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Phage therapy

Nature Reviews Methods Primers volume 5, Article number: 9 (2025) Cite this article

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Abstract

Bacteriophage (phages) are viruses that exclusively use bacterial cells for propagation, killing the bacterial host in the process. In phage therapy, phages are used to reduce bacterial numbers, thereby curing bacterial infections. Although this principle is conceptually straightforward, its practical application faces several hurdles. In this Primer, the practical aspects of phage therapy are outlined. We introduce the microbiological methods used to prepare and characterize phages and elucidate their interactions with bacteria. The discussion covers how the information in complete phage genome sequences is used, along with how RNA sequencing can enhance our understanding of phage biology. Selection parameters for therapeutic phages for clinical applications and key elements in industrial-scale phage production are provided. A summary of clinical trials both past and present, phage administration and dosing issues is analysed, as well as limitations associated with phage therapy and mitigation strategies. Finally, we speculate on the future of phage therapy.

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Fig. 1: Mechanism of phage therapy in the human body.
Fig. 2: Phage therapy development in practice.
Fig. 3: Diversity of plaque morphology.
Fig. 4: Typical batch production processes used to make industrial and clinical grade phage products.
Fig. 5: Schematic overview of the production processes for different types of phage-based therapeutics.
Fig. 6: Conceptual schematic of a device for the synthetic production of therapeutic phages.

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Author information

Authors and Affiliations

  1. Human Microbiome Research Program, Department of Bacteriology and Immunology, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Mikael Skurnik & Saija Kiljunen

  2. Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, Hebrew University of Jerusaleum, Jerusalem, Israel

    Sivan Alkalay-Oren & Ronen Hazan

  3. The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center, Jerusalem, Israel

    Sivan Alkalay-Oren, Ronen Hazan & Ran Nir-Paz

  4. Laboratory of Gene Technology, Department of Biosystems, KU Leuven, Leuven, Belgium

    Maarten Boon & Rob Lavigne

  5. Leicester Centre for Phage Research, Department of Genetics and Genome Biology, University of Leicester, Leicester, UK

    Martha Clokie

  6. Center for Evolutionary Hologenomics, Globe Institute, University of Copenhagen, Copenhagen, Denmark

    Thomas Sicheritz-Pontén

  7. Centre of Excellence for Omics-Driven Computational Biodiscovery (COMBio), AIMST University, Bedong, Kedah, Malaysia

    Thomas Sicheritz-Pontén

  8. Laboratory of Phage Molecular Biology, Institute of Immunology and Experimental Therapy, Wrocław, Poland

    Krystyna Dąbrowska

  9. Wrocław University of Science and Technology, Faculty of Medicine, Wrocław, Poland

    Krystyna Dąbrowska

  10. Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA

    Graham F. Hatfull

  11. European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Non-traditional Antibacterial Therapy (ESGNTA), Basel, Switzerland

    Ronen Hazan, Saija Kiljunen, Rob Lavigne, Ran Nir-Paz & Jean-Paul Pirnay

  12. Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland

    Matti Jalasvuori

  13. Chemical Engineering Department, Loughborough University, Loughborough, UK

    Danish J. Malik

  14. Department of Clinical Microbiology and Infectious Diseases, Hadassah–Hebrew University Medical Center Jerusalem, Jerusalem, Israel

    Ran Nir-Paz

  15. Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgium

    Jean-Paul Pirnay

Authors
  1. Mikael Skurnik
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  2. Sivan Alkalay-Oren
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  3. Maarten Boon
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  4. Martha Clokie
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  8. Ronen Hazan
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  9. Matti Jalasvuori
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  10. Saija Kiljunen
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  12. Danish J. Malik
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  14. Jean-Paul Pirnay
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Contributions

Introduction (M.S., M.C., G.F.H., R.H., M.J., S.K., R.L., R.N.-P. and J.-P.P.); Experimentation (M.S., S.A.-O., M.B., M.C., T.S.-P., K.D., G.F.H., R.H., M.J., S.K., R.L., D.J.M., R.N.-P. and J.-P.P.); Results (M.S., M.C., T.S.-P., K.D., G.F.H., R.H., R.L., D.J.M. and J.-P.P.); Applications (M.S., M.C., T.S.-P., K.D., G.F.H., R.H., M.J., R.L., D.J.M., R.N.-P. and J.-P.P.); Reproducibility and data deposition (M.S., G.F.H., R.L., D.J.M. and J.-P.P.); Limitations and optimizations (M.S., M.C., T.S.-P., K.D., G.F.H., R.H., M.J., R.L., R.N.-P. and J.-P.P.); Outlook (M.S., G.F.H., R.L., M.J., R.N.-P. and J.-P.P.); overview of the Primer (M.S., G.F.H., R.L. and J.-P.P.).

Corresponding author

Correspondence to Mikael Skurnik.

Ethics declarations

Competing interests

S.K. and M.J. are shareholders and board members in PrecisionPhage Ltd, Jyväskylä, Finland. The other authors declare no competing interests.

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Peer review information

Nature Reviews Methods Primers thanks Pranita Tamma, Paul Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Actinetobacteriophage Database: https://phagesdb.org/

CLSI: https://clsi.org/

DSMZ collection: https://www.dsmz.de/

EUCAST: https://www.eucast.org/clinical_breakpoints

EUCAST and EDQM workgroups: https://www.eucast.org/organization/subcommittees/phage-subcommittee

Felix d’Herrelle Reference Center for Bacterial Viruses: https://www.phage.ulaval.ca/en/home/

Phage Directory: https://phage.directory/

Phages for Global Health: https://www.phagesforglobalhealth.org/

The Phagistry network: https://www.phagistry.org/

Supplementary information

Glossary

Adsorption rate

The rate at which phage binds to host bacterial cells.

Antiphage defence systems

The various approaches bacteria use to limit phage infections.

Appelmans protocol

Iterative co-cultivation of phage(s) with bacteria to select adapted phages with enhanced (therapeutic) characteristics (for example, increased pathogen clearance or delayed emergence of bacterial phage resistance).

Capsid sequencing

Deep sequencing of individual DNA strands, as packaged into phage capsids, enabling determination of the exact DNA molecule contained and identification of sequences of host origin of transduction events.

Circular permutations

Genomic arrangements in which the linear genome of a phage is organized such that the starting point of the sequence appears to vary between different genomic copies.

Cohesive ends

DNA termini that carry short single-stranded DNA overhangs, allowing genome circularization within the bacterial cell.

Complement system

A part of the innate immune system, which enhances the ability of antibodies and phagocytic cells to clear microorganisms and damaged cells from an organism, promote inflammation and attack the cell membrane of the pathogen.

Co-resistance

A situation in which a bacterial mutant that is selected as resistant to a phage is also resistant to another phage owing to the phages sharing, for example, the same phage receptor.

Double agar overlay method

A method to detect and quantify phages by mixing them with host bacteria in soft agar, overlaying it on a solid agar base and observing plaque formation.

Excipients

Inactive substances formulated alongside the active ingredient of a medication to aid in its manufacturing, stability, delivery or absorption without contributing to its therapeutic effect.

Fill-and-finish process

The end of the manufacturing process. Liquid products are filled in vials using a sterile liquid handling process.

Generalized transduction risk

Phages may package bacterial DNA instead of their own DNA during phage assembly, resulting in an infectious virus particle containing bacterial DNA: the packaged host DNA may contain genes encoding virulence factors, antibiotic resistance or toxins, and there is a risk that these may get established in the target bacterial cell infected by such a transducing phage.

Host range

The diversity of bacterial strains that a given phage is able to infect and replicate in.

Innate immune response

The body’s first line of defence against infections, providing a rapid, non-specific response through physical barriers, immune cells and soluble factors to detect and eliminate pathogens.

Major tailed phage morphotypes

Morphotypes of myoviruses (with long contractile tails), podoviruses (with short tail stubs) and siphoviruses (with long non-contractile flexible tails).

Multiplicities of infection

The ratio of the number of viable phage particles to the number of bacterial hosts when a bacterial culture is infected with a phage.

Oxygen uptake rate

The rate at which oxygen is used by cells for respiration.

Phage bioavailability

The extent and rate at which phages are able to reach and effectively interact with their target bacterial cells in a given environment, such as within the body or in a treatment setting.

Phage cocktails

A mixture of phages combined to increase host range and to maximize effective bactericide while minimizing resistance.

Phage lysates

A culture in which phages are co-cultured with bacteria and grown until most bacteria are lysed and have released propagated phages into the growth medium.

Phage selectivity

The ability of a phage to specifically infect and replicate within certain bacterial strains or species while leaving others unaffected.

Pharmacodynamics

Study of pharmacological actions on living systems, including reaction with and binding to cell constituents and the biochemical and physiological consequences of these actions.

Pharmacokinetics

Process of drug uptake by the body, their resulting biotransformation, distribution of both the drugs and their metabolites in tissues and the elimination of drugs and their metabolites from the body over time.

Plaques

Clear zones formed on a bacterial lawn where phages have infected, replicated and lysed bacterial cells, indicating phage activity.

Single-step growth curve

Enables monitoring of the phage life cycle in vitro, in which a bacterial culture is infected with phage such that each infected bacterium is infected by a single phage. The proliferation of the phages in the bacterial culture is monitored by titrating the phage in samples withdrawn from the culture over time.

Strictly lytic phages

Phages that always enter lytic life cycle upon infection of the bacterial host, leading to eventual lysis of the host cell to release the phage progeny.

Temperate phages

Phages whose genomes reside as a prophage integrated into the host genome or as a plasmid. These phages may enter a lytic cycle spontaneously or via stressors.

Transduction

Transfer of non-phage genetic material from a bacterial cell to another by mis-packaged phage particles.

Virulence

Measure of severity of phage infection on a pathogen. The higher the value, the more effective the phage.

Virulence index

Estimate of the ability of a phage to kill or damage a bacterial host population.

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Skurnik, M., Alkalay-Oren, S., Boon, M. et al. Phage therapy. Nat Rev Methods Primers 5, 9 (2025). https://doi.org/10.1038/s43586-024-00377-5

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