Bacteriophages, also known simply as phages, are prokaryotic viruses that exclusively infect and kill bacteria. While phages were already being explored as antimicrobial agents in the early 1900s ^[1][2][1,2], the rise of antimicrobial chemical agents that became increasingly popular after the discovery of penicillin, overshadowed their use in Western countries [3]. However, after decades of being overlooked, the emergence of bacteria that are resistant to these antibacterial agents has refocused efforts on the use of phages for treating infectious diseases ^[4][5][6][4,5,6]. On the other hand, bacterial resistance to phages can also arise through a variety of molecular mechanisms. Several clinical studies on phage therapy have reported the occurrence of phage-resistant variants, which represents a significant concern for the successful development of phage-based therapies [7]. It is therefore important to carefully address phage resistance within the context of developing anti-bacterial treatments and therapies.

Phages have played a significant role in shaping the evolution of bacterial communities and populations through a co-evolutionary mechanism known as an arms race [8]. Recent studies investigating the fitness costs associated with phage resistance have highlighted how the selection of phage-resistant variants can have significant implications for clinical therapy settings ^[7][9][10][7,9,10]. This concept demonstrates that pathogenic bacteria can potentially develop resistance to phages, but that this occurs at the expense of virulence. Moreover, antimicrobial-resistant bacteria can regain susceptibility to antimicrobial agents as a fitness cost for phage resistance. These findings shed new light on the applications of phages to therapy and other phage-related approaches.

Phages are highly prevalent viruses that specifically infect bacterial cells. The global population of phages is estimated to be greater than 10³¹ particles, making them the most abundant biological entities on Earth [11]. Phages can be found wherever bacteria exist, including within the human body, and they play a crucial role in regulating bacterial populations as predators ^[12][13][14][12,13,14]. The ongoing evolutionary competition between bacteria and phages known as an arms race has driven the development of biological systems including microbial defense mechanisms against phages and anti-phage defense systems, contributing to the remarkable diversity observed in these microorganisms.

All phages package their genetic material within a capsid, and a significant number of phages possess a tail which is essential for attachment to host cells and for injection of their genome into the host ^[4][5][15][4,5,15]. Among the various types of phages, the most common are the tailed phages with double-stranded DNA (dsDNA) [4]. These can be classified into three distinct morphologies ^[15][16][15,16]: Myoviridae (contractile tail), Siphoviridae (long non-contractile tail), and Podoviridae (short tail). While the International Committee on Taxonomy of Viruses (ICTV) updated viral taxonomy in 2022 based on sequence information alone, eliminating morphology-based taxa, they acknowledged that these terms can still be used to describe phage characteristics and maintain their historical reference [17].

Phages primarily exhibit two main life cycle modes: the lysogenic and lytic life cycles [4]. Temperate phages employ both cycles, while virulent phages employ only the lytic cycle. During infection, phages attach to specific receptors on the bacterial surface and inject their genetic material into the bacterium. After gene expression and genome replication within the host, new phage particles are assembled and released by lysing the host cells through the action of endolysin, a peptidoglycan hydrolytic enzyme encoded in the phage genome ^[5][18][5,18]. While both temperate and virulent phages employ this lytic cycle, temperate phages can also enter a lysogenic state. In the lysogenic state, repressor proteins encoded by the temperate phages repress the genes responsible for bacterial lysis. Lysogenic phages integrate into the host chromosome or form independent replicating plasmids known as prophages. Repressors encoded in prophages prevent reinfection by the same phage. Under certain environmental conditions or stressors, the lysogenic phage genome can be excised, initiating the lytic cycle [19]. The characteristics of temperate phages, such as their capacity for horizontal transmission of genes associated with virulence and antimicrobial resistance among bacteria, as well as their tendency for maintaining a high frequency of lysogenic states, generally make them less suitable for therapeutic applications ^{[4][19][20][21]}[4,19,20,21].

Bacterial host factors play a significant role in determining the specificity of phage infections. Bacteria have developed various anti-phage systems to counteract phage infection at various stages of the viral life cycle. For example, restriction-modification systems and CRISPR-Cas systems are well-known defense mechanisms that disrupt the phage genome before replication [22]. On the other hand, the phage host range is primarily restricted by receptors present on the surface of the host bacteria. Various structures on bacterial surfaces, such as outer membrane proteins, polysaccharides, teichoic acids, pili, flagella, and transporters, can act as bacterial receptors ^{[23][24][25][26][27][28]}[23,24,25,26,27,28]. Much of the acquisition of phage resistance is attributable to mutations or deletions in receptor genes.

Although phages were first discovered by Frederick Twort in 1915 through experiments on filter-passing viruses, providing definitive evidence of their existence at the time was difficult [1]. Two years later, Felix d’Herelle isolated the first phage from the intestinal contents of dysentery patients, describing them as invisible living entities that parasitize bacteria [2]. D’Herelle recognized the therapeutic potential of phages and successfully used phages to treat patients with bacillary dysentery in 1921 ^[29][30][29,30]. Furthermore, a large clinical trial conducted in India by D’Herelle and colleagues in 1927 demonstrated a significant reduction in mortality rates among cholera patients treated with the Vibrio cholerae phage [31]. Based on these successes, scientists attempted numerous phage therapies for treatment of other bacterial infections. However, the outcomes of these phage therapy for other bacterial infections were inconsistent ^[32][33][32,33]. This could potentially be explained due to factors such as the lack of adequate controls, variations in phage dose and administration routes, the narrow host range of phages, bacterial-derived substances in phage products, and the influence of body fluids and the immune response [34]. Several of these are still discussed today ^[35][36][35,36]. Thereafter, in Western countries, the commercialization of antibiotics gradually replaced phage therapy. However, in countries such as the Soviet Union, Georgia, and Poland, where access to antibiotics was restricted by the “Iron Curtain” during the Cold War era, the use of phages to treat infectious diseases has continued [6]. Against this background, the emergence and increasing prevalence of antimicrobial-resistant bacteria has become a serious concern. It has been estimated that antimicrobial-resistant bacteria will be responsible for the deaths of approximately 10 million people annually in 2050 unless a global program to reduce antimicrobial resistance is implemented [37]. Consequently, phage therapy has been proposed as a potential “trump card” for treating these infections, given their unique bactericidal mechanisms that differ from those of antibiotics, making them effective for treating antimicrobial-resistant bacteria. In 2017, the Food and Drug Administration (FDA) approved the first application of phage therapy for treating infections caused by multidrug-resistant Acinetobacter baumannii [38]. In this clinical case, despite multiple antibiotics and percutaneous drainage of a pancreatic pseudocyst with suboptimal results, the use of phages significantly improved symptoms and ultimately led to a full recovery. After this, multiple case reports describing the application of phages to the treatment of multidrug-resistant bacterial infections have been published ^[4][39][4,39]. Recently, phage therapy programs have been established in the US, Belgium, France, and Sweden [4]. Notably, Belgium has initiated the implementation of a practical framework for phage therapy, focusing on creating personalized phage treatments using magistral preparations ^[40][41][40,41]. In addition, human clinical trials have been conducted for a variety of bacterial infections, as shown in Table 1. While one trial for treating otitis externa caused by Pseudomonas aeruginosa infection has shown positive therapeutic outcomes [42], other trials have failed to demonstrate conclusive efficacy ^[43][44][43,44]. In most clinical trials, the investigation and documentation of bacterial resistance to phage therapy have generally been inadequate, largely due to the lack of prior phage sensitivity tests. Taken together, phage therapy is a promising alternative approach to treating antimicrobial-resistant bacterial infections, and ongoing research and clinical trials are being conducted to further explore the efficacy of this treatment modality and establish its role in modern medicine.

Despite receiving considerable attention as a promising approach against antimicrobial-resistant bacterial infections, phage therapy faces challenges due to bacterial phage resistance; this is similar to the challenges facing antibiotics. Notably, several clinical studies on phage therapy have documented the occurrence of phage-resistant variants, and these findings have raised concerns about the successful development of phage therapies. Based on this perspective, researchers present a summary of phage therapy cases where phage-resistant variants were encountered, particularly instances that resulted in inadequate therapeutic outcomes or instances that necessitated changes in phage therapy protocols (Table 2).

In these clinical cases of phage therapy, each patient was treated with a combination of phages known as a “phage cocktail”. Generally, the purpose of using phage cocktails is to prevent the development of phage-resistant variants [4]. It has been observed that using a combination of phages that target different receptors can effectively suppress or delay the emergence of phage resistance ^[27][51][52][27,51,52]. In these cases, although evaluating the exact effectiveness of these phage cocktails in suppressing resistance was challenging, primarily due to the lack of clarity regarding the specific receptors targeted by each phage in the cocktail, phage resistance was still observed. After phage resistance was observed, in five out of eight cases at the University of California San Diego and Eliava Phage Therapy Center (Cases 1, 2, 3, 5, and 6; Table 2), the introduction of additional phages targeting the phage-resistant variants was required ^{[38][39][45][46][48]}[38,39,45,46,48]. In two separate cases in China and Spain (Cases 4 and 8; Table 2), the emergence of phage-resistant variants mandated exploring alternative therapeutic strategies, which may or may not have incorporated phages ^[47][50][47,50]. In Case 7 shown in Table 2, Dedrick et al. demonstrated that, although phage resistance was not specifically concluded to be a major factor limiting the effectiveness of treatment, the development of a phage-resistant variant during treatment and the failure of intravenous mycobacteriophage therapy to cure refractory Mycobacterium abscessus lung disease was observed [49]. Taken together, these clinical outcomes strongly suggest that phage resistance had a significant negative impact on successful treatment. Notably, according to a report by Schooley et al. (Case 1), a phage-resistant variant was isolated from a patient just eight days after the initial administration of phage cocktails for A. baumannii infection [38]. Additionally, Bao et al. and Blasco et al. (Cases 4 and 8) described cases in which phage resistance developed within five days and one week from the beginning of phage administration against Klebsiella pneumoniae and P. aeruginosa infections, respectively ^[47][50][47,50]. These cases suggest that phage-resistant variants can emerge in a relatively short timeframe, even within approximately a week after administration in clinical phage therapy. However, as seen in the cases managed at the Eliava Phage Therapy Center (Cases 2 and 3), it appears that phage resistance can also develop gradually during relatively long-term phage administration spanning several months ^[45][46][45,46].

To address the challenge of phage-resistant variants, most cases required specific interventions, such as the preparation of alternative phage cocktails. In fact, personalized cocktails prepared during phage therapy are more likely to be effective, but a major concern is the time required to isolate (or screen), amplify, and purify new phages for phage resistance. In addition, difficulties in accessing sufficient phage banks or isolating novel phages against resistant variants would further complicate the production of personalized cocktails during phage therapy. Consequently, a proactive strategy that relies on a comprehensive molecular understanding of how phage-resistant variants develop could be a key for successful phage therapy. As the fitness trade-offs between phages and antibiotics are becoming clearer, phages have been identified as “old and new” approaches to resensitizing bacteria to antibiotics. From this perspective, phages are not solely antimicrobial agents for treating bacterial infections, but also powerful agents that could extend the lifespan of classical antibiotics and contribute to effective infection control of antimicrobial-resistant bacteria. It will be essential to clarify the molecular basis of these fitness trade-offs and design appropriate clinical trials based on their applicability. This type of phage-based therapy is expected to develop as a promising approach in combination with antibiotics, providing robust options in this era of phage “re”-discovery in the domain of modern medicine.