**2.5 Phage therapy**

Phage therapy, which is also termed viral phage therapy, is the utilization of bacteriophages as medicaments for controlling and treating diseases brought by pathogenic bacterial infections [145]. Bacteriophages, like other viruses, are obligate intracellular parasites that utilize the enzymatic machinery of their hosts for establishing their physiological functions and replication [131]. The hosts for bacteriophages are bacteria, and phages have unique ability to specifically infect bacterial hosts resulting in their lysis [8].


#### **Table 1.**

*Bacteriocins produced from different sources and exhibit antimicrobial and antibiofilm activity.*

**201**

*New Approaches for Competing Microbial Resistance and Virulence*

There are many conceivable usages for phage therapy in the treatment of crucial diseases in plants, animals as well as human [8, 145]. An outstanding advantage of utilizing bacteriophages over commonly used antibiotics, during treating infectious diseases, is their selectivity and specificity to infect and lyse infectious bacteria only without harming the host [9]. Besides, bacteriophages cause no harm to other organisms that live in a commensalism within hosts, such as the normal flora in human, which decreases significantly the incidence of superinfections or other opportunistic infections [10]. Moreover, due to their mode of action that phages replicate *in vivo* within their bacterial hosts, they can be used in modicum concentrations, which results in decreasing any side effects may rise during therapy and giving them a high therapeutic index [9, 10]. In addition, the capability of bacteriophages to penetrate bacterial biofilms that act as shields during the conventional antibiotic therapy, gives phages a superiority in controlling and treating diseases brought by pathogenic bacterial infections [146]. As living organisms, the capability of bacteriophages of continuous evolution, gives them the ability to overcome any resistance that can be developed by the evolution of pathogenic bacteria [146, 147]. All these tremendous advantages put the bacteriophage treatment as a superior alternative for treating diseases brought about multidrug resistant MDR bacterial pathogens [132]. On the other hand, the high bacterial host specificity of bacteriophages is encountered as a disadvantage during therapy, where, a phage can kill only its specific bacterial strain. However, this drawback can be solved by utilizing mixtures of bacteriophages, which is termed phage cocktails that have different pathogenic specific bacterial hosts as targets, to enhance the opportunities of unguis complete treatment [148]. Attention must be given, during the preparation of these cocktails, to the fact of continuous evolution of new MDR strains, so the cocktails must be updated periodically to be sufficient enough to treat infections

Historically, the first trials for the utilization of bacteriophages as medicaments

It has been estimated that there are more than 100 different phage species and at least 10 phages for each bacterium. The International Committee for the Taxonomy of Viruses (ICTV) was affirmed at 1971 with the objective to always bring to date the taxonomic guidelines of viruses. The ICTV classified tailed bacteriophages (bacterial infecting phages) under the order of viruses which is termed *Caudovirales*. In this respect, three main families are involved within this order named *Siphoviridae*, *Myoviridae* and *Podoviridae*. The main difference between bacteriophages belonging to each of these families is the characteristics of the tail. Phages under the *Siphoviridae* family have long and non-contractile tails, and those belong to *Myoviridae* family have long and contractile tails, while those belong to the *Podoviridae* family have short, stubbed tails and a striking lack of features. Each

of these three families can also be divided into different genera [8].

for treating bacterial pathogens was reported in the Eastern world before the discovery of marvelous medicaments so-called antibiotics; however, there was any report of their usage in the Western world [150, 151]. The ability of bacteriophages to infect and lyse pathogenic bacteria was discovered by the scientists Frederick Twort and Felix D'Hérelle, who worked on *Shigella dysenteriae* [152]. They found that the cultures of stool specimens recovered from convalescent patients who were suffering from *Shigella* dysentery always depicting a high titer of phages [153]. Subsequently, they recorded that phages are the most abundant organisms in the environment and there are many sources where they can be found combined with their bacterial hosts; including gut and feces of convalescent patients as well as sewages [153]. Thereafter, due to their ubiquity especially in sewages, bacteriophages were widely utilized as medicaments for controlling and eradication of diseases

*DOI: http://dx.doi.org/10.5772/intechopen.90388*

brought by these strains [148, 149].

brought by pathogenic bacteria [8].

#### *New Approaches for Competing Microbial Resistance and Virulence DOI: http://dx.doi.org/10.5772/intechopen.90388*

*Microorganisms*

diterpene [133, 134].

**2.4 Bacteriocins**

marized in **Table 1**.

**2.5 Phage therapy**

bacterial hosts resulting in their lysis [8].

Nisin A *Lactococcus lactis* subsp. lactis

*gallinarum*

*Staphylococcus epidermidis*

*amyloliquefaciens* GA1

*philippinensis* MA7347

Sonorensin *Bacillus sonorensis* MT93 *Listeria monocytogenes* and

Gallidermin *Staphylococcus* 

Amylolysin *Bacillus* 

Philipimycin *Actinoplanes* 

Epidermicin NI0

Guaijaverin, a flavonoid obtained from the leaves of *Psidium guajava*, has been shown to prevent the attachment of *S. mutans* to smooth surfaces by 83.7% at a concentration of 500 μg/mL. Eembelin, which is isolated from *Embelia ribes*, has been shown to inhibit biofilm formation in *S. mutans* [130]. Macelignan, isolated from the nutmegs of *Myristica fragrans*, was shown to reduce more than 50% of *S.* 

Terpenes are a large class of natural hydrocarbons that are synthesized in microorganisms, plants and animals. Bakuchiol, isolated from the seeds of *Psoralea corylifolia*, has been shown to inhibit the adherence of *S. mutans* [132]. Other examples for terpenes that inhibit biofilm formation in *S. mutans*, are Xanthorrhizol (in combination with chlorhexidine gluconate) and casbane

Bacteriocins are proteins or peptides that are produced by bacteria or archaea, and are usually active against strains of bacteria that are related or unrelated to the producer strain [135]. Several bacteriocins are reported to exhibit antibiofilm activity and/or antimicrobial activity. The results of some these reports are sum-

Phage therapy, which is also termed viral phage therapy, is the utilization of bacteriophages as medicaments for controlling and treating diseases brought by pathogenic bacterial infections [145]. Bacteriophages, like other viruses, are obligate intracellular parasites that utilize the enzymatic machinery of their hosts for establishing their physiological functions and replication [131]. The hosts for bacteriophages are bacteria, and phages have unique ability to specifically infect

**Bacteriocin Source Antimicrobial activity Antibiofilm activity** Mutacin 1140 *Streptococcus mutans* Oral biofilm-associated

> *Enterococcus faecalis* and *Streptococcus gordonii* [137]

> *Staphylococcus aureus* [141]

*Listeria monocytogenes*, *Staphylococcus aureus* and *Staphylococcus epidermidis* [143]

MRSA [144]

*Bacteriocins produced from different sources and exhibit antimicrobial and antibiofilm activity.*

MRSA, *Enterococci* [142] *Staphylococcus* 

with *Streptococcus sobrinus*, *Streptococcus oralis* [136]

*Listeria monocytogenes* [138]

*Staphylococcus aureus* and *Staphylococcus epidermidis*

*Staphylococcus aureus* [141]

[139, 140]

*epidermidis* [142]

*mutans* biofilm at a concentration of 10 μg/mL [131].

**200**

**Table 1.**

There are many conceivable usages for phage therapy in the treatment of crucial diseases in plants, animals as well as human [8, 145]. An outstanding advantage of utilizing bacteriophages over commonly used antibiotics, during treating infectious diseases, is their selectivity and specificity to infect and lyse infectious bacteria only without harming the host [9]. Besides, bacteriophages cause no harm to other organisms that live in a commensalism within hosts, such as the normal flora in human, which decreases significantly the incidence of superinfections or other opportunistic infections [10]. Moreover, due to their mode of action that phages replicate *in vivo* within their bacterial hosts, they can be used in modicum concentrations, which results in decreasing any side effects may rise during therapy and giving them a high therapeutic index [9, 10]. In addition, the capability of bacteriophages to penetrate bacterial biofilms that act as shields during the conventional antibiotic therapy, gives phages a superiority in controlling and treating diseases brought by pathogenic bacterial infections [146]. As living organisms, the capability of bacteriophages of continuous evolution, gives them the ability to overcome any resistance that can be developed by the evolution of pathogenic bacteria [146, 147]. All these tremendous advantages put the bacteriophage treatment as a superior alternative for treating diseases brought about multidrug resistant MDR bacterial pathogens [132]. On the other hand, the high bacterial host specificity of bacteriophages is encountered as a disadvantage during therapy, where, a phage can kill only its specific bacterial strain. However, this drawback can be solved by utilizing mixtures of bacteriophages, which is termed phage cocktails that have different pathogenic specific bacterial hosts as targets, to enhance the opportunities of unguis complete treatment [148]. Attention must be given, during the preparation of these cocktails, to the fact of continuous evolution of new MDR strains, so the cocktails must be updated periodically to be sufficient enough to treat infections brought by these strains [148, 149].

Historically, the first trials for the utilization of bacteriophages as medicaments for treating bacterial pathogens was reported in the Eastern world before the discovery of marvelous medicaments so-called antibiotics; however, there was any report of their usage in the Western world [150, 151]. The ability of bacteriophages to infect and lyse pathogenic bacteria was discovered by the scientists Frederick Twort and Felix D'Hérelle, who worked on *Shigella dysenteriae* [152]. They found that the cultures of stool specimens recovered from convalescent patients who were suffering from *Shigella* dysentery always depicting a high titer of phages [153]. Subsequently, they recorded that phages are the most abundant organisms in the environment and there are many sources where they can be found combined with their bacterial hosts; including gut and feces of convalescent patients as well as sewages [153]. Thereafter, due to their ubiquity especially in sewages, bacteriophages were widely utilized as medicaments for controlling and eradication of diseases brought by pathogenic bacteria [8].

It has been estimated that there are more than 100 different phage species and at least 10 phages for each bacterium. The International Committee for the Taxonomy of Viruses (ICTV) was affirmed at 1971 with the objective to always bring to date the taxonomic guidelines of viruses. The ICTV classified tailed bacteriophages (bacterial infecting phages) under the order of viruses which is termed *Caudovirales*. In this respect, three main families are involved within this order named *Siphoviridae*, *Myoviridae* and *Podoviridae*. The main difference between bacteriophages belonging to each of these families is the characteristics of the tail. Phages under the *Siphoviridae* family have long and non-contractile tails, and those belong to *Myoviridae* family have long and contractile tails, while those belong to the *Podoviridae* family have short, stubbed tails and a striking lack of features. Each of these three families can also be divided into different genera [8].

Compared with antibiotics and other therapeutic regimens, the steps and cost of production of bacteriophages are much easier and cheaper, respectively [10]. The easiest process for capturing of bacteriophages is done through collecting samples that seem to involve high titers of phages like sewage water samples. The collected samples are inoculated with the host bacterium, which seems to be infected by phages, on suitable growth medium. The successful isolation of certain lytic phage is depicted by the presence of clear inhibition zones in which bacteria cannot grow termed plaques; which indicates the lytic power of the isolated phage. Thereafter, the titer of isolated phage is increased by passing the phage in its specific bacterial strain several times to increase its concentration. Then, the pure supernatants containing phages are gained by centrifugation of bacterial/phage mixture, filtered through bacterial filters to remove any bacterial debris and pure phages are participated using special solutions containing NaCl and polyethylene glycol 8000 (PEG8000) [154].

Caution must be given during isolation of phages as a type called lysogenic bacteriophage may be isolated rather than the required bacterial pathogen killing type, which is called lytic bacteriophage. Lysogenic bacteriophages do not lyse bacterial cells, but they perform as tools for transfer of genetic elements of the nucleic acid between bacteria; including the genes responsible for antibiotic resistance. Fortunately, the most abundant phages are of the lytic type not the lysogenic [8, 145, 150].

Practically, bacteriophages can be dispensed and used through many routes including; less commonly oral or systemic route and most commonly topical route as sprays, liquid solutions or their application on surgical dressings for the treatment of wound infections [154]. The possibility of their clearance during the presence in blood stream by immune system or presence of any trace hazards of chemicals or parts of the bacterial host used during their production, made bacteriophage usage as intravenous injections uncommon and very rare [148, 149]. Lyophilization of bacteriophages and their production as solid dosage forms as pills or tablets do not decrease their potency and increase their shelf life as oral dosage forms [155, 156]. The supplementation of oral forms of phages, either solid or liquid, with antacid increases its stability, as it protect them from the high acidity during their bypassing in the stomach [155, 156].

The application of bacteriophages as therapeutic medicaments has been extensively reported. For example, in the field of human health promotion and food protection, different bacteriophages have been employed to eradicate common bacterial pathogens that may cause food spoilage as *Listeria* sp. and *Campylobacter* sp. [157, 158]. In the fields of veterinary medicine and agriculture different bacteriophages were employed to control and eradicate bacterial pathogens like *Xanthomonas*, *Escherichia*, *Campylobacter* and *Salmonella* [159]. Moreover, in the field of fish production and aquacultures, different bacteriophages were employed to control and eradicate bacterial pathogens like *Vibrio* sp. [160]. In the field of human medicine, different bacteriophages were employed to control and eradicate bacterial pathogens including *P. aeruginosa*, *Staphylococci*, *Streptococci*, *E. coli*, *Vibrio* and *Shigella* and *Mycobacterium* sp. [161, 162]. Most recent application of bacteriophages in human medicine is their utilization as drug delivery system, which is very interesting as they can be used for the delivery of common antibiotics [163, 164] or antitumor agents [165].

A more recent policy, termed enzybiotic, for using phages as therapeutic agents is the utilization of their enzymes only, which are produced by recombinant technology, combined with other antibacterial agents or as a separate antibacterial agents [166].

As other therapeutic regimens for controlling bacterial pathogens, the patients may develop extensive fever and shock, when the bacteria are lysed due to the release of what is called pyrogens or endotoxins within the patient [167]. This

**203**

**Table 2.**

*New Approaches for Competing Microbial Resistance and Virulence*

and the other bacterial structures into harmless products [168].

*aeruginosa*

*aureus*

*parahaemolyticus*

*aeruginosa*

*baumannii*

*faecium*

Ileocecitis Hamster *Clostridium difficile* Oral administration of

Dysentery Human *Shigella dysenteriae* Oral administration of

Cholera Human *Vibrio cholerae* Oral administration of

*pneumoniae*

*pyogenes*

*aureus*

*baumannii*

*aeruginosa* and *Salmonella typhimurium*

*In vitro In vitro Bacillus anthracis* Application of PlyG lysin

Meningitis Murine *Escherichia coli* Intraperitoneal

Human *Proteus vulgaris* Topical application of

Typhoid Human *Salmonella typhi* Oral administration of

Human *Staphylococcus* 

problem can be coped during phage therapy through the utilization of genetically modified phages that harbor enzymes having the ability to lyse these endotoxins

Examples of therapeutic approaches of bacteriophages and their enzymes are

**of phages/enzymes**

Oral administration of phages

phages

Topical application of phages

Intraperitoneal and oral administration of phages

Intranasal administration of phages

phages

or subcutaneous administration of phages.

Intraperitoneal administration of phages

Intraperitoneal administration of phages

phages

phages

phages

Intraperitoneal administration of Cpl-1 lysin enzyme

Intraperitoneal administration of PlySs2 lysin enzyme

enzyme

Application of Ply187 lysin as eye drops

Administration of PlyF307 lysin enzyme

Application of ABgp46 lysin enzyme

**Treatment outcomes**

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Successful treatment

Significant reduction in bacterial density

Successful treatment

Successful treatment

Significant reduction in bacterial density

**Reference**

[169]

[170]

[171]

[172]

[154]

[173]

[174]

[175]

[176]

[177]

[178]

[178]

[179]

[179]

[180]

[181]

[182]

[183]

**Model Causative agent Route of administration** 

*DOI: http://dx.doi.org/10.5772/intechopen.90388*

Chronic otitis Human *Pseudomonas* 

Sepsis Murine *Vibrio* 

Pneumonia Murine *Pseudomonas* 

Sepsis Murine *Acinetobacter* 

Bacteremia Murine *Enterococcus* 

Pneumonia Murine *Streptococcus* 

Bacteremia Murine *Streptococcus* 

Endophthalmitis Murine *Staphylococcus* 

Bacteremia Murine *Acinetobacter* 

*In vitro In vitro Pseudomonas* 

*Therapeutic approaches of bacteriophages and their enzymes.*

illustrated in **Table 2**.

**Infection/ disease**

Diabetic foot ulcer

Ulcers and wounds

*Microorganisms*

Compared with antibiotics and other therapeutic regimens, the steps and cost of production of bacteriophages are much easier and cheaper, respectively [10]. The easiest process for capturing of bacteriophages is done through collecting samples that seem to involve high titers of phages like sewage water samples. The collected samples are inoculated with the host bacterium, which seems to be infected by phages, on suitable growth medium. The successful isolation of certain lytic phage is depicted by the presence of clear inhibition zones in which bacteria cannot grow termed plaques; which indicates the lytic power of the isolated phage. Thereafter, the titer of isolated phage is increased by passing the phage in its specific bacterial strain several times to increase its concentration. Then, the pure supernatants containing phages are gained by centrifugation of bacterial/phage mixture, filtered through bacterial filters to remove any bacterial debris and pure phages are participated using special solutions containing NaCl and polyethylene glycol 8000 (PEG8000) [154]. Caution must be given during isolation of phages as a type called lysogenic bacteriophage may be isolated rather than the required bacterial pathogen killing type, which is called lytic bacteriophage. Lysogenic bacteriophages do not lyse bacterial cells, but they perform as tools for transfer of genetic elements of the nucleic acid between bacteria; including the genes responsible for antibiotic resistance. Fortunately, the

most abundant phages are of the lytic type not the lysogenic [8, 145, 150].

during their bypassing in the stomach [155, 156].

Practically, bacteriophages can be dispensed and used through many routes including; less commonly oral or systemic route and most commonly topical route as sprays, liquid solutions or their application on surgical dressings for the treatment of wound infections [154]. The possibility of their clearance during the presence in blood stream by immune system or presence of any trace hazards of chemicals or parts of the bacterial host used during their production, made bacteriophage usage as intravenous injections uncommon and very rare [148, 149]. Lyophilization of bacteriophages and their production as solid dosage forms as pills or tablets do not decrease their potency and increase their shelf life as oral dosage forms [155, 156]. The supplementation of oral forms of phages, either solid or liquid, with antacid increases its stability, as it protect them from the high acidity

The application of bacteriophages as therapeutic medicaments has been extensively reported. For example, in the field of human health promotion and food protection, different bacteriophages have been employed to eradicate common bacterial pathogens that may cause food spoilage as *Listeria* sp. and *Campylobacter* sp. [157, 158]. In the fields of veterinary medicine and agriculture different bacteriophages were employed to control and eradicate bacterial pathogens like *Xanthomonas*, *Escherichia*, *Campylobacter* and *Salmonella* [159]. Moreover, in the field of fish production and aquacultures, different bacteriophages were employed to control and eradicate bacterial pathogens like *Vibrio* sp. [160]. In the field of human medicine, different bacteriophages were employed to control and eradicate bacterial pathogens including *P. aeruginosa*, *Staphylococci*, *Streptococci*, *E. coli*, *Vibrio* and *Shigella* and *Mycobacterium* sp. [161, 162]. Most recent application of bacteriophages in human medicine is their utilization as drug delivery system, which is very interesting as they can be used for the delivery of common antibiotics [163, 164] or

A more recent policy, termed enzybiotic, for using phages as therapeutic agents is the utilization of their enzymes only, which are produced by recombinant technology, combined with other antibacterial agents or as a separate antibacterial

may develop extensive fever and shock, when the bacteria are lysed due to the release of what is called pyrogens or endotoxins within the patient [167]. This

As other therapeutic regimens for controlling bacterial pathogens, the patients

**202**

antitumor agents [165].

agents [166].

problem can be coped during phage therapy through the utilization of genetically modified phages that harbor enzymes having the ability to lyse these endotoxins and the other bacterial structures into harmless products [168].

Examples of therapeutic approaches of bacteriophages and their enzymes are illustrated in **Table 2**.


#### **Table 2.**

*Therapeutic approaches of bacteriophages and their enzymes.*
