1.1. Phage Isolation and Enrichment

The key processes in phage therapy protocols are phage selection and isolation. The wrong choices can have fatal consequences ^[1]. Generally, two methods are used when choosing the appropriate phage for therapy: (1) A phage cocktail, such as Pyophage and Intestiphage. These preparations have a broader spectrum of activity than a single phage component and do not allow resistance to develop within a short time. (2) A pathogen-specific phage. Bacteria are isolated from the infection and tested for susceptibility to particular phages isolated previously ^[1].

The key processes in phage therapy protocols are phage selection and isolation. The wrong choices can have fatal consequences [11]. Generally, two methods are used when choosing the appropriate phage for therapy: (1) A phage cocktail, such as Pyophage and Intestiphage. These preparations have a broader spectrum of activity than a single phage component and do not allow resistance to develop within a short time. (2) A pathogen-specific phage. Bacteria are isolated from the infection and tested for susceptibility to particular phages isolated previously [11].

Samples for phage isolation are taken from environments where the bacterial host can often be found, including soil, plant residues, fecal matter, wastewater and sewage (

Figure 1). Phages against 

2). Phages against 

Shigella dysenteriae 2308 were isolated from the New York City sewage by Dubos et al. ^[2]. B_VpS_BA3 and vB_VpS_CA8 phages against 

 2308 were isolated from the New York City sewage by Dubos et al. [12]. B_VpS_BA3 and vB_VpS_CA8 phages against 

Vibrio parahaemolyticus were isolated from sewage collected in China ^[3]. The vB_KpnP_IME337 phage against carbapenem-resistant 

 were isolated from sewage collected in China [13]. The vB_KpnP_IME337 phage against carbapenem-resistant 

Klebsiella pneumoniae was isolated from hospital sewage in China ^[4]. Li et al. ^[5] isolated 54 novel phages against the same organism from medical and domestic sewage wastewater. The newly isolated phage P545 had a relatively wide host range and strong antibacterial activity.

 was isolated from hospital sewage in China [14]. Li et al. [15] isolated 54 novel phages against the same organism from medical and domestic sewage wastewater. The newly isolated phage P545 had a relatively wide host range and strong antibacterial activity.

Figure 12.

 Stages of phage preparation. The environment (e.g., wastewater, farms and soil) is a source for all types of phages. The presence of phages in the tested sample is determined by different methods such as the double layer agar method, spot assays, the colorimetric method, the enrichment method or electron microscopy. The plaques that indicate lytic activity are picked up and transferred for the determination of phage type, specificity, etc. A phage lysate is prepared. At this stage, multiple procedures are performed to check for sterility (microbial contamination), toxicity (bacterial endotoxin or lipopolysaccharide (LPS) quantification), bacterial DNA contamination and phage titer. The purified phage preparation is stored.

Although there are differences in phage isolation, the basic principle of the methods is the same as that developed by d’Herelle, and they are generally characterized as enrichment procedures ^[6]. First, the presence of phages is detected in the collected sample. Selection of the bacterial host is vital for the isolation of a phage in a recently acquired sample from the environment. Solid samples are mixed with sterile broth or buffer and then subjected to centrifugation and filtration ^[7]. Bacteria of interest are incubated overnight with the environmental sample. The bacteria that have survived the attack of the lytic phages are removed from the mixture by centrifugation or filtration, or both. The presence of phages in the filtrate is then assessed by plaque assay or by qPCR. The isolated phages have to be analyzed for their virulence, i.e., their ability to lyse target bacteria and the range of bacterial types they are able to infect. In an alternate method, samples from the environment are plated directly onto a lawn of particular bacteria and the presence of plaques resulting from bacterial lysis is detected. The latter method has been used to discover phages that lyse 

Although there are differences in phage isolation, the basic principle of the methods is the same as that developed by d’Herelle, and they are generally characterized as enrichment procedures [16]. First, the presence of phages is detected in the collected sample. Selection of the bacterial host is vital for the isolation of a phage in a recently acquired sample from the environment. Solid samples are mixed with sterile broth or buffer and then subjected to centrifugation and filtration [17]. Bacteria of interest are incubated overnight with the environmental sample. The bacteria that have survived the attack of the lytic phages are removed from the mixture by centrifugation or filtration, or both. The presence of phages in the filtrate is then assessed by plaque assay or by qPCR. The isolated phages have to be analyzed for their virulence, i.e., their ability to lyse target bacteria and the range of bacterial types they are able to infect. In an alternate method, samples from the environment are plated directly onto a lawn of particular bacteria and the presence of plaques resulting from bacterial lysis is detected. The latter method has been used to discover phages that lyse 

Escherichia coli and various bacteria from dental plaque and the oral cavity ^[7][8].

 and various bacteria from dental plaque and the oral cavity [17,18].

In the procedure described in detail by Luong et al. ^[9], the target bacterial strain is isolated and incubated with the phages. Then, after several agar plaque isolations, a single plaque is cultivated overnight. The isolated phage genome is sequenced to screen and identify lysogenic and deleterious genes. Phages are grown at liter scale, and the lysate is purified to eliminate any bacteria and cellular debris by pressure-driven filtration through filters of 0.8-, 0.45- and 0.22-µm pore size, followed by cross-flow ultra-filtration to eliminate debris smaller than 100 kD. This process eliminates endotoxin, exotoxins, peptidoglycan, nucleic acids and flagella. The phage particles are purified by CsCl density gradient centrifugation and dialysis to eliminate the CsCl. Any residual endotoxin molecules are removed by lipopolysaccharide-affinity chromatography. The last step ensures that the phage preparations do not cause inflammation or endotoxic shock when administered to patients ^[9].

In the procedure described in detail by Luong et al. [19], the target bacterial strain is isolated and incubated with the phages. Then, after several agar plaque isolations, a single plaque is cultivated overnight. The isolated phage genome is sequenced to screen and identify lysogenic and deleterious genes. Phages are grown at liter scale, and the lysate is purified to eliminate any bacteria and cellular debris by pressure-driven filtration through filters of 0.8-, 0.45- and 0.22-µm pore size, followed by cross-flow ultra-filtration to eliminate debris smaller than 100 kD. This process eliminates endotoxin, exotoxins, peptidoglycan, nucleic acids and flagella. The phage particles are purified by CsCl density gradient centrifugation and dialysis to eliminate the CsCl. Any residual endotoxin molecules are removed by lipopolysaccharide-affinity chromatography. The last step ensures that the phage preparations do not cause inflammation or endotoxic shock when administered to patients [19].

Phages are expected to be found where the host bacteria reside. For example, phages that infect intestinal bacteria can be isolated from fecal material, and phages against epidermal bacteria such as 

Staphylococcus aureus

 are most likely isolated from skin samples or wound exudates. Identifying a phage against a particular bacterium is not straightforward, however. Whereas phages that lyse antibiotic-resistant 

Klebsiella pneumoniae

 and 

Pseudomonas aeruginosa

 were readily isolated from sewage samples, phages against antibiotic-resistant 

Acinetobacter baumannii were not found as frequently ^[10]. Furthermore, phages against methicillin-resistant 

were not found as frequently [20]. Furthermore, phages against methicillin-resistant 

Staphylococcus aureus

 were identified only rarely.

The choice of a host for phage isolation may also depend on the ease of culturing the bacteria, as in the use of 

Mycobacterium smegmatis

 to isolate phages that will infect other 

Mycobacterium

 species. 

M. smegmatis

 grows much faster than 

M. tuberculosis and thus can produce a lawn on the appropriate agar surface for testing phage activity ^[7][10][11]. The isolated phages would then be tested further on the specific target 

 and thus can produce a lawn on the appropriate agar surface for testing phage activity [17,20,21]. The isolated phages would then be tested further on the specific target 

Mycobacterium

 species.

Swanstrom et al. ^[12] investigated the variables contributing to the generation of high-titer phage stocks, using the agar layer method and coli phage T4r. The numbers of virus particles and bacteria per plate, the incubation period, the amount of soft agar in the agar layer as well as the broth volume used for virus extraction from the agar were found to be significant factors. When these factors were optimized, stock concentrations in the range of 10

Swanstrom et al. [22] investigated the variables contributing to the generation of high-titer phage stocks, using the agar layer method and coli phage T4r. The numbers of virus particles and bacteria per plate, the incubation period, the amount of soft agar in the agar layer as well as the broth volume used for virus extraction from the agar were found to be significant factors. When these factors were optimized, stock concentrations in the range of 10

¹¹

–10

¹² infectious particles/mL could be obtained ^[12].

 infectious particles/mL could be obtained [22].

Echeverría-Vega et al. ^[13] used a straightforward protocol for the isolation of bacteriophages from coastal organisms. They also validated the protocol for the isolation of lytic bacteriophages for the fish pathogen bacterium 

Echeverría-Vega et al. [23] used a straightforward protocol for the isolation of bacteriophages from coastal organisms. They also validated the protocol for the isolation of lytic bacteriophages for the fish pathogen bacterium 

Vibrio ordalii. This method has particular utility for the recovery of bacteriophages for use as natural antimicrobial agents in aquacultures. In the enrichment method, samples are added to the host produced in a suitable medium, incubated and then centrifuged. The suspension containing the phage is filtered and applied at different concentrations onto an agar medium with target bacteria. The formed plaques are then counted. Thanks to the enrichment method, phages at low concentration can reach the desired level in culture. Enrichment is an advantageous method in cases where the amount of phages is low ^[14]. Numerous lytic phages were isolated against 

. This method has particular utility for the recovery of bacteriophages for use as natural antimicrobial agents in aquacultures. In the enrichment method, samples are added to the host produced in a suitable medium, incubated and then centrifuged. The suspension containing the phage is filtered and applied at different concentrations onto an agar medium with target bacteria. The formed plaques are then counted. Thanks to the enrichment method, phages at low concentration can reach the desired level in culture. Enrichment is an advantageous method in cases where the amount of phages is low [24]. Numerous lytic phages were isolated against 

Caulobacter

 and 

Asticcacaulis bacteria using the enrichment method ^[15]. Methods such as spot testing, plaque testing, culture lysis and the calorimetric method are used in the detection of newly isolated bacteriophages (

 bacteria using the enrichment method [25]. Methods such as spot testing, plaque testing, culture lysis and the calorimetric method are used in the detection of newly isolated bacteriophages (

Figure 2) ^{[16][17][18][19][20][21]}. In the spot assay ^[16], bacteria are grown in Luria-Bertani broth, and after they are in the early log phase, they are mixed with soft agar and poured onto a Petri plate with previously poured agar. A phage filtrate is then placed on the soft agar and the plates are incubated overnight at 37 °C, after which bacterial lysis zones are counted ^[18]. In the double layer agar method, a bacterial culture in the log phase is mixed with a purified phage preparation and incubated briefly to allow for phage adsorption. This mixture is combined with soft agar and poured onto a previously solidified agar layer to form a homogeneous layer. After incubation at 37 °C for 24 h, plaque formation is observed, indicating phage activity. The plaques are resuspended in Mg (SM) buffer ^[18].

) [26,27,28,29,30,31]. In the spot assay [26], bacteria are grown in Luria-Bertani broth, and after they are in the early log phase, they are mixed with soft agar and poured onto a Petri plate with previously poured agar. A phage filtrate is then placed on the soft agar and the plates are incubated overnight at 37 °C, after which bacterial lysis zones are counted [28]. In the double layer agar method, a bacterial culture in the log phase is mixed with a purified phage preparation and incubated briefly to allow for phage adsorption. This mixture is combined with soft agar and poured onto a previously solidified agar layer to form a homogeneous layer. After incubation at 37 °C for 24 h, plaque formation is observed, indicating phage activity. The plaques are resuspended in Mg (SM) buffer [28].

1.2. Phage Production

Bacteriophages need a host cell to reproduce. Understanding the interactions between host bacteria and bacteriophages is a crucial step in estimating the risks in production, including possible mutations in either microorganism ^[22]. The production process may also be affected by the nutrient composition, oxygenation, temperature and pH ^[23].

Bacteriophages need a host cell to reproduce. Understanding the interactions between host bacteria and bacteriophages is a crucial step in estimating the risks in production, including possible mutations in either microorganism [32]. The production process may also be affected by the nutrient composition, oxygenation, temperature and pH [33].

The substrate and temperature chosen for phage infection and bacterial growth are important factors. Fermentation is an important stage for host bacteria to multiply and produce bacteriophages. The sterilization step is performed to destroy undesired microorganisms. The bioreactor can be sterilized with heat, medium or a combination of these. During the fermentation process, the injected air is filtered through an in-line membrane. The air released after fermentation is filtered after it condenses ^[24].

The substrate and temperature chosen for phage infection and bacterial growth are important factors. Fermentation is an important stage for host bacteria to multiply and produce bacteriophages. The sterilization step is performed to destroy undesired microorganisms. The bioreactor can be sterilized with heat, medium or a combination of these. During the fermentation process, the injected air is filtered through an in-line membrane. The air released after fermentation is filtered after it condenses [34].

Phages are grown basically in shaker flasks or stirred tank bioreactors. The latter are used to carry out industrial-scale production of bacteriophages, which has been divided into three different systems: batch, semi-continuous and continuous ^[25]. Each system has brought about its distinct benefits and drawbacks, discussed earlier by Merabishvili et al. ^[26]. Mancuso et al. ^[27] developed a production process that makes it possible to obtain high titers of 

Phages are grown basically in shaker flasks or stirred tank bioreactors. The latter are used to carry out industrial-scale production of bacteriophages, which has been divided into three different systems: batch, semi-continuous and continuous [35]. Each system has brought about its distinct benefits and drawbacks, discussed earlier by Merabishvili et al. [36]. Mancuso et al. [37] developed a production process that makes it possible to obtain high titers of 

E. coli

 T3 phages at high concentrations (10

¹¹

 PFU mL

⁻¹

) using two continuous stirred tank bioreactors. The first bioreactor is just for propagation of the host bacteria at a steady-state growth rate by using controllable dilution rates and growth-limiting substrate (glucose). The second bioreactor is used for bacteriophage production and is fed from the host bacteria of the first bioreactor. Besides achieving high phage productivity of bacteriophages via the production process, the mutation risk of the host bacteria potentially caused by bacteriophages is suppressed.

1.3. Phage Purification and Quality Control

For the pharmaceutical application of phages, it is necessary to first carry out the purification process. The bacteriophage of interest is separated from host bacteria cells and debris by centrifugation, microfiltration or by using these methods together. The potential presence of any toxins in the preparations would be detrimental to the final pharmaceutical product. A Chamberland filter of 0.1–1 µm was used for bacteriophage preparations to be used in human trials ^[28]. It was recently clarified with a 0.2-μm filter pore size. Purification procedures of phages should follow the Critical Quality Attributes (CQA) specification ^[24]. The process of removing endotoxins from phages is complex because lipopolysaccharide forms micelles that have approximately the same size as phages. Therefore, extra purification methods such as ion exchange, affinity chromatography and solvent extraction are needed for lysates of phage-infected Gram-negative bacteria ^[23].

For the pharmaceutical application of phages, it is necessary to first carry out the purification process. The bacteriophage of interest is separated from host bacteria cells and debris by centrifugation, microfiltration or by using these methods together. The potential presence of any toxins in the preparations would be detrimental to the final pharmaceutical product. A Chamberland filter of 0.1–1 µm was used for bacteriophage preparations to be used in human trials [38]. It was recently clarified with a 0.2-μm filter pore size. Purification procedures of phages should follow the Critical Quality Attributes (CQA) specification [34]. The process of removing endotoxins from phages is complex because lipopolysaccharide forms micelles that have approximately the same size as phages. Therefore, extra purification methods such as ion exchange, affinity chromatography and solvent extraction are needed for lysates of phage-infected Gram-negative bacteria [33].

Endotoxin. In bacteriophage products, endotoxin measurement is critical. Gel clot, turbidimetric and chromatic methods are used for endotoxin determination in bacteriophage products. The 

Limulus amoebocyte lysate (LAL) assay is the most commonly used method ^[29].

 amoebocyte lysate (LAL) assay is the most commonly used method [39].

Transmission electron microscopy (TEM). The specific morphology of phages in a final product can be viewed by transmission electron microscopy. Merabishvili et al. ^[30] used TEM for confirmation of the presence of the expected virion morphologic particles as well as their specific interaction with the target bacteria.

Transmission electron microscopy (TEM). The specific morphology of phages in a final product can be viewed by transmission electron microscopy. Merabishvili et al. [40] used TEM for confirmation of the presence of the expected virion morphologic particles as well as their specific interaction with the target bacteria.

Titer. The process of determining phage concentration by dilution and plating with susceptible cells is called titering or the plaque assay. A bacteriophage capable of productively infecting a cell is named a plaque-forming unit (PFU/mL) ^[31].

Titer. The process of determining phage concentration by dilution and plating with susceptible cells is called titering or the plaque assay. A bacteriophage capable of productively infecting a cell is named a plaque-forming unit (PFU/mL) [41].

pH. In a therapeutic formulation, the pH value is very important. According to the European Pharmacopoeia, the pH should be in the range 6.0–8.0 ^[32].

pH. In a therapeutic formulation, the pH value is very important. According to the European Pharmacopoeia, the pH should be in the range 6.0–8.0 [42].

Nucleic Acid Contaminants. Because phages break down bacterial DNA, the presence and concentration of nucleic acid residues in final products should be determined. qPCR can be used for this purpose ^[23].

Nucleic Acid Contaminants. Because phages break down bacterial DNA, the presence and concentration of nucleic acid residues in final products should be determined. qPCR can be used for this purpose [33].

1.4. Phage Stability and Storage Conditions

Once solutions of phages are prepared, the biological properties of the phages have to be preserved during storage. Freeze-drying, spray-drying or encapsulation methods can be used to increase phage stability, as well as adding stabilizing additives to their solutions ^{[33][34][35][36]}. The quality, safety and storage conditions of phages to be prepared for use in treatment should be validated ^[37]. González-Menéndez et al. ^[38] investigated different preservation techniques for the storage of 

Once solutions of phages are prepared, the biological properties of the phages have to be preserved during storage. Freeze-drying, spray-drying or encapsulation methods can be used to increase phage stability, as well as adding stabilizing additives to their solutions [43,44,45,46]. The quality, safety and storage conditions of phages to be prepared for use in treatment should be validated [47]. González-Menéndez et al. [48] investigated different preservation techniques for the storage of 

Staphylococcus phages (phiIPLA88, phiIPLA35, phiIPLA-RODI and phiIPLA-C1C). They evaluated the stability of phages at different temperatures (−20, −80 and −196 °C) and time periods (1, 6, 12 and 24 months). They also investigated various stabilization enhancing agents, including disaccharides, glycerol, sorbitol and skim milk. They showed that at −80 and −196 °C, all phages showed good viability after 24 months, regardless of the stabilizer ^[38].

 phages (phiIPLA88, phiIPLA35, phiIPLA-RODI and phiIPLA-C1C). They evaluated the stability of phages at different temperatures (−20, −80 and −196 °C) and time periods (1, 6, 12 and 24 months). They also investigated various stabilization enhancing agents, including disaccharides, glycerol, sorbitol and skim milk. They showed that at −80 and −196 °C, all phages showed good viability after 24 months, regardless of the stabilizer [48].

1.5. Therapeutic Phages

Hyman et al. ^[7] proposed the following characteristics of phages to be used for therapeutic purposes: (a) The phage should be virulent and be able to cause complete cytotoxicity to the target bacterium. (b) It should be exclusively lytic and should not become temperate (i.e., lysogenic). (c) The phage should have the potential to transduce the host bacteria. (d) It should have the desired host range. (e) It should be screened for toxin genes that can affect the patient. 

Hyman et al. [17] proposed the following characteristics of phages to be used for therapeutic purposes: (a) The phage should be virulent and be able to cause complete cytotoxicity to the target bacterium. (b) It should be exclusively lytic and should not become temperate (i.e., lysogenic). (c) The phage should have the potential to transduce the host bacteria. (d) It should have the desired host range. (e) It should be screened for toxin genes that can affect the patient. 

Myoviridae, Siphoviridae and Podoviridae families are used commonly for phage therapy ^[39][40]. There are approximately 800 phages against pathogens such as 

 families are used commonly for phage therapy [49,50]. There are approximately 800 phages against pathogens such as 

Escherichia

, 

Morganella

, 

Klebsiella

, 

Enterococcus

, 

Pseudomonas

, 

Staphylococcus

 and 

Salmonella ^[21].

 [31].