Next Article in Journal
Multiple Strategies for the Application of Medicinal Plant-Derived Bioactive Compounds in Controlling Microbial Biofilm and Virulence Properties
Previous Article in Journal
The Patterns and Appropriateness of Systemic Antifungal Prescriptions in a Regional Hospital in Hong Kong
Previous Article in Special Issue
Bacteriophage Therapy on an In Vitro Wound Model and Synergistic Effects in Combination with Beta-Lactam Antibiotics
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 14
Issue 6
10.3390/antibiotics14060553
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Current Clinical Laboratory Challenges to Widespread Adoption of Phage Therapy in the United States

by
Ahnika Kline
1,2,*,
Ana G. Cobián Güemes
1,3,
Jennifer Yore
2,3,
Chandrabali Ghose
4,
Daria Van Tyne
5,6,
Katrine Whiteson
7 and
David T. Pride
1,2,3
1
Department of Pathology, University of California, San Diego, CA 92093, USA
2
Department of Medicine, University of California, San Diego, CA 92093, USA
3
Center for Innovative Phage Applications and Therapeutics, University of California, San Diego, CA 92093, USA
4
Bioharmony, Inc., New York, NY 10016, USA
5
Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
6
Center for Innovative Antimicrobial Therapy, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
7
Department of Biology, University of California, Irvine, CA 92697, USA
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(6), 553; https://doi.org/10.3390/antibiotics14060553
Submission received: 28 February 2025 / Revised: 9 May 2025 / Accepted: 15 May 2025 / Published: 29 May 2025
(This article belongs to the Special Issue Bacteriophage Therapy a Renaissance Weapon Recent Developments and Application, 2nd Edition)
Browse Figure
Versions Notes

Abstract

:
The resurgence of phage therapy in Western societies has been in direct response to recent increases in antimicrobial resistance (AMR) that have ravaged many societies. While phage therapy as a concept has been around for over 100 years, it has largely been replaced by antibiotics due to their relative ease of use and their predictability in spectrum of activity. Now that antibiotics have become less reliable due to greater antibiotic resistance and microbiome disruption, phage therapy has once again become a viable and promising alternative, but it is not without its challenges. Much like the development of antibiotics, with deployment of phage therapeutics there will be a simultaneous need for diagnostics in the clinical laboratory. This review provides an overview of current challenges to widespread adoption of phage therapy with a focus on adoption in the clinical diagnostic laboratory. Current barriers include a lack of standard methodology and quality controls for phage susceptibility testing and selection, the absence of phage-antibiotic synergy testing, and the absence of standard methods to assay phage activity on biofilms. Additionally, there are a number of lab-specific administrative and regulatory barriers to widespread phage therapy adoption including the need for pharmacokinetic (PK) and pharmacodynamic (PD) assays, methods to account for changes in phages after passaging, an absence of regulatory guidance on what will be required for agency approvals of phages and how broad that approval will apply, and the increased need for lab personnel or automation to account for the work of testing large phage libraries against bacteria isolates.

1. Introduction

What is phage therapy? Phage therapy is the treatment of clinical bacterial infections with bacteria-targeting viruses called bacteriophages. It is a treatment that dates back over 100 years [1,2,3]. Phages were not commonly used to treat bacterial infections until the early 1940s when they were deployed primarily in Eastern Europe and in the Soviet Union for the treatment of bacterial infections [4]. Phage clinical trials even predate the deployment of the first antibiotics [5]: sulfonamides in 1932 [6] and penicillin in 1943 [7]. Critical reviews of phage products [8] and the widespread adoption of antibiotics in the 1950s, caused a shift away from phages [9]. From there, the development of phage therapy lagged behind antibiotics significantly, particularly in the United States, with phages confined to only a few enclaves in Eastern Europe by the late 1900s [10,11].
Only recently has phage therapy seen a rebirth in the United States, largely due to antibiotic resistance reaching a critical mass [12,13]. The number of antibiotics in the pipeline that deploy new mechanisms to kill bacteria are insufficient to address the growing threat of increasing antimicrobial resistance (AMR) [14]. This is significant because many of the antibiotics currently in use in medicine, animal husbandry, and agriculture use similar mechanisms of action [15]; because of the shared mechanisms, resistance to one antibiotic within a class frequently results in resistance to multiple antibiotics within that class [16]. Thus, new therapeutics that can treat infections caused by antibiotic-resistant bacterial pathogens are desperately needed.

2. Existing Milestones in Phage Therapy

2.1. Phages Are Abundant

It is not difficult to identify phages that are capable of killing many bacterial pathogens. Because phages are naturally occurring, they are often found in the environment. They are also readily available in the human virome, where they likely exist in the trillions [17,18,19]. Just as many bacterial pathogens are found in the human body, their phages are also found in the body. Thus, in searching for phages capable of killing human pathogens, it is useful to examine sites such as sewage, which is often a rich source of phages that target human-associated bacteria [20]. Rich sources of phages typically include sewage, rivers, ponds, lakes, run-off water, soil, animal feces, and human specimens such as body swabs, urine, and saliva [21,22,23,24].

2.2. Successful Trials and Experience with Phage Therapy

There exists a significant dichotomy in the wide adoption of phage therapy between Russia and Georgia compared to Europe and the United States. The Eliava Institute in Georgia continued to push phage therapy forward with wide adoption after the interest in phages subsided in the West [25]. Today, pre-prepared cocktails can be purchased in Georgia and Russia without a prescription [26,27]. Outside of these countries, phage therapy remains restricted to compassionate use and clinical trials [28]. However, the collective continued publication of outcomes of patients treated in countries where phages are widely adopted and of clinical trial results supports the clear utility of phage therapy [29,30,31,32]. In the USA, the most advanced ongoing trial is an active phase 2/3 randomized double-blind active-controlled trial for phage therapy for urinary tract infections caused by drug resistant E. coli [33]. There are now a handful of phage therapy centers in the USA, matched by those in Europe and Australia (Table 1) [26]. There are also now many companies in the US and Europe emphasizing clinical phage development [34,35].

2.3. Promising Results for Phage Therapy for Treating Resistant Bacteria

Now that AMR (Antimicrobial Resistance) has reached a critical mass, physicians commonly observe AMR bacteria in their clinical practice. For example, there are seven pathogens called the ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) that are known to commonly harbor AMR and are responsible for the preponderance of nosocomial infections [36,37]. While some of these infections can be treated with commonly used antibiotics, their increasing acquisition of AMR and increasing virulence has necessitated a search for alternative means for treatment [38,39]. There now are a number of institutes across the world that specialize in providing services to individuals who are seeking phages to treat infections caused by these multi-drug resistant (MDR) or extensively drug resistant (XDR) and hypervirulent bacteria [40,41,42,43,44,45]. Many of these institutions interface directly with physicians and hospitals to provide phages for treatment, but some work directly with patients.
In the United States and Western Europe, the concept of phage therapy has seen a rebirth in the past decade [46], largely due to the need impressed upon physicians by the crisis of AMR. While the microbiology observed at most centers likely represents local AMR and bacteria trends, some patterns are consistent across centers. A retrospective review of 100 cases treated with phage therapy throughout Europe demonstrates that most phage therapy interventions targeted patients with P. aeruginosa infections, followed by S. aureus [47]. At our center in the Western U.S., the majority of requests are for phages targeting P. aeruginosa, followed by S. aureus, K. pneumoniae, E. coli, and A. baumannii (Table 2). Not all of these requests result in successful identification of lytic phages and the initiation of treatment, but they highlight the bacterial pathogens that are emerging as target organisms driving the need for phage therapy. Even mycobacteria, which can replicate intracellularly, develop AMR and often require three to four drug regimens over many months of therapy, have shown promise as a target for phage therapy [48,49,50,51].

2.4. The Existence of a Variety of Laboratory Methods for Phage Selection

With over one hundred years of experience with phage biology, it is not surprising that reliable laboratory methods for isolating and testing phage have been developed. The existing literature is rich with primary publications and reviews detailing phage selection and susceptibility testing methodologies [52]. Most phage research laboratories employ solid media plaque assays and/or liquid media growth kinetic assays to assess phage activity [53]. Both methods have been successful in identifying phages for clinical use. Whether one chooses to examine susceptibility via solid media or broth testing or both may depend on the personal preference or the available equipment at a particular site [53,54,55]. Additionally, there is wide support for the use of efficiency of plating (EOP) analysis to determine whether a bacterial host is phage-susceptible when performing solid media assays [56]. EOP depends upon using a phage production host where there is high lytic capacity and comparing the efficiency of lysis of the phage on the production host to a bacterial isolate acquired from an infected patient. If the EOP is >0.001 then that phage is considered to have great enough efficiency to be used in the patient for phage therapy [56,57]. While this theory has not been rigorously tested in clinical trials, it represents a current guideline for the use of phages in the treatment of patients [48]. A caveat of using EOP values is that a phage with low titer on both the production host and the target clinical isolate can still produce an EOP > 0.001, but the phage may not be effective therapeutically [58].

3. Methodology Challenges to Phage Testing in the Clinical Microbiology Laboratory

3.1. Analysis in Liquid Solution Compared to Solid Medium

Some phages may show greater lytic activity on solid media compared to liquid media, and vice versa [59], yet there is currently nothing known about which circumstance might predict greater efficacy in the human host. Most studies and clinical trials take an either/or approach to examining phage lytic potential [60]. In general, when we examine the lytic potential of a phage for a host, we examine not only whether the phage is capable of lysing its host on solid medium by examining EOP, but we also examine its ability to inhibit bacterial growth longitudinally in liquid culture [58]. We took this approach in a recent outbreak of extensively drug-resistant P. aeruginosa found in contaminated eyedrops which took four lives and rendered at least 14 people blind [58,61,62]. We were able to identify highly effective jumbo phages, which historically have very little activity in liquid media, through this dual method approach [63]. Jumbo phages have large >200 kp genomes shielded by nucleus-like structures which helps protect them from anti-phage defense mechanisms employed by bacteria, such as restriction–modification systems, and CRISPR-Cas systems, which often leads to broader host ranges [64,65,66,67,68]. While this is an intensive workup of a single isolate for each patient, in the absence of guidelines to support a single assay, the safer choice is to evaluate phage susceptibility against the host via multiple different means to assess activity until consensus methods are established for specific phage/host pairs [69]. Phage plaque assays on solid medium are likely more easily adapted to clinical microbiology laboratory workflows due to the lack of reliance on sophisticated equipment but can be subject to user variability in interpretation of results. They can be adapted to imaging protocols to remove user variability in interpretation of plaque lysis [70], though such a step would incorporate additional expenses and time.

3.2. Establishment of Quality Control Bacteria and Phages

The absence of standardization is already problematic for the relatively small number of institutions that currently offer phage therapy, and many have identified that minute differences in protocols can lead to significant differences in phage susceptibilities. Two laboratories using the exact same protocols and host strains will often find that the same phages have different host ranges [59]. There are also differences in protocols regarding whether/when to evaluate if a pathogen has changed its phage susceptibility [71,72]. Standardization of approaches is critical to reduce variability between laboratories, and to achieve consensus in how phage activity is described [55,73]. The variability between laboratories is expected at this moment, as most laboratories often work with different bacterial isolates, different phages, and different testing modalities. Use of different bacterial isolates is difficult, if not impossible to change, as each institution has its own unique patient population with their own pathogens. However, positive and negative quality control bacteria and standard quality control phages to be used in assays can be established across institutions.

3.3. The Need for a Standards Committee

Developing a universal standard for phage testing could be accomplished by putting together an advisory board of phage experts with experience in different testing modalities with the goal to develop practical and reproducible testing standards. We applaud that EUCAST (the European Committee on Antimicrobial Susceptibility Testing) recently convened a subcommittee dedicated to developing standards for susceptibility testing of phages with member representatives from 16 different countries [74]. The Antibacterial Resistance Leadership Group (ARLG) also recently published suggested answers to a number of clinical questions related to the use of phage therapy in clinical practice, including what parameters phage susceptibility testing platforms should consider. The group identified similar issues with standardization of methodology, controls, and even storage temperatures of reagents [75]. Consensus methods must ultimately be viable to be reproduced in a clinical microbiology facility, i.e., experiments that require sophisticated and expensive equipment, and high-level scientific training would not be preferred.

3.4. The Need for Antibiotic Synergy Testing

It is currently required that patients receive standard-of-care antibiotics during treatment with phage therapy [76]. It is currently unclear how much of a patient’s response is due to phages, antibiotics, or the additive/synergistic effect of combining them together. A number of different studies indicate that synergistic interactions between antibiotics and phages frequently occur [77,78,79,80,81,82,83,84,85], but when it comes to treatment protocols for patients, these concepts appear to be rarely considered. Antibiotics and phages could also act antagonistically. The primary reasons are that: (1) protocols for synergy testing are generally not standardized across laboratories, (2) it is unknown whether in vitro antibiotic–phage synergy correlates with in vivo efficacy, and (3) most clinical laboratories are not properly equipped to perform synergy testing. As is generally the protocol in clinical microbiology laboratories globally, standardization of assays when possible is the key to assay development. Nonetheless, several available assays for phage-antibiotic synergy testing hold promise.
Liquid-based assays have generally been the mainstay for assessing phage-antibiotic synergy [80,86,87]. These assays involve setting up a plate (often a 96-well plate) with a grid of antibiotics and phages at different concentrations along the different axes on the plate, allowing the user to evaluate whether there is greater inhibition of growth of a target pathogen treated with antibiotic and phage together compared to control wells containing either agent alone. This technique has been used to demonstrate synergistic interactions that occur between antibiotics and phages for pathogens such as Enterococcus spp. [77,88], S. aureus [89], E. coli [79], A. baumannii [90], Salmonella spp. [91], and P. aeruginosa [78]. Liquid-based assays have also been used to demonstrate synergy between phages and the anticancer drug, mitomycin C, which highlights their utility in new antimicrobial discovery [92]. While these assays can be useful for demonstrating that antibiotics and phages can work together in vitro and suggest that they may work together in vivo as well, they are cumbersome for clinical laboratories to perform. Most clinical laboratories do not have the necessary equipment, such as plate readers, to perform such assays.
More recently, members of our group have demonstrated that synergistic interactions between antibiotics and phages could be observed in a more simplistic fashion, using a solid media assay rather than more sophisticated liquid-based assays [72]. The benefits of such an assay would be that it would not require expensive equipment to develop, would be relatively easy to interpret, would follow a straightforward model for predicting synergy, additivity, antagonism, and indifference, and would fit within the bounds of what could easily be developed in clinical microbiology labs. Using this protocol, our group found that trends in synergy could be established for both Gram-positive and Gram-negative pathogens and confirmed that pre-existing antibiotic susceptibility was not required for synergy to be observed. Specifically, we confirmed the previous observation made using liquid cultures that vancomycin-resistant Enterococcus became susceptible when combined with phage [77,88,93]. The drawback of this assay is that it requires phage strips and antibiotic strips to be constructed by a manufacturer to ensure quality and reproducibility. We recommend that pathogens be evaluated for antibiotic-phage synergy, even in cases where resistance to those antibiotics exists in the source pathogen, as this will provide more information to the treating physician.

4. Phage Therapy Treatment Differences That May Influence Laboratory Methodology

4.1. Cocktails vs. Individual Phages

There are many strategies to deliver phage therapy to individuals, but two have emerged as the primary strategies employed by most treatment centers. In the first strategy, multiple different phages often targeting different receptors are combined into a cocktail and employed simultaneously to attack the same bacterial pathogen. The rationale behind this strategy is to create a higher barrier to mutation in any single bacteria that would render it resistant to all of the cocktail phages at once and may also better account for heterogeneity in even clonal bacterial populations [94,95,96,97]. As quickly as phages for human bacterial pathogens can be found, bacterial resistance to those phages can develop, and may explain why phage treatment protocols often employ cocktail therapies [5,98]. In the second strategy, an individual phage is employed against a bacterial pathogen and is then replaced by another individual phage only if the pathogen develops resistance to the first phage in a staged approach, with the advantage of potentially eradicating the infection with fewer phages and reduced cost [99,100]. The use of a single phage may decrease the chance of an adverse host immune response compared to the use of a phage cocktail [101]. In each of these scenarios, laboratory testing is necessary to identify whether the bacterial pathogen in question is susceptible to the phages being used. Additional information is also needed, including the mechanism of action and/or the receptors that each phage employs to infect the bacterial host, to ensure that resistance to one phage does not convey resistance to multiple phages within the cocktail or resistance to phages that may be used subsequently in staged approaches. Unfortunately, both approaches can require significant labor for a clinical laboratory to determine susceptibilities for multiple different phages among single or multiple bacterial pathogen hosts.
Phages use a number of bacterial virulence factors as receptors [102]. However, identification of the receptors for phages prior to using them creates a significant burden for most laboratories given the expense and labor involved in developing phages for clinical use. Particularly when using phage cocktails where each phage may employ a different receptor, each receptor should be identified separately prior to use. Even genetically unrelated phages might employ the same receptors [103,104], and thus, even when employing a technique such as a staged approach for phage therapy, receptors likely need to be identified beforehand to reduce the potential for cross-resistance [105].
One prominent issue for evaluating phage therapy for both cocktail and staged approaches is the potential for mechanisms that reduce likelihood of lysis across multiple phage types. For example, a prior study characterizing resistance to a group of Enterococcus phages found that phage resistance developed via membrane mechanisms that generally made the bacteria less accessible to the phages [106]. While these mechanisms were not likely to represent the actual receptors for the phages, the lack of access to their receptors resulted in cross-resistance to a number of different types of phages. When evaluating the development of phage resistance, it is important to note whether mutations might occur that result in resistance to a variety of phages through mutations in similar genes in the same biosynthetic pathways [107,108].

4.2. Phage Therapy for Multispecies Infections

Phages are thought to be attractive for single species infections because their specificity may limit killing of normal microbial flora [109]. Because many infections, such as intraabdominal infections, are caused by multiple bacteria simultaneously, phage therapy approaches that target multiple bacteria simultaneously have been investigated. These approaches generally combine multiple phages targeting different bacteria into a single cocktail [110,111,112,113,114]. There also exist some polyvalent phages, which target multiple bacteria species or genera [115,116]. For example, polyvalent phages have been discovered that target multiple diarrheal pathogens simultaneously [117], and some that target multiple Salmonella serovars [118,119]. Recent improvements in sequential multi-host isolation methods make the selection of polyvalent phages more feasible [120,121]. The presumption is that a polyvalent phage would be tested in vitro against each of the desired multiple bacterial targets from a polymicrobial infection individually prior to its selection for use. The clinical laboratory would then test each host pathogen for susceptibility to the polyvalent phage prior to use in phage therapy. Phages with broad host ranges may lose a key advantage of phage therapy over antibiotics in sparing the host bacterial microbiome, so polyvalent phages also need to be evaluated against known commensal organisms, to ensure they do not increase adverse outcomes caused by disruption of normal flora, such as C. difficile colitis [122].

4.3. Assays for Phages Used to Treat Biofilms

Biofilms remain largely impenetrable to standard antimicrobial therapy. In clinical practice, the mainstay of treatment for infections predominated by biofilms like catheter-associated infections or prosthetic hardware infections remains removal of the material containing the biofilm [123]. Numerous in vitro and ex vivo models have shown biofilm disruption by phages [124]. A number of clinical cases have shown promise with the addition of phages to existing device removal strategies in prosthetic joint infections [125]. Nonetheless, strategies to harness phages to disrupt biofilms are under investigation [126]. No FDA approved diagnostics exist to assay biofilm degradation by phages. Most researchers assay biofilm lysis by measuring crystal violet uptake in 96 well plates after phage exposures [127,128], which could be adapted to the clinical lab, but generally requires equipment that is not always present. Automated confocal microscopy images hold promise in research labs, but are unlikely to be developed into clinical assays [129,130]. As many others have highlighted, the development of an alternative assay for biofilm measurement that does not require expensive equipment, is necessary for biofilm assays to become routine measurements to characterize phage activity in the clinical laboratory [131].

4.4. Accounting for Phage Passaging

Because phages are biological entities, propagation of the “same” phage across different laboratories can lead to the accumulation of mutations and divergence, which has implications for phage activity. Optimal phage amplification methods also vary depending on the phage [132]. Clinical laboratories need to be able to assay for lot-to-lot variability caused by phage passaging as part of phage optimization. For example, to produce phages with broadened host ranges, laboratories may perform phage training experiments [120,133]. The Appelman’s protocol involves passaging phages iteratively on bacteria isolates, including resistant ones, until the phage evolves with an improved ability to lyse the bacterial host [134]. Many of the phages used in the 100-phage therapy case study set in Belgium [47] used a modified Appelman’s protocol [135]. Another method used in vitro involves co-evolution of phage and host over successive generations, where both the host and the phage evolve together. While the method can result in extinction events for either the host or the phage, it can also result in a stalemate between host and phage where neither is capable of eradicating the other. The important part of this method is that when the trained phage is taken from this experiment and is exposed to the untrained host, the untrained host is often incapable of adapting to the trained phage [136]. By accelerating evolution between host and phage without recombinant manipulation, researchers were able to generate phages with superior abilities to eradicate untrained E. coli hosts and with expanded phage host ranges. Phage optimization experiments could become a standard part of phage development, particularly as researchers seek to extend host ranges of phages to mirror those of antibiotics. These tools show how rapidly phages can evolve through successive passaging, and highlight the need for assays in the clinical laboratory to verify that prepared phages still perform as expected prior to their clinical use, including evaluating standard quality control phages for consistency. This type of rapid evolution does not exist with antibiotics, where laboratories and clinicians need not usually worry about batch-to-batch variation.

4.5. The Need for Lytic Phages

Though lysogenic phages may hold future promise [137], therapeutic phages used currently are strictly lytic, which means they do not have the capacity of integrating into the bacteria genome. Genome sequencing and annotation are needed to confirm that lysogenic genes or pathogenicity factors are not present in candidate phage genomes, and that the phages are appropriate for therapeutic use [138,139]. This sequencing need creates a barrier to the creation of new phage libraries outside of a handful of centers, but we feel that pre-developed phage libraries could still be tested for susceptibility within clinical labs.

5. Organization and Regulatory Barriers to Widespread Phage Testing in Clinical Microbiology Laboratories

5.1. Automation and Personnel Issues

A trend that is occurring throughout the world is the development of total laboratory automation (TLA) [140]. Utilization of automation protocols, particularly in the processing of traditional cultures, has helped to reduce the hands-on time necessary for the processing of cultures [141], accelerate the time to results [142], and allows hospital systems to adjust to aging work forces that are difficult to replace [143]. Automation has helped to reduce manual tasks in many labs across the world, which is in stark contrast to the development of phage testing in the clinical laboratory. To adapt the clinical laboratory to phage testing, the laboratory would either need dedicated research staff to perform this type of testing or have the ability to automate phage testing. Right now, there are no known clinical laboratories that have automated phage susceptibility testing. The approved assays for routine antibiotic susceptibility that have been developed for total lab automated platforms are based on disk diffusion assays on solid medium [144]. While such assays could theoretically be developed for phages to measure zones of inhibition, no universal standards have been developed. Broth microdilution modules for TLA are in the development pipeline, but will be only compatible with susceptibility plates developed by the parent company [145]. Therefore, it is incumbent upon regulatory bodies such as EUCAST and CLSI (the Clinical Lab Sciences Institute) to begin to develop standardized processes that can then be automated with results available to clinicians in the same timeframe as standard antibiotic testing (24 h), and available to repeat if the patient continues to grow the bacteria despite therapy.

5.2. Concurrent Development of PK/PD and Anti-Phage Antibody Assays

In any clinical drug development, Pharmacokinetic and Pharmacodynamic (PK/PD) assays are part of dose finding studies. In many antimicrobials therapy regimens, such as aminoglycosides, vancomycin, and triazoles, measurement of blood levels is a standard of therapy [146,147,148], and usually performed in a chemistry laboratory. Phages are self-replicating with complex PK/PD profiles [149,150]. As such standardizing the methods for assessing phage concentrations in blood is also an area of research, with many labs preferring qPCR methodology [151]. Given the existing qPCR assays are often performed in the clinical microbiology laboratory, it should be expected that the laboratory may need to simultaneously bring on phage qPCR assays to help determine individualized phage dosing, and will require collaboration with clinical chemists and pharmacists. At the same time, phage specific immunity may hinder treatment efficacy and clinical assays to look for phage neutralizing antibodies are likely to be necessary.

5.3. Regulatory Hurdles

At present, phages are only approved in the United States for pesticide applications (by the EPA), for food applications (by the FDA and USDA) and are used in FDA-approved phage based diagnostic assays [152]. Phages are not FDA approved for human use in the USA and are only available through clinical trials and through expanded access programs [153]. Despite the development of polyvalent phages, phage cocktails, and phage-antibiotic synergy approaches, all of which may protect against the development of phage resistance and allow for phage development centers to consolidate applications to the FDA, we believe that the eventual breadth of available phages for treatment will surpass the number of antibiotics available. The best current correlate to this individuality and simultaneous breadth of product comes from gene therapy treatments such as CAR-T cell therapies, in which regulatory agencies determine how much clinical and pre-clinical data are required to approve each successive treatment [154,155]. We envision that individual phages may be regulated for clinical use in a similar fashion, and support that the FDA has entertained alternative trial designs for phages [156]. The need for high throughput assays to help providers select the correct individual phages will be paramount. If the sheer breadth of phages that have already been used in the current models of phage therapy come to market, it may make the eventual regulatory approval of widely deployable susceptibility testing assays for phages quite costly. Even if susceptibility testing is able to be implemented as a lab-developed test (LDT), the work to validate LDTs presents a barrier to widespread deployment. This increases the likelihood that phage selection, testing and therapy remains restricted to a few individual centers. As a start, recognizing the novelty of phage therapy and moving towards a different model of regulatory approval for phage susceptibility testing would allow for a wider network of phage susceptibility testing and better access to phage therapy for patients (Figure 1) [157].

6. Conclusions

Phage therapy holds significant promise for changing the landscape of how infections caused by bacterial pathogens are treated. Developing virulence and AMR in the community and in hospitals is widely driven by the use and availability of antibiotics [158]. A substantial decrease in the use of antibiotics resulting from the use of phages as an alternative could cause a dramatic shift in the current growth projections of AMR. To achieve this, physicians must be convinced to change their antimicrobial prescription practices and shift towards more widespread use of phages. Currently, however, the phage field has not yet proven the utility of phage therapy in the USA through large, randomized controlled trials. Additionally, phage host ranges generally do not rival antibiotics, though the disruption to normal microbial flora may be less as a result [159]. Production of phages is challenging at most facilities [160]. Furthermore, general skepticism remains about the ability of phages to cure infections. Phage research lacks investment as a field as compared to other pharmaceuticals, but is gaining ground in the antibiotic space [161]. Finally, the general public is only now beginning to appreciate the looming AMR crisis [162]. Once these issues are addressed, the more practical challenges to implement phage testing in the clinical laboratory, as described in this manuscript, will be front and center.

Author Contributions

A.K., A.G.C.G., J.Y., C.G., D.V.T., K.W. and D.T.P. contributed to the writing and editing of this manuscript. J.Y. and D.T.P. contributed data to create Table 1. A.K created Figure 1. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study.

Conflicts of Interest

A.G.C.G., J.Y., and D.P. are members of the Center for Innovative Phage Applications and Therapeutics at the University of California San Diego. D.V.T. is a member of the Center for Innovative Antimicrobial Therapy at the University of Pittsburgh. C.G. is the Founder and CEO of Bioharmony, Inc.

References

  1. Twort, F.W. AN INVESTIGATION ON THE NATURE OF ULTRA-MICROSCOPIC VIRUSES. Lancet 1915, 186, 1241–1243. [Google Scholar] [CrossRef]
  2. d’Hérelles, F. BACTERIOPHAGE: Sur un Microbe Invisible Antagoniste des Bacilles Dysentériques; Comptes Rendus Hebdomadaires des Séances de l’Académie des Sciences; Gauthier-Villars: Paris, France, 1917; Volume 165. [Google Scholar]
  3. Altamirano, F.L.G.; Barr, J.J. Phage Therapy in the Postantibiotic Era. Clin. Microbiol. Rev. 2019, 32, e00066-18. [Google Scholar] [CrossRef] [PubMed]
  4. Chanishvili, N. Chapter 1—Phage Therapy—History from Twort and d’Herelle Through Soviet Experience to Current Approaches. In Advances in Virus Research; Łobocka, M., Szybalski, W., Eds.; Academic Press: Cambridge, MA, USA, 2012; Volume 83, pp. 3–40. [Google Scholar]
  5. Marongiu, L.; Burkard, M.; Lauer, U.M.; Hoelzle, L.E.; Venturelli, S. Reassessment of Historical Clinical Trials Supports the Effectiveness of Phage Therapy. Clin. Microbiol. Rev. 2022, 35, e00062-22. [Google Scholar] [CrossRef] [PubMed]
  6. Otten, H. Domagk and the development of the sulphonamides. J. Antimicrob. Chemother. 1986, 17, 689–690. [Google Scholar] [CrossRef]
  7. Gaynes, R. The Discovery of Penicillin—New Insights After More Than 75 Years of Clinical Use. Emerg. Infect. Dis. 2017, 23, 849–853. [Google Scholar] [CrossRef]
  8. Straub, M.E. Studies on Commercial Bacteriophage Products. JAMA J. Am. Med. Assoc. 1933, 100, 110–113. [Google Scholar] [CrossRef]
  9. Summers, W.C. The strange history of phage therapy. Bacteriophage 2012, 2, 130–133. [Google Scholar] [CrossRef]
  10. Kirby, T. Nina Chanishvili—Keeping bacteriophages in the limelight. Lancet Infect. Dis. 2021, 21, 926. [Google Scholar] [CrossRef]
  11. Venturini, C.; Petrovic Fabijan, A.; Fajardo Lubian, A.; Barbirz, S.; Iredell, J. Biological foundations of successful bacteriophage therapy. EMBO Mol. Med. 2022, 14, e12435. [Google Scholar] [CrossRef]
  12. Brives, C.; Pourraz, J. Phage therapy as a potential solution in the fight against AMR: Obstacles and possible futures. Palgrave Commun. 2020, 6, 100. [Google Scholar] [CrossRef]
  13. Strathdee, S.A.; Hatfull, G.F.; Mutalik, V.K.; Schooley, R.T. Phage therapy: From biological mechanisms to future directions. Cell 2023, 186, 17–31. [Google Scholar] [CrossRef] [PubMed]
  14. Alas Portillo, M.; Gómez Rodríguez, I.M.; Gradmann, C.; Kirchhelle, C.; Leisner, J.J.; Martinenghi, L.D.; Paterson, E.L.; Santesmases, M.J.; Skender, B.; Vagneron, F. Paradoxes of the antibiotic pipeline. Humanit. Soc. Sci. Commun. 2024, 11, 678. [Google Scholar] [CrossRef]
  15. Kasimanickam, V.; Kasimanickam, M.; Kasimanickam, R. Antibiotics Use in Food Animal Production: Escalation of Antimicrobial Resistance: Where Are We Now in Combating AMR? Med. Sci. 2021, 9, 14. [Google Scholar] [CrossRef] [PubMed]
  16. Soulsby, E.J. Resistance to antimicrobials in humans and animals. BMJ 2005, 331, 1219–1220. [Google Scholar] [CrossRef]
  17. Cobian Guemes, A.G.; Youle, M.; Cantu, V.A.; Felts, B.; Nulton, J.; Rohwer, F. Viruses as Winners in the Game of Life. Annu. Rev. Virol. 2016, 3, 197–214. [Google Scholar] [CrossRef]
  18. Liang, G.; Bushman, F.D. The human virome: Assembly, composition and host interactions. Nat. Rev. Microbiol. 2021, 19, 514–527. [Google Scholar] [CrossRef] [PubMed]
  19. Pride, D.T.; Salzman, J.; Haynes, M.; Rohwer, F.; Davis-Long, C.; White, R.A., 3rd; Loomer, P.; Armitage, G.C.; Relman, D.A. Evidence of a robust resident bacteriophage population revealed through analysis of the human salivary virome. ISME J. 2012, 6, 915–926. [Google Scholar] [CrossRef]
  20. Jurczak-Kurek, A.; Gasior, T.; Nejman-Falenczyk, B.; Bloch, S.; Dydecka, A.; Topka, G.; Necel, A.; Jakubowska-Deredas, M.; Narajczyk, M.; Richert, M.; et al. Biodiversity of bacteriophages: Morphological and biological properties of a large group of phages isolated from urban sewage. Sci. Rep. 2016, 6, 34338. [Google Scholar] [CrossRef]
  21. Cobian Guemes, A.G.; Le, T.; Rojas, M.I.; Jacobson, N.E.; Villela, H.; McNair, K.; Hung, S.H.; Han, L.; Boling, L.; Octavio, J.C.; et al. Compounding Achromobacter Phages for Therapeutic Applications. Viruses 2023, 15, 1665. [Google Scholar] [CrossRef]
  22. Strancar, V.; Marusic, M.; Tusar, J.; Pracek, N.; Kolenc, M.; Suster, K.; Horvat, S.; Janez, N.; Peterka, M. Isolation and in vitro characterization of novel S. epidermidis phages for therapeutic applications. Front. Cell. Infect. Microbiol. 2023, 13, 1169135. [Google Scholar] [CrossRef]
  23. Lewis, R.; Clooney, A.G.; Stockdale, S.R.; Buttimer, C.; Draper, L.A.; Ross, R.P.; Hill, C. Isolation of a Novel Jumbo Bacteriophage Effective Against Klebsiella aerogenes. Front. Med. 2020, 7, 67. [Google Scholar] [CrossRef] [PubMed]
  24. Rahimzadeh, G.; Resch, G.; Rezai, M.S.; Nemati Hevelaee, E. Characterization and Lytic Activity of Isolated Escherichia coli Bacteriophages against Escherichia coli in vitro. Iran J. Med. Sci. 2020, 45, 298–303. [Google Scholar] [PubMed]
  25. Parfitt, T. Georgia: An unlikely stronghold for bacteriophage therapy. Lancet 2005, 365, 2166–2167. [Google Scholar] [CrossRef]
  26. Yang, Q.; Le, S.; Zhu, T.; Wu, N. Regulations of phage therapy across the world. Front. Microbiol. 2023, 14, 1250848. [Google Scholar] [CrossRef]
  27. Dabizheva, A.N.; Voroshilova, N.N.; Prisada, T.V.; Efimova, M.G. Phages Attack: A History of Bacteriophage Production and Therapeutic Use in Russia. Science First Hand 2017 [cited 46 N1]. Available online: https://scfh.ru/en/papers/phages-attack/ (accessed on 5 May 2025).
  28. Uchechukwu, C.F.; Shonekan, A. Current status of clinical trials for phage therapy. J. Med. Microbiol. 2024, 73, 001895. [Google Scholar] [CrossRef]
  29. Zaldastanishvili, E.; Leshkasheli, L.; Dadiani, M.; Nadareishvili, L.; Askilashvili, L.; Kvatadze, N.; Goderdzishvili, M.; Kutateladze, M.; Balarjishvili, N. Phage Therapy Experience at the Eliava Phage Therapy Center: Three Cases of Bacterial Persistence. Viruses 2021, 13, 1901. [Google Scholar] [CrossRef] [PubMed]
  30. Międzybrodzki, R.; Hoyle, N.; Zhvaniya, F.; Łusiak-Szelachowska, M.; Weber-Dąbrowska, B.; Łobocka, M.; Borysowski, J.; Alavidze, Z.; Kutter, E.; Górski, A.; et al. Current Updates from the Long-Standing Phage Research Centers in Georgia, Poland, and Russia. In Bacteriophages: Biology, Technology, Therapy; Harper, D.R., Abedon, S.T., Burrowes, B.H., McConville, M.L., Eds.; Springer International Publishing: Cham, Switzerland, 2021; pp. 921–951. [Google Scholar]
  31. Nir-Paz, R.; Onallah, H.; Dekel, M.; Gellman, Y.N.; Haze, A.; Ben-Ami, R.; Braunstein, R.; Hazan, R.; Dror, D.; Oster, Y.; et al. Randomized double-blind study on safety and tolerability of TP-102 phage cocktail in patients with infected and non-infected diabetic foot ulcers. Med 2025, 6, 100565. [Google Scholar] [CrossRef]
  32. Hitchcock, N.M.; Devequi Gomes Nunes, D.; Shiach, J.; Valeria Saraiva Hodel, K.; Dantas Viana Barbosa, J.; Alencar Pereira Rodrigues, L.; Coler, B.S.; Botelho Pereira Soares, M.; Badaró, R. Current Clinical Landscape and Global Potential of Bacteriophage Therapy. Viruses 2023, 15, 1020. [Google Scholar] [CrossRef]
  33. Biosciences, L. A Study of LBP-EC01 in the Treatment of Acute Uncomplicated UTI Caused by Drug Resistant E. Coli (ELIMINATE Trial). Available online: https://clinicaltrials.gov/study/NCT05488340 (accessed on 22 April 2025).
  34. Abedon, S.T. Phage Companies. 2023. Available online: https://companies.phage.org/ (accessed on 22 April 2025).
  35. BiomX, Inc. BiomX Announces Closing of the Acquisition of Adaptive Phage Therapeutics and Concurrent $50 Million Financing; BiomX, Inc.: Gaithersburg, MD, USA, 2024. [Google Scholar]
  36. Santajit, S.; Indrawattana, N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Res. Int. 2016, 2016, 2475067. [Google Scholar] [CrossRef]
  37. Rajput, A.; Seif, Y.; Choudhary Kumari, S.; Dalldorf, C.; Poudel, S.; Monk Jonathan, M.; Palsson Bernhard, O. Pangenome Analytics Reveal Two-Component Systems as Conserved Targets in ESKAPEE Pathogens. mSystems 2021, 6, e00981-20. [Google Scholar] [CrossRef]
  38. De Oliveira, D.M.P.; Forde, B.M.; Kidd, T.J.; Harris, P.N.A.; Schembri, M.A.; Beatson, S.A.; Paterson, D.L.; Walker, M.J. Antimicrobial Resistance in ESKAPE Pathogens. Clin. Microbiol. Rev. 2020, 33, e00181-19. [Google Scholar] [CrossRef] [PubMed]
  39. Song, L.; Yang, X.; Huang, J.; Zhu, X.; Han, G.; Wan, Y.; Xu, Y.; Luan, G.; Jia, X. Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene. Front. Microbiol. 2021, 12, 739319. [Google Scholar] [CrossRef]
  40. Page, M.L. Phage Therapies for Superbug Infections are Being Tested in Belgium; New Scientist: London, UK, 2022. [Google Scholar]
  41. Naval Medical Research Center (NMRC). NMRC Maintains a Library of Diverse Bacteriophages to Fight Multidrug Resistant Infections; Naval Medical Research Center (NMRC): Silver Spring, MD, USA, 2019. [Google Scholar]
  42. Locus Biosciences. Locus Biosciences Announces $23.9 Million in Funding from BARDA to Support First Phase 2 Trial of CRISPR-Engineered Bacteriophage Therapy; Locus Biosciences: Morrisville, NC, USA, 2024. [Google Scholar]
  43. Center for Innovative Phage Applications and Therapeutics. MicrobiotiX Co., Ltd. and the Center for Innovative Phage Applications and Therapeutics (IPATH) Sign Collaborative Agreement to Improve Access to Phages for IPATH’s Phage Therapy Patients; Division of Infectious Diseases & Global Public Health, University of California San Diego: San Diego, CA, USA, 2022. [Google Scholar]
  44. Aslam, S.; Roach, D.; Nikolich, M.P.; Biswas, B.; Schooley, R.T.; Lilly-Bishop, K.A.; Rice, G.K.; Cer, R.Z.; Hamilton, T.; Henry, M.; et al. Pseudomonas aeruginosa ventricular assist device infections: Findings from ineffective phage therapies in five cases. Antimicrob. Agents Chemother. 2024, 68, e0172823. [Google Scholar] [CrossRef] [PubMed]
  45. Onallah, H.; Hazan, R.; Nir-Paz, R.; Brownstein, M.J.; Fackler, J.R.; Horne, B.; Hopkins, R.; Basu, S.; Yerushalmy, O.; Alkalay-Oren, S.; et al. Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series. Med 2023, 4, 600–611.e4. [Google Scholar] [CrossRef] [PubMed]
  46. Aslam, S.; Schooley, R.T. What’s Old Is New Again: Bacteriophage Therapy in the 21st Century. Antimicrob. Agents Chemother. 2019, 64, e01987-19. [Google Scholar] [CrossRef]
  47. Pirnay, J.P.; Djebara, S.; Steurs, G.; Griselain, J.; Cochez, C.; De Soir, S.; Glonti, T.; Spiessens, A.; Vanden Berghe, E.; Green, S.; et al. Personalized bacteriophage therapy outcomes for 100 consecutive cases: A multicentre, multinational, retrospective observational study. Nat. Microbiol. 2024, 9, 1434–1453. [Google Scholar] [CrossRef]
  48. Dedrick, R.M.; Smith, B.E.; Cristinziano, M.; Freeman, K.G.; Jacobs-Sera, D.; Belessis, Y.; Whitney Brown, A.; Cohen, K.A.; Davidson, R.M.; van Duin, D.; et al. Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients with Drug-Resistant Mycobacterial Disease. Clin. Infect. Dis. 2023, 76, 103–112. [Google Scholar] [CrossRef]
  49. Hosseiniporgham, S.; Sechi, L.A. A Review on Mycobacteriophages: From Classification to Applications. Pathogens 2022, 11, 777. [Google Scholar] [CrossRef]
  50. Dedrick, R.M.; Guerrero-Bustamante, C.A.; Garlena, R.A.; Russell, D.A.; Ford, K.; Harris, K.; Gilmour, K.C.; Soothill, J.; Jacobs-Sera, D.; Schooley, R.T.; et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat. Med. 2019, 25, 730–733. [Google Scholar] [CrossRef]
  51. Śliwka, P.; Ochocka, M.; Skaradzińska, A. Applications of bacteriophages against intracellular bacteria. Crit. Rev. Microbiol. 2022, 48, 222–239. [Google Scholar] [CrossRef]
  52. Glonti, T.; Pirnay, J.P. In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success. Viruses 2022, 14, 1490. [Google Scholar] [CrossRef]
  53. Daubie, V.; Chalhoub, H.; Blasdel, B.; Dahma, H.; Merabishvili, M.; Glonti, T.; De Vos, N.; Quintens, J.; Pirnay, J.P.; Hallin, M.; et al. Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective. Front. Cell. Infect. Microbiol. 2022, 12, 1000721. [Google Scholar] [CrossRef]
  54. Xie, Y.; Wahab, L.; Gill, J.J. Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence. Viruses 2018, 10, 189. [Google Scholar] [CrossRef]
  55. Yerushalmy, O.; Braunstein, R.; Alkalay-Oren, S.; Rimon, A.; Coppenhagn-Glazer, S.; Onallah, H.; Nir-Paz, R.; Hazan, R. Towards Standardization of Phage Susceptibility Testing: The Israeli Phage Therapy Center “Clinical Phage Microbiology”—A Pipeline Proposal. Clin. Infect. Dis. 2023, 77 (Suppl. S5), S337–S351. [Google Scholar] [CrossRef] [PubMed]
  56. Kutter, E. Phage host range and efficiency of plating. Methods Mol. Biol. 2009, 501, 141–149. [Google Scholar]
  57. Khan Mirzaei, M.; Nilsson, A.S. Isolation of phages for phage therapy: A comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. PLoS ONE 2015, 10, e0118557. [Google Scholar] [CrossRef] [PubMed]
  58. Cobian Guemes, A.G.; Ghatbale, P.; Blanc, A.N.; Morgan, C.J.; Garcia, A.; Leonard, J.; Huang, L.; Kovalick, G.; Proost, M.; Chiu, M.; et al. Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections. Antimicrob. Agents Chemother. 2023, 67, e0065423. [Google Scholar] [CrossRef] [PubMed]
  59. Parmar, K.; Komarow, L.; Ellison, D.W.; Filippov, A.A.; Nikolich, M.P.; Fackler, J.R.; Lee, M.; Nair, A.; Agrawal, P.; Tamma, P.D.; et al. Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing. J. Clin. Microbiol. 2023, 61, e0061423. [Google Scholar] [CrossRef]
  60. Tamma, P.D.; Souli, M.; Billard, M.; Campbell, J.; Conrad, D.; Ellison, D.W.; Evans, B.; Evans, S.R.; Greenwood-Quaintance, K.E.; Filippov, A.A.; et al. Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: Study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial. Trials 2022, 23, 1057. [Google Scholar] [CrossRef]
  61. Grossman, M.K.; Rankin, D.A.; Maloney, M.; Stanton, R.A.; Gable, P.; Stevens, V.A.; Ewing, T.; Saunders, K.; Kogut, S.; Nazarian, E.; et al. Extensively Drug-Resistant Pseudomonas aeruginosa Outbreak associated with Artificial Tears. Clin. Infect. Dis. 2024, 79, 6–14. [Google Scholar] [CrossRef]
  62. Sundermann, A.J.; Rangachar Srinivasa, V.; Mills, E.G.; Griffith, M.P.; Waggle, K.D.; Ayres, A.M.; Pless, L.; Snyder, G.M.; Harrison, L.H.; Van Tyne, D. Two Artificial Tears Outbreak-Associated Cases of Extensively Drug-Resistant Pseudomonas aeruginosa Detected Through Whole Genome Sequencing-Based Surveillance. J. Infect. Dis. 2024, 229, 517–521. [Google Scholar] [CrossRef]
  63. Chan, B.K.; Stanley, G.L.; Kortright, K.E.; Modak, M.; Ott, I.M.; Sun, Y.; Würstle, S.; Grun, C.; Kazmierczak, B.; Rajagopalan, G.; et al. Personalized Inhaled Bacteriophage Therapy Decreases Multidrug-Resistant Pseudomonas aeruginosa. MedRxiv 2023. [Google Scholar]
  64. Iyer, L.M.; Anantharaman, V.; Krishnan, A.; Burroughs, A.M.; Aravind, L. Jumbo Phages: A Comparative Genomic Overview of Core Functions and Adaptions for Biological Conflicts. Viruses 2021, 13, 63. [Google Scholar] [CrossRef] [PubMed]
  65. Yuan, Y.; Gao, M. Jumbo Bacteriophages: An Overview. Front. Microbiol. 2017, 8, 403. [Google Scholar] [CrossRef]
  66. Chaikeeratisak, V.; Nguyen, K.; Khanna, K.; Brilot, A.F.; Erb, M.L.; Coker, J.K.; Vavilina, A.; Newton, G.L.; Buschauer, R.; Pogliano, K.; et al. Assembly of a nucleus-like structure during viral replication in bacteria. Science 2017, 355, 194–197. [Google Scholar] [CrossRef]
  67. Malone, L.M.; Warring, S.L.; Jackson, S.A.; Warnecke, C.; Gardner, P.P.; Gumy, L.F.; Fineran, P.C. A jumbo phage that forms a nucleus-like structure evades CRISPR-Cas DNA targeting but is vulnerable to type III RNA-based immunity. Nat. Microbiol. 2020, 5, 48–55. [Google Scholar] [CrossRef]
  68. Costa, A.R.; van den Berg, D.F.; Esser, J.Q.; Muralidharan, A.; van den Bossche, H.; Bonilla, B.E.; van der Steen, B.A.; Haagsma, A.C.; Fluit, A.C.; Nobrega, F.L.; et al. Accumulation of defense systems in phage-resistant strains of Pseudomonas aeruginosa. Sci. Adv. 2024, 10, eadj0341. [Google Scholar] [CrossRef] [PubMed]
  69. Kolenda, C.; Jourdan, J.; Roussel-Gaillard, T.; Medina, M.; Laurent, F. Phage susceptibility testing methods or ‘phagograms’: Where do we stand and where should we go? J. Antimicrob. Chemother. 2024, 79, 2742–2749. [Google Scholar] [CrossRef]
  70. Trofimova, E.; Jaschke, P.R. Plaque Size Tool: An automated plaque analysis tool for simplifying and standardising bacteriophage plaque morphology measurements. Virology 2021, 561, 1–5. [Google Scholar] [CrossRef]
  71. Gordillo Altamirano, F.L.; Kostoulias, X.; Subedi, D.; Korneev, D.; Peleg, A.Y.; Barr, J.J. Phage-antibiotic combination is a superior treatment against Acinetobacter baumannii in a preclinical study. EBioMedicine 2022, 80, 104045. [Google Scholar] [CrossRef]
  72. Khong, E.; Oh, J.J.; Jimenez, J.M.; Liu, R.; Dunham, S.; Monsibais, A.; Rhoads, A.; Ghatbale, P.; Garcia, A.; Cobian Guemes, A.G.; et al. A simple solid media assay for detection of synergy between bacteriophages and antibiotics. Microbiol. Spectr. 2024, 12, e0322123. [Google Scholar] [CrossRef] [PubMed]
  73. Abedon, S.T.; Danis-Wlodarczyk, K.M.; Wozniak, D.J. Phage Cocktail Development for Bacteriophage Therapy: Toward Improving Spectrum of Activity Breadth and Depth. Pharmaceuticals 2021, 14, 1019. [Google Scholar] [CrossRef] [PubMed]
  74. The EUCAST Subcommittee on Phage Susceptibility Testing. Available online: https://www.eucast.org/ast-of-phages (accessed on 25 October 2024).
  75. Suh, G.A.; Lodise, T.P.; Tamma, P.D.; Knisely, J.M.; Alexander, J.; Aslam, S.; Barton, K.D.; Bizzell, E.; Totten, K.M.C.; Campbell, J.L.; et al. Considerations for the Use of Phage Therapy in Clinical Practice. Antimicrob. Agents Chemother. 2022, 66, e0207121. [Google Scholar] [CrossRef]
  76. Aslam, S.; Lampley, E.; Wooten, D.; Karris, M.; Benson, C.; Strathdee, S.; Schooley, R.T. Lessons Learned from the First 10 Consecutive Cases of Intravenous Bacteriophage Therapy to Treat Multidrug-Resistant Bacterial Infections at a Single Center in the United States. Open Forum Infect. Dis. 2020, 7, ofaa389. [Google Scholar] [CrossRef] [PubMed]
  77. Stellfox, M.E.; Fernandes, C.; Shields, R.K.; Haidar, G.; Hughes Kramer, K.; Dembinski, E.; Mangalea, M.R.; Arya, G.; Canfield, G.S.; Duerkop, B.A.; et al. Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia. mBio 2024, 15, e0339623. [Google Scholar] [CrossRef]
  78. Nikolic, I.; Vukovic, D.; Gavric, D.; Cvetanovic, J.; Aleksic Sabo, V.; Gostimirovic, S.; Narancic, J.; Knezevic, P. An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection. Viruses 2022, 14, 1542. [Google Scholar] [CrossRef]
  79. Gu Liu, C.; Green, S.I.; Min, L.; Clark, J.R.; Salazar, K.C.; Terwilliger, A.L.; Kaplan, H.B.; Trautner, B.W.; Ramig, R.F.; Maresso, A.W. Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry. mBio 2020, 11, e01462-20. [Google Scholar] [CrossRef]
  80. Knezevic, P.; Curcin, S.; Aleksic, V.; Petrusic, M.; Vlaski, L. Phage-antibiotic synergism: A possible approach to combatting Pseudomonas aeruginosa. Res. Microbiol. 2013, 164, 55–60. [Google Scholar] [CrossRef]
  81. Neter, E. Inhibitory Effect of Sulfamido Compounds upon Development and Growth of Phage-Resistant Bacteria. Proc. Soc. Exp. Biol. Med. 1941, 47, 20–23. [Google Scholar] [CrossRef]
  82. Hagens, S.; Habel, A.; Bläsi, U. Augmentation of the antimicrobial efficacy of antibiotics by filamentous phage. Microb. Drug Resist. 2006, 12, 164–168. [Google Scholar] [CrossRef]
  83. Himmelweit, F. Combined Action of Penicillin and Bacteriophage on Staphylococci. Lancet 1945, 246, 104–105. [Google Scholar] [CrossRef]
  84. Diallo, K.; Dublanchet, A. Benefits of Combined Phage-Antibiotic Therapy for the Control of Antibiotic-Resistant Bacteria: A Literature Review. Antibiotics 2022, 11, 839. [Google Scholar] [CrossRef] [PubMed]
  85. Kim, M.K.; Chen, Q.; Echterhof, A.; Pennetzdorfer, N.; McBride, R.C.; Banaei, N.; Burgener, E.B.; Milla, C.E.; Bollyky, P.L. A blueprint for broadly effective bacteriophage-antibiotic cocktails against bacterial infections. Nat. Commun. 2024, 15, 9987. [Google Scholar] [CrossRef] [PubMed]
  86. Kebriaei, R.; Lev, K.; Morrisette, T.; Stamper Kyle, C.; Abdul-Mutakabbir Jacinda, C.; Lehman Susan, M.; Morales, S.; Rybak Michael, J. Bacteriophage-Antibiotic Combination Strategy: An Alternative against Methicillin-Resistant Phenotypes of Staphylococcus aureus. Antimicrob. Agents Chemother. 2020, 64, e00461-20. [Google Scholar] [CrossRef]
  87. Van Nieuwenhuyse, B.; Galant, C.; Brichard, B.; Docquier, P.-L.; Djebara, S.; Pirnay, J.-P.; Van der Linden, D.; Merabishvili, M.; Chatzis, O. A Case of In Situ Phage Therapy against Staphylococcus aureus in a Bone Allograft Polymicrobial Biofilm Infection: Outcomes and Phage-Antibiotic Interactions. Viruses 2021, 13, 1898. [Google Scholar] [CrossRef]
  88. Ghatbale, P.; Sah, G.P.; Dunham, S.; Khong, E.; Blanc, A.; Monsibais, A.; Garcia, A.; Schooley, R.T.; Cobián Güemes, A.G.; Whiteson, K.; et al. In vitro resensitization of multidrug-resistant clinical isolates of Enterococcus faecium and E. faecalis through phage-antibiotic synergy. Antimicrob. Agents Chemother. 2024, 69, e0074024. [Google Scholar] [CrossRef]
  89. Loganathan, A.; Bozdogan, B.; Manohar, P.; Nachimuthu, R. Phage-antibiotic combinations in various treatment modalities to manage MRSA infections. Front. Pharmacol. 2024, 15, 1356179. [Google Scholar] [CrossRef]
  90. Mukhopadhyay, S.; Zhang, P.; To, K.K.W.; Liu, Y.; Bai, C.; Leung, S.S.Y. Sequential treatment effects on phage–antibiotic synergistic application against multi-drug-resistant Acinetobacter baumannii. Int. J. Antimicrob. Agents 2023, 62, 106951. [Google Scholar] [CrossRef]
  91. Ma, D.; Li, L.; Han, K.; Wang, L.; Cao, Y.; Zhou, Y.; Chen, H.; Wang, X. The antagonistic interactions between a polyvalent phage SaP7 and β-lactam antibiotics on combined therapies. Vet. Microbiol. 2022, 266, 109332. [Google Scholar] [CrossRef]
  92. Pacios, O.; Fernández-García, L.; Bleriot, I.; Blasco, L.; González-Bardanca, M.; López, M.; Fernández-Cuenca, F.; Oteo, J.; Pascual, Á.; Martínez-Martínez, L.; et al. Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 2021, 65, e0090021. [Google Scholar] [CrossRef]
  93. Shlezinger, M.; Coppenhagen-Glazer, S.; Gelman, D.; Beyth, N.; Hazan, R. Eradication of Vancomycin-Resistant Enterococci by Combining Phage and Vancomycin. Viruses 2019, 11, 954. [Google Scholar] [CrossRef]
  94. Molina, F.; Menor-Flores, M.; Fernandez, L.; Vega-Rodriguez, M.A.; Garcia, P. Systematic analysis of putative phage-phage interactions on minimum-sized phage cocktails. Sci. Rep. 2022, 12, 2458. [Google Scholar] [CrossRef]
  95. Aslam, S.; Courtwright, A.M.; Koval, C.; Lehman, S.M.; Morales, S.; Furr, C.L.; Rosas, F.; Brownstein, M.J.; Fackler, J.R.; Sisson, B.M.; et al. Early clinical experience of bacteriophage therapy in 3 lung transplant recipients. Am. J. Transplant. 2019, 19, 2631–2639. [Google Scholar] [CrossRef]
  96. Uyttebroek, S.; Chen, B.; Onsea, J.; Ruythooren, F.; Debaveye, Y.; Devolder, D.; Spriet, I.; Depypere, M.; Wagemans, J.; Lavigne, R.; et al. Safety and efficacy of phage therapy in difficult-to-treat infections: A systematic review. Lancet Infect. Dis. 2022, 22, e208–e220. [Google Scholar] [CrossRef] [PubMed]
  97. Bradley, J.S.; Hajama, H.; Akong, K.; Jordan, M.; Stout, D.; Rowe, R.S.; Conrad, D.J.; Hingtgen, S.; Segall, A.M. Bacteriophage Therapy of Multidrug-resistant Achromobacter in an 11-Year-old Boy with Cystic Fibrosis Assessed by Metagenome Analysis. Pediatr. Infect. Dis. J. 2023, 42, 754–759. [Google Scholar] [CrossRef] [PubMed]
  98. Moulton-Brown, C.E.; Friman, V.P. Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments. Evol. Appl. 2018, 11, 1630–1641. [Google Scholar] [CrossRef]
  99. Gainey, A.B.; Burch, A.K.; Brownstein, M.J.; Brown, D.E.; Fackler, J.; Horne, B.; Biswas, B.; Bivens, B.N.; Malagon, F.; Daniels, R. Combining bacteriophages with cefiderocol and meropenem/vaborbactam to treat a pan-drug resistant Achromobacter species infection in a pediatric cystic fibrosis patient. Pediatr. Pulmonol. 2020, 55, 2990–2994. [Google Scholar] [CrossRef] [PubMed]
  100. Chan, B.K.; Turner, P.E.; Kim, S.; Mojibian, H.R.; Elefteriades, J.A.; Narayan, D. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol. Med. Public Health 2018, 2018, 60–66. [Google Scholar] [CrossRef]
  101. Dedrick, R.M.; Freeman, K.G.; Nguyen, J.A.; Bahadirli-Talbott, A.; Smith, B.E.; Wu, A.E.; Ong, A.S.; Lin, C.T.; Ruppel, L.C.; Parrish, N.M.; et al. Potent antibody-mediated neutralization limits bacteriophage treatment of a pulmonary Mycobacterium abscessus infection. Nat. Med. 2021, 27, 1357–1361. [Google Scholar] [CrossRef]
  102. de Melo, A.G.; Morency, C.; Moineau, S. Virulence-associated factors as targets for phage infection. Curr. Opin. Microbiol. 2024, 79, 102471. [Google Scholar] [CrossRef]
  103. Kortright, K.E.; Chan, B.K.; Turner, P.E. High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria. Proc. Natl. Acad. Sci. USA 2020, 117, 18670–18679. [Google Scholar] [CrossRef] [PubMed]
  104. Gencay, Y.E.; Gambino, M.; Prussing, T.F.; Brondsted, L. The genera of bacteriophages and their receptors are the major determinants of host range. Environ. Microbiol. 2019, 21, 2095–2111. [Google Scholar] [CrossRef]
  105. Borin, J.M.; Lee, J.J.; Gerbino, K.R.; Meyer, J.R. Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages. Evol. Appl. 2023, 16, 152–162. [Google Scholar] [CrossRef] [PubMed]
  106. Wandro, S.; Ghatbale, P.; Attai, H.; Hendrickson, C.; Samillano, C.; Suh, J.; Dunham, S.J.B.; Pride, D.T.; Whiteson, K. Phage Cocktails Constrain the Growth of Enterococcus. mSystems 2022, 7, e0001922. [Google Scholar] [CrossRef]
  107. Yu, J.; Zhang, H.; Ju, Z.; Huang, J.; Lin, C.; Wu, J.; Wu, Y.; Sun, S.; Wang, H.; Hao, G.; et al. Increased mutations in lipopolysaccharide biosynthetic genes cause time-dependent development of phage resistance in Salmonella. Antimicrob. Agents Chemother. 2024, 68, e0059423. [Google Scholar] [CrossRef] [PubMed]
  108. Adler, B.A.; Kazakov, A.E.; Zhong, C.; Liu, H.; Kutter, E.; Lui, L.M.; Nielsen, T.N.; Carion, H.; Deutschbauer, A.M.; Mutalik, V.K.; et al. The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella. Microbiology 2021, 167, 001126. [Google Scholar] [CrossRef]
  109. Paule, A.; Frezza, D.; Edeas, M. Microbiota and Phage Therapy: Future Challenges in Medicine. Med. Sci. 2018, 6, 86. [Google Scholar] [CrossRef]
  110. Nir-Paz, R.; Gelman, D.; Khouri, A.; Sisson, B.M.; Fackler, J.; Alkalay-Oren, S.; Khalifa, L.; Rimon, A.; Yerushalmy, O.; Bader, R.; et al. Successful Treatment of Antibiotic-resistant, Poly-microbial Bone Infection with Bacteriophages and Antibiotics Combination. Clin. Infect. Dis. 2019, 69, 2015–2018. [Google Scholar] [CrossRef]
  111. Alt, V.; Gessner, A.; Merabishvili, M.; Hitzenbichler, F.; Mannala, G.K.; Peterhoff, D.; Walter, N.; Pirnay, J.P.; Hiergeist, A.; Rupp, M. Case report: Local bacteriophage therapy for fracture-related infection with polymicrobial multi-resistant bacteria: Hydrogel application and postoperative phage analysis through metagenomic sequencing. Front. Med. 2024, 11, 1428432. [Google Scholar] [CrossRef]
  112. Onsea, J.; Soentjens, P.; Djebara, S.; Merabishvili, M.; Depypere, M.; Spriet, I.; De Munter, P.; Debaveye, Y.; Nijs, S.; Vanderschot, P.; et al. Bacteriophage Application for Difficult-To-Treat Musculoskeletal Infections: Development of a Standardized Multidisciplinary Treatment Protocol. Viruses 2019, 11, 891. [Google Scholar] [CrossRef]
  113. Exarchos, V.; Tkhilaishvili, T.; Potapov, E.; Starck, C.; Trampuz, A.; Schoenrath, F. Successful bacteriophage treatment of infection involving cardiac implantable electronic device and aortic graft: A Trojan horse concept. EP Eur. 2020, 22, 597. [Google Scholar] [CrossRef] [PubMed]
  114. Rubalskii, E.; Ruemke, S.; Salmoukas, C.; Boyle, E.C.; Warnecke, G.; Tudorache, I.; Shrestha, M.; Schmitto, J.D.; Martens, A.; Rojas, S.V.; et al. Bacteriophage Therapy for Critical Infections Related to Cardiothoracic Surgery. Antibiotics 2020, 9, 232. [Google Scholar] [CrossRef] [PubMed]
  115. Malki, K.; Kula, A.; Bruder, K.; Sible, E.; Hatzopoulos, T.; Steidel, S.; Watkins, S.C.; Putonti, C. Bacteriophages isolated from Lake Michigan demonstrate broad host-range across several bacterial phyla. Virol. J. 2015, 12, 164. [Google Scholar] [CrossRef]
  116. Göller, P.C.; Elsener, T.; Lorgé, D.; Radulovic, N.; Bernardi, V.; Naumann, A.; Amri, N.; Khatchatourova, E.; Coutinho, F.H.; Loessner, M.J.; et al. Multi-species host range of staphylococcal phages isolated from wastewater. Nat. Commun. 2021, 12, 6965. [Google Scholar] [CrossRef]
  117. Kim, S.H.; Adeyemi, D.E.; Park, M.K. Characterization of a New and Efficient Polyvalent Phage Infecting E. coli O157:H7, Salmonella spp., and Shigella sonnei. Microorganisms 2021, 9, 2105. [Google Scholar] [CrossRef] [PubMed]
  118. Mondal, P.; Mallick, B.; Dutta, M.; Dutta, S. Isolation, characterization, and application of a novel polyvalent lytic phage STWB21 against typhoidal and nontyphoidal Salmonella spp. Front. Microbiol. 2022, 13, 980025. [Google Scholar] [CrossRef]
  119. Fong, K.; Wong, C.W.Y.; Wang, S.; Delaquis, P. How Broad Is Enough: The Host Range of Bacteriophages and Its Impact on the Agri-Food Sector. PHAGE 2021, 2, 83–91. [Google Scholar] [CrossRef]
  120. Yu, P.; Mathieu, J.; Li, M.; Dai, Z.; Alvarez, P.J.J. Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches. Appl. Environ. Microbiol. 2016, 82, 808–815. [Google Scholar] [CrossRef]
  121. Jensen, E.C.; Schrader, H.S.; Rieland, B.; Thompson, T.L.; Lee, K.W.; Nickerson, K.W.; Kokjohn, T.A. Prevalence of broad-host-range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa. Appl. Env. Microbiol. 1998, 64, 575–580. [Google Scholar] [CrossRef]
  122. Federici, S.; Kredo-Russo, S.; Valdés-Mas, R.; Kviatcovsky, D.; Weinstock, E.; Matiuhin, Y.; Silberberg, Y.; Atarashi, K.; Furuichi, M.; Oka, A.; et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation. Cell 2022, 185, 2879–2898.e24. [Google Scholar] [CrossRef]
  123. Yousif, A.; Jamal, M.A.; Raad, I. Biofilm-Based Central Line-Associated Bloodstream Infections. In Biofilm-Based Healthcare-Associated Infections: Volume I; Donelli, G., Ed.; Springer International Publishing: Cham, Switzerland, 2015; pp. 157–179. [Google Scholar]
  124. Hassan, A.Y.; Lin, J.T.; Ricker, N.; Anany, H. The Age of Phage: Friend or Foe in the New Dawn of Therapeutic and Biocontrol Applications? Pharmaceuticals 2021, 14, 199. [Google Scholar] [CrossRef] [PubMed]
  125. Kovacs, C.J.; Rapp, E.M.; McKenzie, S.M.; Mazur, M.Z.; McHale, R.P.; Brasko, B.; Min, M.Y.; Burpo, F.J.; Barnhill, J.C. Disruption of Biofilm by Bacteriophages in Clinically Relevant Settings. Mil. Med. 2024, 189, e1294–e1302. [Google Scholar] [CrossRef]
  126. Liu, S.; Lu, H.; Zhang, S.; Shi, Y.; Chen, Q. Phages against Pathogenic Bacterial Biofilms and Biofilm-Based Infections: A Review. Pharmaceutics 2022, 14, 427. [Google Scholar] [CrossRef] [PubMed]
  127. Shafique, M.; Alvi, I.A.; Abbas, Z.; ur Rehman, S. Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa. APMIS 2017, 125, 579–584. [Google Scholar] [CrossRef]
  128. El-Atrees, D.M.; El-Kased, R.F.; Abbas, A.M.; Yassien, M.A. Characterization and anti-biofilm activity of bacteriophages against urinary tract Enterococcus faecalis isolates. Sci. Rep. 2022, 12, 13048. [Google Scholar] [CrossRef]
  129. Mountcastle, S.E.; Vyas, N.; Villapun, V.M.; Cox, S.C.; Jabbari, S.; Sammons, R.L.; Shelton, R.M.; Walmsley, A.D.; Kuehne, S.A. Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images. npj Biofilms Microbiomes 2021, 7, 44. [Google Scholar] [CrossRef] [PubMed]
  130. Pallavali, R.R.; Degati, V.L.; Narala, V.R.; Velpula, K.K.; Yenugu, S.; Durbaka, V.R.P. Lytic Bacteriophages Against Bacterial Biofilms Formed by Multidrug-Resistant Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus Isolated from Burn Wounds. PHAGE 2021, 2, 120–130. [Google Scholar] [CrossRef] [PubMed]
  131. Suh, G.A.; Patel, R. Clinical phage microbiology: A narrative summary. Clin. Microbiol. Infect. 2023, 29, 710–713. [Google Scholar] [CrossRef]
  132. Skaradzińska, A.; Ochocka, M.; Śliwka, P.; Kuźmińska-Bajor, M.; Skaradziński, G.; Friese, A.; Roschanski, N.; Murugaiyan, J.; Roesler, U. Bacteriophage amplification—A comparison of selected methods. J. Virol. Methods 2020, 282, 113856. [Google Scholar] [CrossRef]
  133. Borin, J.M.; Avrani, S.; Barrick, J.E.; Petrie, K.L.; Meyer, J.R. Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance. Proc. Natl. Acad. Sci. USA 2021, 118, e2104592118. [Google Scholar] [CrossRef]
  134. Burrowes, B.H.; Molineux, I.J.; Fralick, J.A. Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol. Viruses 2019, 11, 241. [Google Scholar] [CrossRef] [PubMed]
  135. Merabishvili, M.; Pirnay, J.P.; De Vos, D. Guidelines to Compose an Ideal Bacteriophage Cocktail. Methods Mol. Biol. 2018, 1693, 99–110. [Google Scholar] [PubMed]
  136. Borin, J.M.; Lee, J.J.; Lucia-Sanz, A.; Gerbino, K.R.; Weitz, J.S.; Meyer, J.R. Rapid bacteria-phage coevolution drives the emergence of multiscale networks. Science 2023, 382, 674–678. [Google Scholar] [CrossRef]
  137. Monteiro, R.; Pires, D.P.; Costa, A.R.; Azeredo, J. Phage Therapy: Going Temperate? Trends Microbiol. 2019, 27, 368–378. [Google Scholar] [CrossRef]
  138. Grigson, S.R.; Giles, S.K.; Edwards, R.A.; Papudeshi, B. Knowing and Naming: Phage Annotation and Nomenclature for Phage Therapy. Clin. Infect. Dis. 2023, 77 (Suppl. S5), S352–S359. [Google Scholar] [CrossRef] [PubMed]
  139. McNair, K.; Zhou, C.; Dinsdale, E.A.; Souza, B.; Edwards, R.A. PHANOTATE: A novel approach to gene identification in phage genomes. Bioinformatics 2019, 35, 4537–4542. [Google Scholar] [CrossRef]
  140. Antonios, K.; Croxatto, A.; Culbreath, K. Current State of Laboratory Automation in Clinical Microbiology Laboratory. Clin. Chem. 2022, 68, 99–114. [Google Scholar] [CrossRef]
  141. Yu, H.-Y.E.; Lanzoni, H.; Steffen, T.; Derr, W.; Cannon, K.; Contreras, J.; Olson, J.E. Improving Laboratory Processes with Total Laboratory Automation. Lab. Med. 2019, 50, 96–102. [Google Scholar] [CrossRef]
  142. Trigueiro, G.; Oliveira, C.; Rodrigues, A.; Seabra, S.; Pinto, R.; Bala, Y.; Gutiérrez Granado, M.; Vallejo, S.; Gonzalez, V.; Cardoso, C. Conversion of a classical microbiology laboratory to a total automation laboratory enhanced by the application of lean principles. Microbiol. Spectr. 2024, 12, e02153-23. [Google Scholar] [CrossRef]
  143. Heggeness, M.L.; Roman, C.E.Z.; Ginther, D.K. Workforce Trends: The Future of Microbial Sciences; American Society for Microbiology: Bethesda, MD, USA, 2023. [Google Scholar]
  144. Cherkaoui, A.; Schrenzel, J. Total Laboratory Automation for Rapid Detection and Identification of Microorganisms and Their Antimicrobial Resistance Profiles. Front. Cell Infect. Microbiol. 2022, 12, 807668. [Google Scholar] [CrossRef]
  145. Snyder, J.W.; Thomson, G.K.; Heckman, S.; Jamros, K.; AbdelGhani, S.; Thomson, K.S. Automated preparation for identification and antimicrobial susceptibility testing: Evaluation of a research use only prototype, the BD Kiestra IdentifA/SusceptA system. Clin. Microbiol. Infect. 2021, 27, e781.e1–e783.e5. [Google Scholar] [CrossRef]
  146. Hodiamont, C.J.; van den Broek, A.K.; de Vroom, S.L.; Prins, J.M.; Mathôt, R.A.A.; van Hest, R.M. Clinical Pharmacokinetics of Gentamicin in Various Patient Populations and Consequences for Optimal Dosing for Gram-Negative Infections: An Updated Review. Clin. Pharmacokinet. 2022, 61, 1075–1094. [Google Scholar] [CrossRef] [PubMed]
  147. Tsutsuura, M.; Moriyama, H.; Kojima, N.; Mizukami, Y.; Tashiro, S.; Osa, S.; Enoki, Y.; Taguchi, K.; Oda, K.; Fujii, S.; et al. The monitoring of vancomycin: A systematic review and meta-analyses of area under the concentration-time curve-guided dosing and trough-guided dosing. BMC Infect. Dis. 2021, 21, 153. [Google Scholar] [CrossRef]
  148. McCreary, E.K.; Davis, M.R.; Narayanan, N.; Andes, D.R.; Cattaneo, D.; Christian, R.; Lewis, R.E.; Watt, K.M.; Wiederhold, N.P.; Johnson, M.D. Utility of triazole antifungal therapeutic drug monitoring: Insights from the Society of Infectious Diseases Pharmacists: Endorsed by the Mycoses Study Group Education and Research Consortium. Pharmacotherapy 2023, 43, 1043–1050. [Google Scholar] [CrossRef]
  149. Nang, S.C.; Lin, Y.-W.; Petrovic Fabijan, A.; Chang, R.Y.K.; Rao, G.G.; Iredell, J.; Chan, H.-K.; Li, J. Pharmacokinetics/pharmacodynamics of phage therapy: A major hurdle to clinical translation. Clin. Microbiol. Infect. 2023, 29, 702–709. [Google Scholar] [CrossRef]
  150. Międzybrodzki, R.; Kasprzak, H.; Letkiewicz, S.; Rogóż, P.; Żaczek, M.; Thomas, J.; Górski, A. Pharmacokinetic and Pharmacodynamic Obstacles for Phage Therapy from the Perspective of Clinical Practice. Clin. Infect. Dis. 2023, 77 (Suppl. S5), S395–S400. [Google Scholar] [CrossRef] [PubMed]
  151. Kłopot, A.; Zakrzewska, A.; Lecion, D.; Majewska, J.M.; Harhala, M.A.; Lahutta, K.; Kaźmierczak, Z.; Łaczmański, Ł.; Kłak, M.; Dąbrowska, K. Real-Time qPCR as a Method for Detection of Antibody-Neutralized Phage Particles. Front. Microbiol. 2017, 8, 2170. [Google Scholar] [CrossRef] [PubMed]
  152. Schofield, D.A.; Sharp, N.J.; Westwater, C. Phage-based platforms for the clinical detection of human bacterial pathogens. Bacteriophage 2012, 2, 105–283. [Google Scholar] [CrossRef]
  153. Huang, Y.; Wang, W.; Zhang, Z.; Gu, Y.; Huang, A.; Wang, J.; Hao, H. Phage Products for Fighting Antimicrobial Resistance. Microorganisms 2022, 10, 1324. [Google Scholar] [CrossRef]
  154. Marks, P.; Witten, C. Toward a new framework for the development of individualized therapies. Gene Ther. 2021, 28, 615–617. [Google Scholar] [CrossRef]
  155. Food and Drug Administrations. Human Gene Therapy Products Incorporating Human Genome Editing. In Guidance for Industry; Center for Biologics Evaluation and Research, Ed.; FDA: Silver Spring, MD, USA, 2024. [Google Scholar]
  156. Food and Drug Administrations. Adaptive Designs for Clinical Trials of Drugs and Biologics. In Guidance for Industry; Center for Biologics Evaluation and Research, Ed.; FDA: Silver Spring, MD, USA, 2019. [Google Scholar]
  157. Palma, M.; Qi, B. Advancing Phage Therapy: A Comprehensive Review of the Safety, Efficacy, and Future Prospects for the Targeted Treatment of Bacterial Infections. Infect. Dis. Rep. 2024, 16, 1127–1181. [Google Scholar] [CrossRef] [PubMed]
  158. Salam, M.A.; Al-Amin, M.Y.; Salam, M.T.; Pawar, J.S.; Akhter, N.; Rabaan, A.A.; Alqumber, M.A.A. Antimicrobial Resistance: A Growing Serious Threat for Global Public Health. Healthcare 2023, 11, 1946. [Google Scholar] [CrossRef] [PubMed]
  159. Loc-Carrillo, C.; Abedon, S.T. Pros and cons of phage therapy. Bacteriophage 2011, 1, 111–114. [Google Scholar] [CrossRef] [PubMed]
  160. Bretaudeau, L.; Tremblais, K.; Aubrit, F.; Meichenin, M.; Arnaud, I. Good Manufacturing Practice (GMP) Compliance for Phage Therapy Medicinal Products. Front. Microbiol. 2020, 11, 1161. [Google Scholar] [CrossRef]
  161. Henein, A. What are the limitations on the wider therapeutic use of phage? Bacteriophage 2013, 3, e24872. [Google Scholar] [CrossRef]
  162. Hopkins, C. We Have Arrived in the Post-Antibiotic Era’: WHO Warns of Too Few New Drugs for Deadly Superbugs; NBC News: New York, NY, USA, 2023. [Google Scholar]
Antibiotics 14 00553 g001
Figure 1. A proposed schematic for phage susceptibility testing in the clinical laboratory. Phage products will be tested against patient isolates for activity. Methods may depend on the phage product makeup. Phage resistance should lead to secondary testing with additional standardized assays, QC assurance that the phage has not mutated through passaging, and/or selection of new phage(s) for testing. Automated methods are necessary to assure sufficiently rapid test result times. Breakthrough infection will require repeat testing to assure no development of isolate resistance, synergy testing aligned with the antibiotics used in the patient’s care, measurement of phage serum levels (through qPCR assays) and confirmation that there are not additional organisms contributing to the infection. Red triangles highlight current needs and challenges to this proposed clinical testing workflow.
Figure 1. A proposed schematic for phage susceptibility testing in the clinical laboratory. Phage products will be tested against patient isolates for activity. Methods may depend on the phage product makeup. Phage resistance should lead to secondary testing with additional standardized assays, QC assurance that the phage has not mutated through passaging, and/or selection of new phage(s) for testing. Automated methods are necessary to assure sufficiently rapid test result times. Breakthrough infection will require repeat testing to assure no development of isolate resistance, synergy testing aligned with the antibiotics used in the patient’s care, measurement of phage serum levels (through qPCR assays) and confirmation that there are not additional organisms contributing to the infection. Red triangles highlight current needs and challenges to this proposed clinical testing workflow.
Antibiotics 14 00553 g001
Table 1. Phage centers.
Table 1. Phage centers.
CenterAssociated University or HospitalLocationWebsite
Center for Innovative Phage Therapeutics and Applications (IPATH)University of California, San DiegoSan Diego, CA, USAhttps://sites.medschool.ucsd.edu/som/medicine/divisions/idgph/research/center-innovative-phage-applications-and-therapeutics/Pages/default.aspx (accessed on 5 May 2025)
Center for Phage Technology (CPT)Texas A&M UniversityCollege Station, TX, USAhttps://cpt.tamu.edu/history-and-mandate/ (accessed on 5 May 2025)
TAILOR LabsBaylor College of MedicineHouston, TX, USAhttps://www.bcm.edu/research/research-centers/tailor (accessed on 5 May 2025)
Center for Phage Research and Therapy at YaleYale UniversityNew Haven, CT, USAhttps://phage.yale.edu/ (accessed on 5 May 2025)
Pittsburgh Phage ProgramChildren’s Hospital of Pittsburgh Pittsburgh, PA, USAhttps://www.i4kids.org/research/pittsburgh-phage-progam-p3 (accessed on 5 May 2025)
Eliava Phage Therapy CenterEliava Institute of Bacteriophages, Microbiology, and VirologyTbilisi, Georgiahttps://eptc.ge/ (accessed on 5 May 2025)
Phage Therapy Unit of the Medical Centre of the Institute of Immunology and Experimental Therapy PASPolish Academy of SciencesWroclaw, Polandhttps://hirszfeld.pl/en/structure/iitd-pan-medical-center/phage-therapy-unit/ (accessed on 5 May 2025)
Israeli Phage Therapy Center of HadassahHebrew University of Jerusalem and Ein Kerem HospitalJerusalem, Israelhttps://phageil.com/the-israeli-phage-therapy-center/ (accessed on 5 May 2025)
Phage Therapy Institute at Waseda University Waseda UniversityTokyo, Japanhttps://www.waseda.jp/inst/cro/en/institutes-list/phage-therapy-institute/ (accessed on 5 May 2025)
Phage Center at Queen Astrid Military HospitalQueen Astrid military hospitalBrussels, Belgiumhttps://www.hopitalmilitaire.be/home_fr.php (accessed on 5 May 2025)
Monash Phage FoundryMonash UniversityMelbourne, Australiahttps://www.monash.edu/impact-amr/phage-therapy (accessed on 5 May 2025)
Shanghai Institute of PhageShanghai Public Health Clinical Center of Fudan UniversityShanghai, China
Phage AustraliaThe Westmead Institute for Medical ResearchWestmead, New South Waleshttps://www.phageaustralia.org/ (accessed on 5 May 2025)
Table 2. Top phage therapy requests for pathogens at IPATH UCSD.
Table 2. Top phage therapy requests for pathogens at IPATH UCSD.
BacteriumNumber of RequestsNumber of Phages FoundNumber of Cases Treated
Pseudomonas aeruginosa3486714
Staphylococcus aureus17574
Escherichia coli131175
Klebsiella pneumoniae125153
Acinetobacter baumannii721212
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kline, A.; Cobián Güemes, A.G.; Yore, J.; Ghose, C.; Van Tyne, D.; Whiteson, K.; Pride, D.T. Current Clinical Laboratory Challenges to Widespread Adoption of Phage Therapy in the United States. Antibiotics 2025, 14, 553. https://doi.org/10.3390/antibiotics14060553

AMA Style

Kline A, Cobián Güemes AG, Yore J, Ghose C, Van Tyne D, Whiteson K, Pride DT. Current Clinical Laboratory Challenges to Widespread Adoption of Phage Therapy in the United States. Antibiotics. 2025; 14(6):553. https://doi.org/10.3390/antibiotics14060553

Chicago/Turabian Style

Kline, Ahnika, Ana G. Cobián Güemes, Jennifer Yore, Chandrabali Ghose, Daria Van Tyne, Katrine Whiteson, and David T. Pride. 2025. "Current Clinical Laboratory Challenges to Widespread Adoption of Phage Therapy in the United States" Antibiotics 14, no. 6: 553. https://doi.org/10.3390/antibiotics14060553

APA Style

Kline, A., Cobián Güemes, A. G., Yore, J., Ghose, C., Van Tyne, D., Whiteson, K., & Pride, D. T. (2025). Current Clinical Laboratory Challenges to Widespread Adoption of Phage Therapy in the United States. Antibiotics, 14(6), 553. https://doi.org/10.3390/antibiotics14060553

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop