the data from the articles is given in Table 2.

Table 2.

Summary of studies used phage lysins effects on biofilm

References	Antimicrobial agent	Bacterial target	Biofilm model	Key findings
Drilling et al. [37]	P128 Protein	S. aureus clinical isolates, including MRSA	MBEC assay using a 96-well plate	The P128 protein demonstrates antimicrobial activity against S. aureus, including methicillin-resistant strains
Tyagi et al. [14]	Phage endolysins T7L and T4L	Gram-positive bacteria: S. aureus and B. thuringiensis, and Gram-negative bacteria: E. coli and P. aeruginosa	Static biofilm assays in 96-well plates; the outer membrane of Gram-negative bacteria was sensitized with 0.1 M EDTA	Endolysins T7L and T4L exhibited efficacy against a range of bacterial pathogens, with the exception of B. thuringiensis. Enhanced antimicrobial effects were observed when these endolysins were combined with antimicrobial peptides, specifically colistin, polymyxin B (PMB), and nisin
Sosa et al. [23]	PlySs2	Xen 36 MSSA	24-well plates	PlySs2 effectively reduces biofilms of varying maturity levels
Golosova et al. [13]	Endolysin LysSte	S. aureus	Not specified	The study focused on the expression and purification of the endolysin LysSte from the bacteriophage St 134, highlighting its ability to hydrolyze staphylococcal peptidoglycans
Idelevich et al. [15]	Endolysin HY-133	S. aureus strain SH1000	Vascular graft surface	Endolysin HY-133 displayed moderate activity against S. aureus that were attached to vascular grafts, particularly against mature biofilms
Jiang et al. [24]	Phage WV	Clinical isolates of S. aureus	Not specified, but includes a biofilm model	Phage WV successfully eliminates most of the tested clinical isolates of S. aureus. When used in combination with streptomycin, phage WV demonstrated enhanced antibiofilm and bactericidal effects compared to the use of either agent alone
Gutierrez et al. [16]	Lytic proteins: LysH5, CHAP-SH3b, and HydH5-SH3b	S. aureus strains: 15981, ISP479r, IPLA1, and Sa9	Static biofilms evaluated using a real-time cell analyzer (RTCA)	LysH5 exhibited the most effective antibiofilm activity among the tested proteins. The study emphasizes the utility of RTCA technology for the efficient and reproducible screening of antibiofilm agents
Duarte et al. [25]	Phage phiIPLA-RODI	S. aureus strains: 15,981, V329, Newman, JE2, and IPLA16	Static biofilm assays in 96-well plates	Phage phiIPLA-RODI, in combination with depolymerase Dpo7, exhibited synergistic antibiofilm effects against a variety of S. aureus strains
Duarte et al. [26]	Lytic protein CHAPSH3b	S. aureus strains: 15981, IPLA1, and V329	Static assays in 24-well plates and an ex vivo pig skin model	The combined application of phage phiIPLA-RODI and CHAPSH3b resulted in synergistic effects against S. aureus biofilms. This combination also demonstrated promising results in an ex vivo pig skin wound infection model
Schuch et al. [27]	CF-301	S. aureus strains (both methicillin-susceptible and methicillin-resistant), coagulase-negative staphylococci, S. pyogenes (group A), and S. agalactiae (group B)	Static and in vitro catheter biofilm models	CF-301 demonstrated potent activity against biofilms formed by a range of staphylococcal strains and also exhibited efficacy against other Gram-positive bacteria, including S. pyogenes and S. agalactiae
Olsen et al. [17]	Endolysin LysK	S. aureus strains: SA113, Newbould, RN6911, SA001, R174, R177, 95, 319, 2971, R191, and R192	Static biofilm assays using 96-well plates and dynamic biofilm assays using a Biostream flow cell	LysK demonstrated high efficacy against biofilms formed by the majority of S. aureus strains that were tested. PNAG depolymerases, especially DA7, also exhibited notable antibiofilm activity against S. aureus
Golosova et al. [28]	Endolysin LysAP45	Multidrug-resistant strains of S. aureus, S. haemolyticus, and S. epidermidis	Static biofilms grown on coverslips	LysAP45 exhibited potent activity against biofilms formed by multidrug-resistant staphylococci. The enzyme effectively hydrolyzed peptidoglycans from various staphylococcal species. Notably, LysAP45 retained its activity even after exposure to high temperatures (80 °C for 30 min), highlighting its thermostability

Drilling et al. examines the effectiveness of the bacteriophage-derived muralytic enzyme P128 in eradicating S. aureus biofilms. The researchers tested P128 on biofilms from the reference strain S. aureus ATCC 25923, two methicillin-sensitive, and one methicillin-resistant S. aureus clinical isolates from patients with chronic rhinosinusitis (CRS). Using both minimum biofilm eradication concentration (MBEC) and Alamar Blue assays (AB), the researchers demonstrated that P128 significantly reduced S. aureus biofilm biomass and planktonic cell presence in all tested strains. Concentrations of P128 at or above 12.5 µg/mL effectively reduced biofilm levels in all clinical isolates, while concentrations at or above 25 µg/mL were effective against the reference strain. The study concluded that P128 is a promising candidate as an antimicrobial and anti-biofilm agent, particularly for treating CRS, due to its ability to target S. aureus specifically. Further research is needed to determine the optimal method for delivering P128 as a treatment for nasal infections [12]. Tyagi et al. investigates the synergistic potential of combining bacteriophage endolysins (T7L and T4L) with antimicrobial peptides (AMPs) to combat bacterial biofilms. Researchers found that while endolysins were effective against planktonic cells of Pseudomonas aeruginosa, Escherichia coli, and S. aureus, AMPs showed strain-specific inhibition. Combining T7L with Polymyxin B or Colistin effectively eradicated P. aeruginosa biofilms, demonstrating a synergistic effect. Similarly, combining T4L with Nisin showed synergy against S. aureus biofilms. Microscopic analysis revealed that these combinations disrupted biofilm structure and damaged bacterial cells. The study emphasizes the potential of endolysin–AMP combinations as an effective strategy against drug-resistant bacterial biofilms, offering a promising alternative to conventional antibiotics [14]. Sosa et al. examines the effectiveness of PlySs2 lysin, a bacteriophage-derived enzyme, in reducing S. aureus biofilms, a major challenge in prosthetic joint infections (PJIs). The study found that PlySs2 significantly reduced both 1-day and 5-day-old biofilms in vitro, outperforming vancomycin, a commonly used antibiotic. PlySs2 also exhibited rapid and sustained bactericidal activity against planktonic S. aureus. Furthermore, a combination of PlySs2 and vancomycin demonstrated synergistic activity, reducing the bacterial load in periprosthetic tissue and on implant surfaces in a murine model of PJI. The study suggests that PlySs2, with its ability to target both biofilm and planktonic bacteria, and its synergistic action with vancomycin, holds promise as a novel treatment approach for PJIs [23]. Golosova et al. describes a newly isolated bacteriophage, vB_SepP_134 (St 134) and its potential as an antimicrobial agent, specifically its endolysin LysSte134_1, against Staphylococcus infections. St 134 is a podophage capable of infecting various strains of 12 coagulase-negative Staphylococcus species and one clinical strain from the S. aureus complex. The researchers identified two genes in the St 134 genome that encode for lytic enzymes: endolysin (LysSte134_1) and tail tip lysin (LysSte134_2). LysSte134_1 exhibited catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus (S. haemolyticus), and Staphylococcus warneri (S. warneri). Further testing revealed that LysSte134_1 effectively destroyed both planktonic cells and biofilms formed by clinical strains of S. aureus and S. epidermidis. The study emphasizes the potential of phage-derived endolysins, such as LysSte134_1, as a promising alternative to traditional antibiotics for treating staphylococcal infections, including those caused by MDR strains [13]. Idelevich et al. investigates the in vitro activity of bacteriophage endolysin HY-133 against S. aureus attached to vascular graft surfaces, comparing its efficacy to daptomycin and rifampin. The results demonstrated that HY-133 had a moderate effect against S. aureus attached to vascular graft surfaces, particularly against mature biofilms. Daptomycin, on the other hand, exhibited a rapid bactericidal effect against biofilm-embedded S. aureus. While rifampin has been reported to have high anti-biofilm activity, this study found it did not achieve a bactericidal effect. Notably, none of the tested antimicrobial agents, even at the highest concentrations, could completely eradicate the surface-adherent bacteria. The researchers utilized both vital cell counts and ATP measurement to quantify bacterial attachment to the graft surface and observed similar trends in anti-biofilm activity assessment using both methods [15]. Jiang et al. investigates the effects of a newly isolated lytic S. aureus phage WV on S. aureus biofilms. Researchers found that phage WV was effective at killing a number of clinical isolates of S. aureus that had been isolated from patients in the First People's Hospital of Yunnan Province. They tested the bactericidal effects of phage WV, the antibiotic streptomycin, and a combination of both agents on S. aureus biofilms. The study found that streptomycin was more effective at killing S. aureus than phage WV in low-concentration cultures. However, in high-concentration cultures, phage WV was more effective than streptomycin. The combination of phage WV and streptomycin was the most effective treatment, showing an improved bactericidal effect compared to either agent alone. Researchers concluded that phage WV could be a viable alternative to antibiotics for treating S. aureus infections, especially considering the rise of antibiotic-resistant strains [24]. Gutierrez et al. examines the use of a real-time cell analyzer (RTCA) to assess and compare the antibiofilm activity of four phage-derived proteins against S. aureus biofilms. Researchers used three lytic proteins: LysH5, CHAP–SH3b, and HydH5–SH3b, and one exopolysaccharide depolymerase: Dpo7. They treated preformed biofilms of four S. aureus strains, 15981, ISP479r, IPLA1, and Sa9, with the phage-derived proteins and monitored biofilm disruption using the xCelligence RTCA system. The researchers calculated several antibiofilm parameters including the MBEC50 needed to remove 50% of the biofilm, and the lowest observed antibiofilm effect (LOABE). The results revealed that LysH5 was the most effective antibiofilm protein, showing statistically significant differences in biofilm removal compared to the other proteins tested. All four proteins exhibited a dose-dependent antibiofilm effect, as demonstrated by a gradual reduction in normalized cell index (CI) values with increasing protein concentration. Interestingly, the antibiofilm activity of the proteins was not dependent on the initial robustness of the biofilm, as similar activity levels were observed against both strong and weak biofilms. The researchers concluded that the RTCA system provides a rapid and standardized method for assessing and comparing the antibiofilm activity of phage-derived proteins. In addition, the study emphasizes the potential of these proteins as effective antibiofilm agents against S. aureus biofilms, which are associated with recurrent contamination of food products and pose a significant risk to human health [16]. Duarte et al. investigates the synergistic effect of combining the bacteriophage Kayvirus rodi with the exopolysaccharide depolymerase Dpo7 to remove S. aureus biofilms. The researchers found that the combination of the phage and Dpo7 resulted in a significant decrease in both biofilm biomass and the number of viable cells compared to either treatment alone. This synergistic effect was observed against a variety of S. aureus strains, including those with different biofilm-forming abilities and matrix compositions. While the exact mechanism of synergy is not fully understood, the study suggests that Dpo7 may loosen the biofilm structure, allowing the phage particles to more easily access and infect the bacterial cells. The researchers also found that Dpo7 treatment did not significantly affect phage adsorption to biofilm cells. This study highlights the potential of combining phages with depolymerases to enhance the effectiveness of phage therapy against S. aureus biofilms [25]. Duarte et al. investigates the synergistic effects of combining the bacteriophage phiIPLA–RODI with the phage-derived chimeric lytic protein CHAPSH3b to remove S. aureus biofilms. Researchers tested this combination therapy on biofilms of three S. aureus strains: V329, 15981, and IPLA1, which have different biofilm formation abilities and matrix compositions. They found that the combination of phage and lysin was significantly more effective at reducing both biofilm biomass and the number of viable cells than either treatment alone. The synergistic effect was attributed to several factors, including the lysin’s ability to rapidly disrupt the biofilm structure, allowing phage particles to more effectively access and infect bacterial cells. In addition, the lysin helped to increase the phage-to-bacteria ratio (MOI) and killed phage-resistant mutants, further enhancing the phage’s effectiveness. The study concluded that the combination of phiIPLA–RODI and CHAPSH3b is a promising strategy for the development of improved anti-biofilm products [26]. Schuch et al. examines the effectiveness of the bacteriophage lysin CF-301 as a treatment for S. aureus biofilms. The researchers found that CF-301 was highly effective at disrupting mature biofilms formed by a wide range of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. CF-301 was also effective against biofilms formed by coagulase-negative staphylococci, Streptococcus pyogenes (S. pyogenes), and Streptococcus agalactiae (S. agalactiae). The researchers tested CF-301 on biofilms formed on a variety of surfaces, including polystyrene, glass, surgical mesh, and catheters. They also tested CF-301 in the presence of human serum, plasma, and whole blood, and in human synovial fluid. The researchers found that CF-301 was effective at disrupting biofilms and killing biofilm bacteria in all of these conditions. They also found that CF-301 was synergistic with lysostaphin, another cell wall hydrolase, in disrupting biofilms. The study concluded that CF-301 is a promising new agent for treating staphylococcal infections with a biofilm component. The researchers noted that CF-301 could be a good candidate for antibiotic lock therapies as it remained 99.9% active over 3 weeks at 37 °C in lactated Ringer’s (LR) solution, a commonly used vehicle in this type of treatment [27]. Olsen et al. investigates the effectiveness of combining the bacteriophage endolysin LysK with the polysaccharide depolymerase DA7 in removing S. aureus biofilms. The study used both static and dynamic biofilm models to assess the efficacy of these enzymes, alone and in combination. LysK was found to be effective against biofilms formed by multiple S. aureus strains, showing activity at concentrations as low as 40 nM in the static model. In the dynamic model, LysK removed a significant amount of biofilm from glass surfaces at concentrations of 0.63 µM and 1.25 µM. DA7, an Aggregatibacter actinomycetemcomitans-derived depolymerase, was even more potent, eradicating biofilms at nanomolar concentrations in both static and dynamic models. Importantly, the researchers found that combining LysK and DA7 resulted in a synergistic effect, leading to significantly greater biofilm removal compared to either enzyme alone. This synergy was observed in both static and dynamic models and was confirmed using confocal laser scanning microscopy. The study concluded that the combination of LysK and DA7 is a promising strategy for the development of new anti-biofilm agents [17]. Golosova et al. describes the characterization of a thermostable endolysin called LysAP45, derived from the Aeribacillus phage AP45, and its potential as a biofilm-removing agent against Staphylococcus species. The researchers found that LysAP45 effectively hydrolyzed peptidoglycans from various Staphylococcus strains, including methicillin-resistant S. aureus (MRSA), S. epidermidis, and S. haemolyticus, despite the fact that its parent phage, AP45, does not infect these species. LysAP45 demonstrated a significant reduction in colony-forming units (CFUs) of S. aureus and completely eradicated S. aureus cells when supplemented with zinc chloride (ZnCl2). The study revealed that Zn2 + ions enhanced the lytic activity of LysAP45, confirming it as a zinc-dependent amidase. Furthermore, LysAP45 effectively disrupted biofilms formed by multidrug-resistant S. aureus, S. epidermidis, and S. haemolyticus. Importantly, LysAP45 exhibited thermostability, retaining its enzymatic activity even after being incubated at 80°C for 30 min. The researchers concluded that LysAP45, with its broad-spectrum activity against staphylococcal peptidoglycans, its biofilm-disrupting capabilities, and thermostability, holds promise as a novel antimicrobial agent for combating infections caused by Staphylococcus species, particularly those associated with biofilms [28].

Many of the studies discussed in this review demonstrate the effectiveness of phage-derived enzymes, particularly lysins and depolymerases, in disrupting and removing S. aureus biofilms in vitro. However, a common limitation is the reliance on laboratory strains and simplified biofilm models, which might not accurately reflect the complexity of clinical infections. Future research should focus on testing these enzymes against a wider range of clinical isolates, including those with diverse resistance profiles and genetic backgrounds. In addition, more complex in vivo models that mimic the physiological conditions of infections, such as those utilizing pig skin or catheters, should be employed to better assess the therapeutic potential of these enzymes. Moreover, exploring combination therapies involving lysins, depolymerases, and conventional antibiotics could lead to enhanced biofilm eradication and reduced risk of resistance development. Finally, optimizing the delivery methods of these enzymes to target sites of infection is crucial for their clinical translation.

Effect of phage on biofilm

Exploring the application of whole bacteriophages to manage biofilms produced by S. aureus presents an exciting research opportunity that capitalizes on the distinctive traits of these viruses, which exclusively target and eliminate bacteria. Investigating whole phages as a therapeutic option for S. aureus biofilms offers a novel and potentially transformative strategy for tackling stubborn bacterial infections [29, 30]. This method's targeted action, capacity to break down biofilm structures, and potential for integration with other treatments have positioned it as a key area of focus in current research initiatives. A summary of the data from the articles is given in Table 3.

Table 3.

Summary of studies used phage effects on biofilm

References	Biofilm model	Treatment	Outcome
Archana et al. [56]	–	Bacteriophage therapy and phage-antibiotic combinations	–
Seth et al. [34]	In vitro catheter model, in vitro biofilm culture systems, murine lungs, rabbit ear model	Bacteriophage therapy, phage “cocktail”, combined with clinical wound care strategies, carbon monoxide releasing molecule	Reduction of biofilm formation demonstrated in vitro. In vivo clearance of P. aeruginosa biofilm. Notes need for optimization of phage delivery
Gharieb et al. [35]	In vitro microtiter plate assay with S. aureus isolates from milk and milking equipment	Lytic phages, different MOIs (1 and 10) of phage suspension	Phages can prevent biofilm formation. The study also assesses antibiotic susceptibility
Alves et al. [29]	In vitro microtiter plate assay with S. aureus strains, biofilms formed in TSB supplemented with glucose	Single phage K, single phage DRA88, and a mixture of the two phages, at MOIs of 1 and 10	Phage mixture showed a decrease in biofilm biomass compared to single phages. Higher MOIs resulted in a more rapid reduction in biofilm density
Taha et al. [36]	In vitro biofilm-like aggregates formed in synovial fluid	Bacteriophage and vancomycin combination	Not specified in detail
Manoharadas et al. [30]	Biofilm formation ability of the strains was tested on an immersed glass cover slip	Bacteriophage and AgNPs combination	The synergistic activity of the nanoparticles and bacteriophages causes the loss of viability of the biofilm entrapped bacterial cells thus preventing establishment of a new infection and subsequent colonization
Abdulamir et al. [57]	In vitro biofilm formation by MRSA and MSSA on polystyrene microtiter plates, using the TCP method	Lytic phages, with or without chemical adjuvants (e.g., ethanol)	Phage-based control of biofilms was assessed, with higher effectiveness in some cases, using the TCP method. MRSA showed a higher tendency for biofilm formation than MSSA
Kosznik-Kwaśnicka et al. [33]	In vitro mature MDRSA biofilms	Lactoferrin, bacteriophages (vB_SauM-A, vB_SauM-C, vB_SauM-D), and phage-lactoferrin cocktail	Lactoferrin decreased biofilm biomass and viability. Phage-lactoferrin cocktail showed statistical significance in decreasing bacterial numbers compared to phage alone for some strains
Drilling et al. [37]	–	Bacteriophage alone	Reports reduction in biofilm of S. aureus from chronic rhinosinusitis patients
Kumaran et al. [38]	In vitro S. aureus biofilm formation in 48-well plates	Bacteriophage, antibiotics, and combination therapies	Investigates the order of phage and antibiotic treatment on biofilm eradication
Wang et al. [39]	In vitro biofilm formation on porous glass beads	Single antibiotics and phage-antibiotic combinations. Evaluates simultaneous and staggered application of phage and antibiotics	Determined minimum biofilm bactericidal concentration (MBBC). Staggered application of phage followed by antibiotic was more effective
Ponce Benavente et al. [40]	In vitro biofilm formation using porous glass beads	Evolutionary approach to enhance phage activity against biofilms, using a panel of S. aureus strains	Phage activity against pre-established S. aureus biofilms was enhanced
Kaźmierczak et al. [41]	In vitro biofilm formation, in vivo Galleria mellonella larva model	Bacteriophages (vB_SauM-A, vB_SauM-C, vB_SauM-D) and antibiotics (SXT, TE, GM, FA, VAN)	Phage therapy resulted in a statistically significant increase in survival rate in in vivo model. Also compares phage and antibiotic effectiveness in biofilm eradication
Kebriaei et al. [42]	In vitro 24-h bead biofilms	Individual phages, daptomycin (DAP), ceftaroline (CPT), and combinations	Antibiofilm effects of single phages and a combination of antibiotics and phages are tested. It is noted that well turbidity is used to screen for treatment success rather than quantifying CFUs
Kelly et al. [43]	In vitro biofilm formation in microtitre plates	Phage K and modified derivatives of phage K	The study showed a reduction in biofilm formation when phages were added
Lungren et al. [44]	In vitro biofilm on silicone discs (central venous catheter material)	Staphylococcal bacteriophage K	Significant reduction of bacterial colonization and biofilm presence
Dickey et al. [45]	In vitro biofilm model	Phage, antibiotics (including RIF), and combinations, with simultaneous and sequential treatments	Combined action of phage and RIF on biofilms were studied. Cell densities and phage densities are presented. Antibiotic and phage treatment of S. aureus biofilms were studied
Song et al. [46]	–	Reports on an antimicrobial peptide, SDQ that inhibits biofilm formation	SDQ inhibited biofilm formation. No specific results on phage treatment
Kebriaei et al. [47]	In vitro biofilm on beads	Bacteriophage (Sb-1), daptomycin (DAP), ceftaroline (CPT) and combinations	Phage proliferation was increased in biofilms, and phage was effective in combination with antibiotics

In a study by Archana et al., researchers investigated the potential of bacteriophages using Galleria mellonella (waxworm) as a model organism. They infected the larvae with S. aureus and treated them with two phages, vB_Sau_Saa90 and vB_Sau_Saa165, combined with oxacillin. Treatments were applied in three sequences—before, simultaneously with, and after phage administration—with 90-min intervals in between. Results showed that both pre-phage and post-antibiotic treatments were effective, with survival rates varying based on biofilm strength. The phages demonstrated significant effectiveness when paired with antibiotics, highlighting the potential of combination therapy for biofilm infections. This research confirms the utility of the waxworm model in studying bacteriophage therapies and suggests further exploration with larger animal models to advance treatment strategies for clinical use [31]. In another study researchers explored a novel strategy using highly lytic phages to combat biofilms formed by methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA), both with and without chemical agents that disrupt the extracellular matrix. The study focused on two types of biofilm matrices: polysaccharide intercellular adhesion (PIA) and fibronectin-binding protein A (FnBPA). Biofilms were grown in microtiter plates under controlled conditions, and their characteristics were analyzed using scanning electron microscopy (SEM) and PCR assays to identify relevant genes. Results indicated that phages treated with benzethonium chloride were effective in eliminating MSSA biofilms entirely and reducing MRSA biofilms by about 78%. Phage treatments combined with chemical disruptants proved significantly more effective than those without. The study found that FnBPA biofilms were more common in MRSA, while MSSA had more PIA biofilms. FnBPA biofilms in MRSA showed the highest resistance to phage treatment, achieving only a 50% reduction. The researchers concluded that using agents such as PIA and protein-denaturing alcohol can improve phage access to host cell walls, enhancing the prevention and treatment of biofilms associated with both MRSA and MSSA [32]. In research conducted by Kosznik-Kwaśnicka et al., staphylococcal phages were combined with lactoferrin, a protein known for its anti-biofilm capabilities. Through the examination of biofilm biomass and metabolic activity, the study revealed that incorporating lactoferrin into phage lysate enhances the effectiveness of the phages against biofilms and inhibits their regrowth. Consequently, lactoferrin may be a promising agent for use in strategies aimed at eliminating biofilms in medical environments [33]. In a particular study, researchers assessed the effectiveness of a species-specific bacteriophage against wounds infected with S. aureus biofilms, utilizing a validated, quantitative rabbit ear model. The team created 6-mm dermal punch wounds on the ears of New Zealand rabbits, which were inoculated with either wild-type or mutant strains of biofilm-deficient S. aureus. An in vivo biofilm was established by following previously published methods for wound biofilm models. The wounds were either left untreated or received treatment every other day with topical S. aureus-specific bacteriophage, sharp debridement, or a combination of both. Following the harvest, the researchers conducted histological assessments of wound healing, viable bacterial count analysis, and scanning electron microscopy. The results showed no significant differences in healing or bacterial viability for wounds infected with wild-type S. aureus when treated with either bacteriophage or sharp debridement alone. However, the combination of these two treatments led to significant improvements in all measured parameters of wound healing (P < 0.05) and decreased bacterial counts (P = 0.03), as corroborated by scanning electron microscopy. Similarly, treating the biofilm-deficient S. aureus mutant wounds with bacteriophage alone also demonstrated comparable trends for both endpoints (P < 0.05) [34]. In a study by Gharieb et al., researchers isolated two new lytic phages, vB_SauM_ME18 and vB_SauM_ME126, from 40 multidrug-resistant (MDR) S. aureus isolates, with only 10% of the isolates being susceptible to these phages. Both phages belong to the Myoviridae family and display specific structural characteristics, including icosahedral heads and long contractile tails, with genome sizes around 20 kb. The phages demonstrated a latent period of approximately 15 min and a growth period of about 30 min, achieving burst sizes of 114 PFU for ME18 and 140 PFU for ME126. The study found that treatment with these phages significantly reduced both biofilm formation and biomass in S. aureus compared to controls (P < 0.05). They effectively eliminated MDR S. aureus in ultra-treated milk at 25 °C, and at 37 °C, ME126 completely eradicated S. aureus, while ME18 reduced CFU/ml by 87.2% (P < 0.05). These findings suggest that lytic phages could serve as potential natural antimicrobials to control MDR S. aureus in milk and help in the biocontrol of foodborne pathogens [35]. In their research, Alves et al. identified a novel phage designated as DRA88, which exhibits a broad host range among S. aureus strains. This phage is classified within the Myoviridae family and possesses a substantial double-stranded DNA (dsDNA) genome, consisting of 141,907 base pairs. When combined with phage K, DRA88 formed a high-titer mixture that demonstrated potent lytic activity against various S. aureus isolates, encompassing both MRSA international clones and coagulase-negative staphylococci. The phage mixture's efficacy was assessed in both planktonic cultures and in the treatment of biofilms formed by three different S. aureus isolates, with a notable decrease in biofilm biomass observed within 48-h post-treatment in all instances. Ultimately, the authors suggested that this phage mixture might serve as a promising therapeutic strategy for infections resulting from S. aureus biofilms [29]. Taha et al. utilized a clinical isolate of S. aureus, designated BP043, which is a methicillin-resistant strain known for its ability to form biofilms and associated with periprosthetic joint infections (PJI). The Rhemus phage, effective against S. aureus, was selected for the treatment regimen. BP043 was cultured in human synovial fluid, where it formed aggregates. The structure and size of the S. aureus aggregates were analyzed using scanning electron microscopy (SEM) and flow cytometry, respectively. The formed pellets were treated for a duration of 48 h with: (a) Rhemus phage at approximately 10⁸ plaque-forming units (PFU/ml), (b) vancomycin at a concentration of 500 mg/ml, or (c) a combination of Rhemus phage (approximately 10⁸ PFU/ml) followed by vancomycin (500 mg/ml). Bacterial survival was assessed by measuring colony-forming units (CFU/ml). The effectiveness of Rhemus phage and vancomycin was evaluated both as individual treatments and in combination in an in vivo model using Galleria mellonella larvae infected with BP043 aggregates suspended in synovial fluid. SEM imaging and flow cytometry revealed that human synovial fluid facilitated the formation of S. aureus aggregates. Treatment with Rhemus phage resulted in a significant decrease in viable S. aureus within the aggregates compared to untreated aggregates (P < 0.0001). Rhemus phage treatment showed greater efficacy in eradicating viable bacteria from these aggregates than vancomycin (P < 0.0001). Furthermore, the combination of Rhemus phage followed by vancomycin proved to be more effective in lowering the bacterial load than either treatment used alone (P = 0.0023 and P < 0.0001, respectively). In the in vivo analysis, this combination treatment also led to the highest survival rate of 37% at 96-h post-treatment, in contrast to the 3% survival rate observed in untreated larvae (P < 0.0001). Ultimately, the study demonstrated that the collaboration between Rhemus phage and vancomycin resulted in a synergistic effect against MRSA biofilms, both in vitro and in vivo [36]. In a particular study, researchers highlighted the combined effectiveness of green synthesized silver nanoparticles and bacteriophages in eliminating established S. aureus biofilms from glass surfaces. The study revealed a time-dependent ineffectiveness of single treatments. Notably, it marked the first time rapid dispersal of bacterial biofilms was observed. Furthermore, the synergistic effects of the nanoparticles and bacteriophages led to a reduction in the viability of the bacterial cells embedded in the biofilms, thereby preventing new infections and inhibiting further colonization. This work established a novel combined treatment strategy using various nanoparticles alongside bacteriophages to target both single and multiple bacterial biofilms [30]. Drilling et al. confirms that the NOV012 phage cocktail is effective against a wide array of S. aureus isolates, including MRSA isolates, in patients with CRS. Researchers applied the NOV012 cocktail to the frontal sinuses of sheep twice daily for 20 days and found that this longer-term application was safe. There were no changes observed in the sheep’s general well-being, and an examination of the sinus mucosal lining revealed no changes to the tissue’s architecture or an increased presence of immune cells. In addition, no infectious phages were found in the sheep’s bloodstream during treatment. The study also investigated the host range of NOV012 in CRS S. aureus infections. Researchers found that using a cocktail of phages, as opposed to a single phage, helps overcome the issue of matching phage to bacteria. Phage K710 was effective against 59% of the S. aureus strains, but the addition of phage P68 increased the effectiveness to 85%. Using phage cocktails also reduces the development of bacteria resistant to phage infection [37]. Kumaran et al. investigated whether bacteriophages could augment the activity of antibiotics against biofilm-forming S. aureus. Researchers tested five antibiotics (cefazolin, vancomycin, dicloxacillin, tetracycline, and linezolid) against the biofilm-positive S. aureus strain ATCC 35556 in conjunction with the lytic S. aureus phage SATA-8505. They found that treatment of biofilms with either SATA-8505, antibiotics, or both simultaneously resulted in minimal reduction of viable biofilm-associated cells. However, a significant reduction in viable bacterial cells (up to 3 log CFU/mL) was observed when the phage treatment preceded the antibiotic treatment. This effect was most pronounced with vancomycin and cefazolin, particularly at lower antibiotic concentrations. The researchers determined that this enhanced bacterial reduction was likely due to the phage's ability to disrupt the biofilm matrix, allowing the antibiotic to penetrate deeper into the biofilm. The study also noted that sub-lethal concentrations of antibiotics affecting cell wall integrity can up-regulate phage replication and cell lysis, further enhancing the anti-biofilm effect. These findings demonstrate that the order in which phage and antibiotics are administered is a key determinant of biofilm reduction outcomes and that phage treatment preceding antibiotic exposure can lead to synergistic interactions and significant biofilm eradication [38]. Wang et al. examined the effectiveness of combining the staphylococcal bacteriophage Sb-1 with various antibiotics in eradicating biofilms of rifampin-resistant S. aureus (RRSA). Researchers tested ten clinical RRSA isolates (four MRSA and six MSSA) and two laboratory standard strains (MRSA ATCC 43300 and MSSA ATCC 29213). They found that all tested strains were susceptible to higher concentrations of antibiotics when grown as biofilms compared to their planktonic counterparts. Combining Sb-1 with daptomycin, either simultaneously or in a staggered fashion, demonstrated the highest activity against all MRSA biofilms. Notably, the staggered administration of Sb-1 followed by flucloxacillin, cefazolin, or fosfomycin improved the antibiofilm activity in four out of six MSSA strains. This study highlights that the order of administration when combining antibiotics and phages can significantly impact their effectiveness. Overall, the research suggests that combining Sb-1 with specific antibiotics could offer a potential treatment strategy for RRSA biofilms, but the choice of antibiotic and the order of administration are crucial factors to consider [39]. Ponce Benavente et al. explored a novel approach to enhance the activity of bacteriophages against biofilms formed by antibiotic-resistant S. aureus. Researchers developed an in vitro evolutionary assay involving serial passaging of phages against pre-established biofilms. This method, monitored in real time using isothermal microcalorimetry (IMC), resulted in the isolation of evolved phages with improved antimicrobial capabilities. Notably, these evolved phages demonstrated an expanded host range, with one phage infecting 83% of the tested strains compared to the widest host range (44%) observed among the ancestral phages. IMC data revealed that the evolved phages were more effective at suppressing bacterial growth than their ancestral counterparts. A phage cocktail comprising two of the most effective evolved phages achieved over 90% suppression of bacterial growth even after 72 h of monitoring. Furthermore, RT-qPCR analysis confirmed the enhanced antibiofilm performance of the evolved phages, showing no biofilm regrowth up to 48 h in treated MRSA strains [40]. Kaźmierczak et al. examined the effectiveness of bacteriophages vB_SauM-A, vB_SauM-C, and vB_SauM-D as antibiofilm agents against multidrug-resistant S. aureus (MDRSA) in vitro and in vivo. Researchers found that application of these bacteriophages to 24-h-old MDRSA biofilms reduced the number of adhered bacteria by 2–3 logs in most of the strains tested. The study found that bacteriophages were more efficient at removing biofilm biomass and reducing staphylococci count when compared to antibiotics. In addition, the bacteriophages significantly increased the survival rate and extended the survival time of Galleria mellonella larvae infected with MDRSA [41]. Kebriaei et al. focused on identifying phage–antibiotic combinations that could eradicate pre-formed S. aureus biofilms, particularly in methicillin-resistant (MRSA) and daptomycin–non-susceptible vancomycin–intermediate (DNS–VISA) strains. Researchers screened a library of lytic phages and selected a three-phage cocktail based on host range, genetic diversity, and the ability to infect phage-resistant bacterial mutants. They found that biofilms of two strains, D712 (DNS–VISA) and 8014 (MRSA), were the most resistant to killing by single phages, even at high concentrations. However, when these biofilms were treated with combinations of phages and antibiotics, bacterial regrowth was prevented at significantly lower phage and antibiotic concentrations than when using the single agents alone. The study highlights that phage–antibiotic synergy may be more effective in eradicating biofilms compared to single-agent treatments, but the choice of phage and antibiotic, their concentrations, and the order of administration are crucial factors to consider [42]. Kelly et al. investigates the effectiveness of a phage cocktail, consisting of phage K and six modified derivatives, in preventing and reducing established S. aureus biofilms. The study utilizes the bioluminescent S. aureus Xen29 strain and a static microtitre plate assay to assess biofilm formation and treatment. Results show that the phage cocktail completely inhibits biofilm formation over a 48-h period, as confirmed by both spectrophotometric analysis and bioluminescence monitoring. In addition, the cocktail significantly reduces established biofilm biomass in a time-dependent manner, with greater reduction observed after 72 h compared to 24 or 48 h. The study suggests that the phage cocktail's efficacy in disrupting biofilms is likely due to cell lysis rather than enzymatic degradation of the biofilm matrix [43]. Lungren et al. investigated the effectiveness of bacteriophage therapy in reducing S. aureus biofilm on central venous catheter material. Researchers inoculated silicone discs with S. aureus for 24 h to allow for biofilm formation. The discs were then randomized into two groups: a control group bathed in sterile phosphate-buffered saline and an experimental group bathed in a solution containing bacteriophage K. After 24 h, the discs were processed for quantitative culture. The results showed a statistically significant decrease in mean colony-forming units (CFU) in the experimental group compared to the control group (control 6.3 × 10⁵ CFU, experimental 6.7 × 10¹, P ≤ 0.0001). This suggests that the application of bacteriophage K to central venous catheter material significantly reduced S. aureus colonization and biofilm presence [44]. Dickey et al. examined the combined effect of bacteriophages and antibiotics on S. aureus biofilms. Researchers tested nine antibiotics with varying pharmacodynamic properties at two concentrations (2 × and 10 × MIC) alongside a single phage isolated from the Eliava PYO phage cocktail. They found that most antibiotics alone were ineffective at the lower concentration, but the addition of phage significantly improved their efficacy against the biofilms. At the higher concentration, antibiotics generally performed well on their own, and the addition of phage did not yield substantial improvements. The study also highlighted that using phage in conjunction with rifampin effectively suppressed the emergence of resistant strains during treatment [45]. Song et al. examines the potential of bacteriophage vB_SauM_SDQ (SDQ) as a disinfectant to control S. aureus biofilms, which are a major contributor to mastitis in dairy cows and food contamination. SDQ belongs to the Myoviridae family and effectively sterilized S. aureus cultures within 12 h while multiplying itself 1000-fold. The study found that SDQ significantly reduced biofilms on various surfaces, including polystyrene, milk, and mammary gland tissue. Microscopic analysis confirmed the destruction of the biofilm structure by SDQ. Importantly, SDQ retained its activity in the presence of nonionic detergents, tap water, and organic materials, even demonstrating enhanced biofilm removal when combined with Triton X-100 [46]. Kebriaei et al. investigates the efficacy of phage–antibiotic combinations (PAC) against S. aureus biofilms, specifically focusing on methicillin-resistant S. aureus (MRSA). The researchers found that while some PACs were effective against both planktonic and biofilm bacteria, the most effective treatment against both was a combination of phage Sb-1, daptomycin, and ceftaroline. This particular PAC was bactericidal, meaning it reduced bacterial counts by at least 3 log10 CFU/mL. Notably, the researchers observed synergy between Sb-1 and daptomycin against a daptomycin–non-susceptible (DNS) strain in biofilms, indicating that PACs can be effective even against antibiotic-resistant strains. The study also found that phage resistance, which was observed in phage-only treatments, was avoided when phages were combined with antibiotics. In addition, phage Sb-1 appeared to propagate more efficiently in biofilms compared to planktonic cultures, suggesting potential advantages for phage therapy in biofilm-associated infections [47].

A common limitation across the studies is the reliance on in vitro models and the use of a limited number of S. aureus strains. While these studies provide valuable insights into the potential of phages and phage–antibiotic combinations, translating these findings to clinical settings requires further investigation using in vivo models that more accurately represent the complexity of human infections [34, 37, 45]. Future research should focus on testing the efficacy of phage treatments in animal models and eventually in human clinical trials. Another limitation is the lack of standardized protocols for phage therapy. Establishing guidelines for phage selection, dosage, administration routes, and treatment duration is crucial for developing safe and effective therapies. The sources also suggest that the choice of phage and antibiotic, their concentrations, and the order of administration are crucial factors influencing treatment outcomes [47]. Future studies should focus on understanding the mechanisms underlying phage–antibiotic synergy and optimizing treatment regimens to maximize bacterial killing and minimize the emergence of phage or antibiotic resistance [42]. In addition, the impact of environmental factors, such as the presence of organic materials and shear stress, on phage–biofilm interactions needs further investigation [46]. Addressing these limitations and incorporating future research suggestions will be crucial for advancing phage therapy as a viable treatment option for biofilm-associated infections.

Phage resistance in bacteria manifests through various mechanisms, including receptor modification, CRISPR–Cas systems, restriction–modification systems, and abortive infection systems, each of which disrupts distinct stages of the phage life cycle [48, 49]. This resistance is situated within an ongoing “arms race” between phages and bacteria, wherein phages evolve counter-strategies, such as mutating receptor-binding proteins or recombining with other viral entities, to circumvent bacterial defenses [49]. This dynamic interaction poses significant challenges for the long-term efficacy of phage therapy, as the emergence of resistance is inevitable; however, it can be managed through strategic interventions. Phage cocktails represent a promising solution for mitigating resistance by simultaneously targeting multiple bacterial receptors or pathways, thereby reducing the likelihood of resistance development [50]. For instance, the combination of phages with diverse host ranges or counter-defense capabilities can exploit evolutionary trade-offs, where bacteria resistant to one phage may exhibit increased vulnerability to antibiotics or other phages—a phenomenon referred to as “collateral sensitivity” [51]. Moreover, pre-adapted or “trained” phages can enhance therapeutic success by proactively overcoming bacterial resistance mechanisms [49]. In addition, cocktails may benefit from synergistic effects, wherein certain phages inhibit bacterial defenses, thereby augmenting the effectiveness of others [50].

The ecological and evolutionary dynamics of phage–bacteria interactions provide further insights into therapy design. For example, fluctuating selection dynamics (FSD) and arms-race dynamics (ARD) shape resistance outcomes, with ARD facilitating rapid phage adaptation and FSD imposing greater fitness costs on bacteria [49]. Personalized phage therapy, specifically tailored to the bacterial strain and resistance profile of an individual patient, can enhance durability by taking these dynamics into account [49]. Furthermore, the integration of phages with antibiotics can leverage trade-offs between phage and antibiotic resistance, aiding in the restoration of antibiotic susceptibility in resistant strains [51]. In conclusion, although phage resistance poses a substantial challenge, a comprehensive understanding of its mechanisms and evolutionary dynamics enables the design of effective therapeutic strategies. Phage cocktails, when combined with adaptive and personalized approaches, provide a viable method for sustaining long-term efficacy in the fight against antibiotic-resistant infections [48]. Future research should prioritize real-time monitoring of co-evolutionary processes and the optimization of phage–antibiotic combinations to maximize therapeutic outcomes.

Limitation and challenges

Phage therapy development faces several critical challenges, including the emergence of bacterial resistance, which is influenced by treatment duration, phage concentration, and environmental conditions [52]. Pharmacokinetic complexities arise from varying absorption rates based on administration routes (intravenous, oral, or inhalation) and distribution challenges due to organ-specific accumulation and microbiome interactions [53]. In addition, phage inactivation can occur from environmental pH, immune responses, and metabolic processes, while formulation and storage issues further complicate delivery [54]. Environmental factors such as biofilm age and flow conditions also affect efficacy, and inconsistent outcome assessments add to the difficulty. Despite these hurdles, researchers are optimizing protocols through dose adjustments, route selection, and treatment timing, alongside developing standardized methods and specialized therapy centers to advance clinical applications [52].

Clinical feasibility and commercialization are hindered by the need for specialized facilities, high production costs, and limited accessibility, particularly in Western countries, where regulatory approval lags behind Eastern Europe. The biological and personalized nature of phage therapy complicates mass production, as phages must be matched to individual bacterial strains and updated to counter resistance [52]. Regulatory barriers remain a major obstacle, though progress has been made in Belgium with magistral phage preparations and in Australia and the U.S. through clinical trial approvals. Establishing dedicated centers such as PTC and IPATH has helped advance clinical and regulatory frameworks, suggesting gradual progress toward broader adoption despite persistent challenges [55].

Conclusion

Our research investigated the application of bacteriophages as a treatment for S. aureus biofilms. We explored the efficacy of phage-derived enzymes, including lysins and depolymerases, in disrupting and eliminating these biofilms. In addition, we examined the potential of whole bacteriophages, particularly in combination with antibiotics, for biofilm management. Numerous studies have demonstrated the effectiveness of phage therapy, both independently and in conjunction with conventional treatments, in reducing bacterial populations and accelerating wound healing. While the majority of research has been conducted in vitro, animal models have provided encouraging evidence of the therapeutic potential of these enzymes in vivo. However, further investigation is required to optimize treatment protocols, including phage selection, dosage, and administration methods, for clinical application. Future research should delve into the mechanisms underlying phage–antibiotic synergy, the influence of environmental factors, and the long-term safety of phage therapy. In conclusion, bacteriophages present a promising alternative or complementary approach to traditional antibiotics for the management of S. aureus biofilm infections. Nevertheless, additional research is necessary to fully realize their therapeutic potential in clinical settings. Also focus on developing advanced in vivo models that more accurately replicate human infections, moving beyond traditional animal models. This includes using larger animals, such as pigs, for wound infection studies and creating device-related infection models with catheter implantation. There is also a need to expand models incorporating physiological fluids, which will enhance understanding of specific infection sites. Comparative studies on phage therapy efficacy across different animal models will help identify the most applicable systems for humans. In addition, employing omics-based approaches is essential for understanding the molecular interactions in phage therapy, particularly regarding phage–biofilm interactions and the mechanisms of phage–antibiotic synergy. Investigating bacterial resistance mechanisms in biofilms and the influence of environmental factors on phage activity will further deepen insights into these interactions. To translate findings into clinical practice, well-designed large-scale clinical trials are necessary. These trials should standardize phage selection and administration for specific staphylococcal biofilm infections and assess the safety and long-term efficacy of phage therapy, both alone and with antibiotics. Investigating the pharmacokinetics and pharmacodynamics of phages in human subjects will be critical, along with evaluating the environmental impact of widespread phage therapy to ensure its sustainable application.

Author contributions

A.E: Methodology, Investigation, Writing—original draft, Writing—review and editing Z.M: Methodology, Investigation, Writing—original draft, Writing—review and editing S.A: Methodology, Writing—review and editing Z.M: Methodology, Supervision, Project administration, Writing—review and editing.

Funding

This research received support from the Mashhad University of Medical Sciences, Iran (Grant No. 4031724).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.