**2. Phage library and sensitivity**

Availability of a library with a range of therapeutic bacteriophages is the foundation for the success of phage therapy. Bacteriophages are observed to show off a narrow to broad host specificity [30–32]. The lytic bacteriophages ought to have the capacity to kill strange bacterial species even as a range of bacteriophages can kill the identical bacterial strain (**Figures 1**–**4**) [33]. For a safe medical use of the phage, genes of toxins, antibiotic-resistance, and multidrug-resistant genes should not be present in the genome of the phage', whilst the lytic phage must have the potential to kill the multidrug-resistant bacteria such as *Acinetobacter baumannii, Enterococcus, Escherichia coli*, *Klebsiella pneumoniae, Pseudomonas aeruginosa,* and *Staphylococcus aureus* [34–38]. Confirmation of phage-sensitive bacteria is the prerequisite for

### **Figure 1.**

*Transmission electron micrograph (TEM) bacteriophages infected Bacillus. Note the capsid, nucleic acid, tail fibers [2].*

### **Figure 2.**

*A magnified portion of a Bacillus infected with bacteriophages is seen in this Transmission electron micrograph (TEM). The capsid, nucleic acid, tail fibers should all be noted [2].*

initiation of antibacterial therapy. First, the bacterium inflicting the infection in the patient has to be received and identified; second, phage in the library that are effective against the pathogenic bacterium need to be screened, and selected for therapeutic use. If there are specific phage or cocktails of phage in the library that kill the identical bacterial strain, are preferred for the therapy [39, 40]. Reports have shown that phage cocktail preparations would possibly decorate bactericidal efficacy and additionally limit the chance of the emergence of phage-resistant isolates for the duration of the therapy [30–32]. Bacteriophages (phage) have a unique sorting mechanism for their target bacteria since they have a number of necessary characteristics such as inherent natural specificity, ease of use of cell signalling and receptor molecules, and simple phage or phage-derived product processing. These characteristics make bacteriophages more suitable for use as bacterial detectors and

### **Figure 3.**

*TEM showing* V. vulnificus *(VV-1) bacteriophage particles (Bp) within the cytolyzed cytoplasm (c) of the host cell bacterium* Vibrio *sp. Note the presence of phage within the cytoplasm [10].*

### **Figure 4.**

*Electron micrograph showing* V. vulnificus *(VV-2) bacteriophage (Bp) particles within the lysed cytoplasm (c) of the host cell bacterium* Vibrio *sp [10].*

as aids in the detection of human pathogens. Phage-based systems are currently being used to diagnose *Staphylococcus aureus, Mycobacterium tuberculosis, Bacillus anthracis*, and *Yersinia pestis* in the clinical setting.

### **3. Production and purification of bacteriophages**

Appropriate cultural media used for the growth, proliferation, and fermentation process of cells of bacterial hosts of bacteriophages for therapeutic applications. The fundamental processing of bacteriophages consists of several stages of purification (**Table 1**). These are broth specifications with low-speed centrifugation or filtering, cell removal, and cellular debris. Chloroform will be added to the lysate to form lysis and release the phage from non-lytic cells. Specified bacteriophage lysate can be used in many applications but clinical applications require additional purification of the lysate to eliminate endotoxins, metabolites, hydrophilic O-specific polysaccharide, phosphorylated oligosaccharides, phage, bacterial cells, and other wastes. Impure preparations of bacteriophages should not be used for injection [41, 47]. Occurrences of such as endotoxins in the bacteriophage preparations may aggravate



**Table**

**1.**

the immune system responses viz. fever, leucocytosis, leukopenia, fatal endotoxin shock, can open up macrophages, and release inflammatory mediators such as TNFα, IL-6, and IL-1 and cause serious side effects. The final limit of endotoxins recommended for intravenous administration is 5 Endotoxin Unit (100 pg) (EU)/ kg. The elimination of endotoxin from bacteriophages is a multidisciplinary procedure. Two-Phase fluid extraction processes viz. LPS affinity resins, ultrafiltration, and chromatographic methods for removal of well-charged endotoxin proteins. Ion exchange, size exclusion chromatography, interaction with histidine or polymyxin B, and anion-exchange chromatographic exchange were methods used for further phage purification. Diafiltration was used to exchange phage particles from lysate media with a suitable buffer. Cesium chloride density gradient centrifugation, ultracentrifugation, PEG precipitation, and ultrafiltration used for the removal of endotoxins and purification of phages. Bacteriophage CM8-1/SJT-2 stock solution mixed with bacterial culture in the mid-log phase spread on a double-layer agar Petri plate, Sodium magnesium (SM) buffer added after the bacteriophage had grown of the entire Petri plate and placed on a shaker at 120 rpm/min for 2 hours. The SM-bacteriophage lysate solution was filtered through a 0.22 μm filter, PEG8000 (10%) was once added to the bacteriophage stock solution, left the solution overnight at 4°C, and centrifuged at 10,000 g for 10 min to obtain bacteriophage precipitation [46]. By contrast, T4 bacteriophages had prepared by using a stepwise gradient of anion-exchange quaternary amine (QA) CIM column and NaCl elution buffer [48, 49]. Purified bacteriophages of *Mycobacterium smegmatis* and *S. aureus* were prepared by using columns such as QA CIM and diethylamine (DEAE) while QA and DEAE CIM columns, were employed to remove endotoxins from pre-purified phage preparations by using the Endotrap HD column (Cambrex BioScience, EndoTraptm Blue) [50]. Enterococcal bacteriophages, viz. ENB6 and C33 were prepared from the raw wastewater by using caesium chloride density gradient centrifugation and stored at 4°C. Thus, there was a great deal of variation in the elution conditions between the different phages. A common operating procedure for varied phage preparations, storage, and transport is lacking [51]. Standard operating procedure (s) for large scale-bacteriophage cultivation, isolation, titration, and purification and to produce sufficient plaqueforming units of bacteriophages (PFUs) per milliliter of *Pseudomonas, Klebsiella, and Serratia* were established [41, 52]. Such a universal process and production of the final phage preparations for use could reduce endotoxins, might be pivotal in alleviating fears and the phage therapy shall be readily accepted all the world over.
