Current Drug Delivery

3

Current Drug Delivery

Send Orders for Reprints to [email protected] Current Drug Delivery, 2018, 15, 3-20

REVIEW ARTICLE ISSN: 1567-2018 eISSN: 1875-5704

Impact Factor: 1.478

An Overview on Bacteriophages: A Natural Nanostructured Antibacterial Agent BENTHAM SCIENCE

Vaibhav Rastogi1,*, Pragya1, Navneet Verma1, Arun Kumar Mishra1, Gopal Nath2, Praveen Kumar Gaur3 and Anurag Verma1 1

Department of Pharmacy, IFTM University, Moradabad, Uttar Pradesh, 244001, India; 2Department of Microbiology, Institute of Medical Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India; 3Department of Pharmaceutics, I.T.S. Paramedical College (Pharmacy), Muradnagar, Ghaziabad, Uttar Pradesh, 201206, India

ARTICLE HISTORY Received: June 01, 2015 Revised: January 22, 2016 Accepted: April 05, 2016 DOI: 10.2174/1567201813666160406115744

Abstract: Background: Recent advances in the field of bionanomedicine not only enable us to produce biomaterials but also to manipulate them at molecular level. Viruses particularly bacteriophages are a promising nanomaterial that can be functionalized with great precision. Bacteriophages are the natural antimicrobial agents that fight against antibiotic resistant bacteria which cause infections in animals, humans, or in crops of agricultural value. The idea of utilizing bacteriophages as therapeutic agents is due to their ability to kill bacteria at the end of the infectious cycle. Objective: This paper reviewed the general biology of bacteriophages and the presence of receptors on the bacteria which are necessary for the recognition and adsorption of bacteriophages. Pharmacokinetics and therapeutic potential of bacteriophages administered through various routes in treating diverse bacterial infections is also reviewed along with the problems associated with bacteriophage therapy. Conclusion: Among various routes of administration, parenteral route is found to be the most thriving route for the treatment of systemic infections whereas oral route is meant to treat gastrointestinal infections and; local delivery (skin, nasal, ears) of phages has proven its potency to treat topical infections.

Keywords: Bacterial receptors, bacteriophage, bionanomedicine, delivery routes, pharmacokinetics, antibacterial agents. 1. INTRODUCTION In the last decade, several papers and many books have been published on the use of novel nanomaterials in the areas of medicine and technology. Meanwhile, with the advancement of phage display technique, a new and a promising nanomaterial named bacteriophage has emerged which has attracted many researchers to reveal their application in the field of nanomedicines. Bacteriophages have a distinguished feature which is totally opposite to other nanomaterials, i.e. their structure and function are encrypted in their genomic DNA. This feature of bacteriophages renders them easily modifiable or even rewritten using routine genetic engineering techniques [1]. DNA or RNA is the known genetic material for the bacteriophages but many of them have doublestranded DNA and can be placed as per the morphological groups: tailless icosahedral phages, viruses with contractile tails, viruses with non contractile tails, filamentous phages and enveloped phages. T-even phages of E. coli are among *Address correspondence to this author at the Department of Pharmacy, IFTM University, Moradabad, Uttar Pradesh, 244001, India; Tel: +919457041148; E-mail: [email protected]

1875-5704/18 $58.00+.00

the most complex forms of phages with contractile tails. Some morphological characteristics of certain bacteriophages are given in Table 1 [2, 3]. In particular, multivalent displayed foreign peptides have allowed the construction of landscape phage with tremendous surface architectures and spurt properties. Recently, phage, as a novel nanostructured material gather the attention of specialists working in the broad areas of genetics and molecular biology, such as, material science, pharmaceutical science, microelectronics, detection, biosensors, environmental sciences, etc. [1]. This review work highlights the biology of the bacteriophage and their potential in treating various infections caused by antibiotic resistant bacteria. In the early 1920s, bacteriophages as antibacterial agents were explored by English bacteriologist Fredrick Twort in 1915 and also by French Canadian scientist Felix d’Herelle in 1917. d’Herelle observed a microbe that was antagonistic to bacteria and resulted in lysis and eventual bacterial cell death, later that microbe was termed as bacteriophage. 24 months earlier, a similar discovery was recorded by Fredrick Twort, but he never considered that the bacterial cell death was due to phage therapy but d’Herelle devoted his scientific © 2018 Bentham Science Publishers

4

Current Drug Delivery, 2018, Vol. 15, No. 1

Table 1.

Rastogi et al.

Morphological characteristics of certain bacteriophages. Particle Dimensions (nm)

S. No.

Bacteriophage

1

T1

2

Host

Structure(s)

Nucleic Acid (Mol. Wt.) 106 Daltons

Head

Tail

E.coli

50

10 150

Hexagonal head, Simple tail

DNA (ds*) 27

T2, T4, T6

E.coli

80110

25110

Prolate, Icosa, Hedral Head, Complex, tail with fibres

DNA (ds) 105-120

3.

T3, T7

E.coli

60

1015

Hexagonal head, Short tail

DNA (ds) 25

4.

T5

E.coli

65

10170


DNA (ds) 66

5.

Phase

E.coli

54

10140


DNA (ds) 31

6.

SPO1

B. subtilis

90

30 120

Hexagonal Head, Complex Tail

DNA (ds) 105

7.

PM2

Pseudomonas

60

None

Hexagonal Head

DNA (ds) 6

8.

X174, S13

E.coli

27

None

Icosahedral

DNA (ss)** 1.7

9.

f1, fd, M13

E.coli

5-10800

None

Filamentous

DNA (ss) 1.3

10.

M16

Pseudomonas

65

None

Polyhedral head

RNA (ds) 9.5

11.

MS2, f2, Q

E.coli

24

None

Icosahedral

RNA (ss) 1.2

*ds= Double Stranded; **ss = Single Stranded.

life in the study of bacteriophages only [4, 5]. Clinical applications of phages is renowned [6, 7]. Discovery of penicillin commenced the antibiotic era and the phage therapy was largely expelled from the developed world, with the exemption of a number of Eastern bloc countries. Significance of bacteriophages as therapeutic agents has recently gained renaissance by the troublesome and bothering debut of antibiotic resistant bacteria on a global scale [6, 8-10]. Phages are a monarchy of viruses that are different from the plant and animal viruses, but possess either a “lytic” or a “lysogenic” life cycle. For phage therapy the lytic phages are the most appropriate agents as they quickly reproduce within the bacteria, lyse their host and can be grown exponentially in number during the infection cycle. Approximately 200 “daughter phages” per lytic cycle each “parent” phage can produce on average. This approximate number will grow exponentially if each daughter phage infects and kills a host bacterium so that at the end of the 2nd cycle there will be 40,000 progeny; 8 million after the completion of 3rd cycle and 1.6 billion at the end of the 4th cycle; and so on [10]. Due to the resistance offered by bacteria against multi drugs a major threat is continuously posing on the health of human beings and the long term significance of conventional antibiotics [11, 12]. Infections caused by these drug resistant bacteria were turned into 25,000 deaths per year, in the European Union alone, out of which 2/3 of the deaths were occurred due to Gram-negative bacteria, such as Acinetobacter baumannii, P. aeruginosa and Enterobacteriaceae, including E. coli and K. pneumonia [13]. Pharma industry is facing number of hurdles for investing in the synthesis and development of new antibiotics. Firstly, the presence of generic antibiotics that is still thought to be effective in the management of infections due to bacteria. Secondly, antibiotics are less profitable than many drugs and the duration of

antibiotic regimens is also limited. Thirdly, the resistance showed by the bacteria which shortens the lifespan of antibiotics is enough to affect its profitability [13, 14]. Just because of these hurdles, the vigilant approach of gigantic pharmaceutical companies towards the establishment of new antibiotics has prompted a renewed interest in phage therapy. Table 2 enlists the prophylactic and/or therapeutic use of Bacteriophages and Antibiotics [15]. 2. PRINCIPLES OF PHAGE BIOLOGY Bacteriophages are present in all habitats of the globe where bacteria bloom and there are ten bacteriophages for each bacterial cell. Recently, the presence of viruses’ specific for archaebacteria (archeophages) has also become evident. The taxonomy of bacteriophage is relying on their shape, size and also to their nucleic acid. Many phages have dsDNA, however, some have ssDNA, dsRNA or ssRNA. Upon infection on the host bacterium different phages show dissimilar fates, as some phages follow the lytic infection cycle and some uses lysogenic pathway. In lytic cycle, phages will multiply in the bacterial cell and lyse the bacterial cell and at the end of the cycle, newly formed phage particles are released, whereas in the lysogenic pathway the phage genome will integrate, replicate as a part of host genome, and stay in a dormant state as a prophage for extended time duration. This prophage may become activated under the environmental stress and trigger the lytic cycle, at the end of which the newly formed phage particles will lyse the host cell [16]. The following phases can be distinguished in the lytic bacteriophage developmental cycle (Fig. 1): 1. The phage gets adsorbed on the bacterial cell by binding to a specific receptor. 2. The nucleic acid of phage is injected into the bacterium.

An Overview on Bacteriophages: A Natural Nanostructured Antibacterial Agent

Table 2.


5

Comparison of the prophylactic and/or therapeutic use of bacteriophages and antibiotics. Bacteriophages

Antibiotics

Comments

Bacteriophages are very specific to their host bacteria; therefore, dysbacteriosis and chances of developing secondary infections can be avoided.

Antibiotics act on both pathogenic as well as normal microflora of the body which alters the microbial balance and may lead to serious secondary infections.

Host specificity is the disadvantage of bacteriophage because the identification of disease-causing bacterium need to assess before the initiation of phage therapy. Antibiotics has high probability of being efficient than phages when the identity of the causing agent has not been determined but it may also imbalance the normal microbial flora.

They replicate at the site of infection and hence they are promising targeting agent.

Their fate (metabolism and elimination) in the body do not render them to concentrate at the site of infection.

Due to "exponential growth" at the site of infection, phages require less frequent administration which is contrary to antibiotics in order to attain the optimal therapeutic effect.

No serious adverse effects have been described.

Multiple side effects such as allergies, intestinal disorders, and secondary infections (e.g., yeast infections) have been reported.

Minor side effects are reported for therapeutic phages due to the release of endotoxins from the phage lysed bacteria. Such effects also may be observed when antibiotics are used.

Phage-resistant bacteria can also be lysed by other phages that have a similar target range.

Not only the targeted bacteria got resistant to antibiotics but other may too.

Selection of a new phage for phage resistant bacteria is comparatively easier to alteration in the functional moiety of the antibiotic or synthesis of new antibiotic.

Selecting new phages for phage resistant bacteria is a rapid process that can be achieved in days or week.

Developing a new antibiotic is a time consuming process, very costly and the success rate is also very less.

Evolutionary arguments support the idea that active phages can be selected against every antibiotic-resistant or phage-resistant bacterium by the ever-ongoing process of natural selection.

5. The formation of new phage particles and the lysis of host bacterium occurred through the expression of the phage late proteins. 1 Attachment (adsorption)

l teria Bac ll e c

1 2

2 Entry

3 High-level gene expression (protein synthesis)

Transcription Translation

3

4

Virus proteins

Shift to synthesis of linear DNA

4 Genome replication 5 Assembly

6 Release

l l cel teria Bac Linear DNA enters and circularizes Synthesis of multiple copies of circular DNA and further gene expression

5 6 Cell lysis

Fig. (1). The infection cycle of bacteriophage [17].

3. Expression of the phage early genes and synthesis of early proteins is occurred which shutdown the host bacterium systems but triggers the phage genome replication. 4. In this step, the phage genome is replicated.

6. Assembly of the phage heads, tails and packaging of the genome is occurred. 7. Lastly, the lysis of host bacterium and the release of new phage progenies occurred. The idea of utilizing specific phages as therapeutic agents is due to their ability to kill specific bacterial cells at the end of the infectious cycle. However, for a positive incorporation of phages in the list of therapeutic agents all the above listed steps in the phage infectious cycle need to take place. Already in the few thoroughly studied phage bacterium interactions many examples of complicated molecular mechanisms have been revealed. Therefore, one cannot expect that all the phage/host systems will behave identically under the conditions met in-vivo; on the converse, it would be surprising to find two identically behaving systems [16]. 3. BACTERIOPHAGE RECEPTORS ON BACTERIAL CELLS Phage adsorption onto the bacterium cell is the crucial step in phage infection. For this, phages may use capsule on the bacteria, flagella, lipopolysaccharide (LPS), fimbriae and surface proteins as receptors. In order to reach the cell wall of bacterium, the breakdown of capsule is necessary and for this bacteriophages may use enzymes present on the bacterial surface. The nature of attraction between receptors and bacteriophages are different for varied taxonomic groups and is principally explained by composition of host cell wall and surface structures.

6


Both gram positive and negative bacteria are quiet distinct from each other in context to outer and inner membrane [18]. Proteins and enzymes localized in membrane and various LPS sites may act as receptors for bacteriophage in case of gram negative bacteria [19]. Following are the some examples of types of receptors present on Gram-negative bacteria: •

Transmembrane protein OmpA was characterized as receptors serving for virus adsorption. OmpA inhibits bacteriophage K3, while mutants defective OmpA are resistant to phage infection [20-22].

•

Phage tulb was inhibited by OmpA proteinlipopolysaccharide (LPS) complex and this is a reversible inhibition but precipitating the complex with Mg+2 leads to irreversible phage attachment [22, 23].

•

Porins are the first outer membrane proteins of Gramnegative microorganisms which are characterized and among all OmpC and OmpF are the major proteins. Phages Hy2, ss4, Tulb and T4 recognizes OmpC receptor for adsorption [21, 24]. Protein gp37 present in tail fibers of phage T4 helps in recognizing receptors (OmpC along with LPS) on host cell. It has been observed that efficiency of infection is reduced in the absence of at least one receptor, whereas loss of both receptors induced bacteriophage resistance. [25, 26]. Whereas, OmpF is a receptor for phage T2 [27, 28]. The receptor recognizing site in T2 is situated within the hyper variable region of protein gp38 attached to terminal part of protein gp37 [22, 29].

•

LamB (selective transport protein) is the receptor for phage . Phage recognizes LamB via protein gpJ- the factor defining host range of this phage [22, 30].

•

Proteases (Ompt and OmpX) are the enzymes localized in the outer membrane of bacteria which acts as receptors for T-like phages with host range mutations M1 and Ox2, respectively [31, 32]. Proteins TonA or FhuA and TonB serve as receptors for phages T5, 80 and T7 [22].

•

•

Lipopolysaccharides (LPS) in Gram-negative are the receptors for bacteriophage adsorption. Smooth (S) type and rough (R) type are two types of LPS which act as the sites for phage adsorption however, some phages might adsorb on both the types. S-type specific phage shows extremely narrow host range as defined by large variability of O-antigen in bacteria of varied taxonomic groups. On the other hand, phage recognizing R-type shows a wide host range as the organization of LPS core is rather conventional in various species and genera of gram-negative bacteria [33]. Receptors for T-phages, specifically T3, T4 and T7 are resembled to R-type LPS of Shigella and Escherichia [22, 34, 35]. A ordinary feature of bacteriophages fixing to LPS Ochain is the enzymatic cleavage of polysaccharide chain. 15 and P22 may be referred to such phages possessing endo rhamnosidase activity and ability to lyse the bond Rha-1 3-Gal in O-antigen of Salmonella anatum and Salmonella typhimirium, respectively [36-39]. Bacteriophage 8 adsorbed on the surface of E.coli O8 shows endo mannosidase activity, breaking down Man-1 3Man link between repeating oligosaccharides and re-

Rastogi et al.

leases prevailing levels of hexa- and nonsaccharides [40]. In common phages adsorbing to O-antigen chain of LPS in gram negative bacteria recognizes it via enzyme restricted on the tail end, which upon recognition and attachment hydrolyzes one of the bonds in polysaccharide chain of O-antigen [22]. There is a difference in the cell wall structure and chemical composition of Gram-positive bacteria in comparision to Gram-negative bacteria. Peptidoglycan is the main component which constitutes 40 to 90% of cell dry weight. Teichoic acids are among the other crucial elements of grampositive microorganisms which constitute the bulk of bacterial surface antigen but are water soluble polymers comprising of glycerol or ribitol moieties bonded together by phosphodiester bond and traversing peptidoglycan layer at 90° to the surface of plasma membrane. Following are the some examples of types of receptors present on gram-positive bacteria: •

Bacteriophages 3C, 52A, 71, 77, 79 and 80 are specific to S. aureus which are inactivated irreversibly by a complex of teichoic acids and peptidoglycan supported by tetrapeptide attached to muramic acid. Reversible adsorption can also be achieved if phage binds with teichoic acids which are connected with glycan fibers. Presence of NAG (N-acetylglucosamine) in teichoic acid and O-acetyl groups in muramic acid residue is also essential for adsorption [22, 41-44].

•

GamR protein present in the cell wall of B. anthracis is concerned in adsorption of phage [22, 45].

•

Phages for Lactobacillus delbrueskii are inactivated by lipoteichoic acids [22, 46].

•

Phages infecting Lactococcus lactis initially adsorb to polysaccharide cell wall [22, 47].

Apart from receptors mentioned above in Gram-negative and Gram-positive bacteria there are some receptors present in capsular polysaccharides, pili and flagella. Phage AcM4 and AcS2 infect Asticcacaulis biprosthecum by adsorbing on the flagella via site connecting head and phage tail whereas the distal region of the tail remains open for adsorption to the surface of bacterial cell. The attached phage is capable to shift along flagella in the direction of cell and may also adsorb to the surface of fellow cell [22, 48]. Capsule and slime layer may obstruct the contact of phage to receptors on the cell wall or may be used for adsorption of phages, predominantly those which fail to attach to bacteria that are devoid of capsules [49, 50]. One of the phage receptors positioned in capsules of gram-negative bacteria is Vi-antigen distinctive for representatives of Salmonella, Citrobacter and E.coli. [22, 51, 52]. Adsorption of other phages to capsular polysaccharides is also connected with enzymatic activity but sometimes it is aimed at depolymerisation of main chain. Enzymes displaying endo-glucosidase activity were characterized for phages of Klebsiella K11 and E.coli K29 [22]. Viruses of two types are present among bacteriophages that are able to adsorb to pili of bacteria, these are: RNAcontaining viruses with isometric capsid and DNAcontaining viruses in the form of filaments. These Phages


uses sex pili of bacteria and are able to adsorb in hundreds of number which is mediated by protein A (a second capsid component responsible for recognition and adsorption of virion to pili) [22]. Phage P17, fr, M12, Q, f4, f2 are the RNA containing viruses that utilizes sex pili in infecting E. coli [53, 54]. DNA-containing filamentous phages recognizing pili as receptors may be subdivided into two groups: Ff and If phages adsorbing to terminal parts of F and I pili, respectively. Only few viruses may adsorb to one pilus which is also mediated by protein A [22, 55]. 4. PREREQUISITES FOR PHAGE THERAPY The renaissance of phage therapy across the globe was due to the fueled antibiotic crisis and also due to the resistance in bacteria against antibiotics. Bacteriophages are very specific to their host that means they are able to lyse specific strains of bacteria. This high specificity of the phages results in generally narrow bacterial host range than that found for the antibiotics that have been chosen for clinical applications. Due to this limited host range phage the normal microbiota and ecology of the body was less harmed than the generally used antibiotics, which frequently upset the usual gastrointestinal flora and result in opportunistic secondary infections [16]. Before attempting phage therapy several, sometimes rather demanding, prerequisites should be met: 1. Complete biological study of the therapeutic phage and host bacteria is mandatory before attempting for phage therapy due to the complexity of bacteriophage-host interaction [16]. 2. There should be no bacteria in the phage preparations and it should meet all the safety requirements. Secondly storage procedures must be validated for phage [16]. 3. Thorough knowledge of phage receptor is necessary. A population of 106-108 bacteria there is a great likelihood of spontaneous phage-resistant bacteria deficient in the receptor or with an altered receptor. It can be assumed that a mutation eliminating the receptor that functions as a virulence factor of a pathogen (such as LPS) would satisfy the bacterium and then it would be easier for the host immune system to eliminate the bacteria [16]. 4. Reliability on results obtained from the in-vitro experiments only should not be practiced as each phage may behave differently in-vivo. The in-vivo propensity of bacterial pathogens to bacteriophages should be tested in an animal model too [16]. Before a phage in the form therapeutic agent reaches the end user, many bureaucratic and regulatory hurdles need to be cleared even when the above prerequisites are properly met. 5. THERAPEUTIC APPLICATIONS OF BACTERIOPHAGES In the last few years a large number of new bacteriophage research directions have evolved surrounding the delivery routes for phage. Oral and parenteral are among the most popular ones. However, a significant amount of work has been done in local phage delivery i.e. topical, otic and inha-


7

lation. The following paragraphs will describes the possible routes of administration of phages for attaining the desired therapeutic effects. 5.1. Oral Delivery of Phages The treatment of gastrointestinal infections from the orally administered bacteriophages has proven successful but the major concerned with the oral delivery is the stability of phage in the highly acidic proteolytically active milieu of the stomach. Depending on the acid sensitivity each phage needs to be characterized for the efficacy against bacteria. In order to avoid such problem, polymeric microencapsules of bacteriophages helps to provide protection from gastric acidity and may improve the efficacy of the orally administered phages [10]. The pathogenic bacteria in the gut are the principal target to orally administered phages but data obtained from the researches has also reveals the ability of some orally given phages to be absorbed in the blood. Chibani-Chennoufi and their co-workers observed the invivo lytic effect of orally administered phages (JS4, JS94.1, JSD.6, JSL.6) on the intestinal E. coli (isolated from pediatric diarrhea patients) population in laboratory mice. The phage cocktail was given to 10 mice in their drinking water (doses: 103 PFU/ml, 105 PFU/ml and 107 PFU/ml), separated by 3 days of phage-free drinking water. Two hypotheses were drawn after seeing negligible effect in faecal bacterial count. Firstly, the gastric acidity degrades the phages and thus not be able to reach the intestine. Secondly, the endogenous intestinal E. coli resists phage infection. The second hypotheses was re-evaluated by exploring the gastrointestinal passage of orally administered phages and the determination of the lowest phage concentration leading to stable faecal phage excretion [56]. Tanji et al. investigated the therapeutic utility of phage cocktail for curbing gastrointestinal E. coli O157:H7 cells by conducting in-vitro and in-vivo experiments. They observed a marked decrease (five log) of E. coli concentration after accumulation of phage cocktail (SP15, SP21, SP22) in vitro. From the in-vitro experiment, phage cocktail suspension in 0.25% CaCO3 buffer was orally administered to the mice. They concluded that frequent oral delivery of phage cocktail was effective for rapid emigration of E. coli O157:H7 from feces and GIT of mice [57]. Brussow et al. orally given T4 coliphages (sub groups: T4-, RB4 9- and JS98-like phages) and found no negativity on the murine gut microbial flora. Cecum and colon has high titer of T4 while small intestine has low, but none of them were found in the blood, liver or spleen. No side/adverse effects were observed after an exposure to phage for about one-month and neither serum anti-T4 antibodies were detected [58]. A safety test of T4 bacteriophage was also performed by these researchers on healthy adult volunteers. In their study fifteen healthy adult volunteers received a lower T4 dose (103 PFU/ml), a higher T4 dose (105 PFU/ml) in their drinking water and a placebo. Dose dependent detection of coliphage in feces was observed after 1 day in all volunteers receiving the higher bacteriophage dose however no phage was detectable in faeces a week after a two day course of oral phage administration and no adverse events were observed. Serum transaminase levels remained in the normal

8


Rastogi et al.

range, and neither T4 phage nor T4-specific antibodies were observed in the subjects at the end of the study [59].

than those achieved with the lower cocktail concentrations [62].

Efficacy of bacteriophage (KPP10) against gut sepsis caused by Pseudomonas aeruginosa in mice was assessed by Matsumoto and co-workers. In this study, lytic phage strain (KPP10) was orally administered at a dose of 1x1010 PFU to the mice challenged with Pseudomonas aeruginosa and a control group provided with saline. The survival rate was 66.7% in the phage protected group which is significantly different from the unprotected mice with 100% mortality. Phage protected mice groups has lesser numbers of viable P. aeruginosa in the blood, liver, spleen and also in fecal matter than in the saline-treated control mice [60].

Campylobacteriosis is a zoonosis (passed to human via animals or animal products) which is caused by the bacterium, usually Campylobacter jejuni or E. coli. The bacteria are extensively dispersed and found in most warm blooded wild and domestic animals. Campylobacteriosis is mainly occurred in the gastrointestinal tract and are responsible to a severe form of diarrhea. Consumption of contaminated food such as under cooked meats, contaminated water or raw milk by the people exposed them to the bacteria. In a study, the efficacy of a phage cocktail (phi CcoIBB35, phi CcoIBB37, phi CcoIBB12) in reducing the levels of colonization of both C. coli and C. jejuni in broiler birds was studied. Phages were administered by two routes (by oral gavage and in feed). The result showed that even at the highest dose of Campylobacter challenge, no sign of disease or adverse sign was ilicit. This was attributed to the broad host range of the novel phage cocktail which enable it to target both C. jejuni and C. coli strains. Moreover, the reduction of Campylobacter by approximately 2log10 CFU/g, could lead to a 30 fold reduction in the incidence of campylobacteriosis associated with consumption of chicken meals [63].

Filho et al. evaluated the effectiveness of four dissimilar bacteriophages isolated from commercial broiler houses (CB4) and 45 bacteriophages from municipal wastewater treatment plant (WT45) against Salmonella entrica serovar enteritidis in broiler chickens. In one experiment, the ability of bacteriophages at concentrations of 105 to 109 PFU/ml to reduce S. enteritidis in the simulated crop environment was evaluated. The findings reveal that after 2 hours at 37 °C, CB4 or WT45 reduced S. enteritidis recovery by 1.5 or 5 log, respectively, as compared with control. However, S. enteridis recovers after 6 hours in the group treated with CB4, whereas WT45 showed 6-log reduction of S. enteritidis. In experiment 2, hatch chicks were challenged orally with 3 103 CFU/chick Salmonella enteritidis and were supplemented cloacally with 1 109 WT45 PFU/chick 1 hour post challenge. The results showed significant reduction in Salmonella enteritidis recovery from cecal tonsils at 24 hours as compared with controls, with no adverse effect). In experiment 3, day-of-hatch chicks were challenged orally with 9 103 CFU/chick S. enteritidis and treated via oral gavage with 1 108 CB4 PFU/chick, 1.2 108 WT45 PFU/chick, or a combination of both, 1 hour post challenge. All treatments greatly reduced Salmonella enteritidis from cecal tonsils at 24 hour as compared with untreated controls, but no significant differences were observed at 48 hours following treatment. These data suggest that some bacteriophages can be efficacious in reducing Salmonella enteritidis colonization in poultry during a short period [61]. The efficacy of virulent bacteriophagic cocktail in targeting O104:H4 55989str enteroaggregative strain of E. coli in the mouse intestine was assessed by Debarbieux et al. The bacterial strain was cultured in Luria Bertani broth or agar at 37˚C. Phages CLB_P1, CLB_P2 and CLB_P3 infecting the bacteria were isolated from waste water. Colonization of mouse intestine with the O104:H4 55989str was achieved in 3 and 7 days after oral administration at a mean concentration ranging from 103 to 109 CFU/g. After colonization mice were divided into two groups, one group received placebo drinking water, whereas other received phage cocktail in their drinking water at 3x108 PFU/ml concentrations. Only one-eighth of the control mice yielded 55989str concentrations in the ileum after 24 hours of phage treatment. However, no viable 55989str cells was detected in the ileum of 40% of these mice. The author also concluded that increase in the concentration of the cocktail (100 times greater concentration of the initial cocktail concentration) resulted in a decrease in ileal 55989str concentrations 70 times greater

5.2. Parenteral Delivery of Bacteriophages Due to the instantaneous distribution of phages into the blood, parenteral administration of bacteriophages in animals has proven to be one of the most successful and popular among all delivery methods. However, recent studies also revealed the success of bacteriophage therapy from the site specific phage administration i.e., intramuscular (IM), subcutaneous (SC) or intraperitoneal (IP) [10]. Escherichia coli is the cause of 80-85% of community acquired UTIs (urinary tract infections) that travel up the urethra to the bladder. Nishikawa et al. examined bacteriophage therapy for UTIs caused by the UPEC strains as an alternative to chemotherapy. They isolated and purified seven coliphages from environmental water using 12 uropathogenic E. coli strains as indicators. In addition to the wellknown T4 phage, KEP10 (newly isolated T4 resembling phage) showed 67% of a broad bacteriolytic spectrum for UPEC strains than T4 (14%). Following i.p injection phages T4 and KEP10 were rapidly distributed in all organs at a high titer for upto 24 hours. Both phages were stable in the mice urine as well as of humans for 24 hours at 37°C. A distinguished fall in the mortality of phage treated mice with no adverse effect was observed which was challenged transurethrally with a uropathogenic strain, whereas many mice among the control group died within a few days of microbial infection [64]. Chhibber et al. isolated phage SS forKlebsiella pneumoniae B5055 from sewage. Phage SS was evaluated for K. pneumoniae-mediated lobar pneumonia in mice. A single i.p. administration of 1010 PFU ml-1 phage to mice immediately after i.n. challenge (108 CFU ml-1) was adequate to rescue 100 % of animals from K. pneumoniae-mediated respiratory infections but the treatment fails when the phage was administered after 6 hour of post infection however 3 hour prior administration of phage showed significant protection to the infected mice. The results suggested that phage therapy tim-


ing after instigation of infection significantly contributes towards the success of the treatment. No toxicity was observed for phage SS in mice and also the mean rectal temperature of all the phage treated mice was 36.7°C which was analogous to that of control group 37.1°C [65]. The ability of T4 bacteriophage and its sub-strain Hap1 to bind to cancer cells and to state antimetastaic activity in a mouse B16 melanoma model was studied by Opolski et al. They found there was a momentous decrease in the number of murine melanoma metastasis in the lungs when both of the bacteriophages were administered intravenously. The antimetastatic effect was significantly stronger for HAP1 as compared to the strain T4. The binding capacity of bacteriophages to cancer cell membrane was also studied using confocal and electron microscopy and showed that the binding was inhibited by 3 integrins ligands and anti- 3 antibodies. They also proposed mechanism of interactions T4 capsid proteins with 3 integrins on target cells and observed that probably gp24 that contain KGD-amino acid motifs i.e., RGD homologs of T4 phage proteins interact with 3 receptors. The ligands which binds with these cells targets are able to block the integrin functions and to thus reduces the tumorigenicity of the cells [66, 67]. With the obtained data, they again reported the potential phage anticancer activity in primary tumor models. The tumor model was prepared by inducing tumor in mice by inoculation with B16 or LLC cells (subcutaneously) while bacteriophages T4 and HAP1 were injected intraperitoneally daily with a dose of 8x108 PFU/mouse. They found that there is significant reduction in the size of tumor which is dependent on the dose given. The antimetastatic effect was confirmed when it was observed that the tumor weight was decreased by 26.5% and 8% at a concentration of 1010 and 109, respectively, on 16th day of experiment. They also found that activity of HAP1 phage was found to be more effective in the treatment of mice bearing melanoma tumors which was concentration dependent. The tumor volume was reduced by 73% at 1010 PFU/mouse concentration on 16th day of experiment while the most effective concentration was 109 where the inhibition is 45% as compared to T4 phage in which the inhibition was observed 8% only [68]. An approach, named ‘‘Step-by-Step’’ (SBS), has been established by Han et al., a phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) lytic for Klebsiella pneumoniae was established by this method. The phage cocktail considerably decrease the transmutation frequency of Klebsiella pneumoniae and effectively rescued K. pneumoniae bacteremia in a murine K7 strain challenge model. 3 x 104 PFU is the minimal protective dose of the cocktail which was adequate to protect bacteremic mice from lethal K7 infection. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge. These findings suggest that the phage cocktail prepared by SBS Method has great therapeutic potential for multidrug-resistant bacteria infection by efficiently reducing the bacterial mutation frequency [69]. The use of lytic bacteriophage for controlling E. coli septicemia and meningitis in chickens and calves was examined by Barrow et al. The proposed mechanism for the lysis of E. coli H247 by Phage R is the attachment of phage to the K1


9

capsular antigen which ultimately helps to prevent meningitis as well as septicemia in chickens. Intramuscular suspension containing 106 CFU of E. coli H247 was used to inoculate healthy chickens followed by IM administration of 50 l dilutions (106 PFU) of phage preparations. 100% moratality was observed in 3-week old and newly hatched chickens as both of them were not treated with phage, however the profound result was clearly visible in the phage treated chickens in which none of the hatched chicken was dead. The administration of phage at 104 PFU concentrations shows considerable protection, whereas 102 PFU produced a bit less protection. The similar experiment was conducted with calves in which they were orally inoculated with 3x1010 CFU of E. coli H247 and 2 ml suspension of diluted Phage R by IM route (upper thigh). From the experiment it was clear that calves (received phage) showed late onset of symptoms associated with E. coli bacteremia which were directly proportional to the concentration of phage against infection [70]. Gulig et al. reported the possible use of three bacteriophages (CK-2, 153A-5 and 153A-7) as curative agents against Vibrio vulnificus in an iron-dextran treated model of mouse. To determine the efficacy of phages against V. vulnificus infection, mice were first given iron-dextran (i.p) and then inoculated s.c with 106 CFU (100 times the lethal dose) of V. vulnificus MLT403, and immediately injected (i.v) with 108 PFU of phage suspension in BSG (0.1ml). No treatment of phage was given to the mice of control group and the results are very significant that the control mice showed a mean of 108 CFU/g in lesion tissue and their livers contained a mean of 105 CFU/g whereas the treated group shows the normal outcomes. After analyzing data it was concluded that for getting optimal protection against infections caused by V. vulnificus, phages should be administered at high doses (108 PFU/ml) within 3 hours of bacterial infection [71]. In a prophylaxis study by Soothill et al. in New Zealand white rabbits received s.c 8x107CFU of Staphylococcus aureus 2698 and 2x109 PFU of LS2a bacteriophage. After 4 days, 1/8th of phage treated rabbits had an abscess (area=64mm2), whereas untreated rabbits had abscesses of 32 to 144 mm2 area. Each rabbit was received 8x107 CFU of S. aureus 2698 and either 6x107, 6x106, or 6x105 PFU of LS2a or a control suspension. The results were depended on the dose of phage given. At low dose the median areas and bacterial count in the abscesses increased than the abscesses of the groups receiving the two higher doses of phage and the largest abscesses was seen for the control group [72]. In a study by Fralick et al., burned mice were subjected to a lethal infection with Pseudomonas aeruginosa (PAO1). These infected and wounded mice were treated with phage cocktail (Pa1, Pa2, Pa11) to ensure therapeutic efficacy of the phages. Three routes were chosen to administer the phage cocktail viz. i.m, s.c and i.p., and the outcomes of the study indicateds that a single dose (3x108 PFU) of the P. aeruginosa phage cocktail appreciably decreased the death rate of thermally wounded, P. aeruginosa-infected mice (94% moratlity in groups of without treatment to 13-78% mortality in groups provided with phage cocktail). Among the routes of administration, the i.p route provides the most significant efficacy (87%) as the administered phage rapidly increases its titre and rapidly distributes to the tissues which

10


is not possible by s.c or i.m route as there are various barriers to be faced by the phage to reach the site of action [73]. The efficacy of bacteriophages (Msa) against Staphylococcus aureus and methicillin-resistant staphylococcal strains was observed by Iannelli et al. In this study, 20 S. aureus strains were isolated from patients and were grown in Luria Bertani medium. Selected strains were resuspended in saline (107 to 109 CFU/ml). A control group was injected 108 CFU/mouse of S. aureus A170 intravenously. Three groups were given (i.v) phage Msa at final concentration of 107, 108 and 109 PFU/mouse, respectively immediately after infection. The therapeutic efficacy of phages was dose dependent for control group, whereas the mice given lowest phage dose (107 PFU/mouse) died within 4 days. On the other hand, the mice treated with the intermediate dose (108 PFU/mouse) were somewhat protected and the mice with the highest dose (109 PFU/mouse) were all protected. After getting the positive outcomes from the therapy the phage Msa was then studied against methicillin-resistant S. aureus. Two groups of mice were intravenously challenged withe methicillinresistant A352 strain of S. aureus (108 CFU/mouse). One group is served as untreated controls; the second group was given the phage Msa (109 PFU/mouse) intravenously immediately after infection. The survival rate was 20% (2/10) for the control group and 100% (10/10) for the treated group. Thus, it may be suggested that phage Msa has antimicrobial efficacy against human infections with S. aureus or methicillin-resistant S. aureus [74]. Matsuzaki et al. evaluate the potency of novel broad spectrum bacteriophage MR11 in providing protection to the mice Staphylococcus aureus. Eventual death was observed in mice due to bactermia after intraperitoneal injections (8 x 108 CFU) of S. aureus, including methicillin-resistant bacteria. However, subsequent i.p. administration of purified MR11 (MOI > or = 0.1) suppressed the S. aureus-induced lethality which may be attributed to the rapid emergence of MR11 in the blood which was remained at steady levels until the bacteria was eliminated. These results suggest the efficacy of MR11 phage therapy against pernicious S. aureus infections in humans [75]. Gastric acidity or pH may alter the activity of many bacteriophages hence providing protection from the gastric mileu, which seems to be necessary for orally administered phages so that their efficacy may not compromised. In lieu of this, a study utilized encapsulation technique for protecting phages from the gastric acidity. Four phages, wV8, rV5, wV7, and wV11 were encapsulated in polymeric matrix and exposed to pH 3.0 for 20 min which resulted in 13.6% recovery of phages after release, whereas those phages which were not polymer encapsulated had a complete loss of activity under similar conditions. Six pens of four steers (n=24) received 1011 CFU of naladixic acid resistant E. coli O157:H7 on day 0 out of which two pens were considered as control i.e. they were given naladixic acid resistant E. coli O157:H7 only whereas the remaining others received polymer encapsulated phages (Ephage) on days -1, 1, 3, 6, and 8. Two pens were given Ephage orally in the form of bolus gelatin capsules at the concentration of 1010 PFU per steer per day, and the remaining others received Ephage top-dressed on their feed (feed; estimated 1011 PFU per steer per day). Chances of

Rastogi et al.

E. coli O157:H7 in faeces was continuously monitored by collecting faeces and hide swab samples. Acceptable activity of mixed phages on delivery to steers was found for bolus and feed, averaging 1.82 and 1.13 x 109 PFU/g, respectively. However, Ephage did not reduce shedding of naladixic acidresistant E. coli O157:H7, although duration of shedding was reduced by 14 days (P < 0.1) in bolus-fed steers as compared with control steers [76]. 5.3. Transdermal Delivery of Bacteriophages Transdermal delivery of bacteriophages may help to nullify the drawback associated with its parentral therapy as well as aim to attain the therapeutic response not only to the gastrointestinal infection but also to the other possible infected areas caused by bacteria. However, this route of administration for bacteriophage delivery is least explored but it will bypass the requirement of experienced medical practitioner for the successful delivery of the phage through i.v route which will lead to patient incompliance. A hollow array of microneedles composed of polycarbonate was prepared by Ryan et al. and for the very first time in the history of bacteriophage therapy the successful therapeutic achievement against Escherichia coli was obtained by the transdermal delivery of E. coli specific T4 phages both in vitro and in vivo. They showed that bacteriophage can be delivered through microneedles across dermatomed and full thickness skin. In vitro experiments revealed that 2.67 106 PFU/ml of phage was perceived in the receiver compartment when delivered across dermatomed skin whereas 4.0 103 PFU/ml of phage was detected when delivered across full thickened skin. On the other hand in vivo study resulted in 4.13 103 PFU/ml of T4 in blood after 30 min to the microneedle mediated phage administration. They also reported that in the presence of infection the clearance of phages from the systemic circulation was very low due to exponential growth of phages against bacterial challenge whereas in the absence of infection phages were completely cleared within 24hrs from the blood. They also concluded that microneedle mediated delivery allows successful systemic phage absorption [77]. 5.4. Topical Administration of Bacteriophages Goode et al. studied the efficacy of phages to decline the contamination of poultry skin with Salmonella and Campylobacter species. They adopted a laboratory approach and induced infection on chicken skin with these two microorganisms, the skin was removed and stored at -20 °C. Lytic bacteriophage (phage type 4 strain P125589), applied to the contaminated chicken skin. In their study, they utilized low and a high multiplicity of infection (MOI) for the enumeration of bacteria and bacteriophages. AT MOI of 1, they observed increased in titer and pathogen numbers was reduced by less than 1 log10 unit while at MOI of 100 to 1,000 the pathogen numbers was reduce rapidly by up to 2 log10 units over 48 hours. Based on the obtained results they also suggested that it was also possible to eliminate other highly resistant strains of Salmonella [78]. Since wound infection remains the main cause of morbidity in burn patients, therefore Kumari et al. in their study evaluated the efficacy of natural products (aloevera and


honey) in the treatment of infection caused by Klebsiella pneumonia B5055 in mice and compared it with the Klebsiella specific phage therapy. They induced K. pneumoniae B5055 infection via topical route in mice which resulted in a full thickness burn wound. The infected mice with the burn wound were treated daily with natural antimicrobial agents (honey and aloevera gel) applied topically anf their comparison was made with the efficacy of phage Kpn5 suspended in hydrogel which was applied topically only one time. The result of their study clearly indicated that there was significant reduction in mortality on phage treated mice. Application natural agents also provided significant protection but there is no additional advantage when compared with phage therapy. They concluded that phage Kpn5 has is more effective in treating burn wound infection in mice caused by K. pneumoniae B5055 in a single topical application as compared to multiple applications of honey and aloevera gel [79]. 5.5. Intranasal Delivery of Bacteriophages One of the recent advancements in phage therapy is the application of inhalation technologies to treat some lung infections. Since previous literatures suggest the potential of phage therapy in systemic and local applications, therefore the use of bacteriophages to combat lung infections seems to be a rational step. Using a bioluminescent P. aeruginosa strain, Debarbieux and co-worker monitored the potency of a bacteriophage in mice having lung infection. Bacteriophage PAK-P1 specific to P. aeruginosa was isolated from sewage water. Both bacteriophage and bacteria were given through intranasal route. After a preliminary experiment they observed that the PAKP1 delayed the death of highly infected animals while untreated mice are died within 48 hours bacterial challenge. They also studied the consequence of ratio of phage-tobacterium on the survival of mice and found that mice treated with phage in a phage to bacterium ratio of 1:10 died within 5 days while mice treated with higher phage to bacterium ratios (1:1 and 10:1) survived till the end of the experiment (12 days). Moreover, they also determine the safety of the phage therapy to animals and concluded that treatment was harmless and the therapy had no adverse effect on them as no mice showed erratic behavior even after administration of an intranasal dose that was 10 times higher than the effective dose. [80, 81]. The avian pathogenic Escherichia coli (APEC) strains cause serious infections which affect mainly the poultry industry. In a study, Oliveira et al. explored the in vivo efficacy of a cocktail of three lytic coliphages (phiF78E, phiF258E and phiF61E) against severe respiratory E. coli infections in experimentally contaminated birds and naturally infected flocks. The coliphages were combined in a 5.0 x 107 PFU/ml cocktail which was used against naturally infected flocks, while the experimentally infected birds were treated with phiF78E at two different concentrations (107 PFU/ml and 109 PFU/ml) in a single application. They administered phage orally and by spray and recorded the results in terms of morbidity, mortality and pathology scores which were compared with control group of birds (i.e., untreated birds). They observed that the results were dosage dependent


11

and only higher concentration of phage (phiF78E) causes significant reduction in chickens mortality and morbidity. However in case of phage cocktail even a lower concentration (107 PFU/ml) was found sufficient in decreasing the flocks mortality. They concluded that based on the results phage therapy might be used as a valuable alternative to control APEC infections in poultry [82, 83]. The efficacy of bacteriophage (SPRO2 and DAF6) on aerosol administration was studied by Huff et al., to prevent E. coli respiratory infection in broiler chickens. They isolated E. coli from municipal sewage facilities or poultry plants and conducted three different studies to examine the efficacy. They increased the concentration of phage (administered in aerosol spray form) in each study, while the infection was induced by injecting 0.1 ml of a 2.5 h old culture of E. coli (5.6 x 105 CFU/ml) into the thoracic air sac at 7, 8 or 10 days old in each study. They observed marked decrease in death rate compared to the control group. Maximum protection was observed with the second study with phage titers of 2.6 x 108 and 2.35 x 109 PFU/ml for SPR02 and DAF6, respectively while the least protection was seen in third study where the concentration was less. So, they concluded that aerosol administration of phage can be done for the treatment of respiratory infection and the results depend on the phage concentration used [84]. Similarly, the Golshahi et al. studied the practicability of bacteriophages KS4-M and KZ to be given in aerosolized powder form for lung delivery and treatment of pulmonary Burkholderia cepacia complex and P. aeruginosa infections. They formulated respirable powders of the phages and then it was aerosolized using an Aerolizer(®) capsule inhaler. They used idealized mouth-throat replica and examined the viability of bacteriophages delivered from bioassays of samples collected on filters placed after the idealized replica. The percentage of inhaler load was measured which and found to be 33.7 ± 0.3% for KS4-M and 32.7 ± 0.9% for KZ. The results so obtained clearly suggested that both the phages can be lyophilized without significant loss of viability in a lactose/lactoferrin 60:40 w/w matrix and the resulting powders can be aerosolized to deliver viable phages to the lungs [85]. Padmanabhan et al. stated the use of bacteriophages in an in vivo model of nasal carriage. They examined the bactericidal competence of two virulent staphylococcal phages, K and 44AHJD, against S. aureus [86]. In their study, they observed that phage cocktail administered intranasally protect all animals inoculated with S. aureus at 5, 6 and 7days of treatment. Overall, they reported that the phage cocktail lysed approximately >85% of the clinically isolated S. aureus. 5.6. Otic Delivery of Bacteriophages A randomized, double blind, placebo-controlled phase I/II clinical trial approved by UK Medicines and Healthcare Products Regulatory Agency (MHRA) and the central office for Research Ethics Committees (COREC) was conducted by Wright and Co-workers on 24 patients with chronic otitis caused due to antibiotic resistant Pseudomonas aeruginosa. They randomized the 24 patients into two groups of 12 each. One group was provided a single dose of Biophage-PA, while other function as control group which was treated as placebo. They monitored the effect of phage therapy at 7, 21 and 42

12


days after treatment. They found that phage treated group was clinically improved as compared to the placebo group. Also the counts of P. aeruginosa were lowered significantly in the phage treated groups. They also calculated the mean recovery of phage from the swabs taken from the ears of phage treated groups and it was found to be 1.27 x 108 pfu which was 200 times more than the input dose of 6 x 105 pfu. During infection P. aeruginosa was organized as a biofilm which makes it resistant to both antibiotics and immune cells but phages have evolved their approach to breakdown the biofilm through specific enzymes and are therefore able to reach and kill their targets [87]. 6. PHARMACOKINETICS OF BACTERIOPHAGE THERAPY When a conventional drug is administered orally to a patient, it is characteristically absorbed into the blood stream where the concentration reaches to a maximum. When this maximum peak is reached the drug will subjected to metabolism and eliminate from the system, leading to a fall in blood levels. This kinetic profile requires the monitoring of subsequent doses to be administered to maintain drug therapeutic concentrations in the blood [88]. However, bacteriophages do not work in the way as mentioned above; instead, the pharmacokinetics is intricate due to the self replicating nature of phage. Moreover, there is no in vitro-in vivo correlation in the data i.e., in vitro data for one type of phage cannot be directly applied to their in vivo situation; in addition the in vivo data for a phage cannot be converted to/for another phage [16, 89]. Success of phage therapy depends on the generation of phages in the proximity of the target or host bacteria to begin their clearance from the body at an adequate rate or degree. The density of phages will be increased either due to in-situ replication (active treatment) or as an outcome of conventional dosing (passive treatment). These approaches of increasing phage bulk must be in such a way that they are able to oppose the mechanisms of phage loss [90]. Other important factors that affects phage therapy include phage adsorption rate, burst size, latent period, phage treatment timing and clearance rate of the phage by the reticuloendothelial system (RES) [16, 91]. The phage pharmacokinetics was presented in detail by Dabrowska and co-authors [92]. They described that phages are seen as potential invaders (viruses) by the immune system and are rapidly cleared from the blood by RES and tends to accumulate in spleen and liver or/and inactivated by adoptive immune defense mechanisms involving immunoglobulins [92, 93]. Such rapid clearance affects the concentration of phages, as it acts as self-limiting mechanism for phage titer in biological fluids and it is majorly depends on the capsid protein structure. However, in an experiment reported by Matsuzaki et al. in which mice were given MR11 lysogen phage, showed that the bactericidal outcome of the phage was the principle determinant of the protective effect rather than phage stimulated indirect immune response [75]. They determined the lowering of bacterial load in the blood of mice which was treated with phage as compared to that group which received only bacteria. They also observed that phage titers in mice challenged with bacteria remained higher than the titers in mice that received only the phage,

Rastogi et al.

this may be attributed to the replication of phage in the infected mice [75, 94, 95]. In 1940s, mouse experiments were conducted to affirm notable pharmacological features of phage. When phages at a concentration of 105 pfu were injected intraperitoneally into control mice, only 102 pfu reaches the brain. However, in mice which were intracerebrally challenged with bacteria, 109 phages were detected in the brain. No such in vivo amplification was observed in infected mice that were not liable to phage infection [97]. It is therefore clear that specificity of phage to its host bacteria along with time of phage administration is a critical parameter in phage treatment. Phage therapy is thus an exclusive medicine, which challenges the existing pharmacokinetic thoughts. In the absence of host bacteria, phage represents a quickly diluted medicine and rapidly clears from the body, thus minimizing adverse effects. In the presence of the host bacteria, phage acts as a biologically amplifiable drug. When the target bacterial population is exhausted, phages are quickly eliminated from the body [96, 97]. 7. RECENT PATENTS THERAPEUTICS

ON

BACTERIOPHAGE

Various researchers filed patents on bacteriophages as therapeutic agents; however, in this review, the main emphasis is given to the recent patents only. The patents filed in the last four years are listed in Table 3. 8. POTENTIAL PROBLEMS FOR USE OF PHAGE AS ANTIBACTERIAL THERAPY Apart from the ability of phage to kill the bacterial cells, the phage therapy imposes low toxicity and therefore it is relatively safe. The low toxicity may be attributed to composition of phage, like in tailed phages the composition is totally protein and DNA. The interaction of phage with the metabolism of body is common and it can result in the degeneration of phage virions but there is no production of toxic byproducts as it may be seen in certain drugs [108]. However, the intact phages are not effective in interacting with other body functioning such as specifically binding to or manipulating body tissues. Furthermore, since there is a presence of numerous endogenous phages in the body itself, therefore exposure of phages at therapeutic concentration is not beyond the normal body flora. Thus, phage therapy can be considered as safe [109, 110]. Despite the above discussed advantages, the reintroduction of bacteriophage therapy into contemporary medicine faces various problems. The problems are mainly technical (e.g., specificity of phages) and nontechnical (i.e., the “uniqueness” of the phage therapy approaches and strict regulatory constraints) in nature. The technical hurdles include limited range of host for phages, development of bacterial resistance to phages, challenges in manufacturing, systemic side effects associated with phage therapy, and delivery [111]. Phages are not effective against a wide range of bacteria due to their precise spectrum of antimicrobial activity. Therefore, a clear understanding of their limited spectrum of infectivity is needed for


Table 3.


13

Recent patents filed dealing with Phage therapy.

Inventor(s) Yun-Jeong Heo, Yu-Rim Lee, HyunHee Jung, You-Hee Cho

Patent Number US 8282920 B2 9 Oct 2012

Objective Phage therapy against Pseudomonas aeruginosa

Bacteria Pseudomonas aeruginosa (PAO1)

Bacteriophage MPK6

Repercussion of the Study

Refs.

MPK6 a novel bacteriophage belonging to the order Cau- [98] dovirales conferred resistance to mouse peritonitis-sepsis induced by an intraperitoneal infection of P. aeruginosa strain, PAO1. MPK6 and its progeny bacteriophage had an anti-bacterial activity using a mammalian and nonmammalian infection model. Phage samples of MPK6 at a concentration of 5107 PFU in PBS were administered intraperitoneally (i.p.) or intramuscularly (i.m.) into the infected mice. Since i.p. administration can delivers the phages more directly or effectively to the infection site, therefore, phage administration through this route showed better efficacy for MPK6 in combating the PAO1.

Obligate anaerobes such as: CD-HS1, CD-HM6, CDHM5, CD-HM4, CD-HM3, CDHM2, CD-HM1

Five Hamsters were used in this study (CI, C2, Tl, T2, T3). [99] CI and C2 were used as controls and were not treated with phage while other animals were given bacteriophage at 108 PFU dose. Each phage was found to be capable of lysing C. difficile.

Vibrio anguillarum, V. ordalii, V. parahaemolyticus

Polish Collection of Microorganisms (PCM) of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences submitted bacteriophages namely:F/00072, F/00073, F/00074 and F/00075

The antibacterial formulation is prepared in a buffered [100] medium at a pH between 4 and 10, liquid or semisolid and whose active ingredients are concentrated in order to permit broad applications such as its incorporation into the food, or liberating matrixes, among others. The formulation of the antibacterial composition includes, F/OOO72: between 0 PFU/ml and 11010 PFU/ml; F/00073: between 0 and 11010 PFU /ml; F/OOO74: between 0 PFU /ml and 11010 PFU /ml; F/00075: between 0 PFU /ml and 11010 PFU /ml. The compositions and formulations are added directly to the water in which the species to be treated are being cultivated. All the phages are capable of infecting some tested strains of V. anguillarum and V. ordalii, but none is capable of infecting tested strains of V. parahaemolyticus.

US 20140219968 Novel salmoA1 nella bacteriophage compo7 Aug 2014 sitions and uses thereof

Salmonella

UAB_Phi20, UAB_Phi78 and UAB_Phi87

The cocktail of phages was administered orally to chicks [101] when the digestive tract of chicks was fully colonized by Salmonella (values in the cecum were of 108 cfu/g). The administration schedule of phage cocktail at a dose of 1010 PFU/animal was twice per day on days 4th and 5th after Salmonella infection. A decrease of Salmonella count was observed during the first two days post-treatment indicating a high efficacy of phage cocktail in the first stages of Salmonella colonization. Furthermore, a significant reduction of Salmonella concentration over the time was observed with the administration schedule.

US 8475787 B2

Pseudomonas aeruginosa.

NCIMB (National collection of Industrial and Marine Bacteria) 41174, NCIMB 41175, NCIMB 41176, NCIMB 41177, NCIMB 41178 and NCIMB 41179 and the mixture of these phages was named BioVet-PA

This invention is based on the induction of sensitivity to [102] chemical antibiotics by the use of bacteriophage treatment in vivo in humans or in animals, where such sensitivity is heritable, does not rely on active bacteriophage metabolism and does not relate to the destruction of bio film to induce such sensitivity, along with the preparation of medicaments to permit the sequential use of bacteriophages and antibiotics so as to take advantage of such induction in sensitivity in the control of bacterial disease, especially for example a Pseudomonas aeruginosa infection. BioVet-PA was stored at 80° C. Immediately prior to administration, the product was thawed and warmed in the hand. 0.2 ml (containing 1105 infectious units of each of the 6 bacteriophages) was administered drop-wise using a sterile 1 ml capacity syringe into the ear. antibioticresistant Pseudomonas aeruginosa ear infections treated with BioVet-PA showed improvement in clinical symptoms within two days of treatment and reductions in bacterial numbers over the same timescale.

Martha Clokie WO 2014030020 A1

Therapeutic Clostridium bacteriophages difficile

27 Feb 2014

US 20140105866 Romilio Hernan Espejo A1 Torres, Jaime 17 Apr 2014 Moises Romero Ormazabal, Roberto Andres Bastias Romo, Gaston Ariel Higuera Guajardo

Montserrat Llagostera, Jorge Barbé, Carlota Bardina, Maria Pilar Cortés, Denis Augusto Spricigo David Harper

2 Jul 2013

Bacteriophages useful for the prophylaxis and therapy of Vibrio anguillarum

Beneficial effects of bacteriophage treatments

(Table 3) Contd….

14


Inventor(s)

Objective

Bacteria

Bacteriophage


Warren H. US 8889105 B2 Finlay, Jonathan J. Dennis, 18 Nov 2014 Helena Orszanska, Kimberley D. Seed, Karlene Heather Lynch

Bacteriophage compositions for treatment of bacterial infections

Burkholderia cepacia complex (BCC) bacteria

KS4-M

A respirable composition for treatment of a bacterial [103] infection includes one or more active bacteriophages in combination with a pharmaceutically acceptable respirable carrier. The composition includes a carbohydrate carrier, and is prepared as fine powder. In another aspect, bacteriophages are provided in a liquid carrier for administration by nebulization. Larvae infected with about 2.5103 CFU of B. cenocepacia K56-2 have nearly a 100% mortality rate 48 hours post-injection. The efficacy and survival rates depend upon the quantity and concentration of phage used in a therapeutic dose. The larvae show a prompt response with respect to survival rates when it was treated with phage KS4-M immediately after infection. The multiplicity of infection (MOI) was found 1 when the optimal dose of bacteriophage KS4-M used in the experiment was approximately 2.5103 PFU. At this optimal dose, 60% of the treated larvae remained alive 48-hours post-injection, while all untreated larvae died at this time point.

Jeremy Math- US 20150044175 ers, Alexander A1 Sulakvelidze 12 Feb 2015

Bacteriophage C. perfringens preparations and methods of use thereof

CPAS-12, CPAS15, CPAS-16 CPLV-42

Develop a purified bacteriophage preparation that is able to [104] lyse effectively a large number of groups of C. perfringens strains, this result in the reduction in the mortality rate of the chicken due this infection. The method include administration of a purified bacteriophage preparation consisting of four or more C. perfringens-specific bacteriophage (108 PFU/ml) in which each of the incorpotrated bacteriophage has lytic activity against at least five C. perfringens strains that is responsible for necrotic enteritis in poultry.

US 20150064156 Vincent A. Fischetti, Anu A1 Daniel, Chad 5 Mar 2015 Euler

StaphyloChimeric bacteriophage coccus aureus lysin with activity against staphylococci bacteria

A novel chimeric lysin called ClyS

Bacteriophage endolysins (lysins) are novel antimicrobial [105] agents that are used for the prophylactic and therapeutic treatment of infections caused due to bacteria. During the infection cycle of double-stranded DNA bacteriophages, cell wall hydrolases are produced which is termed as lysins. Native or recombinant lysins were able to degrade the cell wall of prone bacteria and thus cause rapid lysis of cell, when applied exogenously. The result reveals that 5 micrograms of ClyS corresponded to 1 U (unit) of lytic activity. When 50 U of ClyS was added to exponentially growing 8325-4 cells the OD600 dropped to baseline within 5 min. Transmission electron microscopy was used to visualize the lytic effect 50 U of ClyS for 1-3 min on S. aureus 8325-4 cells. Typical of lysin activity observed previously, localized degradation of the cell wall was observed at single or multiple sites.

EK99P-1

This invention relates to a composition comprising EK99P-1, a bacteriophage isolated from natural source which is capable to kill E. coli type K99, thus emerging out as a novel method for preventing and treating E. coli type K99 infections. The composition comprises of carriers like lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil which are pharmaceutically acceptable. According to the present invention, the bacteriophage EK99P-1 or the variants thereof are included in the composition at the concentration of 110 PFU/ml to 11015 PFU/ml. The results from this invention clearly reveals that the bacteriophage EK99P-1 not only suppresses the growth of E. coli type K99 but also effectively destroys them, so that the phage can be used as an active ingredient in the composition formulated for the purpose of the prevention of E. coli type K99 infections.

Seongjun Yoon, Sooyoun Jun, Hyoungrok Paik, Jeesoo Son, Sanghyeon Kang

Patent Number

Rastogi et al.

US 20150004141 Method for E. coli type A1 prevention and K99 treatment of 1 Jan 2015 Escherichia coli type K99 infections

(Table 3) Contd….

Ref.

[106]


Inventor(s)

Patent Number

Objective

Bacteria

Venigalla B. Rao, Pan Tao

US 20150017198 Mutated and Yersinia A1 bacteriophage pestis CO92 t4 nanoparticle 15 Jan 2015 arrayed f1-v immunogens from Yersina pestis as next generation plague vaccines


Bacteriophage


T4

Techniques developed from two basic approaches namely [107] structure-based immunogen design and phage T4 nanoparticle delivery are used to design novel plague vaccines. The NH2- terminal -strand of F1 of Yersinia pestis is transplanted to the COOH-terminus of F1 of Yersinia pestis and the NH2-- terminus sequence flanking the strand of F1 of Yersinia pestis is duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 is fused to the V antigen of Yersinia pestis to thereby form a fusion protein F1mut-V mutant, which produces a entirely soluble monomer. The fusion protein F1mut-V is then arrayed on phage T4 nanoparticles via a small outer capsid protein, Soc, from a T4 phage or a T4related phage. Both the soluble and T4 decorated F1mut-V are capable of providing approximately 100% protection to mice and rats against pneumonic plague elicited by high doses of Yersinia pestis CO92.

successful use in vivo. Generally, mixture of phages i.e., cocktail approach has been used to overcome the problems associated with the limited host range of any single phage. But, the intricacy of phage cocktails is restricted by regulation and manufacturing issues. Thus, by the use of modern biotechnology and automation techniques, engineering efforts are expanding to develop new strategies to overcome this problem. For example, Marzari et al. in their study found that host range is extended when g3p phage protein of one filamentous phage is grafted to another [112]. Another technical hurdle is evolution of bacterial resistance to phages through a variety of extracellular mechanism including phage adsorption blocking, constraining the injection of phage genomes and intracellular mechanisms (such as restriction-modification systems, and abortive infection systems). The techniques to overcome this problem include use of phage cocktails (traditional approach), phages can be given in combination with other antimicrobials, such as antibiotics, and use of modified phages develop through some engineering techniques to directly target the phage-resistance mechanism [113]. Phage manufacturing is also related to another technical challenge. The manufacturing process (including numerous steps like preparation, purification, formulation, and quality control) of bacteriophage becomes complicated and difficult due to the presence of endotoxins, pyrogens and other cellular toxins (metabolites) which are released during cell lysis. Merabishvili et al. in their study utilized a commercially available endotoxin removal kit to accomplish adequate purity. Thus, they illustrate a complete protocol for the isolation, characterization, manufacturing, purification, and quality control of bacteriophages for clinical use [114]. The toxicity of bacteriophage is a topic of concern when the toxicity is a consequence of cell lysis. Phages, upon death and lysis, can release bacterial toxins (endotoxins) that stimulate inflammatory response which ultimately results in bacterial infections which could appear to be limited as well as mild [115, 116]. These infections might be difficult to treat using any antibacterial agent and can cause remarkable morbidity. In order to decrease the risk of this toxicity, different approaches have been reported in the literature by the scientists. Phages can be selected or engi-

15

Ref.

neered (lysis-deficient and/or non-replicative) and/or purified in a way that they are minimally associated with virulence factors, such as endotoxins or exotoxin-encoding genes [117, 118]. These approaches can improve survival by considerably reducing the amount of bacterial endotoxin and mediators of inflammation produced during phage therapy [119]. For example, in one of the such studies, Hagens and his coworkers engineered filamentous phages to express restriction endonucleases and holins in E. coli [120]. These engineered phages did not cause cell lysis, and thus levels of released endotoxins were found low. In another study, they also designed non-lytic and non-replicative phages against Pseudomonas aeruginosa [121]. They observed that the survival rate of infected mice treated with non-lytic phages was increased as compared with lytic phages due to decreased levels of inflammation. In a similar study, Matsuda et al. reported significantly better survival rate within the lysisdeficient treatment groups at 6 h and 12 h post-treatment, in addition to simultaneous lowering in the levels of inflammatory markers, TNF- and IL-6 at 12 h in a murine peritonitis model [122]. The non-technical hurdles include regulatory approvals, patent protection and market acceptance. The regulatory status for the phage therapy needs to be more defined. Only few preparations or clinical trials involving phages were subjected to the same process of regulatory approval that other antimicrobial agents undergo today, before they are approved for human therapy or other commercial applications. Certainly, regulatory approval process along with patent protection is needed for phage therapy like other antimicrobials. Lastly, market acceptance comprises a fairly broad range of issues, from consumer acceptance to the product’s cost. Despite of the fact that how effective and safe phage-based products, their acceptance in addition with the other approaches and methods for preventing and treating bacterial infections is finally dependent on their status in the market. So, educating the general public about the phage therapy and their application is of paramount importance and is needed for market acceptance [119].

16


Table 4.

S. No.

Rastogi et al.

Commercial companies involved in Phage R&D.

Company

Description

1

Biochimpharm (Georgia)

Company deals with the various phage lysates mix which are used for intestinal problems, for example, Dysentery, salmonellosis, dyspepsia, colitis and enterocolitis and for bacterial infections. Products include: Phagesti, Phagyo, Phagedys, Phagetyph, Phagestaph, Phagesal, Phagepy.

2

Biophage Pharma Inc. (Canada)

Company deals with the environmental therapies and diagnostics, phage products prepared to overcome problems associated with antibacterial resistance and as a weapon against bioterrorism.

3

Biopharm Limited (Georgia)

Company deals with products include Pyobacteriophage and Intesti-bacteriophage that are mixtures of phage lysates for bacterial intestinal and infection control-sold to pharmacies as Over The Counter drugs.

4

BigDNA (UK)

Company deals with the Bacteriophage DNA vaccination delivered through intravenous or oral route via phage encoded DNA.

5

Intralytix (USA)

Company deals with the development and commercialization of therapeutic phages. Products include: ListShield, EcoSchield, SalmoFresh.

6

EBI Food Safety (Exponential Biotherapies) (New York)

Company deals with a food-processing aid that targets Listeria monocytogenes strains on food products. Product include: LISTEX™ P100

7

Gangagen (Bangalore)

Company deals with the phage products against Staphylococcus aureus. The lead product of this company is a proprietary recombinant protein, P128 which is developed from a key protein of bacteriophage, for the topical prevention and treatment of Staplylococcal infections, including infection with methicillin-resistant Staphylococcus aureus (MRSA).

8

Immunopreparat Research Productive Association (Russia)

The company’s subsidiary, Biophag, currently manufactures at least two complex phage preparations (“Bacteriophagum” and “Piobacteriaphagum”) targeting various bacterial pathogens.

9

Special Phage Services Ptv Ltd (Australia)

SPS has an exclusive license with a Georgian-based company for accessing phage production know-how as well as a phage library of over 3,000 strains. The company develops and commercializes tailored phage therapeutics (SmartPhageTM) for use in human and animal health, food and environmental markets in Australia and Asia.

10

Targanta Therapeutics Inc. (Massachusetts)

Company deals with the development of small-molecule antibacterial agents which cause the disruption of the DNA replication and transcription machinery through the targets of bacteriophages.

11

Omnilytics (USA)

The company deals with the phage product named as AgriPhage which is a natural, safe, effective in the prevention and control of infections caused by harmful bacteria on tomato and pepper plants.

12

Novolytics (UK)

Deals with the designing of a bacteriophage cocktail targeted at methicillin resistant Staphylococcus aureus (MRSA). Company develops a gel formulation containing the bacteriophage cocktail that can be applied topically, code named NOV012

9. FUTURE SCOPE There are already multiple companies that produce phage-based antibacterial products for food safety and animal usages. Table 4 enlists the name and description of companies involved in Phage R&D [123, 124]. There are increasing number of researchers and companies which are involved in the phage research and clinical trials. The first product that is approved by FDA is LMP-102™ which is a bacteriophage-based product applied by spray to ready to eat meat and poultry products just prior to packaging.LMP-102™ is a mixture of equal proportions of six individually purified Listeria monocytogenes-specific bacteriophages in phosphatebuffered saline. The phages are lytic double-stranded DNA phages that belong to the family Siphoviridae [125]. LISTEX™ P100, a product of Micreos Food Safety, was the first phage product to be recognized as GRAS by the FDA (Food & Drug Administration, USA). LISTEX™ is a culture of safe micro-organisms (bacteriophage preparation) which is char-

acterized by its broad spectrum towards the strains of Listeria monocytogenes [126]. Recently, SalmoFresh™, a phage product of Intralytix company has also acquired GRAS (Generally Recognized as Safe) recognition with GRAS Notice No. GRN 000435 from the FDA in 2013 for its direct applications onto poultry, fish and shell fish, and fresh and processed fruits and vegetables [127]. Apart from the food safety products like ListShield, EcoSchield, SalmoFresh, Intralytix, at present, has two therapeutic products for human use in various stages of development that will prove successful in numerous crucial and unmet medical needs. One of the prototype phage preparations by Intralytix for treating infected wounds was successfully used during the Phase I human clinical trial in Lubbock, Texas [128]. Fortunately, with advances in biotechnology, molecular techniques and using extensive current knowledge about phage biology, companies now have the leverage in making the second wave of phage therapy a successful one. The


phage therapy would not eradicate any drugs or vaccines present in the market. However, they might provide an alternative, a medicine of choice and a possibility to boost up some types of therapies especially in the case of multi-drug resistant infections. Therefore, despite a controversial beginning, d’Herelle’s phage therapy may at last be about to accomplish its potential.


REFERENCES [1] [2] [3] [4]

CONCLUSION The global emergence of the problems associated with the resistance of pathogenic bacteria to antibiotics makes it crucial to exploit alternative strategies to combat this threat. Several characteristics of bacteriophage like high specificity, ease of replication at the site of infection and less side effects, make them potentially attractive therapeutic agents to overcome the problem associated with the antibiotics. Details discussed above give a brief look of the wide range of applications of phages in treating bacterial infections which are anitibiotic resistant, with a glimpse of assessing the wide range of routes used for the delivery of phages. From our review, it can be clearly observed that parenteral route is the most efficacious and majorly used for delivery of bacteriophages followed by the oral route.Thus, bacteriophages can be considered safe and useful to humans in many ways. By engineering the phages or by making a cocktail of phages it would become easy to treat various bacterial infections which are as such resistant to the current generations of antibiotics available in the market. Based on the few examples presented here, the use of bacteriophages to control bacterial infections shows therapeutic promise. Despite their wide applications, there are some concerns about the phage therapy which make them a challenging antimicrobial therapy. In order to achieve proper clinical use, regulatory and technical impediments of this therapy have to be overcome. The current pharmaceutical regulatory framework and business models are not agreeable with a dynamic and sustainable phage therapy concept. Clarity on the regulatory approval, patent protection and market acceptance of natural as well as engineered phages will allow researchers, companies, and investors to design and develop more appropriate ways to explore their use. Technical hurdles associated with phage therapy such as limited host range of phages, the expansion of phage-resistant bacteria, problems in phage manufacturing, purification, growth optimization, systemic side effects, and phage delivery needed to be addressed.

[5] [6] [7] [8] [9]

[10]

[11]

[12] [13] [14]

[15] [16] [17]

[18] [19] [20]

[21]

CONSENT FOR PUBLICATION Not applicable.

[22]

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

[23] [24]

ACKNOWLEDGEMENTS The authors express their sincere thanks to Prof. R.M. Dubey, Vice Chancellor, IFTM University, Moradabad, Uttar Pradesh, India, for his constant encouragement to carry out this work.

17

[25]

[26]

Petrenko, V.A.; Smith P.G. Phage Nanobiotechnology, Royal Society of Chemistry: United Kingdom, 2011. Prescott, L.M. Microbiology, 5th ed.; McGraw Hill Education: New York, 2002. Tauro, P.; Kapoor, K.K.; Yadav, K.S. An Introduction to Microbiology, New Age International: New Delhi, 2004. d’Hérelle, F. On an invisible microbe antagonistic toward dysenteric bacilli: Brief note by Mr. F. D’Herelle, presented by Mr. Roux. 1917. Res. Microbiol., 2007, 158, 553-554. d’Hérelle, F.; Ackermann, H.W. On an invisible microbe antagonistic to dysentery bacilli. Note by M. F. d’Herelle, presented by Mr. Roux. CR Acad. Sci., 1917, 165, 373-375. Kutter, E.; Sulakvelidze, A. Bacteriophages: Biology and Applications, CRC Press: New York, 2005. Mathews, C.K. Bacteriophage T4, American Society for Microbiology: Washington, DC, 1983. Sulakvelidze, A.; Alavidze, Z.; Morris J.G. Jr. Bacteriophage therapy. Antimicro. Agents. Chemo., 2001, 45, 649-659. Chopra, I.; Hodgson, J.; Metcalf, B.; Poste, G. The search for antimicrobial agents effective against bacteria resistant to multiple antiobiotics. Antimicrob. Agents Chemother., 1997, 41, 497-503. Ryan, E.M.; Gorman, S.P.; Donnelly, R.F.; Gilmore, B.F. Recent advances in bacteriophage therapy: How delivery routes, formulation, concentration and timing influence the success of phage therapy. J. Pharm. Pharmacol., 2011, 63, 1253-1264. Infectious Disease Society of America. IDSA Report: Bad Bugs, No Drugs: As Antibiotic Discovery Stagnates, a Public Health Crisis Brews. [Online] July 2004. http://www.idsociety.org/ badbugsnodrugs.html. (Accessed On: March 19, 2015). Cars, O.; Högberg, L.D.; Murray, M.; Nordberg, O.; Sivaraman, S.; Lundborg, C.S.; So, A.D.; Tomson, G. Meeting the challenge of antibiotic resistance. BMJ., 2008, 337, 726-728. Morel, C.; Mossialos, E. Stoking the antibiotic pipeline. BMJ., 2010, 340, 1115-1118. European Medicines Agency. European Centre for Disease Prevention and Control. Joint technical report: the bacterial challenge – time to react. (Accessed On: March 11, 2015). Phage therapy. http://www.phagetherapycenter.com/pii/Patient Servlet? command=static_phagetherapy (Accessed On: March 11, 2015). Skurnik, M.; Strauch, E. Phage therapy: Facts and fiction. Int. J. Med. Microbiol., 2006, 296, 5-14. Isolation of Bacteriophage from Sewage and Determination of Phage Titer. http://highered.mheducation.com/sites/dl/free/ 0072487445/92720/Exercise37.pdf (Accessed On: March 11, 2015). Nikaido, H. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev., 2003, 67, 593-656. Lindberg, A.A. Bacteriophage receptors. Ann. Rev. Microbiol., 1973, 27, 205-241. VanAlphen, L.; Havekes, L.; Lugtenberg, B. Major outer membrane protein of Escherichia coli K12. Purification and in vitro activity of bacteriophages K3 and f-pilus conjugation. FEBS Lett., 1977, 75, 285-290. Scurray, R.A.; Hancock, R.E.W.; Reeves, P. Con- mutants: Class of mutants in Escherichia coli K-12 lacking a major cell wall protein and defective in conjugation and adsorption of a bacteriophage. J. Bacteriol., 1974, 119, 726-735. Rakhuba, D.V.; Kolomiets, E.I.; Dey, E.S.; Novik, G.I. Bacteriophage receptors, mechanism of phage adsorption and penetration into host cell. Pol. J. Microbiol., 2010, 59(3), 145-155. Datta, D.B.; Arden, B.; Henning, U. Major protein of the Escherichia coli outer cell envelop membrane as bacteriophage receptor. J. Bacteriol., 1977, 131(3), 821-829. Yu, F.; Mizushima, S. Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K12 in the receptor function for bacteriophage T4. J. Bacteriol., 1982, 151(2), 718-722. Montag, D.; Hashemolhosseini, S.; Henning, U. Receptorrecognizing proteins of T-even type bacteriophages. The receptorrecognizing area of proteins 37 of phages T4 TuIa and TuIb. J. Mol. Biol., 1990, 216(2), 327-334. Heller, K.J. Molecular interaction between bacteriophage and gram-negative cell envelope. Arch. Microbiol., 1992, 158(4), 235248.

18 [27]

[28] [29]

[30] [31]

[32]

[33] [34] [35] [36]

[37] [38]

[39] [40]

[41] [42] [43]

[44]

[45] [46]

[47]

[48]

[49] [50]

Current Drug Delivery, 2018, Vol. 15, No. 1 Riede, I.; Degen, M.; Henning, U. The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein. EMBO J., 1985, 4(9), 2343-2346. Hantke, K. Major outer membrane proteins of E. coli K12 serve as receptors for the phages T2 (protein Ia) and 434 (protein Ib). Mol. Gen. Genet., 1978, 164(2), 131-135. Jesaitis, M.A.; Goebel, W.F. The chemical and antiviral properties of the somatic antigen of phase II Shigella sonnei. J. Exp. Med., 1952, 96(5), 409-424. Randall-Hazelbauer, L.; Schwartz, M. Isolation of the bacteriophage lambda receptor from Escherichia coli. J. Bacteriol., 1973, 116(3), 1436-1446. Hashemolhosseini, S.; Holmes, Z.; Mutschler, B.; Henning, U. Alterations of receptor specificities of coliphages of the T2 family. J. Mol. Biol., 1994, 240(2), 105-110. Hashemolhosseini, S.; Montag, D.; Krämer, L.; Henning, U. Determinants of receptor specificity of coliphages of the T4 family. A chaperone alters the host range. J. Mol. Biol., 1994, 241(4), 524533. Wilkinson, S.G. Bacterial lipopolysaccharides-themes and variations. Prog. Lipid Res., 1996, 35(3), 283-343. Michael, J.G. The surface antigens and phage receptors in Escherichia coli B. Proc. Soc. Exp. Biol. Med., 1968, 128(2), 434438. Weidel, W. Bacterial viruses (with particular reference to adsorption penetration). Ann. Rev. Microbiol., 1958, 12, 27-48. Takeda, K.; Uetake, H. In vitro interaction between phage and lipopolysaccharide: A novel glycosidase associated with Salmonella phage 15. Virology, 1973, 52, 148-159. Kanegasaki, S.; Wright, A. Studies on the mechanism of phage adsorption: Interaction between Epsilon 15 and its cellular receptor. Virology, 1973, 52, 160-173. Iwashita, S.; Kanegasaki, S. Smooth specific phage adsorption: endorhamnosidase activity of tail parts of P22. Biochem. Biophys. Res. Commun., 1973, 55(2), 403-409. Eriksson, U.; Svenson, S.B.; Lönngren, J.; Lindberg, A.A. Salmonella phage glycanases: Substrate specificity of the phage P22 endo-rhamnosidase. J. Gen. Virol., 1979, 43(3), 503-511. Reske, K.; Wallenfels, B.; Jann, K. Enzymatic degradation of Oantigenic lipopolysaccharides by coliphage omega 8. Eur. J. Biochem., 1973, 36(1), 167-171. Lindberg, A.A. Bacteriophage receptors. Annu. Rev. Microbiol., 1973, 27, 205-241. Coyette, J.; Ghuysen, J.M. Structure of the cell wall of Staphylococcus aureus, strain Copenhagen. IX. Teichoic acid and phage adsorption. Biochemistry, 1968, 7(6), 2385-2389. Chatterjee, A.N. Use of bacteriophage-resistant mutants to study the nature of the bacteriophage receptor site of Staphylococcus aureus. J. Bacteriol., 1969, 98(2), 519-527. Murayama, Y.; Kotani, S.; Kato, K. Solubilization of phage receptor substances from cell walls of Staphylococcus aureus (strain Ceopenhagen) by cell wall lytic enzymes. Biken J., 1968, 11(4), 269-291. Davison, S.; Couture-Tosi, E.; Candela, T.; Mock, M.; Fouet, A. Identification of the Bacillus anthracis phage receptor. J. Bacteriol., 2005, 187(19), 6742-6749. Räisänen, L.; Draing, C.; Pfitzenmaier, M.; Schubert, K.; Jaakonsaari, T.; von Aulock, S.; Hartung, T.; Alatossava, T. Molecular interaction between lipoteichoic acids and Lactobacillus delbrueckii phages depends on D-alanyl and -glucose substitution of poly(glycerophosphate) backbones. J. Bacteriol., 2007, 189(11), 4135-4140. Monteville, M.R.; Ardestani, B.; Geller, B.L. Lactococcal bacteriophages require host cell wall carbohydrate and a plasma membrane protein for adsorption and ejection of DNA. Appl. Environ. Microbiol., 1994, 60(9), 3204-3211. Pate, J.L.; Petzold, S.J.; Umbreit, T.H. Two flagellotropic phages and one pilus-specific phage active against Asticcacaulis biprosthecum. Virology, 1979, 94, 24-37. Chakrabarty, A.M.; Niblack, J.F.; Gunsalus, I.C. A phage-initiated polysaccharide depolymerase in Pseudomonas putida. Virology, 1967, 32, 532-534. Park, B.H. An enzyme produced by a phage-host cell system. I. The properties of a Klebseilla phage. Virology, 1956, 2(6), 711718.

Rastogi et al. [51] [52] [53] [54] [55] [56]

[57]

[58]

[59] [60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

Taylor, K. Enzymatic deacetylation of Vi-polysaccharide by Viphage. II. Biochem. Biophys. Res. Commun., 1965, 20, 752-756. Taylor, K. Physical and chemical changes Vi-polysaccharide due to Vi-phage II action. Acta. Biochim. Pol., 1966, 13, 79-106. Hohn, T.; Hohn, B. Structure and assembly of simple RNA bacteriophages. Adv. Virus Res., 1970, 16, 43-98. Roberts, J.W.; Steitz, J.E. The reconstitution of infective bacteriophages R17. Proc. Natl. Acad. Sci. USA, 1967, 58(4), 1416-1421. Meynell, G.G., Lawn, A.M. Filamentous phages specific for the I sex factor. Nature, 1968, 217(5134), 1184-1186. Chibani-Chennoufi, S.; Sidoti, J.; Bruttin, A.; Kutter, E.; Sarker, S.; Brüssow, H. In vitro and in vivo bacteriolytic activities of phages: Implications for phage therapy. Antimicrob. Agents Chemother., 2004, 48(7), 2558-2569. Tanji, Y.; Shimada, T.; Fukudomi, H.; Miyanaga, K.; Nakai, Y.; Unno, H. Therapeutic use of phage cocktail for controlling Escherichia coli O157:H7 in gastrointestinal tract of mice. J. Biosci. Bioeng., 2005, 100(3), 280-287. Denou, E.; Bruttin, A.; Baretto, C.; Ngom-Bru, Catherine.; Brussow, H.; Zuber, S. T4 phages against Escherichia coli diarrhea: Potential and problems. Virology, 2009, 388, 21-30. Brussow, H.; Bruttin, A. Human volunteers receiving Escherichia coli phage T4 orally: A safety test of phage therapy. Antimicrob. Agents Chemother., 2005, 49(7), 2874-2878. Watanabe, R.; Matsumoto, T.; Sano, G.; Ishii, Y.; Tateda, K.; Sumiyama, Y.; Uchiyama, J.; Sakurai, S.; Matsuzaki, S.; Imai, S.; Yamaguchi, K. Efficacy of bacteriophage therapy against gutderived sepsis caused by Pseudomonas aeruginosa in mice. Antimicrob. Agents Chemother., 2007, 51(2), 446-452. Andreatti Filho, R.L.; Higgins, J.P.; Higgins, S.E.; Gaona, G.; Wolfenden, A.D.; Tellez, G.; Hargis, B.M. Ability of bacteriophages isolated from different sources to reduce Salmonella enterica serovar enteritidis in vitro and in vivo. Poult. Sci., 2007, 86(9), 1904-1909. Maura, D.; Galtier, M.; Le Bouguénec, C.; Debarbieux, L. Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine. Antimicrob. Agents Chemother., 2012, 56(12), 6235-6242. Carvalho, C.M.; Gannon, B.W.; Halfhide, D.E.; Santos, S.B.; Hayes, C.M.; Roe, J.M.; Azeredo, J. The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers of Campylobacter coli and Campylobacter jejuni in chickens. BMC Microbiol., 2010, 10, 232. Nishikawa, H.; Yasuda, M.; Uchiyama, J.; Rashel, M.; Maeda, Y.; Takemura, I.; Sugihara, S.; Ujihara, T.; Shimizu, Y.; Shuin, T.; Matsuzaki, S. T-even related bacteriophages as candidates for treatment of Escherichia coli urinary tract infections. Arch. Virol., 2008, 153(3), 507-515. Chhibber, S.; Kaur, S.; Kumari, S. Therapeutic potential of bacteriophage in treating Klebsiella pneumonia B5055-mediated lobar pneumonia in mice. J. Med. Microbiol., 2008, 57, 1508-1513. Dabrowska, K.; Opolski, A.; Wietrzyk, J.; Switala-Jelen, K.; Boratynski, J.; Nasulewicz, A.; Lipinska, L.; Chybicka, A.; Kujawa, M.; Zabel, M.; Dolinska-Krajewska, B.; Piasecki, E.; WeberDabrowska, B.; Rybka, J.; Salwa, J.; Wojdat, E.; Nowaczyk, M.; Gorski, A. Antitumour activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of beta 3 integrin signaling pathway. Acta. Virol., 2004, 48(4), 241-248. Gorski, A.; Dabrowska, K.; Wietrzyk, J.; Nasulewicz, A.; Opolski, A. In: Bacteriophage inhibits metastasis in mouse transplantable melanoma model In: American Association for Cancer Research: An AACR Special Conference in Cancer Research "Proteases, Extracellular Matrix, and Cancer" October 9-13, 2002, Hilton Head Island, USA, Conference Proceedings: Abstracts of Poster Presentation n. B33. Dabrowska, K.; Opolski, A.; Wietrzyk, J.; Switala-Jelen, K.; Godlewska, J.; Boratynski, J.; Syper, D.; Weber-Dabrowska, B.; Gorski, A. Anticancer activity of bacteriophage T4 and its mutant HAP1 in mouse experimental tumour models. Anticancer Res., 2004, 24(6), 3991-3995. Gu, J.; Liu, X.; Li, Y.; Han, W.; Lei, L.; Yang, Y.; Zhao, H.; Gao, Y.; Song, J.; Lu, R.; Sun, C.; Feng, X. A method for generation phage cocktail with great therapeutic potential. PLoS One, 2012, 7(3), e31698. Barrow, P.; Lovell, M.; Berchieri, A. Jr. Use of lytic bacteriophage for control of experimental Escherichia coli septicemia and menin-


[71]

[72]

[73] [74]

[75]

[76]

[77]

[78]

[79] [80]

[81]

[82]

[83]

[84] [85]

[86]

[87]

[88] [89] [90]

gitis in chickens and calves. Clin. Diagn. Lab. Immunol., 1998, 5(3), 294-298. Cerveny, K.E.; DePaola, A.; Duckworth, D.H.; Gulig, P.A. Phage therapy of local and systemic disease caused by Vibrio vulnificus in iron-dextran-treated mice. Infect. Immun., 2002, 70(11), 62516262. Wills, Q.F.; Kerrigan, C.; Soothill, J.S. Experimental bacteriophage protection against Staphylococcus aureus abscesses in a rabbit model. Antimicrob. Agents Chemother., 2005, 49(3), 1220-1221. McVay, C.S.; Velasquez, M.; Fralick, J.A. Phage therapy of Pseudomonas aeruginosa infection in a mouse burn wound model. Antimicrob. Agents Chemother., 2007, 51, 1934-1938. Capparelli, R.; Parlato, M.; Borriello, G.; Salvatore, P.; Iannelli, D. Experimental phage therapy against Staphylococcus aureus in mice. Antimicrob. Agents Chemother., 2007, 51(8), 2765-2773. Matsuzaki, S.; Yasuda, M.; Nishikawa, H.; Kuroda, M.; Ujihara, T.; Shuin, T.; Shen, Y.; Jin, Z.; Fujimoto, S.; Nasimuzzaman, M.D.; Wakiguchi, H.; Sugihara, S.; Sugiura, T.; Koda, S.; Muraoka, A.; Imai, S. Experimental protection of mice against lethal Staphylococcus aureus infection by novel bacteriophage phi MR11. J. Infect. Dis., 2003, 187(4), 613-624. Stanford, K.; McAllister, T.A.; Niu, Y.D.; Stephens, T.P.; Mazzocco, A.; Waddell, T.E.; Johnson, R.P. Oral delivery systems for encapsulated bacteriophages targeted at Escherichia coli O157:H7 in feedlot cattle. J. Food Prot., 2010, 73(7), 1304-1312. Ryan, E.; Garland, M.J.; Singh, T.R.; Bambury, E.; O'Dea, J.; Migalska, K.; Gorman, S.P.; McCarthy, H.O.; Gilmore, B.F.; Donnelly, R.F. Microneedle-mediated transdermal bacteriophage delivery. Eur. J. Pharm. Sci., 2012, 47(2), 297-304. Goode, D.; Allen, V.M.; Barrow, P.A. Reduction of experimental Salmonella and Campylobacter contamination of chicken skin by application of lytic bacteriophages. Appl. Environ. Microbiol., 2003, 69(8), 5032-5036. Kumari, S.; Harjai, K.; Chhibber, S. Topical treatment of Klebsiella pneumoniae B5055 induced burn wound infection in mice using natural products. J. Infect. Dev. Ctries., 2010, 4(6), 367-377. Debarbieux, L.; Leduc, D.; Maura, D.; Morello, E.; Criscuolo, A.; Grossi, O.; Balloy, V.; Touqui, L. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J. Infect. Dis., 2010, 201(7), 1096-1104. Morello, E.; Saussereau, E.; Maura, D.; Huerre, M.; Touqui, L.; Debarbieux, L. Pulmonary bacteriophage therapy on Pseudomonas aeruginosa cystic fibrosis strains: First steps towards treatment and prevention. PLoS One, 2011, 6(2), e16963. Oliveira, A.; Sereno, R.; Nicolau, A.; Azeredo, J. The influence of the mode of administration in the dissemination of three coliphages in chickens. Poult. Sci., 2009, 88(4), 728-733. Oliveira, A.; Sereno, R.; Azeredo, J. In vivo efficiency evaluation of a phage cocktail in controlling severe colibacillosis in confined conditions and experimental poultry houses. Vet. Microbiol., 2010,146(3-4), 303-308. Huff, W.E.; Huff, G.R.; Rath, N.C.; Balog, J.M.; Donoghue, A.M. Prevention of Escherichia coli infection in broiler chickens with a bacteriophage aerosol spray. Poult. Sci., 2002, 81(10), 1486-1491. Golshahi, L.; Lynch, K.H.; Dennis, J.J.; Finlay, W.H. In vitro lung delivery of bacteriophages KS4-M and KZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis. J. Appl. Microbiol., 2011, 110(1), 106-117. Narasimhaiah, M.H.; Asrani, J.Y.; Palaniswamy, S.M.; Bhat, J.; George, S.E., Srinivasan, R.; Vipra, A.; Desai, S.N.; Junjappa, R.P.; Sriram, B.; Padmanabhan, S. Therapeutic potential of Staphylococcal bacteriophages for nasal decolonization of Staphylococcus aureus in Mice. Adv. Microbiol., 2013, 3, 52-60. Wright, A.; Hawkins, C.H.; Anggård, E.E.; Harper, D.R. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Clin. Otolaryngol., 2009, 34(4), 349-357. Fraise, A.P.; Maillard, J.P.; Sattar, S. Russelle, Hugo and Ayliffe’s Principles and Practice of Disinfection, Preservation and Sterilization, 5th ed.; Wiley-Blackwell: U.K., 2013. Payne, R.J.; Jansen, V.A. Pharmacokinetic principles of bacteriophage therapy. Clin. Pharmacokinet., 2003, 42, 315-325. Abedon, S.T.; Kuhl, S.J.; Blasdel B.G.; Kutter, E.M. Phage treatment of human infections. Bacteriophage, 2011, 1(2), 66-85.

Current Drug Delivery, 2018, Vol. 15, No. 1 [91]

[92] [93]

[94] [95] [96] [97]

[98]

[99] [100]

[101] [102] [103]

[104] [105]

[106] [107]

[108] [109]

[110] [111] [112] [113] [114]

[115]

[116]

19

Payne, R.J.; Jansen, V.A. Understanding bacteriophage therapy as a density-dependent kinetic process. J. Theor. Biol., 2001, 208, 3748. Dabrowska, K.; Switala-Jelen, K.; Opolski, A.; Weber-Dabrowska, B.; Gorski, A. Bacteriophage penetration in vertebrates. J. Applied Microb., 2005, 98, 7-13. Nungester, W.J.; Watrous, R.M. Accumulation of bacteriophage in spleen and liver following its intravenous inoculation. Proc. Soc. Exp. Biol. Med., 1934, 31, 901-905. Mahony, J; Sinderen, D. Gram Positive Phages: From Isolation to Application, Frontiers Media: SA, 2015. Merril, C.R.; Biswas, B.; Carlton, R.; Jensen, N.C.; Creed, G.J.; Zullo, S.; Adhya, S. Long-circulating bacteriophage as antibacterial agents. Proc. Natl. Acad. Sci., 1996, 93, 3188-3192. Schaechter, M. Encyclopedia of Microbiology, 3rd ed.; Elsevier Publication: U.K., 2009. Drulis-Kawa, Z.; Majkowska-Skrobek, G.; Maciejewska, B.; Delattre, A.S.; Lavigne, R. Learning from Bacteriophages - advantages and limitations of phage and phage-encoded protein applications. Curr. Protein Pept. Sci., 2012, 13(8), 699-722. Heo, Y.J.; Lee, Y.R.; Jung, H.H.; Cho, Y.H. Phage therapy against Pseudomonas aeruginosa. U.S. Patent 8282920 B2, October 9, 2012. Clokie, M. Therapeutic bacteriophages. WO2014030020 A1, February 27, 2014. Hernan, R.; Torres, E.; Ormazabal, J.M.R.; Romo, R.A.B.; Guajardo, G.A.H. Bacteriophages useful for the prophylaxis and therapy of Vibrio anguillarum. U.S. Patent 20140105866 A1, April 17, 2014. Llagostera, M.; Barbé, J.; Bardina, C.; Cortés, M.P.; Spricigo, D.A. Novel salmonella bacteriophage compositions and uses thereof. US Patent 20140219968 A1, August 7, 2014. Harper, D. Beneficial effects of bacteriophage treatments. U.S. Patent 8475787 B2, July 2, 2013. Finlay, W.H.; Dennis, J.J.; Orszanska, H.; Seed, K.D.; Lynch, K.H. Bacteriophage compositions for treatment of bacterial infections. U.S. Patent 8889105 B2, November 18, 2014. Mathers, J.; Sulakvelidze, A. Bacteriophage preparations and methods of use thereof. U.S. Patent 20150044175 A1, February 12, 2015. Fischetti, V.A.; Daniel, A.; Euler, C. Chimeric bacteriophage lysin with activity against staphylococci bacteria. U.S. Patent 20150064156 A1, March 5, 2015. Yoon, S.; Jun, S.; Paik, H.; Son, J.; Kang, S. Method for prevention and treatment of Escherichia coli type K99 infections. U.S. Patent 20150004141 A1, January 1, 2015. Rao, V.B.; Tao, P. Mutated and bacteriophage t4 nanoparticle arrayed f1-v immunogens from Yersina pestis as next generation plague vaccines. U.S. Patent 20150017198 A1, January 15, 2015. Merril, C.R. Interaction of Bacteriophages with Animals. In: Bacteriophage Ecology, Abedon, S.T., Ed.; Cambridge University Press: Cambridge, UK, 2008, pp. 332-352. Gorski, A.; Weber-Dabrowska, B. The potential role of endogenous bacteriophages in controlling invading pathogens. Cell. Mol. Life Sci., 2005, 62(5), 511-519. Abedon, S.T.; Thomas-Abedon, C. Phage therapy pharmacology. Curr. Pharm. Biotechnol., 2010, 11(1), 28-47. Summers, W.C. Bacteriophage therapy. Annu. Rev. Microbiol., 2001, 55, 437-451. Marzari, R.; Sblattero, D.; Righi, M.; Bradbury, A. Extending filamentous phage host range by the grafting of a heterologous receptor binding domain. Gene, 1997, 185, 27-33. Labrie, S.J.; Samson, J.E.; Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol., 2010, 8, 317-327. Merabishvili, M.; Pirnay, J.-P.; Verbeken, G.; Chanishvili, N.; Tediashvili, M.; Lashkhi, N.; Glonti, T.; Krylov, V.; Mast, J.; Van Parys, L. Quality-controlled small-scale production of a welldefined bacteriophage cocktail for use in human clinical trials. PLoS One, 2009, 4, e4944. Górski, A.; Borysowski, J.; Miedzybrodzki, R.; Weber-Dabrowska, B. Bacteriophages in medicine. In: Bacteriophage: Genetics and Microbiology; McGrath, S.; van Sinderen, D., Eds.; Caister Academic Press: Norfolk, UK, 2007, pp. 125-158. Sulakvelidze, A.; Kutter, E. Bacteriophage Therapy in Humans. In: Bacteriophages: Biology and Application, Kutter, E.; Sulakvelidze, A.; Eds.; CRC Press: Boca Raton, Florida, 2005, pp. 381-436.

20 [117] [118]

[119] [120] [121]

[122]

Current Drug Delivery, 2018, Vol. 15, No. 1 Gill, J.J.; Hyman, P. Phage choice, isolation and preparation for phage therapy. Curr. Pharm. Biotechnol., 2010, 11(1), 2-14. Waldor, M.K.; Friedman, D.; Adhya, S. Phages: Their Role in Bacterial Pathogenesis and Biotechnology; ASM Press: Washington, DC, 2005. Lu, T.K.; Koeris, M.S. The next generation of bacteriophage therapy. Curr. Opin. Microbiol., 2011, 14, 524-531. Hagens, S.; Blasi, U. Genetically modified filamentous phage as bactericidal agents: A pilot study. Lett. Appl. Microbiol., 2003, 37, 318-323. Hagens, S.; Habel, A.; von Ahsen, U.; von Gabain, A.; Bläsi, U. Therapy of experimental pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob. Agents Chemother., 2004, 48(10), 3817-3822. Matsuda, T.; Freeman, T.A.; Hilbert, D.W.; Duff, M.; Fuortes, M.; Stapleton, P.P.; Daly, J.M. Lysis-deficient bacteriophage therapy decreases endotoxin and inflammatory mediator release and im-

Rastogi et al.

[123]

[124] [125]

[126] [127] [128]

proves survival in a murine peritonitis model. Surgery, 2005, 137(6), 639-646. Commercial companies involved in Phage R&D. http://www.phagetherapycenter.com/pii/PatientServlet?command=s tatic_ptcompanies&language=0 (Accessed On: May 10, 2015). Companies developing and producing enzybiotics and other phage products. http://www.phibiotics.org/index.php?nav=links (Accessed On: May 10, 2015). LMP-102 - Food and Drug Administration. http://www.fda.gov/ OHRMS/DOCKETS/98fr/02f-0316-bkg0001-Ref-04-FDA-Memo. pdf (Accessed On: May 10, 2015). Listeria and Phages. http://www.listex.eu/Listeria#sthash. Exr2K3Se.dpuf (Accessed On: May 10, 2015). Intalytix: Food safety. http://www.intralytix.com/Intral_Food.htm (Accessed On: May 10, 2015). Wolcott, R.; Rhoads, D.; Kuskowski, M.; Ward, L.; Sulakvelidze, A. Bacteriophage therapy of venous leg ulcers in humans: Results of a Phase I safety trial. J. Wound Care, 2009, 18(6), 237-243.