Overuse of antibiotics has heavily contributed to the prevalence of multidrug-resistant (MDR) and antimicrobial resistant (AMR) pathogens, the result of which, antibiotic resistance has become one of the top threats to humanity, causing approximately 0.7 million annual deaths across the globe, and the number may exceed to cancer-driven mortality in the near future. Gene mutation and horizontal gene transfer can easily lead to ineffective antibiotic treatment of an infection and enable pathogens to evolve the ability to evade most therapeutics. This continuously forces researchers to discover new antibiotics with improved features, even though antibiotic agents have a weak research pipeline and it takes a long time for most antibiotic drugs to enter the market for practical use. Some researchers take this from a different perspective, including finding alternatives to antibiotics that can treat multidrug-resistant pathogens. The most promising one is bacteriophages (or simply phages), the natural predators of bacterial cells.
Bacteriophages are viruses that attack specific bacteria and archaea, which actually had been implemented as therapeutic agents for a century before they were replaced with antibiotics. Now, researchers can better understand phage biology and their genome sequences through genetic tools, genome engineering techniques, as well as synthetic biology. The small sizes of phage genomes make bacteriophages suitable candidates for generating treatments against bacterial infections, and phage therapy might again become of interest to combat the increasingly severe antimicrobial resistance.
However, the main challenges of phage-based treatment targeting MDR pathogens are the limited host range and phage hunting. To address the former issue, researchers have succeeded in broadening the host range of phages by a consortium of phages in a single shot, usually known as a phage cocktail, the efficacy of which has been demonstrated. Moreover, researchers also tried a method for bacteriophage engineering that replaces the viral scaffold to create broader host ranges, in which the tail fiber and other associated genes of an Escherichia coli phage are changed for targeting Yersinia and Klebsiella bacteria.
As for the task of phage hunting, synthetic phage bioengineering techniques, in which genome editing plays a key role, have significantly improved it by effectively tailoring interactions between bacteria and phages. Techniques, including classical homologous recombineering approaches, type I-E clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated protein (Cas)-based counter selection, and yeast-based reconstruction of phage genomes have been demonstrated to be useful to generate recombinant phages. Though innovative, these novel techniques are elusive when it comes to successful applications and gene editing efficiency.
Among all these novel phage-bioengineering approaches, the CRISPR-Cas system is the most promising for developing bioengineered phages against MDR pathogens, given that it applies a successful genome editing tool in eukaryotic and prokaryotic systems. In addition, this technique for target-specific genome editing has proven its ability to overcome problems including complex mechanisms, tedious procedures, low efficiency, and higher probabilities of off-target activities. Based on these advantages, the CRISPR-Cas system has been successfully employed by various research groups to introduce point mutations, reporter gene knock-in, as well as deletions in various phage genomes. Advances in techniques used for genome manipulation and adaptive evolution make it possible to engineer a phage with desired characteristics and improve the robustness and fitness of phages for therapeutic applications. All in all, phages integrated with a programmable endonuclease are a promising therapeutic candidate to combat MDR pathogens.
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