In this era of "superbugs" floods, phage therapy has re-entered public vision. Although there have been many cases of patients with "superbugs" infection being cured by phage, there are still some challenges in phage therapy.

Quality and safety requirements
The success of phage therapy depends on the safety of phage preparation ( that has strict requirements on the preparation processes and formulations. Large-scale production of phages is required in accordance with Good Manufacturing Practices (GMP) approved by regulatory agencies for a wide range of medical applications. Although the production of therapeutic phages must comply with strict regulations applicable to pharmaceuticals to ensure high quality standards, no clear guidelines have been developed for the preparation of phages. To solve this problem, some researchers have put forward some quality and safety requirements for phage therapy products. One of the requirements is to avoid selecting phage that encodes lysogenic genes, virulence genes, and antibiotic resistance genes. But this may limit the use of phage therapy in certain bacteria where lytic phages have not been found, such as Clostridium difficile. The presence of impurities (such as endotoxin) in phage preparations should be avoided, or the endotoxin titer should be controlled below the threshold. Although several methods to remove endotoxins have been developed, none of them achieves optimal results.

Stability of phage preparations
The stability of bacteriophage preparations is a core factor. Potential phage drug candidates should have a good shelf life. Choosing the preparations that will not affect phage activity for storage, and the phage titer will not significantly decrease under processing and long-term storage. Several strategies have been developed and optimized. The most common ones include spray drying, freeze drying, emulsion, and polymerization techniques. However, the stability of different phage types in different formulations (liquid, powder, gel) varies greatly. Coat phages with different matrices, such as cellulose, liposomes, and some polymers can also prolong the shelf life.

Since the therapeutic effect depends on the phage concentration at the site of infection, preventing phage from the harsh conditions (such as low pH and clearance mechanisms associated with the immune system) in the human body is essential to avoid phage inactivation during treatment. Most studies on the immune response of phage focus on the development of phage-specific antibodies. Some studies showed that adaptive immunity can reduce the circulation of phage in most cases. In vitro and in vivo experimental studies indicated that phage capsules can last longer at low pH, thereby enhancing efficacy in oral administration.

Another problem associated with phage stability is that spontaneous mutations occur during the preparation and storage of phage, which can impair the original ability of the phage to infect. Therefore, it is necessary to reduce the mutation rate of phage during the production process.

Speedy approaches to screening phages
Due to the high specificity of phages, it is usually necessary to screen big volumes of phages to find bacteriophages against specific strains. At present, the commonly used method is double-layer agar (DLA) by spotting different phages on the target bacterial plate. Different bacteria have different growth rates, and sometimes it may take up to 48 hours to get the screening results. Therefore, the DLA method can be inconvenient in rapid diagnosis. It is necessary to use high-throughput and rapid screening methods to quickly identify the phages that can effectively infect the target strains.

The effect of phages on biofilms
Bacteria often form biofilms that enhance the resistance of bacteria to antibiotics and adverse environments. In a biofilm, bacteria are protected by a matrix (polysaccharides, lipids, proteins, extracellular DNA), which can adsorb phage or diffuse to form a physical barrier to prevent phage from reaching and infecting living cells in the biofilm.

Bacterial resistance to phages
Bacteria can develop tolerance and become insensitive to phage. The most common method to prevent phage toleration is cocktail phage. The phage can also be used in combination with antibiotics to avoid the emergence of resistant phage strains and improve the therapeutic effect.

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