Can bacteriophages be an alternative to antibiotics?

Siyun Luo

Can Bacteriophages Be An Alterna2ve to An2bio2cs? Siyun Luo

Abstract An#bio#cs, the most well-known an#bacterial agents, have been discovered and used since 1929. With the over-dependence and improper uses of them, an#bio#c resistance has been a serious problem in the globe and thousands of pa#ents have died due to nosocomial infec#ons by an#bio#c resistant bacteria such as vancomycin-resistant enterococci (VRE). To solve this problem, discovering new an#bio#cs is no longer realis#c, and other new types of an#bacterial agents need to be discovered instead: phage, a type of virus became the most poten#al an#bacterial agent. Phage therapy was under serious considera#on of scien#sts in the Soviet Union in the 1940s; aNer the discovery of more and more an#bio#cs, phage research became stagnant. In recent decades, phage therapy has been revisited and the mechanisms of a large number of phages were discovered. In this project, the advantages of phage therapy including high host specificity, limita#ons including narrow host range, in vivo clinical experiments on phages, and in silico research on phage receptors are fully discussed in order to answer the ques#on. In comparison with other possible an#bacterial agents such as probio#cs and predatory bacteria, phages may be the most probable alterna#ve to an#bio#cs although a large amount of work is s#ll required to be researched on.

1. Introduc2on In the 14th century, a disease called the plague, also known as the Black Death, originated in Asia and spread to European countries along the Silk Road. It was the most devasta#ng pandemic disease in the whole human history and it resulted in deaths of an es#mated 75 to 200 million people over 7 years . This bacterial 1

infec#on had killed almost 60% of the whole Europe popula#on . The pathogen 2

which caused the Black Death was iden#fied by scien#sts in south Europe as a bacterium called Yersinia pes.s carried by 3

black rats. Similarly in the 19th century, diseases caused by bacteria including cholera spread globally killing millions of people across all con#nents. 21,000 to 143,000 cases of deaths due to cholera have been determined from all around the world by researchers over 19th century . In 4

1914, more soldiers died because of sep#cemia rather than direct military ac#ons. The turning point of these tragedies occurred in 1928: penicillin, the first discovered class of an#bio#cs, was discovered by a Sco\sh scien#st called Alexander Fleming . 5

1.1.An2bio2cs ANer the discovery of penicillin, more and more classes of an#bio#cs were discovered and introduced. These an#bio#cs were

used in the second world war in 1939 and millions of soldiers were secured from sep#cemia and other diseases caused by bacteria. There are four major types of an#bio#cs which inhibit different metabolic process of bacteria including cell wall synthesis, protein synthesis, DNA/RNA synthesis and membrane synthesis (all summarized in Table 1.1). However, along with the rapid developments of an#bio#cs, an#bio#c resistance has been a more and more serious problem facing and threatening the globe. The difficul#es in discovering new classes of an#bio#cs which are not resistant by bacteria have also been harder and harder. Streptomycin, chloramphenicol, tetracycline and erythromycin, all inhibi#ng protein synthesis, were discovered in 10 years aNer penicillin was discovered. In the next ten years (1950-1960), only rifampicin and vancomycin were discovered and the #meline jumps to 1980s in which quinolones were discovered. Finally, two years ago in 2014, teixobac#n which targets lipids on the bacterial membranes was discovered. It is obvious to state that it is in fact more and more difficult for scien#sts to discover a new, non-resistant an#bio#c. In the recent decade, an#bio#c resistance has become serious and bacterial infec#ons have become a threat again . In the US only, 6

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about 721,800 people died due to nosocomial infec#on in 2011 . 7

Table 1.1: Summary of most known an2bio2c mechanism

Type of an#bio#cs

Example Inhibi#on mechanism Bacterial-resistance mechanism

Ref.

Cell wall synthesis inhibitors

Penicillin - stops the cross-linkage of pep#doglycan (a strong la\ce around both gram-posi#ve and nega#ve bacteria which is formed by complex molecules made up of sugars and polypep#des) - afacking the penicillin-binding enzymes - bacteria with weak cell walls will burst due to osmo#c pressure

- reduce the number of porins or the size of the channels of porins, therefore penicillin cannot pass through the cell membrane (specifically for gram-nega#ve bacteria) - modifica#ons of penicillin-binding proteins (the enzymes that cross-link pep#doglycan), therefore it is not recognized. - produce a penicillin hydrolysis enzyme called beta-lactamase

Protein synthesis inhibitors (30 S inhibitors)

Tetracycline - afaches to the A site of ribosomes - blocks the afachment between tRNA and mRNA - similar to a stop codon

- develop ac#ve efflux pumps which can extrude tetracycline out of the cell - 30s unit of ribosomes develop protec#ons preven#ng ribosome-mRNA complexes

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Protein synthesis inhibitors (50 S inhibitors)

Chloramphenicol, erythromycin

-stops pep#de bonds forma#on. No amino acids can be linked into a polypep#de

- 23s ribosomal rRNA (a 2904 unit long component of 50s subunit of bacteria ribosomes) is methylated - Methyla#ons of A2058 and G748. Monomethyla#on of A2058 shows low resistance developed whereas dimethyla#on shows a much higher resistance. - posfranscrip#onal modifica#ons of mRNA

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1.2.Virus infec2ng bacteria An#bio#c resistance has been developed as penicillin was introduced and the improper overuse and overdependence of an#bio#cs have made this situa#on worse . New 8

an#bacterial agents need to be discovered and scien#sts put the hope on virus: phages. In 1947, during the cold war between Soviet Union and America, phages were commonly researched by scien#sts in Soviet Union. However, developing phages has not been the mainstream research in Western countries at that #me. Over the last couple of decades, the biology of virus has been studied well and many viral genomes sequences were discovered. Experiments and researches have also been done on phages. As a result, the mechanisms, poten#al benefits and

limita#ons of phages have been discovered. In the following context, I am going to discuss these mechanism, benefits and limita#ons of phages.

2. Phage life cycles and bactericidal mechanisms Phages are discovered from the nature and they may have the most diverse forms compared to other organisms on the Earth . 9

Phages are classified into 13 families known to date and most of them can be categorized into two classes – ly#c and lysogenic . Ly#c simply means that the 10

phages will replicate and destroy the host bacteria once they entered the host and lysogenic means the opposite – they will not replicate and lyse the host immediately. The phage with ly#c life cycle is known as

RNA synthesis inhibitors

Rifampin (Rifampicin)

-targets RNA polymerase - binds to the RNA polymerase (RNAP β subunit deep within the DNA/RNA channel) and prevents mRNA from elonga#ng from 2-3 nucleo#des long.

- structure of RNA polymerase is altered due to muta#on - Rifampin can no longer recognize the altered RNA polymerase

DNA synthesis inhibitors

Nalidixic acid - An enzyme called DNA gyrase (an enzyme which involved in NDA replica#on and repair) are inhibited and DNA replica#on process is stopped.

- An altera#on occurs on structure of DNA gyrase (specifically, alpha subunit of DNA gyrase) by muta#on. - can also change the number and size of the channel of porins preven#ng entrance of nalidixic acid

Teixobac#n

Teixobac#n - a totally new class of an#bio#c which was discovered on January 9, 2015 - stop cell wall synthesis but target the lipids instead of proteins - recognizes difference between mammalian lipids and bacterial lipids - selec#vely targets bacterial lipids

- resistance to teixobac#n has not been discovered because lipids molecules are hard to alter and this process usually takes a long #me - does not mean that it will not be resistant by bacteria

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virulent phage and the one with lysogenic is called temperate phage. The working mechanisms and aims of those two types of phages are slightly different. However, for both types of phage, adsorp#on to the bacterial surface and then infec#ng the host bacteria will be the very first steps to start their life cycles.

2.1 Virulent phages: T7 as an example First and foremost, T7 could be a classic example of virulent phage which infects most strains Escherichia coli (E.coli). It belongs to the Family Podoviridae of the Order Caudovirales (represents tailed phages) ; therefore it has a structure of a 11

non-contrac#le short tail afached by six elongated homo trimmer tail fibers. These tail fibers, mostly encoded by gene 17, seem to take the major responsibility of afaching to lipopolysaccharide of Escherichia coli in vitro . In this case, 12 13

rough lipopolysaccharide, a subclass of lipopolysaccharide which lacks the outer O an#gen domain specifically Toll-like 14 15

receptor 4, and acts as a receptor of T718 . 16

Besides Gp17 as an important part of adsorbing on surface of host bacteria, the tail formed by Gp11, Gp12 and Gp7.3 is also a necessary part of the virion as the dsDNA is inserted into the host bacteria through the tail . ANer Gp17 tail fibers afach to 17 18

the outer membrane of E.coli, viral endolysin (Gp15 and Gp16) lyses the pep#doglycan cell wall, tail channel extends through the inner membrane and dsDNA is transferred into the host cytoplasm . The 19 20

genome of T7 with length about 40 kbp produces around 55 proteins is sent into the host cytoplasm. However, only 2% of genome successfully enters the host bacteria firstly but the remaining genome is brought in by transcrip#on done by host and T7 RNA polymerases . As soon as the 21

genome is transferred into the host bacterium, early proteins such as Gp0.3 and Gp0.7 are transcribed and translated from the early promoters by host RNA polymerases to inhibit both the defensive system of the host such as CRISPR and the macromolecular synthesis including replica#on, transcrip#on and transla#on of the host. In addi#on to this, T7 RNA

polymerase is also transcribed by the host RNA polymerase. ANer T7 RNA polymerase is produced, middle and late proteins are transcribed by T7 RNA polymerase instead of host RNA polymerase which means host RNA polymerase is now dispensable. As a consequence, Gp2 is transcribed by T7 RNA polymerase for inhibi#on of host RNA polymerase synthesis27 . The replica#on of 22

T7 DNA in the host cell is complicated and it requires helps of complexes of DNA polymerase, processivity factors from the host and DNA helicases26. The helicases from the gene 4 unwind the double stranded DNA to single stranded templates for DNA polymerases (products of gene 5) to work. DNA polymerases are proteins which require high processivity to incorporate thousands of free nucleo#des together to form nucleo#de stands. They usually do not have high processivity unless they cooperate with accessory proteins to increase protein surface interac#on with the duplex por#on of the primer-template29. With saying that, the con#nuous polymerizing of Gp5 needs the help of E.coli processivity factors called thioredoxin (trx) since Gp5 is not a processive polymerase. Gp5/trx complexes polymerize thousands of daughter DNA strands from the templates using host dNTP26 . As soon as many copies of DNA 23

are produced, they are transcribed and translated to many proteins and the DNA will be surrounded by them. Many parts of T7 are then produced by assembly of these various proteins through complex processes with different pathways, and then they are finally incorporated to T7 phages . During 24

the lysis stage, the most important characteris#c of ly#c phages, the same enzyme before called endolysin is produced doing the same func#on as before which is to hydrolyze pep#doglycan cell wall of host. However, one more enzyme called holin is synthesized and produces lesions on the phospholipid membrane of the bacterium by hydrolysis so that endolysin can pass through the membrane to reach the pep#doglycan layer . T7 is now able to 25

escape from the host and the host is basically destroyed. The ly#c cycle is completed and more than thousands of T7

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phages are produced to infect the next bacterium.

2.2 Temperate phage: lambda phage as an example Similarly, lambda phage is another type of bacteriophage which belongs to the same order as T7 but in a different family called Siphoviridae. As a result, lambda has a slight difference on the structure. Instead of having a non-contrac#le short tail like T7, it has a non-contrac#le but long tail. Lambda phage infects E.coli as well, specifically E.coli K-12. The tail protein is called protein J. The C terminus of protein J, takes the major responsibility of adsorbing to the bacterial surface by interac#ng with LamB, a maltose-specific porin located on the outer membrane of E.coli K-12 . Lambda 26

phage has dsDNA of around 4.8 kbp producing 12-14 different types of proteins . ANer the DNA is ejected from 27

the lambda tail, it enters the host cytoplasm and integrates with the host DNA by gene#c recombina#on through two afachments (phage afachment site called a4P and bacterial afachment site called a4B) and become prophages . The enzymes involved 28

in this process are known as phage integrase, which are responsible for a4P recogni#on and cleavage . In fact, lambda 29

phages can be both lysogenic and ly#c phages. There is a switch deciding the produc#on of repressors (controller of repressor operon or C1) under different circumstances and this decision may induce prophage replica#ons and propaga#ons 30 31

. These repressors decide whether the 32

ly#c genes (Q, S, R, and A-J) should be expressed or lysogenic genes (C1, C2 or C3) should be expressed . 33

3. Advantages and limita2ons of phages Since an#bio#cs have been discovered, the success of an#bio#cs superseded the poten#al usage of bacteriophages . In fact, 34

there are many traits of phages that can outweigh those of an#bio#cs including the ability of clearing biofilms and low cost discovering them . In this review, I would 35 36

like to focus on two more advantages (less poten#al resistance and low toxicity) and the limita#ons (narrow host range and

causing immune response) related to these two advantages which are two main limita#ons of phage therapy.

3.1 High specificity leads to less concern about phage resistance Two well-known advantages of phages due to high specificity are their rela#vely low tendency to introduce resistance and no cross-resistance to an#bio#cs4141 . To 37

explain further, it is really hard for bacteria to develop resistance to bacteriophages because some#mes a pathogenesis receptor is involved in the process of phage infec#on which means bacteria might lose their pathogenesis and toxicity if muta#on occurs and the receptor is altered to prevent infec#on of phages . Bacteria 38 39

without toxicity might not be concerned because they are no longer harmful to human beings and their own survivals might be also threatened. Phages also do not work in the same way as an#bio#cs. Obviously, an#bio#cs work either weakening the cell walls or stopping normal cellular metabolisms in the cells but phages always enter the cells through receptors on the surface of the cells and then destroy the cells by lysis of the hosts. In other words, phages are not resisted by an#bio#c resistance bacteria as they work using totally different mechanisms. However, other techniques developed by bacteria can be used against bacteriophage such as CRISPR (Clustered regularly interspaced short palindromic repeats), an “adap#ve immunity” of short palindromic repeats, and capsule, a physical barrier of receptors . 40

One of the most serious problems high specificity brings alongside is that the host range available to phages is narrower which means fewer species of bacteria can be targeted by phages which indirectly affects the effec#veness in the actual applica#ons of them46 . There is a method to 41

overcome this limita#on as much as possible which is called phage cocktails which will be introduced and discussed in sec#on 4.1.

3.2 Low toxicity Based on the fact that bacteriophages are

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mostly DNA and proteins, they have low toxicity28. However, they can raise immune responses by lysing bacteria in short period of #me in vivo. Rapid lysis results into endotoxins (lipopolysaccharides) and super-an#gens (a class of an#gens which can ac#vate T cells generally causing large number of cytokine being released ) are 42

released43. Endotoxins can ac#vate macrophages and therefore inflammatory cytokines are released . Those cytokines 43

are good to human at a certain concentra#on but can be harmful to human at a high concentra#on. High concentra#on of inflammatory cytokines leads to overac#ng of #ssue cells and therefore cells begin to die and large number of immunity cells begins to work which could cause deaths . The invasion of super-an#gens 44

leads to too many T cells being ac#vated. Only about 0.001% of T cells are ac#ve in a human’s daily life, super-an#gens ac#vate 20% of them . As a result, T helper cells 45

ac#vate macrophages and therefore a large number of inflammatory cytokines is produced such as TNF-alpha. The same consequence is caused similar to the one caused by endotoxins. In conclusion, cytokine storm can cause ineffec#veness of phages and it is dangerous to human . 46

While many papers discovered the danger, there are other ar#cles suggest that it is not a real concern in actual phage treatments 47

. Although phages have the poten#al to 48 49

be dangerous to humans, there is befer method compared to using whole phages which is called extrac#on of endolysins. Although endolysins s#ll have similar probability to raise immune response, it has other advantages which outweigh the usage of whole phages. This method will be discussed in sec#on 4.2.

4. Strategies to overcome limita2ons Since there is a probability that immune response can be raised by cytokine storm and phages have narrow specificity, ar#ficial strategies including phage cocktails and lysins purifica#ons are developed to overcome these problems in order to apply phage therapy to solve actual cases.

4.1 Phage cocktails

As men#oned above, most phages have narrow host range and high specificity, which lead to ineffec#veness to some types of bacteria. Different pa#ents under different circumstances might require different phage therapy. Strategies of phage therapy can be categorized into two types: monophage therapy and polyphage therapy. Monophage therapy means there is a single type of phage involving in the therapy and polyphage therapy (also 50

called phage cocktails) means mul#ple phages are used . Monophage therapy is 51 52

used mostly with wide host range phages if they are available or isolated phage and 53

pathogen combina#on being tested and guaranteed to work. Less phages are used means that there is less risk for phages to raise an immune response which is a significant advantage. However, the number of wide range host phages which can be used clinically is limited. Moreover, the matching of isolated phage and pathogen is always done in vitro; therefore the interac#ons of isolated phage and pathogens in vivo are not fully predictable . 54

Furthermore, although many bacteria can be iden#fied to a species level, bacteriophages cannot be effec#ve to all of them; even if they belong to a single strain46. Therefore, polyphage therapy seems more effec#ve in prac#cal due to its versa#lity in terms of formula#on produc#on (just like an#bio#cs, cocktails of phages can be made into liquids, solids etc. 62). The main idea of phage cocktails is 55

to target as many bacterial targets as possible with the help of an#bio#cs and it 56

is possible and capable to design such therapy in theory28. Many different types of phages can further be mixed together and their spectrum of an#bacterial ac#vity can then be broadened through coopera#ng of them . However, in reality, less complex 57

cocktails containing only 2 or 3 phages are used because large number of them might cause damage to non- targeted bacteria although the impact seems less than usual commercial an#bio#cs . 58

4.2 Extrac2on of endolysins Endolysins (lysins) are a type of hydroly#c

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enzymes produced by the phages to cleave the host cell wall. Endolysins which are able to lyse pep#doglycan cell wall can be extracted and used instead of whole bacteriophages because of high effec#veness and low chance being resistant . Endolysins consist of two 59

domain called Enzyma#cally Ac#ve Domain (EAD) and Cell Wall Binding Domain (CBD) 60

which are responsible for cleavage of 61 62

pep#doglycan bonds and preven#ng subsequent damage to surrounding 63

respec#vely. The EAD can be categorized into five classes func#onally (Endo-β-N-acetylglucosaminidase, N-acetylmuramidase, Endopep#dase, N-acetylmuramoyl-L-alanine amidase, γ-D-glutaminyl-L-lysine endopep#dase) because they target different substrates including N-acetylglucosamine (NAG)/N-acetylmuramic acid (NAM), pep#de bonds between amino acids, amide bond between the sugar and the amino acid and gamma bond between D-glutamine and L-lysine residues . In 64

contrast, CBD has a totally different responsibility as it is variable which means it allows a greater specificity and a reduc#on of bacterial resistance . 65

An experiment is done on rabbits in order to test the immune responses raised by lysins . Rabbits immunized with the Cpl-1 66

(a pneumococcal phage lysin) where the concentra#on of IgG has a significant increase, with no significant anaphylaxis and side effects . 67

In conclusion, although there is s#ll a chance to raise cytokine storm, endolysins have a greater effec#veness and lower probability to be resistant by bacteria.

5. Phage therapy examples

5.1 Review of a study done by Biswas et al : Bacteriophage Therapy Rescues Mice 68

Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium The paper of Biswas et al. shows two experiments of vancomycin-resistant Enterococcus faecium (VRE, a bacterium which infect human intes#ne causing meningi#s and endocardi#s) infec#ons in vivo and in vitro, using mice as experimental models. Two VRE specific phages called ENB6 and C33 were studied and they were isolated and purified from raw sewage at a municipal sewage treatment plant. The two experiments I want to discuss in this paper simply show a successful case of curing a disease caused by an#bio#c resistant bacteria using phage therapy under certain condi#ons including short length of delay of treatments. In experiment one; they aimed to inves#gate the effect of delay in treatment on rescuing mice from VRE bacteremia using phages. A strain of VRE called CRMEN 44 was grown, diluted and minimum lethal dose (MLD, determined before the experiment is carried out) was injected in to mice (in groups of five) in 400 μl aliquots. Only ENB6 was used in the experiment as it is effec#ve to CRMEN 44. The results came out with conclusions: ENB6 can rescue all mice in the experiment even aNer 5 hours delay. However, only 50% of mice survived aNer 18 hours delay and the percentage of survival keeps decreasing as the length of delay increases. See Figure 5.1.1. An experiment on ENB6 about immune response was also done. The result showed that the concentra#on of IgG and IgM against ENB6 rise 3800 fold and 5 fold respec#vely aNer five month injec#on of ENB6. Despite this, no anaphylac#c reac#on and changes in body temperature were observed.

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� Figure 5.1.1: the effect of delay in treatment on rescuing mice from VRE bacteremia using phages74.

The first graph shows the health state of a group of mice aNer being infected by CRMEN 44 for 5 hours and the health state aNer being injected ENB6. The second graph shows similar data but the mice were given ENB6 aNer being infected for 18 hours. The third graph shows the health state of the mice which were given ENB6 aNer being infected for 24 hours.

In experiment two; they aimed to inves#gate the extent of phage rescue depending on bacterial suscep#bility. A new strain of VRE called CRMEN 19 was grown and diluted, and then the minimum lethal dose (MLD) was injected into mice (in groups of five) in 400 μl aliquots. Two phage treatments were carried out separately and

9 × 109 PFU of ENB6 and C33 were injected in the bacteremia mice. It is shown that CREMEN 19 is resistant to ENB6 but not C33 in vitro. As a result, in the experiment which was done in vitro, ENB6 did not form any plague in the bacteria strains which means CRMEN 19 was not affected by ENB6 but it was affected by C33. The experiment in vivo shows that group C (a group was treated by ENB6) has 40% survival rate and group D (a group was treated by C33) has 100%. In addi#on, group E (an untreated control group) has about 50% survival rate which was s#ll significant compared to group E. From this experiment, they concluded that C33 can treat CRMEN 19 significantly. See the Figure 5.1.2 below.

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� Figure 5.1.2: phage rescue depends on bacterial suscep2bility Group A and B show healthy mice treated by ENB6 and C33 respec2vely. Group C and D show mice with MLD treated by ENB6 and C33 respec2vely. Group E shows mice with MLD untreated as a control group74.

2. Review of a report done by Maya Merabishvili et al : Produc2on of a 69

Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials

This thorough report describes a detailed process of a laboratory-based produc#on of a well-defined bacteriophage cocktail. As I have men#oned in Sec#on 4.1, most single species of phages have a narrow host range, and therefore, in actual clinical applica#on, phage cocktails are commonly used instead of a single species of phages with wide host range. In the report, they aimed to make a phage cocktail including phages PNM, 14/1 and ISP targe#ng Pseudomonas aeruginosa (a mul#drug resistant bacterium causing

chronic morbidity) and Staphylococcus aureus (an an#bio#c resistant especially methicillin resistant bacterium causing sinusi#s). This cocktail was named BFC-1 in the rest of the report. The processes of isola#on, separa#on and purifica#on ini#ally and later the tests on cytotoxicity (using kera#nocytes), Stability and the actual applica#on were described in details. In this sec#on, I would like to focus on the selec#on of phages. Different types of phages were used targe#ng both P. aeruginosa and S. aureus (in this case, 88 types of phages for 136 cases of P. aeruginosa and 9 types of phages for 116 cases of S. aureus), these data were collected from Eliava Ins#tute for

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Bacteriophage, Microbiology and Virology (EIBMV), Tbilisi, Georgia and the State Ins#tute of Gene#cs and Selec#on of Industrial Micro-organisms (SIGSIM), Moscow, Russia and they were isolated from different clinical or environmental habitats. Each species of phages was put into the 2% LB agar with the target bacteria (108 cfu/ml LB broth), and the each agar was incubated 16-18 hours with 37°C. The results were scored as cl (confluent lysis), ol (opaque lysis), scl (semi-confluent lysis), sp (several plaques) and – (nega#ve reac#on). The report can strongly support that phage cocktails seems to be a promising alterna#ve to an#bio#cs in actual applica#on in case of diseases caused by S. aureus and P. aeruginosa as many different aspects are considered during tes#ng or producing the phage cocktails and different types of phages show posi#ve results in destroying bacteria with lifle side effect.

3. Review of a report done by W. E. Huff et al : Bacteriophage Treatment of a 70

Severe Escherichia coli Respiratory Infec2on in Broiler Chickens

The aim of this study was to determine and compare the efficacy of single intramuscular (i.m.) injec#on and mul#ple i.m. injec#ons of bacteriophages (SPR02 and DAF6) to treat a severe Escherichia coli respiratory infec#on. 6 x 104 colony-forming

units (cfu) of E. coli were injected into the thoracic air sac of the birds at 7 days of age. Injec#on of an i.m. into the thigh with either heat-killed or ac#ve bacteriophage follows right aNer the previous step. The bacteriophages used in the experiment are specific to a serotype 02, non-mo#le Escherichia coli and were isolated from municipal waste treatment facili#es and poultry processing plants. The design of the experiment was complicated therefore a table was provided. The results were measured as body weights which were also shown in the form of a table. (See Table 5.3.1 and Table 5.3.2). Basically, there were 16 treatments with 3 groups in each treatment and there were 10 birds in each group. The age, gender living condi#ons and diet of birds were controlled. The birds and feed were weighed each week. Any bird that died was weighed and the severity of airsacculi#s was scored: 0, no inflamma#on; 1, opacity and thickening of the inoculated air sac; 2, mild airsacculi#s and mild pericardi#s; 3, moderate airsacculi#s/pericardi#s with spread to liver or abdominal cavity (perihepa##s/peritoni#s); 4, severe fibrinous airsacculi#s and severe pericardi#s; 5, severe airsacculi#s/pericardi#s with spread to liver or abdominal cavity according to the paper.

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Table 5.3.1: Experimental design: E. coli challenged at 7 days of age, ac2ve or heat-killed phage given at various days of age76.

�CONT=untreated control; HKP=heat-killed phage; AP=ac#ve phage; ECC=E. coli challenged; HKP7/ ECC=heat-killed phage at 7 days E. coli challenged; AP7/ECC=ac#ve phage at 7 days E. coli challenged; HKP7-8-9/ECC=heat-killed phage at 7, 8, and 9 days E. coli challenged; AP7-8-9/ECC=ac#ve phage at 7, 8, and 9 days E. coli challenged; HKP8/ECC =heat-killed phage at 8 days E. coli challenged; AP8/ECC=ac#ve phage at 8 days E. coli challenged; HKP8-9-10/ECC=heat-killed phage at 8, 9, and 10 days E. coli challenged; AP8-9-10/ECC=ac#ve phage at 8, 9, and 10 days E. coli challenged; HKP9/ECC=heat-killed phage at 9 days E. coli challenged; AP9/ECC=ac#ve phage at 9 days E. coli challenged; HKP9-10-11/ECC=heat-killed phage at 9, 10, and 11 days E. coli challenged; AP9-10-11/ECC=ac#ve phage at 9, 10, and 11 days E. coli challenged.

Using treatments 4, 5, 6 and treatments 7, 8 from Table 5.3.2 as examples, treatment 4, 5 and 6 represent single bacteriophage dose and treatments 7, 8 represent mul#ple bacteriophage doses. In the single dose treatment, the comparison between treatment 5 (E. coli and non-ac#ve phages) and treatment 4 (E. coli only) is not significant but the comparison between treatment 6 (E. coli and ac#ve phages) and treatment 4 (E. coli only) is significant showing that the single dose of ac#ve phages is actually working. In mul#ple doses treatment (treatment 7, 8), comparison between treatment 7 (E. coli with non-ac#ve phages given for three

days) and treatment 4 (E. coli only) is not significant as expected but treatment 8 (E. coli with ac#ve phages given for three days) shows a significant posi#ve result compared to treatment 4 (E. coli only). Treatment 8 seems to have a slightly befer effect on infec#ng E. coli rather than treatment 6 sta#ng that mul#ple phages doses might be slightly more effec#ve than single dose, despite the comparison is not really significant. The effec#veness of both single and mul#ple doses keeps decreasing as the #me which phages are given is delayed. In other words, phage gets less effec#ve if the treatment is delayed.

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Table 5.3.2: The effect on body weight of single and mul2ple intramuscular injec2ons of bacteriophage SPR02 and DAF6 at 7, 8, and 9 days of age when challenged with E. coli at 7 days of age76.

�CONT=untreated control; HKP=heat-killed phage; AP=ac#ve phage; ECC= E. coli challenged; HKP7/ ECC=heat-killed phage at 7 days E. coli challenged; AP7/ECC=ac#ve phage at 7 days E. coli challenged; HKP7-8-9/ECC=heat-killed phage at 7, 8, and 9 days E. coli challenged; AP7-8-9/ECC=ac#ve phage at 7, 8, and 9 days E. coli challenged; HKP8/ECC=heat-killed phage at 8 days E. coli challenged; AP8/ECC=ac#ve phage at 8 days E. coli challenged; HKP8-9-10/ECC=heat-killed phage at 8, 9, and 10 days E. coli challenged; AP8-9-10/ECC=ac#ve phage at 8, 9, and 10 days E. coli challenged; HKP9/ECC=heat-killed phage at 9 days E. coli challenged; AP9/ECC=ac#ve phage at 9 days E. coli challenged; HKP9-10-11/ECC=heat-killed phage at 9, 10, and 11 days E. coli challenged; AP9-10-11/ECC=ac#ve phage at 9, 10, and 11 days E. coli challenged.

This experiment suggests that bacteriophage can be an effec#ve treatment at the early period of E. coli infec#on according to the data shown, in addi#on, at the early stage, mul#ple doses seems more effec#ve than single dose. However, the efficacy of bacteriophage treatment diminishes as it is delayed, with no difference between single and mul#ple treatments. It is strongly supported that phage therapy can be an alterna#ve to an#bio#cs in farming animals like boiler chickens, with decreasing effec#veness of phage on rescuing animals while the treatment is delayed.

6. Discussion This review tries to answer the ques#on, “Can bacteriophage actually be an alterna#ve of an#bio#cs”. In the previous sec#ons, I have discussed (with examples) the biology of phages, their therapeu#c

uses and the possible limita#ons of their uses. In this sec#on, I am going to evaluate the data presented above along with some original in silico analysis, before I actually answer the ques#on.

6.1 Discussion of Sec2on 3: Advantages and limita2ons of phages In Sec#on 3, the advantages of bacteriophage including the ability of clearing biofilms, low commercial cost in discovery, less poten#al resistance and low toxicity were discussed. Only the last two characteris#cs were discussed in Sec#on 3 as these two characteris#cs seem more important in the actual applica#on of phages. Bacteria are always trying to antagonize an#bacterial agents, and hence the effec#veness of phages can some#mes be limited due to muta#on and evolu#on of targeted bacteria . It could be argued that 71

phages have lower poten#al possibili#es to be resisted by bacteria because bacteria will

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lose their pathogenesis if they alter the receptors which interact with phages. The receptors that interact with phages are not developed for this property obviously, they exist for other func#ons. For instance, the LamB receptor which interacts with lambda phages is a maltoporin, which means that the actual property of the receptor is maltose transport . No maltose can be 72

transported in and out of the membrane if any muta#on or altera#on occurs on the receptors. The bacteria will not be able to survive for long without this receptor as maltose is an important energy source . As 73

a result, bacteria develop CRISPR instead of altering the receptors because some#mes the outcomes of altering receptors might be far more serious than losing pathogenesis. CRISPR afacks and cleaves specifically phage DNA; therefore phage DNA cannot be replicated, transcribed and translated. The way in which CRISPR works is that CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cleave exogenous gene#c elements in a manner analogous to RNA interference . However, 74

methyla#on of phage DNA can easily circumvent this prokaryo#c immune system as the Cas (CRISPR spacer) is not able to recognize methylated DNA sequences. This is because methylated DNA sequences do not fit the ac#ve site of Cas therefore it cannot cleave the DNA. Methyla#on and unchangeable receptors might be the two main reasons why phages have much lower poten#al possibili#es to be resisted by bacteria compared to an#bio#cs. In addi#on, even if bacteria do mutate and be resistant to phages without affec#ng their pathogenesis property, phages can always mutate faster than bacteria due to their faster replica#on rate. In other words, the muta#ons of bacteria leading to phage resistances act as selec#ve pressure, phages can outcompete bacteria through faster muta#on rate. Phages have demonstrated their ability to clear biofilms. The study of biofilms has also shown the effec#veness of phages as an an#microbial agent. Biofilm is a robust layer of mucilage adhering to a solid surface and containing a community of bacteria or other microorganisms like fungi. Biofilms

are always a serious challenge to an#bio#cs because an#bio#cs are not able to penetrate the aggrega#on of microorganisms. In worst case scenario, biofilms can be resistant to an#bio#cs which make the effec#veness of an#bio#cs even lower. Compared to an#bio#cs, phages can clear biofilms more effec#vely because phages can replicate themselves aNer infec#ng the first host and then infect the next one whereas an#bio#cs can only be effec#ve to the few bacteria on the surface of the biofilm and not further as they cannot ac#vely replicate themselves and therefore they can move from host to host. As a result, an#bio#cs can only act on the few surface bacteria but not the one protected by the biofilm while phages can . 75

On the other hand, there are plenty limita#ons of phage therapy men#oned in Sec#on 3 as well, however, in my opinion, the limita#ons cannot outweigh the benefits as phages do have a number of useful characteris#cs which an#bio#cs lack. In addi#on, the methods that can be used to overcome those limita#ons are being developed by humans (refer to Sec#on 4) and they seem mature and usable with careful considera#ons in actual treatments.

6.2 Discussion of Sec2on 5.1: Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium Referring back to the first experiment described in Sec#on 5.1, a graph was shown with clear data demonstra#ng the fact that the effec#veness of phage treatments decreases as the delay of treatment gets longer. However, the unit shown for the y-axis is “state of health” without giving any standard of health states such as bacterial loads in blood or temperature of mice. In other words, the determina#on of health state is subjec#ve which might make the data of the experiment less reliable. The same problem occurs in the second graph represen#ng the data from the second experiment.

The other cri#cism of the study is that in

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both the determina#on step and the actual experiment, a group with five mice is used. This is a small sample size and only two or three mice are rescued in the experiment seems as not being enough evidence to support the conclusion although the data is obviously significant (for n=5) and shows strong correla#on.

However, the paper states that they used a similar approach to determine MLD of CRMEN 19 as for CRMEN 44; they did not show the actual sta#s#c or graph like CRMEN 44. Moreover, they claim that they did the MLD determina#on and used the same MLD as the previous experiment. However, because of the high survival rate of the phage-untreated control group, I consider the MLD used to have been too low.

6.3 Discussion of Sec2on 5.2: Review of a report done by Maya Merabishvili et al : 76

Produc2on of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials In Sec#on 5.2, the experiment described has a large sample size and really organized data showing the effec#veness of phage cocktails in actual human clinical trials with or instead of the use of an#bio#cs.

However, the paper stated that the phage cocktail used contains PNM, 14/1 and ISP targe#ng Pseudomonas aeruginosa and Staphylococcus aureus, it did not state any reason of choosing these three phages. As a consequence, I did a sta#s#cal summary based on the extra data given by the authors (an excel document afached at the very end of the paper and available only in the online format of the paper which was not men#oned and evaluated in the paper). In 116 cases of tests for S. aureus, 105 of ISP phages show confluent lysis which means S. aureus can be targeted and lysed by ISP over 90% of cases. Compared to IrB (66 congluent lysis in 99 cases which gives 66.6% lysis rate), the second effec#ve phage to S. aureus which is tested, ISP has almost 25% higher lysis rate. The reason for choosing ISP as part of the phage cocktail seems obvious. Similarly, the lysis rate

(78.2%, 18 out of 23) of phage 14/1 is the highest among all the tested phages which target P. aeruginosa. However, the reason for choosing PNM is a bit confusing as there are only 6 cases of confluent lysis in 113 cases in total which gives only 5.3% lysis rate. It also seems to be one of the lowest values overall. There is a probability that I thought aNer analyzing the data which is PNM can give out posi#ve results of lysing some pathogenic bacteria while others cannot. 4 cases of confluent lysis out of 6 are shown while most of other phages with higher lysis rate have nega#ve reac#on. There is another poten#al reason of choosing PNM which is that PNM might have very high infec#on rate which allows lysis rate to be compromised. In other words, if PNM have high infec#on rate, it can lyse equal amount of hosts as the other two phages although its lysis rate is rela#vely low. In theory, this thought is possible but there is no evidence to support it in this paper and PNM is not a common phage to be researched.

6.4 Discussion of Sec2on 5.3: Bacteriophage Treatment of a Severe Escherichia coli Respiratory Infec2on in Broiler Chickens The experiment done in Sec#on 5.3 is detailed, straight-forward and objec#ve since they measured the weight changes of broiler chickens instead of general health state. The applica#on of phages on chickens is not recently discovered. They were discovered in the early 20th Century and have been applied in the control of food safety in farming animals . However, 77

during the applica#on of phages is wide spread, a problem emerged. Most ly#c phages used in farming animals have fairly short half-life73 which means that the concentra#on of phages inside the animal body will be decreased due to regula#on in the body. Low concentra#on of phages will not be as effec#ve as high concentra#on and there is no strategy to eliminate this problem other than keeping feeding the animals with phages. This seems not to be a commercial problem to farmer as phages are actually cheaper to develop than an#bio#cs41.

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6.5 Common Themes arising from the experiments in Sec2on 5 In all experiments described in Sec#on 5, it is stated that phage cocktails (two phages are used in the experiment in Sec#on 5.1, three are used in 5.2 and two in 5.3) are effec#ve alterna#ves to an#bio#cs with the appropriate phages chosen by doctors. In addi#on, experiments in Sec#on 5.1 and 5.3 both suggest that delay in treatments has huge nega#ve impact on the lysis rate of phages.

6.6 Original in silico research on the suitability of lambda phage on related enterobacteria Lambda phage is a well-known phage which is discovered by Esther in 1950. It has high successful infec#ve rate but strangely an 78

extremely narrow host range – it is found infec#ng no other bacteria sequenced to date except for E. coli. The reason for this might not firmly be that other bacteria are resistant to lambda; it can just be because most phage experiments are done using E. coli but rarely other bacteria. In this sec#on, I am going to summarize what I have done in order to find out the possible reason behind the narrow host range of lambda phages since they have enormous poten#als to be used in actual human trials. Lambda phages adsorb on the surface of bacteria and inject their DNA in to the bacteria by interac#ng with LamB receptors on the outer membrane of gram-nega#ve bacteria (See Sec#on 2.2). The interac#on mechanism and the binding to LamB receptors are s#ll unclear although the 79

protein J in lambda is known as a phage receptor binding protein34.

A paper inves#ga#ng the shape determining amino acids of LamB receptors inspired me to believe that the 80

difference in shape of LamB receptors in other enterobacteria compared to E. coli may be a poten#al reason of lambda phage resistance. According to this paper, Y118 (Tyrosine 118) in the crystal structure is the most important amino acid which affects the stability of the maltoporin as well as the shape. In fact, in the complete amino acid sequence for LamB receptor, the posi#on of tyrosine should be 143 (and the posi#on number given in the following context will be the posi#on in the complete sequence) because the paper used purified crystal of LamB receptor therefore the amino acid sequence is shorter and the posi#ons of amino acids are slightly different (in fact their posi#on is shiNed by 25). There are other important amino acids in the sequences which can also affect the stability and shape of LamB including R134 (Arginine 134), D136 (Aspar#c acid 136) and D141 (Aspar#c acid 141) which I will compare as well as Y143 (See figure 6.6.1). Any muta#on will lead to either increase or decrease in stability with different shape of the maltoporin but there will be one or more specific muta#on(s) will bring the biggest effect(s). As a consequence, I decided to compare the amino acid sequences which cons#tute LamB receptors in E. coli and other pathogenic bacteria. I used BLINK (a tool in PubMed used to 81

compare amino acid sequences) which cons#tutes LamB receptors in different bacteria including E. coli to do my research. Figure 6.6.2 shows the number of maltose related substrates which are transported by LamB mutants as LamB is also responsible for transpor#ng maltose as men#oned in Sec#on 6.1. However, in my research, ability to transport sugar is not included although it is undeniable that the change in shape of LamB will definitely affect the sugar transport system.

� 15

� � Figure 6.6.1 (lea)86: the shape and stability of LamB determining amino acids. Table 6.6.2 (right)86: the number of maltose related substrates which are transported by LamB mutants.

Example 1: Shigella dysenteriae (a pathogenesis bacterium causing coli#s, arthri#s etc.) The four important amino acids are the same in both Escherichia coli and Shigella dysenteriae. See Figure 6.6.3.

� Figure 6.6.3: amino acid sequence comparison between Escherichia coli str. K-12 and

Shigella dysenteriae. The top line is the amino acid sequence of LamB in E. coli, and the boeom line is the one in Shigella dysenteriae. The comparisons shown in the boxes are the four important amino acids (R134, D136, D141 and Y143).

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� 17

Example 2: Salmonella enterica (a bacterium causing typhoid fever, food poisoning ect.) The four important amino acids are the same in both Escherichia coli and Salmonella

enterica similar to the last example. See Figure 6.6.4.

� Figure 6.6.4: amino acid sequence comparison between Escherichia coli str. K-12 and

Salmonella enterica. The top line is the amino acid sequence of LamB in E. coli, and the boeom line is the one in Salmonella enterica. The comparisons shown in the boxes are the four impotant amino acids (R134, D136, D141 and Y143).

I did the research for three other types of bacteria (Citrobacter freundii, Klebsiella pneumonia and Serra.a marcescens) using similar method and ended up with exactly the same result: there is no difference in the posi#on of the shape or stability determining amino acids in LamB. A conclusion can be drawn that, if those five bacteria is resistant to lambda phages, it is not due to the shape or the stability of the LamB receptor as they have similar shapes as LamB receptor in E. coli. As a result, lambda phage resistance is caused because of other reasons such as different binding sites as there s#ll are differences between amino acid sequence of these five bacteria and E. coli and the func#ons of these differences are s#ll unknown.

7. Conclusion In conclusion, phages absolutely have the poten#al to be the most effec#ve alterna#ve to an#bio#cs but choosing the

right combina#on of phages or the right balance of phages and an#bio#cs is also important. The addi#onal advantages of phages are the especially low resistance rate and ability of clearing biofilms. Although poten#al dangers which arise due to the limita#ons of phage therapy cannot be neglected, many strategies have been developed to tackle those limita#ons. In my opinion, the most important aspect of phage development is to figure out the reasons behind the low specificity of phages. The interac#ons between phages and bacteria receptors are s#ll unclear which poten#ally limit the applica#ons of phages. In the research that I did, LamB receptors in many bacteria do not interact for lambda phages with unknown reasons, as a result, the applica#ons of lambda phages are hugely limited and in fact, lambda phages are not used in anywhere except in theore#cal research.

8. Bibliography

� 18

ABC/Reuters (29 January 2008). "Black death 'discriminated' between vic#ms (ABC News in 1

Science)". Australian Broadcas#ng Corpora#on. Retrieved 3 November 2008.

Aus#n Alchon, Suzanne (2003). A pest in the land: new world epidemics in a global perspec#ve. 2

University of New Mexico Press. p. 21.

Haensch S, Bianucci R, Signoli M, Rajerison M, Schultz M, Kacki S, Vermunt M, Weston DA, Hurst 3

D, Achtman M, Carniel E, Braman# B (2010). Besansky, Nora J, ed. "Dis#nct clones of Yersinia pes#s caused the black death". doi:10.1371/journal.ppat.1001134.

Ali M, Nelson AR, Lopez AL, Sack D. (2015). PLoS Negl Trop Dis 9(6): e0003832. doi:10.1371/4

journal.pntd.0003832.

"Discovery and Development of Penicillin". American Chemical Society. Retrieved 30 August 5

2015.

Spellberg B, Gilbert DN. The future of an#bio#cs and resistance: a tribute to a career of 6

leadership by John Bartlef. Clin Infect Dis. 2014;59 (suppl 2):S71–S75.

hfps://www.cdc.gov/hai/surveillance/.7

Richard J Fair and Yitzhak Tor. An#bio#cs and Bacterial Resistance in the 21st Century. 8

Perspect Medicin Chem. 2014; 6: 25–64.

Stone R: Stalin’s Forgofen Cure. Science 2002, 298(5594):7289

Nicolae Done, Hacard Science Review: Spring 2007, 20-23, Bacteriophages, the revived 10

bacterial killers.

Maniloff J, Ackermann HW. 1998. Taxonomy of bacterial viruses: establishment of tailed 11

virus genera and the order Caudovirales. Arch. Virol. 143:2051–2063

Carmela Garcia-Doval and Mark J. van Raaij, Structure of the receptor-binding carboxy-12

terminal domain of bacteriophage T7 tail fibers. PNAS, June 12, 2012, vol. 109, no.24, 9390-9395.

González-García VA, Bocanegra R, Pulido-Cid M, Mar�n-Benito J, Cuervo A, Carrascosa JL, 13

Characteriza#on of the ini#al steps in the T7 DNA ejec#on process, Bacteriophage 2015 Jun 2;5(3):e1056904.

Lindberg A.A. and C.G Hellerovist 1971. Bacteriophage afachment sites, serological 14

specifity and chemical composi#on of the lipopolysaccharides of semi-rough and rough mutants of Salmonella typhimurium. J. Bact. 105: 67-61.

Koike M, Iida K. Effect of polymyxin on the bacteriophage receptors of the cell walls of 15

gram-nega#ve bacteria, J Bacteriol. 1971 Dec;108(3):1402-11.

Rakhuba DV, Kolomiets EI, Dey ES, Novik GI, Bacteriophage receptors, mechanisms of 16

phage adsorp#on and penetra#on into host cell. Polish Journey of Microbiology 2010, vol.59, no.3, 145-155.

Molineux IJ, No syringes please, ejec#on of phage T7 DNA from the virion is enzyme 17

driven, Mol Microbiol. 2001 Apr;40(1):1-8.

Serwer P, Wright ET, Hakala KW, Weintraub ST, Evidence for bacteriophage T7 tail extension 18

during DNA injec#on, BMC Res Notes. 2008 Jun 26;1:36. doi: 10.1186/1756-0500-1-36.

Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Bacteriophages and phage-derived 19

proteins--applica#on approaches, Curr Med Chem. 2015;22(14):1757-73.

� 19

Lee SJ, Richardson CC, Choreography of bacteriophage T7 DNA replica#on, Curr Opin Chem 20

Biol. 2011 Oct;15(5):580-6. doi: 10.1016/j.cbpa.2011.07.024. Epub 2011 Sep 9.

Dhru# Savalia, William Robins, Sergei Nechaev, Ian Molineux, and Konstan#n Severinov, 21

The Role of the T7 Gp2 Inhibitor of Host RNA Polymerase in Phage Development, J Mol Biol. Author manuscript; available in PMC 2011 Sep 10.

Zuzanna Drulis-Kawa, Grażyna Majkowska-Skrobek, Barbara Maciejewska, Anne-Sophie 22

Delafre, and Rob Lavigne, Learning from Bacteriophages - Advantages and Limita#ons of Phage and Phage-Encoded Protein Applica#ons, Curr Protein Pept Sci. 2012 Dec; 13(8): 699–722.

Donald E. Johnson and Charles C. Richardson, A Covalent Linkage between the Gene 5 23

DNA Polymerase of Bacteriophage T7 and Escherichia coli Thioredoxin, the Processivity Factor, Journal of Biological Chemistry 278(26):23762-72 · July 2003.

Anastasia A. Aksyuk and Michael G. Rossmann, Bacteriophage Assembly, Viruses. 2011 24

Mar; 3(3): 172–203.

Young R, Bacteriophage lysis: mechanism and regula#on, Microbiol Rev. 1992 Sep;56(3):25

430-81.

Wang J, Hofnung M, Charbit A, The C-terminal por#on of the tail fiber protein of 26

bacteriophage lambda is responsible for binding to LamB, its receptor at the surface of Escherichia coli K-12, J Bacteriol. 2000 Jan;182(2):508-12.

Rajagopala SV, Casjens S, Uetz P, The protein interac#on map of bacteriophage lambda, 27

BMC Microbiol. 2011 Sep 26;11:213. doi: 10.1186/1471-2180-11-213.

Sujoy Chaferjee and Eli Rothenberg, Interac#on of Bacteriophage l with Its E. coli 28

Receptor, LamB, Viruses 2012, 4(11), 3162-3178; doi:10.3390/v4113162.

Groth AC, Calos MP, Phage integrases: biology and applica#ons, J Mol Biol. 2004 Jan 29

16;335(3):667-78.

Mark Ptashne, Cell Press and Blackwell Scien#fic Publica#ons, A gene#c switch: Gene 30

control and phage λ, DOI: hfp://dx.doi.org/10.1016/0168-9525(87)90171-5.

Mark Ptashne, Sloan-Kefering Memorial Cancer Center, New York, A Gene#c Switch, Third 31

Edi#on, Phage Lambda Revisited.

Oppenheim AB, Kobiler O, Stavans J, Court DL, Adhya S, Switches in bacteriophage lambda 32

development, Annu Rev Genet. 2005;39:409-29.

Gonzalo Durante-Rodríguez, José Miguel Mancheño, Eduardo Díaz, Manuel Carmona, 33

Refactoring the λ phage ly#c/lysogenic decision with a synthe#c regulator, 14 March 2016, DOI: 10.1002/mbo3.352.

Barrow PA, Soothill JS, Bacteriophage therapy and prophylaxis: rediscovery and renewed 34

assessment of poten#al, DOI: 10.1016/S0966-842X(97)01054-8.

Catherine Loc-Carrillo and Stephen T Abedon, Pros and cons of phage therapy, 35

Bacteriophage, 2011 Mar-Apr; 1(2): 111–114. doi: 10.4161/bact.1.2.14590.

Stephen T. Abedon, Ecology of An#-Biofilm Agents I: An#bio#cs versus Bacteriophages, 36

Pharmaceu#cals (Basel). 2015 Sep; 8(3): 525–558. Published online 2015 Sep 9. doi: 10.3390/ph8030525.

� 20

Franklin L. Nobrega, Anna Rita Costa, Leon D. Kluskens, and Joana Azeredo, Revisi#ng 37

phage therapy: new applica#ons for old resources, Trend in Microbiology, April 2015, Vol.23, No.4.

Capparelli R, Nocerino N, Iannaccone M, Ercolini D, Parlato M, Chiara M, Iannelli D, 38

Bacteriophage therapy of Salmonella enterica: a fresh appraisal of bacteriophage therapy, J Infect Dis. 2010 Jan 1;201(1):52-61. doi: 10.1086/648478.

Skurnik M, Strauch E, Phage therapy: facts and fic#on, Int J Med Microbiol. 2006 Feb;39

296(1):5-14. Epub 2005 Nov 15.

Hyman P, Abedon ST, Bacteriophage host range and bacterial resistance, Adv Appl 40

Microbiol. 2010;70:217-48. doi: 10.1016/S0065-2164(10)70007-1. Epub 2010 Mar 6.

Jessica Nicastro, Shirley Wong, Zahra Khazaei, Peggy Lam, Jonathan Blay, Roderick A. 41

Slavcev, Bacteriophage Applica#ons - Historical Perspec#ve and Future Poten#al, Page 12.

Llewelyn M, Cohen J (March 2002). "Superan#gens: microbial agents that corrupt 42

immunity". Lancet Infect Dis. 2 (3): 156–62. doi:10.1016/S1473-3099(02)00222-0. PMID 11944185.

Lanan Wassy Soromou, Zhichao Zhang, Rongtao Li, Na Chen, Weixiao Guo , Meixia Huo, 43

Shuang Guan, Jing Lu and Xuming Deng.

Riccardo V. D'Elia, Kate Harrison, Petra C. Oyston, Roman A. Lukaszewski, and Graeme C. 44

Clark, Targe#ng the “Cytokine Storm” for Therapeu#c Benefit, Clin Vaccine Immunol. 2013 Mar; 20(3): 319–327.

Li H., Llera A., Malchiodi E.L., Mariuzza R.A. The structural basis of T cell ac#va#on by 45

superan#gens. Annu. Rev. Immunol. 1999;17:435–466. doi: 10.1146/annurev.immunol.17.1.435.

Benjamin Cossefe, Xinyuan Wang, Mingxin Yu, SHOULD PHAGE THERAPY BE APPROVED IN 46

THE U.S.?, August 17, 2016, An Interac#ve Qualifying Project Report Submifed to the Faculty of WORCESTER POLYTECHNIC INSTITUTE. In par#al fulfillment of the requirements for the Degree of Bachelor of Science.

Kutateladze M, Adamia R. Bacteriophages as poten#al new therapeu#cs to replace or 47

supplement an#bio#cs. Trends Biotechnol. 2010;28:591–595.

Carlton RM. Phage therapy: past history and future prospects. Arch Immunol Ther Exp 48

(Warsz) 1999;47:267–274.

Alisky J, Iczkowski K, Rapoport A, Troitsky N. Bacteriophages show promise as an#microbial 49

agents. J Infect. 1998;36:5–15. doi: 10.1016/S0163-4453(98)92874-2.

Goodridge LD. Designing phage therapeu#cs. Curr. Pharm. Biotecgnol.11(1), 15-27 (2010).50

Hall AR, De VD, Friman VP, Prinay JP, Buckling A. Effects of bacteriophages on popula#ons 51

of Pseudomonas aeruginosa in vitro and in waxmoth larvae. Appl. Environ. Mivrobiol. 78(16), 5646-5652 (2012).

Levin BR, Bull JJ. Popula#on and evolu#onary dynamics of phage therapy. Nat. Rev. 52

Mivrobiol. 2(2), 166-173 (2004).

Soni KA, Nanapaneni R, Hagens S. Reduc#on of Listeria monocytogenes on the surface of 53

fresh channel ca�ish fillets by bacteriophage Listex P100. Foodborne Patblr. Dis. 7(4), 427-434 (2010).

� 21

Maura D, Gal#er M, Le BC, Debarbieux L. Virulent bacteriophages can target O104:H4 54

enteroaggrega#ve Escherichia coli in the mouse intes#ne an#microb. Agents Chemother. 56 (12), 6235-6242 (2012).

Górski A, Borysowski J, Miedzybrodzki R, Weber-Dabrowska B. Bacteriophages in medicine. 55

In: McGrath S, van Sinderen D, editors. Bacteriophage: Gene#cs and Microbiology. Norfolk, UK: Caister Academic Press; 2007. pp. 125–158.

Kufer E, De Vos D, Gvasalia G, Alavidze Z, Gogokhia L, Kuhl S, et al. Phage therapy in 56

clinical prac#ce: treatment of human infec#ons. Curr Pharm Biotechnol. 2010;11:69–86.

Goodridge LD. Designing phage therapeu#cs. Curr Pharm Biotechnol. 2010;11:15–27.57

Benjamin K Chan; Stephen T Abedon; Catherine Loc-Carrillo, Phage Cocktails and the 58

Future of Phage Therapy, Future Microbiol. 2013;8(6):769-783.

García E, García JL, García P, Arrarás A, Sánchez-Puelles JM, López R (Feb 1988). "Molecular 59

evolu#on of ly#c enzymes of Streptococcus pneumoniae and its bacteriophages". Proceedings of the Na#onal Academy of Sciences of the United States of America. 85 (3): 914–8. Bibcode:1988PNAS...85..914G. doi:10.1073/pnas.85.3.914. JSTOR 31364. PMC 279667. PMID 3422470

Loessner MJ. Bacteriol endolysins- current state of research and applica#ons. Curr. Opin. 60

Microbiol. 2005;8(4): 480-487.

Borysowski J, Weber-Dabrowska B, Gorski A. Bacteriaophage endolysins as a novel class of 61

an#bacterial agents. Exp Biol. Med. (Maywood) 2006;231(4): 366-377.

Lopez R, Garcia E. Recent trends on the molecular biology of pneumococcal capsules, ly#c 62

enzymes, and bacteriophage. FEMS Microbiol. Rev. 2004; 28(5): 553-580.

Loessner MJ, Kramer K, Ebel F, Scherer S. C-terminal domains of Listeria monocytogenes 63

bacteriophage murein hydrolases determine specific recogni#on and high-affinity binding to bacterial cell wall carbohydrates. Mol. Microbiol. 2002;44(2): 225-249.

Ashok K. Sharma, Sanjiv Kumar, Harish K., Darshan B. Dhakan and Vineet K. Sharma. 64

Predic#on of pep#doglycan hydrolases- a new class of an#bacterial proteins. BMC Genomics 17(1) · May 2016 with 66 Reads DOI: 10.1186/s12864-016-2753-8.

García E, García JL, García P, Arrarás A, Sánchez-Puelles JM, López R (Feb 1988). "Molecular 65

evolu#on of ly#c enzymes of Streptococcus pneumoniae and its bacteriophages". Proceedings of the Na#onal Academy of Sciences of the United States of America. 85 (3): 914–8. Bibcode:1988PNAS...85..914G. doi:10.1073/pnas.85.3.914. JSTOR 31364. PMC 279667. PMID 3422470

Loeffler JM, Djurkovie S, Fische\ VA. Phage ly#c enzyme Cpl-1 as a novel an#microbial for 66

pneumococcal bacteremia. Infect. Immun. 2003; 71(11): 6199-6204.

Mathias Schmerlcher, David M Donocan and Mar#n J Loessner. Bacteriophage endolysins 67

as novel an#microbials. Future Microbiol. 2012 Oct;7(10):1147-71. doi: 10.2217/fmb.12.97.

Biswajit Biswas, Sankar Adhya, Paul Washart, Brian Paul, Andrei N. Trostel, Bradford 68

Powell, Richard Carlton and Carl R. Merril, Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium, Infect Immun. 2002 Jan; 70(1): 204–210. doi: 10.1128/IAI.70.1.204-210.2002.

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Maya Merabishvili, Jean-Paul Pirnay, Gilbert Verbeken, Nina Chanishvili, Marina Tediashvili, 69

Nino Lashkhi, Thea Glon#, Victor Krylov, Jan Mast, Luc Van Parys, Rob Lavigne, Guido Volckaert, Wesley Mafheus, Gunther Verween, Peter De Corte, Thomas Rose, Serge Jennes, Mar#n Zizi, Daniel De Vos and Mario Vaneechoufe, Quality-Controlled Small-Scale Produc#on of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials, PLoS ONE. 2009; 4(3): e4944. Published online 2009 Mar 20. doi: 10.1371/journal.pone.0004944.

Bacteriophage Treatment of a Severe Escherichia coli Respiratory Infec#on in Broiler 70

Chickens. W. E. Huff, G. R. Huff, N. C. Rath, J. M. Balog, and A. M. Donoghue. Published By: American Associa#on of Avian Pathologists. DOI: hfp://dx.doi.org/10.1637/7041.

Sabah A. A. Jassim and Richard G. Limoges, Natural solu#on to an#bio#c resistance: 71

bacteriophages ‘The Living Drugs’, World J Microbiol Biotechnol. 2014; 30(8): 2153–2170. Published online 2014 Apr 30. doi: 10.1007/s11274-014-1655-7, PMCID: PMC4072922.

Winfried Boos and Howard Shuman. Maltose/Maltodextrin System of Escherichia coli: 72

Transport, Metabolism, and Regula#on. Microbiol Mol Biol Rev. 1998 Mar; 62(1): 204–229.

Howard A. Shuman. Ac#ve Transport of Maltose in Escherichia coli K12. THE JOURNAL OF 73

BIOLOGICAL CHEMISTRY Vol. 257, No. 10, Issue of May 25, pp. 545S5461. 1982 Prinfed in U.S..

Marraffini LA, Sontheimer EJ (March 2010). "CRISPR interference: RNA-directed adap#ve 74

immunity in bacteria and archaea". Nature Reviews. Gene#cs. 11 (3): 181–90. doi:10.1038/nrg2749. PMC 2928866.

David R. Harper, Helena M. R. T. Parracho, James Walker, Richard Sharp, Gavin Hughes, 75

Maria Werthén, Susan Lehman and Sandra Morales. Bacteriophages and Biofilms. An#bio#cs (Basel). 2014 Sep; 3(3): 270–284. Published online 2014 Jun 25. doi: 10.3390/an#bio#cs3030270.

Maya Merabishvili, Jean-Paul Pirnay, Gilbert Verbeken, Nina Chanishvili, Marina Tediashvili, 76

Nino Lashkhi, Thea Glon#, Victor Krylov, Jan Mast, Luc Van Parys, Rob Lavigne, Guido Volckaert, Wesley Mafheus, Gunther Verween, Peter De Corte, Thomas Rose, Serge Jennes, Mar#n Zizi, Daniel De Vos and Mario Vaneechoufe, Quality-Controlled Small-Scale Produc#on of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials, PLoS ONE. 2009; 4(3): e4944. Published online 2009 Mar 20. doi: 10.1371/journal.pone.0004944.

Enzybio#cs and phages: sage alterna#ves to an#bio#cs in the control of food safety. 77

Microbiology Today Nov 2012: 212-215.

Fogg, P. C., S. M. Gossage, D. L. Smith, J. R. Saunders, A. J. McCarthy, and H. E. Allison. 78

2007. Iden#fica#on of mul#ple integra#on sites for Stx-phage phi24B in the Escherichia coli genome, descrip#on of a novel integrase and evidence for a func#onal an#-repressor. Microbiology 153:4098-4110.

Winfried Boos and Howard Shuman. Maltose/Maltodextrin System of Escherichia coli: 79

Transport, Metabolism, and Regula#on. Microbiol Mol Biol Rev. 1998 Mar; 62(1): 204–229.

Frank Orlik, Chris#an Andersen, and Roland Benz. Site-Directed Mutagenesis of Tyrosine 80

118 within the Central Constric#on Site of the LamB (Maltoporin) Channel of Escherichia coli. II. Effect on Maltose and Maltooligosaccharide Binding Kine#cs. Biophysical Journal Volume 83 July 2002 309–321.

hfps://www.ncbi.nlm.nih.gov/su#ls/blink.cgi?mode=query81

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