Antibiotics play a vital role in curing bacteria-caused minor and life-threatening infections. Nonetheless, bacteria have become incredibly resilient and are capable of using numerous approaches to survive the attack by most antibiotics (Roach and Donovan, 2015). Globally, there is a growing rate of antibiotic resistance (AR) and the rise of multidrug-resistant superbugs, largely caused by the non-judicious usage of antibiotics. In addition to jeopardizing healthy living, AR has a huge economic impact on both developed and developing countries (Aslam et al., 2021). As per the World Health Organization (WHO), AR is responsible for causing approximately 700,000 deaths every year. Moreover, the number of AR-caused deaths is estimated to rise rapidly in the upcoming years (Ling et al., 2022). On average, patients suffering from AR-associated infections require approximately 13 days of hospital stay, imposing a cost of approximately $29,000 per patient treated for such resistant bacterial infections. This can lead to a total annual potential productivity loss of approximately $35 billion (Khurshid et al., 2017; Aslam et al., 2018; Khurshid et al., 2020). These statistics indicate the global impact of AR and the urgency to find novel and effective antimicrobial therapies to control the AR crisis (Baloch et al., 2018; Aslam et al., 2021; Ling et al., 2022).

Bacteriophages (BPs) or phages are viruses that are natural bacterial killers. Approximately, an incredible number (10³¹) of BPs are present in the world, which outnumber bacteria by an estimated tenfold (Skurnik, 2022). In recent times, BP therapy has gained a lot of attention as a powerful and potential therapeutic antibacterial approach (Taati Moghadam et al., 2020; Wei et al., 2020; Tamma and Suh, 2021). BPs are more specific to their targets than antibiotics, and therefore, BPs can eradicate the target pathogenic bacteria without altering the commensal microflora. On the other hand, broad-spectrum antibiotics kill pathogenic bacteria in infected individuals and disrupt microbes and normal bacterial flora (Rai and Kumar, 2022). Antibiotic-resistant bacterial pathogens are also accountable for causing severe nosocomial infections, particularly in individuals with life-threatening conditions. Furthermore, various pathogens can escape the activity of antibiotics by hiding in safe niches within the host; for instance, S. aureus has the ability to endure in the intracellular environment including abscesses or biofilms, which leads to persistent infections. The deficiency of new antibiotics and the rise of multidrug-resistant bacterial strains necessitate the development of alternative therapies, for example, BP-based therapies (Schmelcher and Loessner, 2021).

This review aims to explore the potential of BPs as an alternative to antibiotics in combating antimicrobial resistance. In addition, the advantages of BP-based therapies over antibiotics, phage-antibiotic synergy, the potential of antimicrobial BP-derived proteins, multiple formulation strategies of BP therapy, preclinical and clinical progress of BP-based therapeutics against clinically significant pathogens, and factors that need to be considered in BP therapy have been highlighted in this review.

Antibiotic resistance (AR) is a global phenomenon that even precedes the development and usage of antibiotics by human beings. Microorganisms can generate numerous biologically active molecules across diverse environments that possess antibacterial properties; some of these molecules have been further developed and are currently used as antibiotics. The levels of these molecules within natural environments are frequently under the clinically relevant thresholds, which indicates that resistance does not solely occur to evade their toxic activity. In nature, the interplay between antibiotics and AR mechanisms plays the role of a communication channel between the microbial community members instead of only antimicrobial agents. In addition, these molecules induce genotypic and phenotypic responses and shape the composition of the community. Therefore, genes conferring AR to modern antibiotics were also detected in ancient microbial communities (Perron et al., 2015). Along with the treatment of human infectious diseases, antibiotics are also utilized to mediate livestock growth and treat fish and crop diseases in aquaculture and agriculture (Ventola, 2015). As high as 180 mg of active antibiotic agent is used per kilogram of produced meat in the United States, an even higher proportion has been reported in other countries (Price et al., 2017; Gordillo Altamirano and Barr, 2019). A major problem of using antibiotics in agriculture is the release of millions of tons of antibiotics into environmental reservoirs and water effluents (Manyi-Loh et al., 2018). A lack of proper water treatment facilities further exacerbates this problem. Moreover, pharmaceutical waste and reduced distance between cities and farmlands also play a role in elevating the extents of antibiotic release and environmental persistence. Indeed, this continuous introduction of environmental microorganisms to a range of antibiotics has resulted in increased evolution and extended the range of AR genes in natural reservoirs. AR can also occur due to the genetic information transfer between organisms through transduction, conjugation, or transformation (Yelin and Kishony, 2018) (Figure 1).

Antibiotic-resistant bacteria found in the guts of animals exposed to antibiotics are a major source of horizontal transfer determinants. In addition, bacterial cells can attain non-genetically encoded, transient resistance through various mechanisms including persistence, metabolic dormancy, swarming adaptation, and growth in biofilms (Xu et al., 2022). Numerous resistance mechanisms can hinder the effectiveness of antibiotics at every step of their passage through the bacterial cell. First, bacteria can modify the structure of their cell envelope to repudiate the entry of the antibiotics or can generate efflux pumps to expel antibiotics (Yelin and Kishony, 2018) (Figure 1). Bacteria can inhibit the production of enzymes needed for the activation of antibiotics, or they can also destroy or alter antibiotics by producing enzymes including β-lactamases. Bacteria can hide, modify, or quantitatively alter the intended targets of the drug. Finally, bacteria can cause the activation of alternative metabolic signaling pathways to evade the toxic effects of antibiotics (Munita and Arias, 2016). Since antibiotic-resistant bacteria are rising at an alarming rate, alternative therapies are urgently required for the control of bacterial infections (Alaoui Mdarhri et al., 2022).

Bacteriophages (BPs) are viruses that can infect bacterial hosts (Skurnik, 2022). Typically, BPs share all the common features of viruses, and they cannot replicate by themselves. They need bacterial hosts for reproduction. BPs are small (50–200 nm), and they transmit genetic instructions for effective and fast replication. Generally, BPs are specific to a particular bacterial host; any one BP might infect multiple different species within a genus and many or most strains within a species. However, occasionally, it may affect very few or only one specific isolate of a species (Aslam et al., 2021). Several BPs infect bacteria from diverse genera and usually can do so only when they are phylogenetically closely associated (Tamma and Suh, 2021). The host range of BP can be broad (numerous distinct species or even distinct genera) or very narrow (where only a limited number of bacterial isolates assist replication). Indeed, the host preference or host range largely determines the therapeutic potential of BPs (Rai and Kumar, 2022).

In general, antibiotics can act against a wide range of microbial species and therefore can be used to treat a broad range of bacterial pathogens. However, numerous bacteria have already developed many mechanisms for obtaining AR. In this regard, genetic alterations including horizontal gene transfer (HGT) and mutation also play a role. In addition, various physiological factors including eradication of the antibiotics and enzymatic modification can play a role in altering efflux pump and cell permeability (Munita and Arias, 2016). Several resistance mechanisms developed by bacteria can be passed to others via HGT. In contrast, distantly related bacteria (non-host) are not likely to have ever been exposed to the BP of interest because of the host specificity of BPs. As a result, these bacteria are not likely to play a role in resistance genes to non-hosts through HGT (Cohan et al., 2020). Similarly, CRISPR-based BP immunity is unlikely to protect other taxa and therefore does not play a role in spreading resistance through HGT (Cohan et al., 2020). Thus, the probability of spread and development of BP resistance is markedly lower than that of the development of AR. Moreover, a synergistic effect can be achieved by the combined use of BPs and antibiotics (Gu Liu et al., 2020), since targeted bacterial strains struggle to simultaneously counter two different types of stress (Burmeister et al., 2020; Khan et al., 2022). A comparative advantage of BP-based therapies over antibiotics in the treatment of infections is presented in Table 1.

Phage-antibiotic synergy (PAS) is defined as the phenomenon where the combined effect of BPs and antibiotics is greater compared with the sum of either BPs or antibiotics alone (Chaudhry et al., 2017; Simon et al., 2021). It has been reported that certain antibiotics induce the generation of BPs via a bacterial host and form larger plaques (Figure 3) in the occurrence of antibiotics (Kamal and Dennis, 2015; Li et al., 2021). In addition, the release of progeny BPs from bacterial cells can occur due to the sublethal concentrations of some antibiotics. PAS can decrease the concentration of antibiotics utilized in therapy and ultimately decrease the emergence of AR in bacterial populations (Jo et al., 2016b). The killing mechanism of bacteria differs between BPs and antibiotics (Chaudhry et al., 2017). PAS involves several mechanisms that enhance phage production and/or antibiotic efficacy. Antibiotics such as β-lactams and quinolones induce cell filamentation, potentially aiding phage lysis enzyme (Knezevic et al., 2013). Some antibiotics, such as linezolid and tetracycline, increase plaque size and phage amplification, while others such as cefotaxime enhance phage burst size leading to higher phage concentrations (Ryan et al., 2012).

Subinhibitory antibiotic concentrations inhibit the emergence of phage and/or antibiotic-resistant mutants (Kebriaei et al., 2020). Phage therapy can sensitize bacteria to antibiotics, lowering the MIC and increasing susceptibility (Gu Liu et al., 2020). BPs can also degrade bacterial polysaccharides, aiding antibiotic diffusion. These mechanisms collectively underscore the potential of combined phage-antibiotic therapies against drug-resistant infections. Therefore, it has been suggested that BPs ought to be combined with antibiotics for even better control of bacteria than BPs alone (Torres-Barceló and Hochberg, 2016). The use of BPs and antibiotics is predominantly suggested for infections caused by Gram-positive bacteria such as multidrug-resistant Enterococcus strains and methicillin-resistant Staphylococcus aureus (MRSA). Nonetheless, in terms of killing S. aureus, PAS was found to be effective against the antibiotic-susceptible strains compared to the antibiotic-resistant ones (Jo et al., 2016a), which further indicates that antibiotics can cause alterations in bacterial membrane proteins that can cause alteration in the BP receptors. In a study, Simon et al. showed synergism between the lytic S. aureus BP Sb-1 at BP multiplicity of infection of 10⁻¹ and 10 and oxacillin at concentrations varying from 5 to 100 μg/mL for most studied isolates of S. aureus. The combination of oxacillin and BP Sb-1 markedly reduced the level of bacteria than oxacillin alone (Simon et al., 2021). A study conducted by Malik S showed that combining an E. coli phage cocktail with antibiotics reduced MIC values, notably from 8 to 2 μg/mL for amikacin and from 32 to 8 μg/mL for fosfomycin. These findings underscore how phages serve as adjuvants, bolstering antibiotic effectiveness against drug-resistant E. coli strains (Malik et al., 2021).

Daptomycin (an antibiotic) is often used to treat infections caused by Enterococcus faecium, since 80–90% of Enterococcus faecium strains are resistant against ampicillin and vancomycin. It has also been reported that PAS synergy including daptomycin was linked with lower BP resistance. In another study, Morrisette et al. (2022) observed bactericidal activity with E. faecium BP cocktail and daptomycin in most regimens. In addition, daptomycin introduced to the BP averted BP resistance against daptomycin-resistant E. faecium (Łusiak-Szelachowska et al., 2022). Elevated BP titers and larger plaques were noticed when used in increasing antibiotic concentrations.

A study observed that the combination of low-dose meropenem and B. cepacia BP elevated the survival rate of Galleria mellonella larvae (Kamal and Dennis, 2015). In another study, Aghaee et al. used a combination of antibiotics and BPs against the P. aeruginosa strain PA14 isolated from a burn (Aghaee et al., 2021). This strain was treated with antibiotics at a sub-minimum inhibitory concentration (MIC) and MIC levels, a combination of BPs, a mixture of two BPs, and a single BP. Subsequently, the four lytic BPs were selected based on their performance in a primary efficiency of plating test. Moreover, out of these four primarily sequenced BPs, those with different infection properties and genetic features were selected. Interestingly, all designated BPs formed plaques on P. aeruginosa. Collectively, these findings suggested that a combination of one antibiotic and two BPs showed the highest killing potential against the strain of P. aeruginosa. In another study involving Citrobacter amalonaticus, synergistic effects were observed with the BP MRM57 at concentrations of 10³ and 10⁶ plaque forming units (PFU/ml), combined with a sublethal dose of antibiotics with a distinct mode of action (including tigecycline, cefepime-tazobactam, meropenem, gentamicin, fosfomycin, colistin, and carbenecillin) at 1/10 × MIC (Łusiak-Szelachowska et al., 2022; Manohar et al., 2022).

BP genomes encode several important enzymes and proteins to eradicate bacteria during infection. Polysaccharide depolymerizing enzymes (PDEs) and virus-associated peptidoglycan hydrolases (VAPGHs) are two such groups of proteins which are required for BPs to adsorb with and infect host bacteria (Roach et al., 2017). VAPGHs are typically found in the outermost layer of the protein shells of BPs, which is a component of the viral structure and plays a role in the local peptidoglycan layer degradation so that the tail tube structures of BPs release their genomes into the host. BP-encoded PDEs can cause the degradation of polysaccharide components found on the bacterial cell membrane surface, for instance, lipopolysaccharides derived from Gram-negative bacteria. Furthermore, PDEs also help BPs reach the host receptors by digesting the polysaccharides present on the bacterial cell membrane, which can further degrade biofilms (Maciejewska et al., 2018). There is a growing interest regarding BP-derived lysins or cell wall hydrolases as potential alternatives to antimicrobials. Lysins are also considered promising clinical candidates because of their differential virulence, low drug resistance, and rapid bactericidal action (Sharma et al., 2018).

BP-derived lysins possess enzymatic functions and can destroy non-divided bacterial cells, which can be a therapeutic option against recurrent or drug-resistant infections. Ectolysins and endolysins are the two major lysine enzymes. Ectolysins play a role in decomposing peptidoglycans externally to bacteria during BP DNA invasion. Endolysins also decompose the peptidoglycans of the cell wall at the end of the replication of BPs (Schmelcher et al., 2012). Various lysine enzymes such as amidases, peptidases, and glycosidases can split distinct chemical bonds according to the selectivity of lysins. The addition of a small quantity of lysin can rapidly decrease the optical density of turbid-suspended Gram-positive bacterial cultures (>10⁸ CFU/mL) within minutes. Moreover, lysine does not exert any effect on eukaryotic cells since murein hydrolases are only present in bacteria. For instance, p128, a chimeric recombinant exolysin derived from BP K, completely inactivated prokaryotic cells and did not exhibit any cytotoxicity on eukaryotic cell lines including Hep 2 and Vero even at a high concentration of 2.5 mg/mL (Ling et al., 2022). The biofilm and capsule structure of bacteria play an important role in resistance that averts the actions of antibiotics. The capsule structures of numerous bacteria can assist pathogens to escape phagocytosis and mediate cell invasion, intravascular survival, and epithelial colonization. Similarly, the biofilm structure can protect the bacterial population against immune functions. Thus, it is possible to remove or target these structures by using BP-derived depolymerases. Moreover, it was confirmed that the synergistic effect of amoxicillin and depolymerase could destroy the biofilms of Klebsiella pneumoniae (Ling et al., 2022).

Bacteriophage therapy to treat bacterial infections encounters several obstacles and opportunities in terms of applicability and future development. As mentioned earlier, these include bacteriophage formulation, understanding its pharmacodynamic properties, the development of phage resistance in bacteria, and finally ethical and legal considerations (Zalewska-Piątek, 2023).

Optimizing the administration of bacteriophage preparations is crucial to maximizing their therapeutic potential. Understanding the pharmacokinetics of phages, including their stability, distribution, and clearance in the body, is essential for determining the most effective dosage regimens and administration routes (Dąbrowska, 2019). Encapsulation technologies, such as microencapsulation or liposome encapsulation, can protect phages from degradation in the gastrointestinal tract and enhance their bioavailability. Finally, advancing clinical experience and monitoring adverse reactions are paramount for refining phage therapy protocols and ensuring patient safety (Loh et al., 2021). Real-time observation of treatment outcomes, coupled with rigorous data collection and analysis, can provide valuable insights into the efficacy and safety of phage therapy in diverse clinical settings. Phage therapy necessitates the use of lytic phages, ensuring high purification, as lysogenic BPs are unable to lyse host bacteria and also hinder the lytic action of other phages upon integration. Moreover, they pose a significant concern as they can transfer toxins and antibiotic-resistance genes to bacteria (Carascal et al., 2022).

Regarding bacterial resistance to BPs, some bacteria develop defense techniques against phages, such as inhibiting surface receptors, altering mechanisms, and secreting extracellular polymeric capsules. These defenses can impair the capacity of bacteriophages to efficiently infect and destroy bacteria (Wang and Leptihn, 2024). Furthermore, BPs have a narrow cleavage spectrum which limits their effectiveness against different bacterial strains. This suggests that some bacteriophages may only be effective against a restricted group or species of bacteria, rendering them ineffective against a wide range of pathogens (Chung et al., 2023). Hence, the emergence of bacterial resistance mechanisms to bacteriophages emphasizes the need for novel techniques. Using a bacterial cocktail, which includes several phage types that target different bacterial strains, can improve treatment efficiency while decreasing the possibility of resistance development (Rahimi-Midani et al., 2021). Genetic engineering of BPs would increase their host range or boost their lytic activity against certain bacteria (Sun et al., 2023).

Additionally, ethical and legal issues encompass various aspects such as patient safety, informed consent, regulatory approval, and research ethics. The strategy is to establish a comprehensive phage bank and streamlined screening protocols to isolate phages that target antibiotic-resistant bacteria effectively (Anomaly, 2020; Zalewska-Piątek, 2023).

It facilitates the swift matching of BPs to bacterial isolates from infected patients, allowing for personalized treatment. Globally, only a few centers, such as those in Georgia, Belgium, Poland, the United States, and Australia, persist in offering phage-based treatments. These centers offer valuable data on therapy efficacy and conduct clinical trials to further enhance treatment outcomes (Zalewska-Piątek, 2023).

The objective is to eliminate bacterial infections in patients via cell lysis while minimizing adverse effects and preserving the natural microflora.

The growing global crisis of AR has inspired the development of BPs as an alternative to antibiotics. Indeed, BPs have great potential as effective, promising, and novel therapeutic agents to fight against AR. Despite the aspirations and novelty of the usage of BPs as an alternative to antibiotics, more studies are required to explore the clinical applications of these therapeutic BPs. As antimicrobial resistance is a global issue now, it is crucial to explore the potentiality of BPs as an effective alternative to antibiotics. However, more preclinical and clinical studies are required on the site-specificity, dosage regimens, half-life, and safety profile of the BPs to demonstrate their safety, efficacy, and potential as alternatives to antibiotics.

AS: Conceptualization, Methodology, Project administration, Formal Analysis, Writing – review & editing, Writing – original draft, Funding acquisition.