Phage therapy: From biological mechanisms to future directions

Abstract

Increasing antimicrobial resistance rates have revitalized bacteriophage (phage) research, the natural predators of bacteria discovered over 100 years ago. In order to use phages therapeutically, they should (1) preferably be lytic, (2) kill the bacterial host efficiently, and (3) be fully characterized to exclude side effects. Developing therapeutic phages takes a coordinated effort of multiple stakeholders. Herein, we review the state of the art in phage therapy, covering biological mechanisms, clinical applications, remaining challenges, and future directions involving naturally occurring and genetically modified or synthetic phages.

Keywords: bacteriophage; bacteriophage therapy; phage; phage therapy.

Figure 1.. Examples of therapeutically useful phages.

Bacteriophages Muddy (A) and Maestro (B) have been used to treat M. abscessus and A. baumannii infections, respectively. Muddy has a siphoviral morphotype with an icsohedral capsid containing the dsDNA genome, and a flexible non-contractile tail; Maestro has a myoviral morphology with a contractile tail. Structures at the tail tips of these phages recognize specific receptors on the bacterial cell surface. Scale bar is 100 nm. Images courtesy of Graham Hatfull and Adriana Carolina Hernandez.

Figure 2.. Phage therapy reports and Phage studies by year listed.

(A) Case reports of phage therapy since 2000. A PubMed search was performed on September 22, 2022 using the search terms “(bacteriophage) AND (therapy) AND (case report)”. Sites of infection in each of the 70 cases reported in 53 manuscripts are depicted. (B) Clinical trials of phage therapy reported to ClinicalTrials.gov since 1999. The registry was queried using the key word “phage” on September 9, 2022.

Figure 3.. Methods used in Phage Engineering.

Commonly used in-vivo homologous recombination methods in combination with CRISPR-Cas system based counter-selection strategy. (A) Rec-A mediated homologous recombination method involves phage DNA recombination with the homology region (shown in blue-red loci) present on plasmid DNA to yield recombinant phages (B) In vivo recombineering method involves recombination between phage genome and electroporated PCR products with homology arms (shown in blue-red fragments) (C) BRED method involves recombination between co-electroporated phage DNA (blue fragments) and PCR products with homology arms (shown in blue-red fragments). Because of different recombination efficiencies, each of these method produce phage progenies made up of recombinant and wild type phages (D). RNA-guided DNA nucleases such as Cas9 and Cas12 or RNA-guided RNA nuclease such as Cas13 counterselection is then applied to selectively remove unedited phages to enrich edited/engineered phages.

Figure 4.. Building synthetic phage genomes.

Using combination of phage genome fragments amplified via PCR and/or built using synthetic oligonucleotides, synthetic phage genomes are assembled into a vector using yeast based assembly or in-vitro assembly methods. Thus assembled genomes are then “rebooted” using suitable permissive bacterial host, cell-wall deprived (L-form) bacterial hosts or by using cell-free transcription-translation (TXTL) systems.

Figure 5.. Potential phage therapy applications from the One Health perspective.

Depicted are phage applications that could be implemented to address AMR arising from interactions between humans, animals and the environment.