By Andy Tay, PhD

Overuse of antibiotics has accelerated the emergence of antimicrobial-resistant (AMR) bacterial species. The World Health Organization named antibacterial resistance as a top public health threat to humanity. In 2022, the Antimicrobial Resistance Collaborators reported that AMR bacteria directly caused 1.3 million deaths and was associated with 5 million deaths.1 The last time a novel class of antibiotics was discovered was in the 1980s, and the first drug of this class, daptomycin, did not receive antibiotic approval until 2003.

Investment in antibiotics research has also been slow. Pharmaceutical companies are financially disincentivized to invest more in this area because the returns are low and because it is challenging to prove that a new drug is significantly better than existing antibiotics. Thus, there is rising interest in developing alternative forms of therapy against AMR bacteria. A promising possibility is phage therapy.

Phages, or bacteriophages, are viruses that infect and replicate within bacteria. Once phages attach to a host, they pursue either a lytic or lysogenic replication strategy.2 In lytic replication, the phage introduces its viral genome into the cytoplasm and uses the host cell’s resources to produce copies of its genome and capsid proteins, which assemble into new phages. The host is then lysed to release the phages, which infect other host cells.

In lysogenic replication, a phage genome is integrated into the bacterial chromosome, allowing the phage genome to be replicated and passed on to bacterial daughter cells. Depending on the environmental conditions, conversion to a lytic replication cycle is possible. Phage therapy uses lytic phages to kill AMR bacteria.

Phage therapy has unique advantages that make it highly attractive for use against AMR bacteria. First, phages can evolve to target AMR bacterial species. Phages, unlike antibiotics, are continuously engaged in an evolutionary arms race with bacteria. In this struggle, phages exploit opportunities to acquire new traits that are more effective against bacteria—even mutated bacteria.

Second, phages have host specificity, which enables them to target pathogenic strains while sparing beneficial parts of the human microbiome. This helps to improve therapeutic efficacy and reduce off-target effects.

Third, there have been studies supporting the idea that phages can sensitize AMR bacterial species to antibiotics that are already widely used in clinics. Finally, phages can disrupt biofilms. By penetrating biofilms, phages can access and kill bacteria before they can evolve and become resistant.

Nevertheless, there are still translational challenges to overcome before phage therapy can become a mainstream clinical activity. Currently, phage therapy remains a last resort—an option that can be justified only on a compassionate use basis for patients who have exhausted all antibiotic options. Several recent efforts to overcome the challenges associated with phage therapy are described below.

Targeted delivery

Most phage therapies in clinics are delivered intravenously. However, bacterial infections are not limited to blood. They can be localized in the lungs and skin, or they can occur at multiple tissue sites. Localized phage delivery using biomaterials such as patches and microparticles can improve phage availability to infected sites while reducing the dose and manufacturing requirements.

Unlike antibiotics, phage therapies lack a well-established dosing regimen that is based on factors such as body weight, pharmacokinetics, pharmacodynamics, and antibiotic susceptibility data. One way to overcome this problem is to build mathematical in silico models to predict the outcome of phage therapy strategies.3 But this approach can be limited by a lack of clinical data. Therefore, it is imperative that phage administration be localized to maximize therapeutic efficacy and reduce adverse side effects.

Hydrogels and microbeads have been developed by researchers at the AO Research Institute to protect phages during delivery.4 These researchers, led by T. Fintan Moriarty, PhD, showed that their material preparation process did not induce physical damage to the phages. However, the phages underwent rapid release, and after five days, most of the phage species they tested had plateaued in percent cumulative release. The researchers concluded that other properties of the hydrogel such as porosity and charges can be further optimized to improve the release profile of phage for a longer-acting treatment.

Baixing Chen
At the AO Research Institute, scientists led by T. Fintan Moriarty, PhD, evaluated bioresorbable materials for their ability to serve as phage delivery systems. Microbeads were designed to enable delayed release; hydrogels, rapid release. In general, the systems performed as expected. However, the researchers warned that their findings pointed to issues that are in need of clarification. For example, it appears that in hydrogels, release kinetics can be “phage dependent and further impacted by charges and hydrophobicity.” Scientists on Moriarty’s team—including Baixing Chen, PhD (shown here)—are working to improve hydrogel technology.

“There are multiple potential advantages of using hydrogels for phage delivery,” said Baixing Chen, PhD, a researcher on Moriarty’s team. “They can be designed to release phages gradually over an extended period. This controlled release can help maintain a therapeutic dose of phages at the infection site, increasing the chances of effectively targeting and eliminating the bacteria. Hydrogels can act as a protective barrier, shielding phages from external factors that could degrade or inactivate them, such as pH changes. They can also be combined with other therapeutic agents, such as antibiotics or wound-healing agents, to create combination therapies.

“We are improving our hydrogel technology to [enhance] formulation so that it is compatible with phage therapy, remains stable long term, and is cost-effective. We also want to further engineer its controlled release mechanisms for phage release in response to specific environmental cues at the infection site. Finally, we are exploring hydrogels that combine phages with other therapeutic agents, such as antibiotics or immunomodulating compounds, in order to enhance treatment outcomes.”

Microneedles can access deep infected tissues, an ability beyond hydrogels, which are suitable as superficial dressings. At the International Iberian Nanotechnology Laboratory, researchers led by Manuel Bañobre-López, PhD, loaded phages into dissolving microneedles made with polyvinyl alcohol.5 The team found that phage release was unaffected by microneedle geometry, and that phage stability was maintained in microneedles stored for six months at 4°C. Nevertheless, phage release was rapid and occurred within a few minutes after the microneedles dissolved. These results suggest that the material is ill-suited for long-term use. However, the material could be repurposed as a wound dressing.

“Chronic wounds have a high prevalence of antibiotic-resistant bacteria living in 3D biofilm structures tolerant to most antibiotics,” said Sanna Sillankorva, PhD, a researcher on Bañobre-López’s team. “Loading microneedles with phages allows phage delivery inside different biofilm layers, surpassing the biofilm matrix and allowing phages to interact more easily with their host bacterium.

“This is particularly useful for phages that do not entail phage depolymerase activity. We are continuously improving microneedle fabrication (for example, by using 3D printing to customize array size and needle shape, geometry, and height), testing different synthetic and natural polymers, and testing combined delivery strategies with other molecules.”

Besides hydrogels and microneedles, there are other strategies to improve localized phage delivery. At the Georgia Institute of Technology, researchers led by Andrés J. García, PhD, developed inhalable phage-loaded microparticles to treat lung infections.6 The scientists showed that the porosity of microparticles is key to adsorbing and depositing sufficient numbers of phages on microparticles. The cream or gel formulations that are extensively used in topical skin applications are also an attractive option for phage delivery, but more evidence in support of this approach is needed.

Phage-antibiotic synergy

Since the 1940s, studies have shown that there is therapeutic synergy between antibiotics and phages.7 Some potential mechanisms include (1) recovery of antibiotic activity against AMR bacteria, (2) phages and antibiotics acting against different bacterial receptors, (3) antibiotics causing morphological changes in bacteria that enhance phage lytic activities, (4) antibiotics inducing better phage production, and (5) phages enabling antibiotics to penetrate biofilms.

Recently, researchers at the University of Minho led by Joana Azeredo, PhD, tested the effect of gentamicin as a co-adjuvant of phages in a dual species–biofilm wound model.8 (Phages targeting Pseudomonas aeruginosa and Staphylococcus aureus were used.) The researchers found that gentamycin is an effective adjuvant of phage therapy particularly when applied simultaneously with phages and in three consecutive doses. The researchers also found that the order and frequency of phage-antibiotic treatment could influence the treatment outcome. From this study, the team suggested that in a multidose treatment with simultaneous application of antibiotics and phages, the bacterial population is subjected to multiple stresses at the same time and cannot recover or evolve resistance.

To realize the promise of antibiotic-phage pairs, researchers need to determine how antibiotic pairs that work synergistically can be identified. According to researchers at the University of Novi Sad led by Petar Knezevic, PhD, this task could be accomplished by applying an optimized checkerboard method.9 In this method, phages and antibiotics are either added at the same time or successively. Then the applicability of different regimens is assessed by the time-kill curve method.

The study found that a phage cocktail performed better than a single phage. This is consistent with previous reports, including one from researchers based at the Westmead Institute for Medical Research and the University of Sydney.10 These researchers described a single-arm noncomparative trial in which 13 patients with severe S. aureus infections were intravenously administered a preparation of three Myoviridae bacteriophages as adjunctive therapy. According to the researchers, there were no adverse reactions. “No phage resistance evolved in vivo,” the researchers added, “and the measurements of bacterial and phage kinetics in blood samples suggest that 12 h dosing of 109 plaque-forming units may be a rational basis for further studies.”

There are also startups, such as Adaptive Phage Therapeutics, that are building large phage banks to screen for specific phage–bacterial host interactions. At these startups, developers are encouraged by the observation that most infections are caused by common bacterial species. Phages that are known to target a particular bacterial species could be manufactured and administered like an off-the-shelf drug.

Synthetically engineered phage

There are biological barriers that prevent the use of naturally occurring phages therapeutically. First, phages that have an undesired tropism may kill a beneficial microbiome. Second, phages that infect bacterial hosts may fail to kill them efficiently. Third, phages may encode proteins that are harmful to human patients. Fourth, the isolation and characterization of clinically useful phages can be challenging.

Although phages exist everywhere, many naturally occurring phages cannot be readily cultured in the laboratory. In addition, the characterization of phages may require extensive testing. For example, it may be necessary to determine host range, phage speciation, and genome-determined traits such as AMR and toxicity.

Synthetic biology offers a way to engineer phages that are more powerful against AMR bacterial species using existing candidates in phage banks. For instance, phages can be engineered to reduce horizontal gene transfer of antibiotic resistance genes (by rapidly degrading the bacterial genome), to enhance the penetration of biofilms, to improve the targeting of intracellular pathogens, or to ensure superior pharmacokinetic and pharmacodynamic properties.

Recently, researchers based at the University of California, San Francisco, combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme to engineer the genome of Pseudomonas aeruginosa ØKZ, a jumbo phage. The researchers, led by Joseph Bondy-Denomy, PhD, reported that they inserted foreign genes, deleted genes, and added fluorescent tags to genes in the ØKZ genome.11

Antimocrobial hourglass
Scientists based at the Innovative Genomics Institute (IGI) and led by Jennifer A. Doudna, PhD, have found a solution to a longstanding problem in phage research: Phages that have had their genomes altered are hard to isolate from phages that have not. The solution depends on CRISPR-Cas13. It is, the IGI team determined, capable of chopping up RNA in a wide range of phages. By engineering a host microbe to contain a CRISPR-Cas13 system that defends against a normal phage genome sequence, the IGI team had a means of eliminating normal (unedited) phages and sparing edited phages. Once the edited phages (orange) get through the sequence-specific counterselection system (a sort of bottleneck), they can undergo enrichment.

In a similar study, one led by the Innovative Genomics Institute’s Jennifer A. Doudna, PhD, CRISPR-Cas13a was used to introduce markerless genome edits to three diverse phages with 100% efficiency, including multigene deletions and a codon replacement.12 These techniques demonstrate the possibility of using CRISPR-Cas tools to create small, precise genome edits and engineer a phage genome to meet therapeutic specifications.

However, CRISPR-Cas systems are limited by bacterial hosts that have genetic toolboxes available for expressing CRISPR-Cas systems and maintaining editing templates. Although phage genomes can also be assembled in vitro and then delivered to transform bacterial cells, this process is inefficient. To overcome this challenge, scientists at the Technical University of Munich led by Gil G. Westmeyer, PhD, made use of cell-free transcription-translation to assemble clinically relevant phages from genomic DNA isolated from purified phage stocks.13

With the severity of AMR worsening, there is rising interest in developing phage therapies. However, before these therapies can earn mainstream status, phage engineering and delivery must be improved. Beyond this, phage manufacturing is currently limited in scale. Workflows and facilities dedicated to phage production have yet to be established.

 

References
1. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 2022; 399(10325): 629–655. DOI: 10.1016/S0140-6736(21)02724-0.
2. Strathdee SA, Hatfull GF, Mutalik VK, Schooley RT. Phage therapy: From biological mechanisms to future directions. Cell 2023; 186(1): 17–31. DOI: 10.1016/j.cell.2022.11.017.
3. Delattre R, Seurat J, Haddad F, et al. Combination of in vivo phage therapy data with in silico model highlights key parameters for pneumonia treatment efficacy. Cell Rep. 2022; 39(7): 110825. DOI: 10.1016/j.celrep.2022.110825.
4. Rotman SG, Post V, Foster AL, et al. Alginate chitosan microbeads and thermos-responsive hyaluronic acid hydrogel for phage delivery. J. Drug Deliv. Sci. Technol. 2023; 79: 103991. DOI: 10.1016/j.jddst.2022.103991.
5. Sillankorva S, Pires L, Pastrana LM, Bañobre-López M. Antibiofilm Efficacy of the Pseudomonas aeruginosa Pbunavirus vB_PaeM-SMS29 Loaded onto Dissolving Polyvinyl Alcohol Microneedles. Viruses 2022; 14(5): 964. DOI: 10.3390/v14050964.
6. Agarwal R, Johnson CT, Imhoff BR, et al. Inhaled bacteriophage-loaded polymeric microparticles ameliorate acute lung infections. Nat. Biomed. Eng. 2018; 2(11): 841–849. DOI: 10.1038/s41551-018-0263-5.
7. Diallo K, Dublanchet A. Benefits of Combined Phage-Antibiotic Therapy for the Control of Antibiotic-Resistant Bacteria: A Literature Review. Antibiotics (Basel) 2022; 11(7): 839. DOI: 10.3390/antibiotics11070839.
8. Akturk E, Melo LDR, Oliveira H, et al. Combining phages and antibiotic to enhance antibiofilm efficacy against an in vitro dual species wound biofilm. Biofilm 2023; 6: 100147. DOI: 10.1016/j.bioflm.2023.100147.
9. Nikolic I, Vukovic D, Gavric D, et al. An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection. Viruses 2022; 14(7): 1542. DOI: 10.3390/v14071542.
10. Fabijan AP, Lin RCY, Ho J. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat. Microbiol. 2020; 5(3): 465–472. DOI: 10.1038/s41564-019-0634-z.
11. Guan J, Oromí-Bosch A, Mendoza SD, et al. Bacteriophage genome engineering with CRISPR-Cas13a. Nat. Microbiol. 2022; 7(12): 1956–1966. DOI: 10.1038/s41564-022-01243-4.
12. Adler BA, Hessler T, Cress BF, et al. Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing. Nat. Microbiol. 2022; 7(12): 1967–1979. DOI: 10.1038/s41564-022-01258-x.
13. Emslander Q, Vogele K, Braun P, et al. Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria. Cell Chem. Biol. 2022; 29(9): 1434–1445.e7. DOI: 10.1016/j.chembiol.2022.06.003.

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