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Jennifer-Jo Kilpatrick Cluff

BIOL 4950 - Senior Seminar SL


Fall 2015
Library Research Paper
December 2, 2015

Bacteriophages and Antibiotic Resistance


ABSTACT
Antibiotic resistance (ABR) is a rapidly evolving problem that is not being adequately
met by new antimicrobial drugs. Therefore, there is a pressing need for effective antibacterial
therapies that can be adapted against antibiotic-resistant bacteria. Before the discovery and
widespread use of antibiotics, treatments administering bacteriophages (phages) were
successfully utilized to treat bacterial infections. Today, phages have once again moved to the
forefront of research for targeting antibiotic resistance. With the current technology of
bioengineering, synthetic bacteriophages are a highly promising resolution to the ABR crisis our
world is facing today. A literary analysis of primary research papers clearly supports the
hypothesis that synthetically engineered bacteriophage are key to overcoming ABR.
INTRODUCTION
Antibiotic resistance results when an antibiotic loses its effectivity to control or kill
bacterial growth. The bacteria become "resistant" to therapeutic levels of that antibiotic and
continue to multiply (APUA, 2014).
ABR is a natural phenomenon. In the presence of an antibiotic, bacteria that are resistant
have a greater chance of persistence than those that are "susceptible." As the susceptible bacteria
are killed or inhibited by the antibiotic, selective pressures result for the resistant strains of
bacteria to survive (APUA, 2014). Resistance also occurs without human intermediation.

Bacteria often produce and use antibiotics against other bacteria, allowing for a low level of
natural selection for ABR. Currently, the higher levels of antibiotic-resistant bacteria are
attributed to the overuse and mistreatment of antibiotics (e.g. using antibiotics to treat viral
infections) (APUA, 2014).
Although some bacteria are naturally resistant to certain types of antibiotics, ABR may
occur in two ways; through a genetic mutation or by acquiring resistance from another
bacterium. Genetic mutations are spontaneous alterations of bacterial genetic material with a
possible occurrence between 1 x 10 -1 x 10 cells (APUA, 2014). Various mutations yield
different types of resistance. Types of resistance include enabling the bacteria to produce
enzymes that inactivate antibiotics, eliminate the cell target that the antibiotic attacks, close up
the entry ports that allow antibiotics into the cell, and manufacture pumping mechanisms that
export the antibiotic back outside the cell (APUA, 2014).
ABR genes can be acquired from other bacteria in several ways. Vertical gene transfer is
the transmission of ABR genes through inheritance in a new generation of cells. Horizontal gene
transfer implements a mating mechanism called conjugation for the exchange of genetic
material found on plasmids or transposon between bacterial cells. This may also occur between
different species of bacteria (APUA, 2014). Viruses are another mechanism for passing
resistance traits between bacteria. ABR genes from one bacterium are packaged into the viral
capsid and then injected into a new bacteria. Bacteria also have the capability to acquire naked,
"free" DNA from their environment (APUA, 2014).
Bacteria that acquire ABR through a spontaneous mutation or genetic exchange with
other bacteria can become resistant to multiple antibiotics. This is due to the ability to collect

multiple resistance traits over time, giving rise to ABR to many different families of antibiotics
(APUA, 2014). It is possible for an ABR bacterial population to revert back to a pre-resistant
state. However, this reversal process occurs much more slowly. If a selective pressure such as
the presence of an antibiotic is removed, over time the ABR bacterial population can become
once again susceptible to antibiotics (APUA, 2014).
In order to preserve the potency of existing antibiotics the following must happen; the
overall antibiotic use must be decreased, antibiotics must be prescribed only for bacterial
infections, prescriptions for the proper dosage and length of time must be applied, narrow
spectrum drugs should be selected whenever possible, and non-therapeutic uses of antibiotics in
farm animals and agriculture should be eliminated (APUA, 2014).
Due to the worlds current epidemic of ABR in pathogenic bacteria, there is a pressing
call to find new, novel antibiotics. Production of a new antibiotic is a time consuming and
expensive process, requiring approximately ten years and $300 million to bring a new antibiotic
to market. Unfortunately, resistance may eventually develop to a new antibiotic, and with heavy
use resistance may occur in as little as 2 years (APUA, 2014).
There are several possibilities to combat ABR. One approach applied by scientists is the
strengthening of existing antibiotics by modification so that bacterial enzymes cannot inactivate
them (APUA, 2014). Decoy molecules can also be used simultaneously with the antibiotic, so
that the decoy molecule is attacked by the bacterium's resistance enzyme instead of the
antibiotic. Clavulanic acid or sulbactam are already in use for blocking the beta-lactamase
enzymes that destroy the penicillin family of drugs (APUA, 2014). Another approach is to
interfere with the mechanisms that allow for attaining ABR, rather than attempting to kill the

bacteria (APUA, 2014). For example, the interference of duplication and movement of genetic
material may be achieved by introducing specifically manipulated genes to bacterial populations.
This altered genetic make-up may encode the inactivation of pilus assembly for conjugation
(Remaut et al., 2006).
Prior to the discovery and widespread use of antibiotics, it was suggested that bacterial
infections could be prevented and/or treated by the administration of bacteriophages or phages.
Early clinical studies with bacteriophages were not vigorously pursued in the United States and
Western Europe, due to the mass production of antibiotics during WWII (Sulakvelidze et al.,
2001). However, post-war relations were so strained, antibiotic accessibility in the former Soviet
Union and Eastern Europe was minimal and bacteriophages continued to be utilized and studied.
The results of these studies were extensively published in non-English (primarily Russian,
Georgian, and Polish) journals and were not readily available to the western scientic
community (Sulakvelidze et al., 2001).
Bacteriophages are bacterial viruses consisting of a DNA or RNA genome surrounded by
a protein coat (capsid). Phages utilize one of two life cycles types. The lysogenic cycle is the
incorporation of the bacteriophage genome into the host genome that is then replicated as part of
the host. The lytic cycle is the multiplication of bacteriophage inside the host cell before lysing
of the cell and releasing new phage particles (Balcazar, 2014).
Bacteriophage therapy is advantageous over antibiotic therapy. Bacteriophages are very
specific to their hosts, thus lessening the chance of secondary infections. Whereas antibiotics
target both pathogens and normal flora of patients, which can cause the secondary infections and
sometimes superinfections (Sulakvelidze et al., 2001). Bacteriophages replicate at the site of

infection where they are most needed to lyse the bacterial cells, however, antibiotics travel
throughout the body and do not concentrate at the site of infection (Sulakvelidze et al., 2001).
No side effects have been reported during or after phage application, but resistant bacteria,
allergies, even fatal anaphylactic reactions, and secondary infections are the common side effects
of antibiotics treatment (Sulakvelidze et al., 2001).
Although bacteria can become resistant to phages, phage resistance is not nearly as
concerning as drug resistance. Like bacteria, phages mutate and therefore can evolve to counter
phage-resistant bacteria (Sulakvelidze et al., 2001). The development of phage resistance can be
arrested altogether if phages are used in cocktails and/or in conjunction with antibiotics (Lu and
Collins, 2009).
In 1958, Arthur Kornberg used the X174 bacteriophage as a model to synthesize DNA
for the first time in vitro by purified enzymes and so ushered in the age of synthetic biology
(Goulian et al., 1967). X174 was also the first DNA-based genome to be sequenced by
Frederick Sanger and his team in 1976 (Sanger et al., 1977). By 2003, researchers of the J. Craig
Venter Institute (JCVI) reported that the genome of X174 was the first to be completely
assembled in vitro from synthesized oligonucleotides (Smith et al., 2003).
With the current technology of bioengineering, synthetic bacteriophages are a highly
promising resolution to the ABR crisis facing medical science today. Synthetically engineered
bacteriophage are key to overcoming ABR.
METHODS AND MATERIAL
To test the hypothesis of this literary research paper, 13 primary scientific research papers
were reviewed and analyzed. The information retrieval procedures included keyword searches

with Galileo and Google Scholar search engines. Keyword searches included the following
words or phrases: Bacteriophages, Phages, Bacteriophages and Antibiotic Resistance, Bacteria
and Antibiotics, Synthetic Bacteriophages, Engineering Bacteriophages, J. Craig Venter Institute,
Eliava Institute, Overcoming Antibiotic Resistance, Microbiology and Antibiotic Resistance,
Microbial Genetics, Bacteriophage Genetics, Cold War and Antibiotics, Medical Science and
USSR, Medical Science and USA and USSR Post-war Relations, Bacteriophage Discovery, Flix
d'Herelle, Frederick Twort.
LITERATURE ANALYSIS
In the first portion of this literary analysis, a background review of published case studies
and early clinical trials was conducted to establish natural phage effectiveness against pathogenic
bacterial infections. The following research in these studies focus on applications of natural
phages. Specifically, phages used in these studies were not biologically modified or synthetic.
It is only appropriate to begin this literature analysis with the publications of FrenchCanadian microbiologist, Flix d' Herelle and English bacteriologist, Frederick Twort. Both are
independently credited with the discovery of bacteriophages; Frederick Twort in 1915 and Flix
d' Herelle in 1917 (Sulakvelidze et al., 2001).
In 1915, Twort published an article reporting his first observations of what is now known
to be the lytic activity of bacteriophages. Twort noted what he described as watery-looking
areas within his bacterial cultures that gradually became glassy and transparent. He then
concluded that the cause of this glassy transformation was an infectious, filterable agent that
killed bacteria and in the process multiplied itself (Twort, 1915). However, due to a lack of
funding and the limitations of science and technology, Twort did not pursue further research on

this anomaly. As a result, his 1915 publication was not recognized for several years (Duckworth,
1976).
The activity described by Twort was also observed by d' Herelle in 1910. However,
where Frederick Twort stopped his investigation of this phenomenon, d' Herelle pursued it
further. By 1922, Flix d' Herelle had identified the virus as the cause of bacterial lysis and
coined the name, bacteriophage (Sulakvelidze et al., 2001). He continued to extensively
research phages and the phage-bacterium relationship. D' Herelle conducted several
experiments, including clinical trials. D' Herelle also developed the earliest techniques for
therapeutic bacteriophage preparation (D' Herelle, 1922 & 1938).
Early clinical trials of bacteriophage therapy by Flix d' Herelle demonstrated the
successful treatment of bacillary dysentery at the Pasteur Hospital. D' Herelle published details
on five of the cases he followed. Each patient presented varying degrees of symptom severity
(e.g. mild to severe). In all of five case studies, Shigella was identified as the causative agents of
disease (D' Herelle, 1922).
Early at the onset of symptoms, a stool sample was taken from the patient and the
isolation of Shigella was achieved. The patient was then administered a phage serum that was
prepared from a stock of hyper-virulent Shigella-specific bacteriophage (D' Herelle, 1922). A
detailed record of symptoms were documented including; number of stools per day and physical
characteristics of stools (e.g. observations of blood or blood and mucus). The stool samples were
analyzed daily by the following procedure: (i.) inoculation of nutrient bouillon with the patients
fecal material, (ii.) overnight incubation of the specimen at 37C to allow bacterial growth, (iii.)
filtration of bacterial growth through a Chamberland filter, (iv.) the filtrate was then added to a

tube of broth, previously inoculated with the Shigella bacillus, and incubated a second time
overnight and finally (v.) the turbidity of the culture was examined for bacterial growth. For the
duration of the patients infection, a daily stool specimen was prepared and analyzed by the same
protocol (D' Herelle, 1922).
To establish the correlation which exist between the patients condition and the virulence
of the bacteriophage specific to this bacterium, d' Herelle first established a scale of phage
virulence (D' Herelle, 1922). No virulence toward a given bacterium was denoted by 0 and
was defined as normal growth in cultures of the bacteria (bouillon or agar), whatever the quantity
of the filtrate from the feces was added. Weak virulence was denoted by a + and was defined
as normal growth in bouillon culture of bacteria to which the filtrate has been added. Transfer of
this culture to agar gave a few minute plaques after incubation. Medium virulence was denoted
by ++ and was defined as some growth decrease in bouillon cultures of the bacteria to which
the filtrate has been added. Transfer of this culture to agar and after incubation gave either a
layer of bacterial growth studded by numerous plaques, or fragments of bacterial colonies due to
high bacteriophage numbers. High virulence was denoted by +++ and was defined as lysis of
a bacterial suspension (significantly decreased turbidity) but secondary cultures still develop.
The re-inoculations on to agar remained sterile or gave only occasional bacterial colonies.
Extreme virulence was denoted by ++++ and was defined as complete lysis of bacterial
suspension with no secondary culture development. Inoculations on to agar always remain sterile
(D' Herelle, 1922).
D' Herelle then charted and compared his results to the patients conditions. Figures 1
through 6 are the graphs published by d' Herelle along with his case studies. The upper part of
the graphs trace the number of patient stools during a given 24 hour period as an indicator of

patient condition; the bold portion indicate stools containing blood and mucus. The lower part of
the graphs trace three types of bacteriophage virulence including; virulence for normal colon
bacteria (colon bacillus) depicted by the dotted line, virulence for a laboratory-maintained stock
strain of Shigella depicted by the broken line, and virulence for the patient strain of Shigella
depicted by the heavy line (D' Herelle, 1922). Fluctuations in virulence for each case study were
reflected in the condition of the patient. In the first three case studies (Figs. 2-4) the dysentery
was mild. The bacteriophages were active at the onset, no resistance to the bacteriophages was
acquire and its growth was quickly suppressed. In the last case (Fig. 6) the bacteria acquired
resistance, which was finally overcome. The condition of this patient was much more serious.
In all cases, complete recovery was reported.

Figure 1. Graph Legend (D' Herelle, 1922).

Figure 2. Germaine Mel, 16 years (D' Herelle, 1922).

Figure 3. Marie Leb, 26 years (D' Herelle, 1922).

Figure 4. Victor Ker, 6 years (D' Herelle, 1922).

Figure 5. Jean Ker, 6 years (D' Herelle, 1922).

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Figure 6. Lans, 70 years (D' Herelle, 1922).

Another successful and more current clinical trial for bacteriophage treatment was
published by Merabishvili et al. and Rose et al. at the Laboratory for Molecular and Cellular
Technology (LabMCT) Burn Center in Brussels, Belgium. In burn wound care, bacterial
infection remains a major therapeutic problem and renders large numbers of thermal injuries
virtually untreatable. Whereas, Staphylococcus aureus is a common cause of early burn wound
infection, Pseudomonas aeruginosa is known as the most common and lethal pathogen in burn
centers, due to acquired antibiotic-resistance (Merabishvili et al., 2009).
To improve burn wound care, this research focused on phage selection for the
development of a highly purified and fully defined bacteriophage cocktail (BFC-1) in a
controlled clinical trial. BFC-1 was active against the P. aeruginosa and the S. aureus strains
that were actually circulating in the LabMCT Burn Center at time of trial. Eight bacteriophages
(PT6, PT8, PNM, 14/1, 9/3, F77, PL and ISP) were selected for 23 P. aeruginosa and 17 S.
aureus strains that were isolated at the Eliava Institute of Bacteriophage, Microbiology and
Virology (Merabishvili et al., 2009).
Nine acute burn wound patients (4 males, 5 females; mean age 61 years; age range, 27 to
88 years; mean TBSA burned, 30%; TBSA burned range, 6-45%) with multidrug resistant

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(MDR) P. aeruginosa and/or S. aureus burn wound colonization, were determined by classical
bacterial culture and species identification and by antibiotic susceptibility testing of routine burn
wound swabs, were included in this clinical trial. Only patients with burn wounds that allowed
for punch biopsy sampling were included. Informed consent was given by all patients or their
legal representatives (Rose et al., 2014).
Just before the BFC-1 application, the colonized burn wound was divided into two
halves. One half received the standard treatment, the other half the phage treatment with BFC-1.
Two biopsies were taken by the MD in charge of the patient using a 4 mm punch biopsy needle;
one in the center of the zone where BFC-1 was to be applied, the other in the center of the zone
where the standard treatment was to be applied (Rose et al., 2014). Tissue biopsies were
preceded by a local anesthesia that had no effect on the result of bacterial culture. The biopsy
sites were sutured with green Ethilon 4/0 (standard treatment site) or with blue Prolene 4/0
(BFC-1 site). The MD in charge of the patient applied a single-dose of approximately 1 ml of
sterile BFC-1 per 50 cm on one half of the burn wound, using a 5 ml syringe with a spray
adapter (Figs. 7 and 8) (Rose et al., 2014). The other half of the burn wound was treated with
antimicrobials according to the standard protocols. Patients with suspected P. aeruginosa burn
wound infection were administered amikacin (single initial dose of 25 mg/kg body weight) in
combination with ceftazidime (single initial dose of 1 g) or meropenem (2 g/8 h) systemically.
Patients with suspected S. aureus burn wound infection were treated with systemic vancomycin
(single initial dose of 1 g) or linezolid (2 x 600 mg/d) (Rose et al., 2014).
A digital photograph was taken of the burn wound. The entire burn wound was then
covered with dressings in accordance with the standard treatment protocols. Two to five hours
later, the burn wound was uncovered. Immediately, two biopsies were taken next to (within

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cm) the previous ones (Rose et al., 2014). A digital photograph was taken a second time of the
burn wound. The entire burn wound was further treated according to standard protocols. The
wound biopsies were immediately weighed and collected into separate, sterile and labeled
microtubes containing 0.5 ml of sterile phosphate buffered saline (PBS) and transported to the
laboratory for bacterial load determination (Rose et al., 2014).
The biopsies were immediately homogenized, on ice, for 1 minute at 30000 rpm. Serial
tenfold dilutions of the homogenized wound biopsy samples were spread, in triplicate, on blood
agar, Manitol Salt Agar (MSA) and cetrimide agar plates. Colony counts were performed after
overnight incubation at 37C. The bacterial load, expressed as colony forming units (cfu) per
gram of tissue, was calculated for each biopsy (Rose et al., 2014).
Bacterial cultures of the homogenized biopsies taken after BFC-1 application showed
only a very small bacterial load (a few colonies) in 8 of the 10 applications. In the two
remaining applications the bacterial loads were much higher (108 cfu) than the other 8 cultures
before BFC-1 application and showed a medium bacterial load (50-60 cfu) after application
(Rose et al., 2014).
Patients were also screened for adverse effects, clinical abnormalities and changes in
laboratory test results that could be related to the application of phages. Clinical abnormalities
that were screened for included cardiovascular, renal, and respiratory complications and pain.
Clinical laboratory tests included the blood formula and standard hemostasis, biochemical,
pharmacological and toxicological parameters (Rose et al., 2014).

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Figure 7. The defined bacteriophage cocktail


(Merabishvili et al., 2009).
Figure 8. Application of BFC-1 on an infected burn
wound using a syringe spray (Merabishvili et al., 2009).

The second portion of this literature analysis focused on research publications involving
synthetic bacteriophages designed to target antibiotic-resistant. Among some of the more recent
scientific breakthroughs is the synthesis of infectious X174 bacteriophage.
In 2003, researchers at the J. Craig Venter Institute created a protocol for the accurate
assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. The procedure
involved three key steps: (i.) gel purication of oligonucleotides, (ii.) ligation of
oligonucleotides, (iii.) assembly of ligation products into full-length genomes, via polymerase
cycling assembly (PCA) (Fig. 9). PCA is a non-exponential reaction in which each terminal
oligonucleotide can be extended only once to produce a full-length molecule. (Smith et al.,
2003).
Synthetic oligonucleotides were obtained from Integrated DNA Technologies in 96-well
format and normalized to 100 M each. To purify the oligonucleotides, the top and bottom
strands were pooled separately, dried, and dissolved in water (Smith et al., 2003). Formamide
was added to the concentrated pools, which was then heated to 95C. The formamideoligonucleotide concentrations were loaded into a sequencing gel and electrophoresed at 1,300 V
for 4 hours. The bands, which migrated closest to the xylene cyanol marker, were visualized

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with a 254-nm UV lamp and excised. The gel was then extruded into TE buffer, frozen
overnight (-20C), eluted and filtered. The recovered oligonucleotides were ethanol-precipitated
and dissolved in water (Smith et al., 2003).
Before ligation, the oligonucleotides were phosphorylated in a reaction mixture
containing the purified oligonucleotides, T4 polynucleotide kinase buffer, ATP, and T4
polynucleotide kinase. This was then incubated at 37C for 1 hour. The reaction was terminated
by phenol-chloroform extraction and ethanol precipitation. The phosphorylation reaction was
repeated a second time. After extraction and precipitation, the oligonucleotides were dissolved in
water (Smith et al., 2003).
The ligation reactions mixture contained the 5'P oligonucleotides (top and bottom
strands), Taq ligation buffer, and water. The mixture was heated to 95C and slow-cooled over
55C. Taq ligase was added to the mixture and incubated at 55C for 18 hours. The reaction was
terminated by phenol chloroform extraction and ethanol precipitation. The products were
dissolved TE buffer (Smith et al., 2003).
PCA was carried out in reaction mixtures containing Advantage 2 buffer, dNTP mixture,
HF polymerase mixture, and the Taq ligation product. The polymerase mixture also contained
both an N-terminal deletion mutant of TaqDNA polymerase that lacks 5'- exonuclease activity
and Deep Vent DNA polymerase with 3'- exonuclease proofreading activity. Cycling parameters
were 94C for 15 seconds, slow cool at -0.1C/sec to 55C, annealing at 55C for 2 minutes, and
extension at 72C for 6 minutes. Thirty-five cycles were carried out (Smith et al., 2003).
The synthetic X174 (synX) DNA molecules produced by PCA were then amplified by
PCR. The PCR mixtures contained Advantage 2 buffer, dNTP, HF polymerase mixture, the PCA

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product, and unpurified oligonucleotides. The parameters were 94C for 15 seconds, 55C for
30 seconds, and 72C for 6 minutes for 25 cycles (Smith et al., 2003). The PCR mixtures were
pooled, phenol-chloroform-extracted, ethanol-precipitated, and re-dissolved in TE buffer. The
pooled PCR-amplified DNA was cleaved with PstI, and the linear DNA was gel-purified to yield
linear DNA, which were circularized by T4 ligase. The ligation mixture was phenol-chloroformextracted, ethanol-precipitated, and re-dissolved in TE/5 buffer in preparation for infectivity
testing (Smith et al., 2003).
To assay for synX DNA infectivity, the ligation product was electroporated into DH10B
cells (E. coli), immediately diluted with S.O.C. broth, and then aliquoted into two screw capped
glass culture tubes (A and B) containing KC broth (Smith et al., 2003). The tubes were rotated at
37C for 40 minutes, and then lysozyme and EDTA (pH 8) were added, followed by 30 minutes
of incubation on ice. The tubes were freeze-thawed twice in dry ice-ethanol to release the
synX phage. Aliquots of the A and B lysates were plated undiluted in 3 ml of top agar
containing 0.3 ml of log phase HF4704 at 5 x 10 cells per ml on LB plates. Phage plaques were
visualized after 6-18 hours of incubation at 37C (Fig. 10). The synX-A lysate yielded 194

plaques (Fig. 10), and the synX-B lysate yielded 181 plaques per 100 l plated. Some
variation in plaque size was observed (Smith et al., 2003).
Several plaques were picked from each plate directly into PCR mixtures for amplification
and sequencing. The A plaques are independent of the B plaques. The 30 cycles of amplification
consisted of 10 seconds at 94C, 30 seconds at 55C, and 6 minutes at 72C. A mixture of
shrimp alkaline phosphatase, exonuclease I, and water was added to the PCR mixture, and
digestion was at 37C for 45 minutes and 45C for 15 minutes, followed by 72C for 15 minutes

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to inactivate the enzymes. The synX DNA was gel-purified before carrying out standard
sequencing reactions and sequencing on a 3730 XL sequencer (Smith et al., 2003).
Representative sequences from four plaques were compared (Fig. 11). Phage DNA from
plaque B3 gave a sequence that was identical to GenBank accession no. J02482. The B1 DNA
sequence contained one silent TC transition, two silent GA transitions, one GA
transition at position 4170 (resulting in a Gly-to-Ser amino acid change in the gene A protein),
and one TG transversion at position 3606 (resulting in a Ser-to-Ala change in the gene H
protein) (Smith et al., 2003). DNA from plaque A4 contained a TC silent mutation at position
1045 and A8 contained a G3A silent mutation at position 446, a CT mutation at position 4399
(resulting in an AlaVal amino acid change in gene A protein), and a CA change at position
5144 (resulting in a Gln-to-Lys change in gene B and silent changes in gene A and A*).
Infectious titers of synthetic phage from plaque B3 were indistinguishable from commercially
available phage (Smith et al., 2003).

Figure 9. Schematic diagram of the steps in the global synthesis of infectious


synX174 bacteriophage from synthetic oligonucleotides (Smith et al., 2003).

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Figure 10. Plaques of synX-A. There appear to


be several plaque morphologies: small plaques with
sharp borders, medium-sized plaques, and large
plaques with fuzzy borders (Smith et al., 2003).

Figure 11. Sequence comparisons of natural


X174 and synX genomes (Smith et al., 2003).

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With the improved methodology and reduced time required (14 days) for accurate
assembly of synthetic DNA, a protocol for bacteriophage synthesis was established.

Researchers at the Sackler School of Medicine in Israel successfully used this same approach
(Smith et al.) to deliver designed genes to reverse bacterial antibiotic resistance (Yosef et al.,
2014).
To sensitize bacteria carrying antibiotic resistance genes, Yosef et al. constructed a
transferable CRISPR-Cas system. This new system was engineered to target and destroy
plasmids encoding genes that confer resistance. Plasmid synthesis was done by GenScript.
Delivery of this designed CRISPR-Cas System was facilitated a Phage (Yosef et al., 2014). The
phage protocol was applied a second time to synthesis T7-N1C1 lytic phages that selectively kill
the non-lysogens. Altogether, the engineered temperate phage delivering the CRISPR-Cas
system was used along with an engineered lytic phage to facilitate the loss of multiple resistance
determinants (Yosef et al., 2014).
In figure 12, the schematics of the procedure to enrich for antibiotic-sensitized bacteria
are depicted. A bacterial culture was mixed with lysogenizing phages, resulting in both lysogens
and non-lysogens in the culture. Lysogens were both antibiotic sensitized (Streptomycin and
Gentamicin) and phage resistant as the CRISPR-Cas system degrades the antibiotic resistanceconferring plasmid and the lytic-phage chromosome. The treated culture was inoculated on agarcontaining T7-N1C1 lytic phages that selectively kill the non-lysogens, thus selecting for
antibiotic-sensitized bacteria (Yosef et al., 2014).

Figure 12. Schematics of the procedure to enrich for


antibiotic-sensitized bacteria (Yosef et al., 2014).

The final publication analyzed was recently published by T.K. Lu and J.J. Collins (2015).
Lu and Collins also used the synthetic phage protocol developed by the researchers at the J.
Craig Venter Institute; and like Yosef et al., these researchers engineered a synthetic
bacteriophage to target antibiotic-resistant bacteria (Lu & Collins, 2015).
Bactericidal antibiotics (e.g., quinolones such as ofloxacin) induce the formation of
hydroxyl radicals which cause DNA, protein, and lipid damage, and ultimately, cell death. DNA
damage initiates the SOS response, which results in DNA repair and cell survival. To suppress
the SOS network and enhance the effect of bactericidal antibiotics, lexA 3 was engineered to
overexpress lexA3, a repressor of SOS (Lu & Collins, 2015).
To test the antibiotic-enhancing effect of lexA 3 , time courses were obtained for killing
of E. coli EMG2 bacteria with phage and/or ofloxacin treatment. Viable cell counts were

calculated by counting colony-forming units (cfu) during treatment. After 6 hours, bacteria
exposed to ofloxacin only were reduced by about 1.7 log (cfu/mL), reflecting the presence of
persister cells not killed by the drug (Lu & Collins, 2015). By 6 hours, lexA3 improved
ofloxacin's bactericidal effect by 2.7 orders of magnitude compared to an unmodified phage,
UNMOD

(-99.8% additional killing) and by over 4.5 orders of magnitude compared to no

phage (-99.998% additional killing). With combination phage and antibiotic treatment, no
significant bacterial regrowth was apparent (Fig. 13) (Lu & Collins, 2015).

Figure 13. Killing curves for no phage (diamonds), unmodified phage (squares), and engineered phage (circles) with 60 ng/mL
ofloxacin (oflox) (solid lines, closed symbols). 108 plaque-forming-units/mL (pfu/mL) phage was used. A growth curve for
EMG2 with no treatment is also shown (dotted line, open symbols). lexA3 greatly enhanced killing by ofloxacin by 4 hours of
treatment (Lu & Collins, 2015).

CONCLUSION
Since their discovery, bacteriophages have contributed enormously to our understanding
of molecular biology as model systems. Bacteriophage-based technologies and their application
to the study of infectious diseases is advancing medical science. This is more important now
than ever with the growth of multidrug-resistant bacteria. Bacteriophages have provided many
tools that have progressed the fields of genetic engineering and synthetic biology (Yang et al.,
2013). New strategies for engineering genomes have the potential to fast-track the design of

novel phages as therapies (Lu & Collins, 2015 and Yosef et al., 2014). Furthermore, the
publications reviewed in this literary research analysis more than sufficiently supports the
hypothesis that synthetically engineered bacteriophage are vital to overcoming ABR.

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DHerelle, F. (1922). The bacteriophage: Its role in immunity (1st ed., pp. 163-282) (G. Smith,

Trans.). Baltimore, MD: Williams & Wilkins.


DHrelle, F. (1938). Preparation of Therapeutic Bacteriophages, Appendix 1 from: Le
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