Table 3.

Comparative analysis of conventional and synthetic approaches to overcome challenges in phage therapy.

	Challenges in Phage Therapy	Conventional Approach	Synthetic Approach
1	Narrow host range	The use of phage cocktail [111,112]	Genetic manipulation of receptor-binding protein [30,34]
2	Emergence of phage-resistant bacteria	Phage cocktail; combined therapy of antibiotic and phage [113,114]	Genetic manipulation of receptor-binding protein [115]; incorporation of small RNAs or CRISPR-Cas system to silence antibiotic resistance determinant [17,49] or delivery of genes encoding proteins to sensitize bacteria against antibiotics [116]
3	Necessitate identification of phage with therapeutic effect against patients’ isolates (personalized medicine)	Establish phage biobanks (isolating large phage collections) [117]	Engineering of phage tail fibers to alter host range [35,36,38]
4	Rapid clearance by reticuloendothelial system (RES)	Multiple phage dosing [118]	Phage capsid protein mutagenesis [41]; PEGylation of phage particles [43]
5	Phage pharmacokinetics (bioavailability through oral administration)	Pharmacological neutralization of gastric acid [119]	Encapsulation of phage in nanoparticles [120,121]
6	Limited accessibility to biofilm-producing bacteria	Use only phages with intrinsic biofilm-degrading properties [122,123], or combined therapy using phage and biofilm-degrading enzymes [124]	Engineered phages expressing biofilm-degrading enzymes [31]
7	Difficulties in defining pharmacokinetics (e.g., MIC)	Standardize routes and dosages of administration (required specified combinations of phage-host for each infection) [125]	Generation of non-proliferative anti-bacterial phage capsids [17]
8	Safety concern: risk of horizontal gene transfer	The use of phage-derived endolysin [126]	Development of well-characterized, non-propagating phages [127], introduction of antibacterial cargo using phagemids [128,129] or phage-inducible chromosomal islands (PICIs) [20]
9	Presence of potential hazardous genes in phage genome (toxin, virulence, antibiotic resistance genes, etc.)	Obligate virulent phage is preferred in therapy [113]; whole-genome analysis should be done in the first place	Custom-made phage can be generated easily using current techniques [30,34,130]; the use of self-destructive engineered phage (conjugation to gold nanorods) [29]
10	Low purity and potential toxin contamination in phage preparation	Purification by CsCl density gradient and ion exchange column [113] or affinity chromatography [131]	The use of cell-free system (cell-free-transcription-translation, TXTL) for phage production [130]