In the lytic cycle, the bacteriophage first inserts its genome into the host bacterial cell. Then, integrate its genetic material into the bacterial chromosomal genome. Integration is followed by the use of bacterial cellular mechanisms by the bacteriophage genome to synthesise numerous copies of its own genetic material and the required proteins for capsomers. Intracellular assembly of bacteriophage particles takes place, followed by their release from the bacterial host cell to infect another one. Bacterial lysis is a result of bacteriophage endolysin breaking the peptidoglycan, followed by its destabilisation and rupture due to osmotic pressure. Bacteriophage assembly is a very complex process, including protein-protein and protein-nucleotide interactions. It is also a model system for the investigation of biological assemblies. The lytic cycle used by the bacteriophage prevents transduction in all circumstances [67–69]. Genetic manipulation of the life cycle of bacteriophages is possible by understanding their genetic regulatory mechanism. Mainly, two bacteriophage genes regulate the phage life cycle. One is the cI gene, which controls the lysogenic life cycle of the phage. The cl gene produces a CI protein through transcription and translation. CI protein acts like a transcriptional repressor. It binds to the two gene operators named pR and pL to inhibit their respective gene expression. The pR promoter gene is responsible for the lytic cycle of bacteriophage by derepressing the cro gene. The second task is to stimulate its expression by interacting with the pRM promoter. The cro gene produces the Cro protein that inhibits the pRM promoter and inhibits the expression of the cI gene [70]. Knocking out the cI gene can be performed to get a phage particle with the characteristic of performing a lytic cycle only. Typical genetic regulation of the bacteriophage replication cycle is well illustrated (Figure 1). BRED (bacteriophage recombineering of electroporated DNA) is an in vivo technique and involves the recombination of very short homologies using the molecular machinery of the bacteriophage itself. First performed in the mycobacteriophage in the year 2008. Che9c, gp60, and gp61 proteins are present in the mycobacteriophage, and it is similar to the RecE/RecT system found in the Rac prophage of E. coli. A substrate (ssDNA) and the prophage DNA template are electroporated into an electrocompetent cell that expresses recombination proteins (gp60 and gp61). Selection can be done with either polymerase chain reaction (PCR) or by plating the competent cell for analysing the plaque. This technique can be used for deletion, knockouts, replacement, and point mutations in the viral genome [71–74]. This technique can be employed for the knockout of repressor genes of the bacteriophage lytic cycle. Genome sequencing and other bioinformatic tools help to search for and identify desirable and undesirable genes. This can be done by homology search of the phage genome with the genes (toxin genes, antibiotic genes, integrase, and repressors associated with the phage lysogeny) existed in the databases [75, 76].

Bacteriophages are very specific for their host cell. Gene editing helps in designing desired bacteria-targeting bacteriophages with selective specificity. Specific bacteriophage tail genes can be expressed in another bacteriophage genome to get the desired host specificity. The end product will be a recombinant bacteriophage that would be able to express the tail structures of interest and can target multiple bacterial species or any specific bacterial species. For example, Bacteriophage T4 (Escherichia coli Virus T4) infects E. coli using tail fibres, and the genes working behind this specificity are gp34 and gp37 (gp stands for gene product). The efficiency of bacteriophage T4 is almost 1.0. Every bacteriophage particle successfully replicates/insert its genetic material after getting adsorbed on the E. coli surface. In the interaction between E. coli and bacteriophage T4, the glucose residue on the core lipopolysaccharide of E. coli serves as a receptor for host recognition [77]. Clustered regularly interspaced short palindromic repeat (CRISPR) was first documented in E. coli in the year 1987 as a defence mechanism against bacteriophage. This can cleave the bacteriophage genome in a sequence-specific manner. Researchers had already used CRISPR as a gene editing tool on Vibrio natriegens TT4P2 in the host bacterium V. natriegens TT4 to achieve gene deletion and replacement. They successfully deleted the orf6 gene of size 292 bp, the orf45 gene of size 162 bp, and replaced the orf6 gene with orf39 [23]. CRISPR is a promising technique to make various recombinant bacteriophages with our desired genetic alterations. This will also counter the act of bacteriophage against human-associated bacterial flora [78, 79]. BRED can also be used for the same. Clinical isolation and identification of the infectious bacteria are crucial for personalised bacteriophage therapy. The phage biobank helps in considering several bacteriophages against the infectious bacterial strain, and high-throughput screening helps in finding specific phage matches with the bacterial host [62].

Biofilm is a fundamental defensive mechanism of bacteria against antibiotics. This prevents antibiotics’ action on bacteria. Biofilms can be observed on places like dental plaques, medical devices, and foodstuffs [80, 81]. It is a slimy layer formed as a result of bacterial extracellular secretions. It mainly comprises exopolysaccharides like N-acetyl-D-glucosamine (dPNAG) exopolysaccharide, alginate exopolysaccharide, psl exopolysaccharide, amylose like glucon. Enzymes like dPNAG glycoside hydrolase (dispersin B), alginate glycoside hydrolase, Psl glycoside hydrolase, and amylose glycoside hydrolase target these exopolysaccharides, respectively [82]. A capsule with the above-integrated enzymes can be developed by using artificial heterodimeric or even monomeric antibodies, which can be fused with the coat protein of a bacteriophage or other capsule-like structures through the constant region of the antibody. This fusion can be achieved by the phage display system [83]. Phage display exploits the screening of a large number of peptides for their binding ability to target molecules. The desired protein (bacterial surface-binding receptor protein or antibody) encoding gene can be inserted between the genes for phage capsids. At the time of phage harvesting, phage particles express the desired protein molecules integrated into their capsid [44]. Use of phage depolymerase is also a good concept for biofilm degradation. Phages infecting mainly E. coli K1, E. coli K20, K. pneumoniae K22, and A. baumannii K26 encode depolymerases [84–86]. The phagemid technique can be utilised for preparing antibodies fused to the coat protein. A phagemid is a genetically engineered DNA molecule with an integrated bacteriophage genome, plasmid replication site, restriction sites, selective markers, and an antibody-encoding gene [87]. The transformation of the phagemid allows it to replicate in a competent bacterial cell to produce a bacteriophage of interest. Not every bacterial cell is competent to receive foreign DNA molecules like plasmids, chromosomal DNA, and free molecules of DNA present in the environment [88]. However, bacteria can be made competent through treatments, such as the calcium chloride method, in which bacterial cells are treated with calcium chloride at low temperatures and suddenly treated with heat (42°C) for 30–60 seconds. The calcium ions neutralise the negative charge present on the bacterial cell membrane and the bacterial DNA. Neutralisation of negative charge and heat shock treatment help bacterial membranes to take up foreign DNA molecules. Almost all divalent cations, such as calcium or magnesium, enhance the transformation. The presence of magnesium ions increases the transformation by 15–20-fold [89, 90]. Electroporation creates temporary pores in the bacterial cell membrane and makes the membrane competent to take foreign DNA molecules [91].

S. aureus, Thermus thermophilus, N. gonorrhoeae, Vibrio cholerae, H. pylori, Haemophilus influenzae, and Bacillus subtilis are naturally competent species for transformation [92]. Prokaryotic antibiotic genes like actinomycin D and mitomycin (which block the prokaryotic translation by interrupting the elongation factor) can be used in the phagemid to kill the bacteria after coming into contact with a phage-infected bacterium [93]. In this way, bacteriophage acts as a specific drug delivery carrier for other bacterial strains present nearby. It restricts its own growth and the growth of surrounding non-infected bacteria through antibiotic production [94]. Researchers enhanced dispersin B digestion by engineering a bacteriophage having a gene of the dispersin B-degrading enzyme. Bacteriophage T7 against biofilm formed by E. coli has been engineered [95]. Schematic diagram showing the bacteriophage covered with a capsule with integrated enzymes (Figure 2). Human innate and acquired immune cells interfere with phage therapy and decrease the effectiveness of the phage on bacteria. In an experiment, the ability of intracellular killing by phagocytes in a patient suffering from chronic bacterial infection is considered. It was found that the intracellular killing ability is higher in patients after phage therapy than before the therapy [96]. This experiment shows that bacteriophage recovers this ability of intracellular killing of pathogens by phagocytic cells. Another experiment resulted in a higher killing effect of an engineered fluorescent K1F-GFP bacteriophage having specificity for E. coli when it entered the T24 human urinary bladder epithelial cell [97]. This showed that bacteriophage can work on bacteria in the presence of the human immune system, and it may even collaborate with the immune system to kill bacteria.

Administration is crucial to the efficacy of a drug, vaccine, or any other antimicrobial substance. Different routes, including oral, intravenous, topical, intrarectal, intraoperative, and nebulization, are available for bacteriophage administration [53, 98–100]. The first successful bacteriophage administration was performed in 1921 in France (Hôpital des Enfants-Malades, Paris) against Shiga dysentery in a child. The effectiveness of bacteriophage therapy is highly controversial [101]. Oral bacteriophage administration has poor stability in the acidic environment of the stomach. HCl present in the gastric secretion and digestive enzymes degrade bacteriophage capsomers (protein coat) [11]. Bacteriophage stays in the intestine for a short time, not long enough for effective host attachment. Therefore, repetitive administration is needed for effective phage therapy [102]. The bile salts and carbohydrates present in the intestine block divalent ions, that’s must for bacteriophage replication. Resulting in the reduction of bacteriophage replication in the host bacterium. Unlike drugs, bacteriophages are generally not absorbed by the intestine and cannot enter the blood circulation. This limits oral administration to treat bacterial infections present only in the gut [51, 53]. Bacteriophage encapsulation (using liposomes, electrospun fibre-encapsulation, and polymers) protects phage particles from gastric secretions. Liposome-encapsulated bacteriophages are covered with a positively charged liposome. It protects the bacteriophage from the acidic pH of the human body, acts as a promoter for the mucosal adhesion of phage particles, and increases the retention time of the bacteriophage in the intestine. The flaw of liposome encapsulation is its nature of conglomeration. Liposomes form large conglomerates through adhesion with each other. Conglomeration compromises the stability of the phage particle and causes non-specific interactions with the target host cell. In electrospun fibers-encapsulated bacteriophage therapy, bacteriophages are attached to the surface of electrospun fibers. This is one of the easiest ways to introduce the bacteriophage to the local site of infection. It helps to identify, detect, capture, kill, and immobilise the targeted bacteria. Electrospun-encapsulation also causes the dehydration of the phage particle and leads to its destruction. Microneedle therapy is another form of bacteriophage administration that involves direct penetration of the needle and introduces the bacteriophages to the site of infection. However, this procedure is very difficult for the patient to accept. Polymers-encapsulated bacteriophages are prepared by the chemical modification of biopolymers like polyamines, polyamides, polycarbonates, alginates, and aliphatic polyesters. Polymer increases the acid stability of bacteriophage in the gastric cavity. For example, alginate polymer encapsulation with the addition of guar gum, pectin, and chitosan. Biopolymer-encapsulated phage does not stimulate proinflammatory cytokines and antibody production. But stimulates phagocytes in the blood circulation [51, 103–112].

The intraoperative route involves direct bacteriophage administration at the site of infection. In the year 2012, a 76-year-old patient was suffering from an aortic aneurysm. During the aortic arch replacement surgery, he got an infection of P. aeruginosa. The infection was diagnosed through a deep wound and blood culture. After the recurrence of the same bacterial infection on the same site. He was treated with a mixture of phage OMKO1 and ceftazidime (0.2 g/mL) into the mediastinal fistula. The patient had never faced the recurrence of P. aeruginosa infection again [113]. A 62-year-old male was suffering from fulminant pleural empyema after LVAD (left ventricular assist device) implant. He was diagnosed with the infection of S. aureus through a wound swab. There was no improvement after the antibiotic treatment with daptomycin (500 mg) that was administered intravenously once per day. Finally, 14 doses of Staphylococcus phage CH1 (1 × 10⁹ PFU/mL) were administered via drainage at the site of infection every 12 hours, along with the same antibiotic regimen. After one week of Staphylococcus phage CH1 administration, eradication of S. aureus from the site of infection was confirmed by microbiological assay. Another 66-year-old female patient was suffering from a sternal wall healing disorder. She had aortocoronary bypass and mitral valve replacement surgery. Wound swab confirmed the infection of E. coli, and the patient’s condition was not improving with antibiotics. 600 mg of clindamycin three times per day was administered orally. A mixture of Escherichia phage V18 and Escherichia phage ECD7 was administered intraoperatively, mixed with fibrin glue. E. coli was not detected in further microbiological assays. Similar case of a patient (13-year-old male) who had a double lung transplant and developed sternal wound abscesses. Infection with P. aeruginosa has been confirmed from a wound swab. The patient did not recover after using antibiotic treatment of 187.5 mg avibactam, 750 mg ceftazidime intravenously three times per day, and 2 MIU colistin twice per day. He recovered after the administration of Pseudomonas phage PA5 and Pseudomonas phage PA10 intraoperatively mixed with fibrin glue [114].

The intravenous route of administration involves the introduction of medicine, drug, or food supplement (vitamins and calcium, etc.) directly into the blood vessels. A 77-year-old male patient got infected with multi-drug-resistant A. baumannii (a gram-negative coccobacillus) after the craniectomy. A collection of 104 A. baumannii bacteriophages was analyzed against the isolate of A. baumannii collected from the patient and cultured in the tryptic soy broth by NMRC (Naval Medical Research Center-Frederick). The screening of the isolate and bacteriophage collection was also performed at NMRC. Only 5 phages were found to be active against the A. baumannii. The bacteriophage (2 × 10¹⁰ PFU/mL, endotoxin level of 3.5 × 10⁵ endotoxin units/mL) in 4 mL of lactated Ringer’s (intravenous saline) was administered intravenously using a central catheter (inserted peripherally). The frequency of administration was once every two hours for 8 days. There were no further signs of infection, and the craniectomy site healed. Further urine, blood, and respiratory cultures were analysed and found negative for A. baumannii [115].

Bacteriophage can also be administered through oral and topical routes (directly on the surface or in the layer of skin) to make its availability easy, non-invasive, and with high levels of patient compliance [116]. A 16-year-old boy suffering from Netherton syndrome (a rare congenital skin disorder) was treated with an undefined mixture of bacteriophage. He showed a reduction in atopic eczema (a chronic inflammation of the skin characterised by swelling, dryness, itchiness, and redness). Used bacteriophage mixtures contained phage for Proteus, Streptococcus, Staphylococcus, Pseudomonas, Escherichia, and Enterococcus. The patient’s health improved with increased joint mobility and reduced skin irritation [117, 118].

Another challenge is the mass production of bacteriophage. Organisations are continuously trying to produce bacteriophage-based products. AmpliPhi Biosciences Corporation has focused on its product named Phagoburn for the treatment of wounds inhabited by E. coli or P. aeruginosa. This organisation has also been working on bacteriophage-based treatment against skin infection of S. aureus. Countries like Russia, Poland, Belgium, and Georgia have used phage therapy for decades due to its positive results [36, 119]. AgriPhage™ is for agricultural lands against plant infections. LISTEX™ P100 and EcoShield™ are used to prevent the growth of L. monocytogenes and E. coli O157:H7, respectively [120]. Traditionally, shaker flasks or stirred tank bioreactors were used for bacteriophage cultivation. Batch, semi-continuous, and continuous modes of cultivation are now optimised for bacteriophage cultivation in bioreactors [121]. A semi-continuous two-stage self-cycling process has been developed to overcome the disadvantages of the above mode of cultivation. In this process, the host bacteria are used to cultivate in the first bioreactor in batch mode. Just before the stationary phase, half of the media from the first bioreactor was introduced into the second bioreactor for bacteriophage cultivation [122]. Host bacterial growth has a crucial impact on the bacteriophage cultivation. Changing the physiological condition (temperature, pH and substrate concentration for bacterial growth) of host bacteria provides different concentrations of bacteriophage [119, 123]. The generalised procedure for phage cultivation is difficult to develop. The literature mentioned three pipelines of phage production (off-the-shelf, pharmacy compounding, and on-demand de novo). Off-the-shelf pipelines constitute the formulation of fixed phage compositions under good manufacturing practices, either against a single bacterial strain or multiple bacterial strains [124]. When microbial isolates of patients are found to be insensitive to the phage used in the fixed phage products. Then the second pipeline is used by the licensed pharmacist. A physician can manufacture a phage product according to the personalised needs of the patient [125]. In this pipeline, the sensitivity of the phage screened against the infectious isolate of the patient from the phage collection and libraries maintained by the institution (Queen Astrid Military Hospital) [126]. In an on-demand de novo pipeline, the sensitive phage particles against the infectious isolates are isolated from the site of infection itself. This pipeline provides good results and better efficacy [40, 127, 128].