**1.1 Market potential of therapeutic bасteriорhаges**

Bacteriophages are found all over the world, have many uses, and have contributed significantly to medicine, biotechnology, and molecular biology. Traditional antibiotic treatments are often replaced or supplemented by bacteriophage therapies and such alternative therapies has had a significant effect on revenues. In 2017, the global bacteriophage market was worth \$567.9 million, and it is projected to grow at a 3.9% annual rate annual rate from 2018 to 2026. Globally, 600 million people are believed to be affected by foodborne diseases, with 420,000 people dying each year. In contrast, foodborne disease is said to affect 40% of children, resulting in 125,000 deaths per year [24]. The fastest-growing market will be for clinical applications of bacteriophages in phage therapy, diagnostics, drug development and manufacturing, phage display technology, antibacterial, vaccines, and biocontrol agents. Food and beverages currently hold the largest share of the global bacteriophage industry. Lytic bacteriophages are commonly used to control the spread of harmful infectious agents in foods such as fruits, vegetables, dairy products, and meals. Increased use of bacteriophages in such safe and healthy food items increased market potential. As a result, bacteriophages are being accepted for use in food safety applications in greater numbers. Companies are developing bacteriophage platforms and phagebanks (The Israeli Phage Bank (IPB) is a member of a global network of phage banks that provides a large assortment of purified bacteriophages) to treat multidrug-resistant bacteria in emergency situations [24]. Microgen, Amplify Bioscience Corporation, Ambiotics, and Phage Biotech Ltd. are

some of the leading players in the bacteriophage industry. According to Amplify Biosciences Corporation, clinical trials for phage therapy against *Pseudomonas aeruginosa* infection in cystic fibrosis have begun in the United States [24].

### **1.2 Isolation and identification of bacteriophages**

Seclusion, identification and propagation of the patient's infecting bacterial strain are critical for successful phage treatment. In medical practice, once a patient is suspected of having a contagious incurable infection, effective bacteriophages should be isolated, identified and purified from isolates of pathogenic bacteria occurring in the samples of urine, blood, and chronic wounds of patients. The bacterial colonies shall be picked up from selective Agar/LB agar plates according to their colony morphology, size**,** and pigmentation variability**.** The isolates are subjected to the staining procedures, biochemical and molecular tests and are cultured in various media to identify the genus and species of bacteria with the help of *Bergey's Manual of Determinative Bacteriology* and Bergey's manual of systematic bacteriology [25, 26]. Each of the bacterial isolates shall be transferred to LB broth at 37°C for 18 hours and then be stored at 20°C after the addition of 20% glycerol for further studies. With the development of diagnostic techniques, nonculturemethods such as 16S rRNA, PCR, RT-PCR, microarray, DNA/RNA sequencing, proteomics, ELISA and immunological methods and MALDI-TOP MS are used in clinical laboratories for microbial testing, identification and classification [1, 26–29]. If patients are opting for phage treatment, the foremost step is to isolate the diseasecausing pathogenic bacteria using traditional methods. Subsequently the pathogens can be identified by using non-traditional methods. Bacteriophages uninfected host cells of bacteria multiply in Nutrient/LB agar plate to form a confluent film of bacterial growth over the surface of the plate at 37°C. In contrast, bacteriophages of infected cells of pathogenic bacterium if occur, bursts of such cells take place and release offspring bacteriophages. A visible, circular area of clearing zone in the confluent bacterial growth is known as a plaque, occurring after 8-10 hours of incubation and halos, zones of secondary lysis around plaques, can be identified after 24 hours. A suspension consisting of incubated samples of phage and cells of bacterial isolates shall be poured on to an appropriate LB/Nutrient agar medium to form a thin 'top layer'. Lastly, sensitivity and specificity of phage to the pathogenic bacteria is tested therapeutically. A key option in the treatment of infection is to use standard antibacterial drug therapy based on an anti-bacterial profile and / or physician experience [27]. Therefore, phage therapy will only be recommended if antibacterial drugs are unsuccessful and/or as soon as the infection is triggered by multidrug-resistant or pandrug-resistant bacteria.
