They’re Everywhere!

: Study on
Bacteriophage Discovery and Application
Andrew Choe
Independent Research G/T I
1 May 2018

Advisor: Dr. Daniel Nelson

Instructor: E. Leila Chawkat

They’re Everywhere!: Study on Bacteriophage Discovery and Application

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Abstract

The purpose of this paper is to inform the reader on what bacteriophage are. Specifically,

this paper will focus on concepts such as bacteriophage discovery, functions in medicine and

agriculture, and the issues of antibiotic-resistant bacteria. The research method used in this

paper is an experiment. In this experiment, the isolation a potential bacteriophage of Escherichia

coli (E. coli), Klebsiella, and Acinetobacter baumannii (Acineto) bacteria. After analyzing the

plates, two potential bacteriophage plaques were found for E. Coli in a dirt sample from the front

of a house.

Table of Contents

Introduction……………………………………………………………………………....Pages 1-2

Literature Review…………………………………………...............................................Pages 2-9

Data Rationale, Data, Analysis, and Conclusions…………………………………...….Pages 9-13

Conclusions…………………………………………………………………………...……Page 14

References……………………………………………………………………………..Pages 15-16

Introduction

Patients with illnesses caused by bacteria depend on antibiotics. However, more and

more bacteria are developing resistance to antibiotics. These bacteria are known as superbugs.

Since antibiotics are too broad in the range that kill bacteria, an alternative solution,

bacteriophage needs to be applied. Bacteriophage, or phage for short, are viruses that target

bacteria cells. Receptors on the phage sense nearby bacteria that match the host of the

bacteriophage. After finding and connecting to a host bacterial cell, the phage will inject its own

genome into the bacteria. During this process the phage genome can go into one of two phases, a

lytic or a lysogenic cycle. A lytic cycle is where the phage starts to replicate within the bacterial
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cell, and the bacterial cell eventually bursts. The lysogenic cycle is where the bacteriophage

DNA integrates with the bacteria DNA. The phage’s state can be described as dormant, but the

bacteriophage can sense harm or stress to the bacteria cell. If this happens, then the lysogenic

phage will go into a lytic cycle. Through the genetic sequencing of bacteriophage, many

principles of molecular biology have been discovered. For example, George Salmond, a writer

for Nature Reviews Microbiology, describes how “phages were crucial in establishing the central

dogma of molecular biology — information is sequentially passed from DNA to RNA to

proteins… from sequencing and genome engineering”. There are many aspects to the uses of

bacteriophage, such as medicine and agriculture. In order to prevent disease, bacteriophage are

more commonly used in phage therapy. The purpose of this paper is to describe the uses of

bacteriophage mainly in the prevention of disease in humans from harmful bacteria. The future

use of bacteriophage can assist in the fight against antibiotic-resistant bacteria, such as

Methicillin-resistant Staphylococcus aureus (MRSA).

Literature Review

Phage are everywhere; there are approximately 10^30 bacteria globally. So, there are at

least 10^31 bacteriophage globally. Finding phage is not an obstacle in phage therapy, but

isolating the correct phage that is specific to a specific bacteria is sometimes challenging.

Bacteriophage are abundant in areas where bacteria is present. Where there are bacteria, there

are definitely phage. These areas with higher numbers of bacteria increase the chance of finding

the correct phage to a specific disease. Holly Ganz, a writer for Plos One, a scientific journal

explains, “We obtained isolates of the siphovirus from two carcass sites in Etosha, a 22,915 km2

national park in northern Namibia with abundant wildlife populations that exhibit regular

occurrences of anthrax infections”. Ganz was able to find vB_BanS-Tsamsa phage for anthrax
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infections in areas where anthrax infections are common in wildlife, livestock, and humans.

Tarek F El-Arabi is able to isolate the B. cereus phage vB_BceM_Bc431v3 (Bc431v3) in an area

where bacterial numbers are higher as well. El-Arabi describes how “Phage vB_BceM_Bc431v3

was isolated from sludge samples collected from a local wastewater management plant, and

plaque purified”. The phage is present in this area, for a wastewater management plant is very

likely to contain high numbers of bacteria, which increase the chance of finding specific phage.

These two cases where phage are isolated illustrates how places with sufficient numbers of

bacteria increase the chance of isolating bacteriophage.

However, going to areas like a sewer is not necessarily required to find phage, for they

are everywhere. Specific bacteriophage can simply be found in the dirt from the back of one’s

house. After obtaining samples, buffer is added, and the sample is spun to separate the phage

from the solid sample. In the solution suspended above the pelleted soil, there are millions of

different bacteriophage. In order to discover the phage specific to a certain bacteria, the phage

suspended in the solution is mixed with the wanted bacteria. After letting the bacteria with

phage incubate on a petri dish, the bacteria will grow into a “lawn”, and little clearings may be

present. The clearings, known as plaques, are areas where the phage is killing the bacteria.

Since bacteriophage are fairly easy to find, many wonder why phage therapy is not

common. One of the factors that provide limitations on the treatment is diagnosing the illness on

time. Figuring out what type of infection a patient has, and finding the correct phage that is

appropriate to that bacterial infection takes time. The process to find the right bacteriophage

may not be enough time to help the patient, so antibiotics are used since they have a broader

range in the types of bacteria they kill. However, doctors can use a bacteriophage that contains

more than one different type of phage. This is known as a phage cocktail. If a doctor can
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identify what kind of infection a patient has, there are certain types of infections that doctors

know are caused by a certain type of bacterium. Pawan Parajuli, a scientist researching Gram-

negative bacteria that cause shigellosis shares how “using hybrid sequencing approach produced

a high-quality reference genome. S. flexneri serotype 1c is an important serotype responsible for

more recent shigellosis outbreaks in the developing nations”. Parajuli used different types of

Shigellosis phage in order to discover the right bacteriophage. Different phages of certain

bacterial infections can be combined to fight a certain strain of bacteria. These different phages

can be found in databases. For example, Daniel Russell wrote a paper on the Actinobacter

bacteriophage database on Oxford Academic. He conveys, “The Actinobacteriophage Database

(PhagesDB) is a comprehensive, interactive, database-backed website that collects and shares

information related to the discovery, characterization and genomics of viruses that infect

Actinobacterial hosts” (Russell). Databases such as this assist doctors and scientists to use other

people’s discoveries in order to help treat patients by using the already discovered phage.

An example of an experiment where phage are isolated from the environment and

characterized based on what bacteria the phage attacks is one by Sophio Rigvava, a scientist

studying biological and physical properties of Enterococcus faecalis bacteriophage and

Streptococcus mitis bacteriophage. In the experiment, Rigvava isolated an Enterococcus faecalis

bacteriophage (vB_EfaS_GEC-EfS_3) and a Streptococcus mitis bacteriophage

(vB_SmM_GEC-SmitisM_2) from the Mtkvari River. Rigvava finds, “Bacteriophages are often

suggested as an alternative therapeutic agent against these infections. In this study, E. faecalis

and S. mitis strains were isolated from female patients with urinary tract infections.

Bacteriophages active against these strains were isolated from sewage water from the Mtkvari

River. Two phages, designated vB_EfaS_GEC-EfS_3 (Syphoviridae) and vB_SmM_GEC-

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SmitisM_2 (Myoviridae), were specific for E. faecalis and S. mitis, respectively”. By collecting

water from a river, Rigvava is able to isolate two phage that can fight or prevent bacterial

infections in the gastrointestinal and genitourinary tracts. This experiment is one of many

experiments that demonstrate how isolating phage is not a very complicated process, and shows

how phage can be found anywhere.

Before the invention of penicillin, diseases caused by bacteria were fought with

bacteriophage. Even before scientists could see phage, they knew that bacteriophage eat

bacteria. However, penicillin enabled doctors to treat patients quicker and more efficiently

through the use of antibiotics. A.B Monk, a writer for the Letters in Applied Microbiology

explains, “Bacteriophages are bacterial viruses and have been used for almost a century as

antimicrobial agents. In the West, their use diminished when chemical antibiotics were

introduced… Increasing antibiotic resistance in bacteria has driven the demand for novel

therapies to control infections and led to the replacement of antibiotics in animal husbandry”.

While antibiotics are good for killing bacteria on a larger range, Monk explains how bacteria

have increasingly become immune to certain antibiotics, and he also explains how phage therapy

has become less common after the invention of penicillin.

In the fight against antibiotic-resistant bacteria, superbugs, doctors are looking for a

solution to the new threat. Karl Thiel, a writer for the Nature Biotechnology Journal highlights,

“A US government document released in June 2000 warned of ‘a growing menace to all people.’

The reference was not to terrorism or foreign dictators, but to the increasing prevalence of

antibiotic-resistant microbes.” The discovery of penicillin is what led to the decrease in use of

phage therapy. Since penicillin has a broad spectrum, the antibiotic could kill almost all bacteria,

so scientists took new interest in the drug. However, the war on bacteria is not over. Recently,
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there have been reports on several cases where bacteria have been growing resistance to

antibiotics. Jyoti Madhusoodanan, a writer for The Scientist describes, “in the last decade,

antibiotic resistance has grown from a concern to a crisis. In addition to the deadly incident at the

NIH, a multidrug-resistant form of methicillin-resistant Staphylococcus aureus (MRSA) in a UK

neonatal unit infected 12 babies in 2011… Even when antibiotics do work, they’re not always

the best option, as they wipe out beneficial bacteria as well as pathogenic ones, with potentially

long-lasting health consequences”. The concern Madhusoodanan has on the safety from

antibiotic-resistant bacteria can be supported through the works of Diana Pires, a researcher who

writes for the Journal of Virology. Pires is researching the treatment for Pseudomonas

aeruginosa, and highlights, “Antimicrobial resistance constitutes one of the major worldwide

public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and

nowadays, a common infection can be fatal… phages for the treatment of bacterial infections has

been extensively studied as an alternative therapeutic strategy”. Through the works of these

authors, the disturbance of bacteria immune to antibiotics is described, and informs readers on an

alternative solution to the problem, bacteriophage. Antibiotic-resistant bacteria are real, and are

increasingly causing problems. In order to fight against the superbugs, scientists and doctors

must revisit the process of phage therapy.

While phage therapy has certain limitations set by the FDA and NIH, there have been

cases where phage therapy was successful. Before testing the use of bacteriophage on humans,

an experiment was done on mice. Laurent Debarbieux, a scientist researching bacteriophage in

animals, ran an experiment on mice with lung infections. In his findings, Debarbieux conveys,

“Using a bioluminescent Pseudomonas aeruginosa strain, we monitored and quantified the

efficacy of a bacteriophage treatment in mice during acute lung infection. Bacteriophage

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treatment was not only effective in saving animals from lethal infection, but also was able to

prevent lung infection when given 24 hours before bacterial infection, thereby extending the

potential use of bacteriophages as therapeutic agents to combat bacterial lung infection”. While

the use of phage therapy was on mice, the same process can apply to humans. These mice were

saved from lethal lung infections, and were able to prevent lung infections due to receiving the

phage before exposure to the bacteria. In another phage experiment with mice, Biswajit Biswas,

a scientist with the National Institutes of Health did an experiment with mice that have infections

in the digestive tract. Biswas describes, “Colonization of the gastrointestinal tract with

vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and

nursing homes in the United States… The emergence of antibiotic-resistant bacterial strains

requires the exploration of alternative antibacterial therapies, which led our group to study the

ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia…

administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals.

Even when treatment was delayed to the point where all animals were moribund, approximately

50% of them were rescued by a single injection of this phage preparation”. Biswas’ experiment

is similar to the one by Debarbieux, for both experiments were able to safely rescue mice from

diseases caused by bacteria through the use of phage therapy. While the outcomes of phage

therapy can be anecdotal, there are many cases where bacteriophage therapy has saved lives in

humans.

Even though bacteriophage are more commonly known to be used in medicine, there are

many applications of phage everywhere. Agriculture is an example where phage can be used to

keep consumers safe through maintaining the safety of foods that are sold. For example, Dr.

Lawrence Goodridge, an associate professor in food microbiology and food safety explains, “The
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scientific literature demonstrates the possibility of using phage therapy to effectively reduce the

presence of foodborne pathogens in food producing animals and in fresh and processed foods”.

Goodridge recognizes the use of phage to benefit humans through agriculture. Since phage are

found in nature, they are a natural way to keep food safe without harmful steroids and chemicals

used to fight bacteria. Some places the bacteriophage can be applied in agriculture are in water,

sprayed on plants, or put in the food for animals. Irshad Haq, a writer for the BMC Virology

Journal describes, “Phages can be used as biocontrol agents in agriculture and petroleum

industry… Phages could be used as predators of pests (bacteria) found in association with plants,

fungi or their products [55, 56]. Phage mediated biocontrol of plant pathogens has successfully

been attempted against Xanthomonas pruni associated bacterial spot of peaches to control

infections of peaches, cabbage and peppers.” Many consumers conscious of their health worry

about what is going into their bodies. In order to prevent pathogenic bacteria in foods, farmers

would apply steroids or antibiotics to their food. Consumers have concern over this process, for

the chemicals and medicine used on the plants and animals are entering their bodies. Since

phage are found in nature and not man made, customers do not have to worry about consuming

harmful medicines or chemicals. Martha Clokie explains, “In all environments phages exist as

part of a complex microbial ecosystem which may be either a free living environment, such as

the ocean, or a microbial environment within a macro-organism”. All three authors agree that

phages are not just used to help society through medicine, but can also keep consumers safe

through an all-natural solution in order to keep food free from pathogenic bacteria.

Data Rationale, Data, Analysis, and Conclusions

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Samples: 1: Pond (Western Regional)

2: Sewer (Bushy Park)

3: Soil (Western Regional)

4: Soil (House)

5: Wet Dirt (House)

6: Dirt (Western Regional)

Procedures

Day 1:

1. Grow 20 ml (tryptic soy broth) of Escherichia coli (700728), Klebsiella pneumoniae

(43883), and Acinetobacter baumannii (1605) at 37 degrees C overnight.

Day 2:

2. Place liquid samples (#1, #2), soil samples (#3, #4), and dirt samples (#5, #6) into 50

ml tubes. Mix 5ml of PBS in 50 ml tubes.

3. Centrifuge samples at 3000 RPM for 10 minutes.

4. Pour supernatants into separate dishes. Sterile filter into clean 50 ml tubes.

5. Mix 1ml of each bacteria with 10 ul of each sample in separate 15 ml tubes. Add 5 ml

of LB (luria broth) top agar (at 50 degrees C), mix, and pour into Petri dishes containing

LB agar.

6. Let solidify and put all Petri dishes into incubator at 37 degrees C overnight.

Day 3:
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7. View each Petri dish for clearing zone (plaque formation).

Results/Analysis

This experiment to discover bacteriophage of the Escherichia coli (E. coli), Klebsiella,

and Acinetobacter baumannii (Acineto) bacteria was conducted. The bacteria were grown at

37°C overnight before the experiment. Six samples were collected from a residential areas,

school sewers, and public parks. Specifically, there are two dirt samples, two soil samples, and

two water samples. The two dirt samples come from a pond in a park and a sewer next to Bushy

Park Elementary School. The two dirt samples were collected from Western Regional park and

the front lawn of a house as well as the soil samples, one from the park and the other from the

same house. Usually, with environmental samples, an enrichment process is done to increase the

chances of discovering the right kind of phage. However, with the interest of time, the

enrichment process was skipped; this leaves a 95% chance of finding nothing.

After the six samples were collected, they were put into six different 50 ml tubes. 5 ml of

PBS, a salt solution, was added to each of the dirt and soil samples. The PBS allows for easier

separation for phage from the dirt and soil. After being placed in each test tube, the samples

were ready for centrifuge. The process of centrifugation is to separate the dirt and particles from

the phage since phage are less dense. The six samples were spun at 3000 RPM for ten minutes.

After ten minutes, the phage in the dirt and soil samples were suspended in the layer of PBS

above a layer of dirt or soil. After centrifugation, the samples were filtered to separate any

remaining bacteria in the sample from phage. The filtered samples were put into sterile 50 ml

test tubes. In 15 ml test tubes, the filtered samples were mixed with the three bacteria and a top
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agar, and they were poured into a petri dish. The molten agar solidifies on the dish, which

allows the bacteria to grow into a lawn covering the entire surface of the dish. After spending 24

hours in an incubator at 37°C, the bacteria lawn had fully developed. The final step in this

experiment was to find clearing zones, or plaques, to see where phage may be killing the specific

bacteria. Unfortunately, 17 samples were negative for E. coli, Klebsiella, and Acinetobacter

bacteriophage. However, sample five, the dirt sample from the house, showed a possibility of

two plaques in the lower right side of the Petri dish. The possibility of finding a plaque was 5%,

but the two clearings that were found are likely bacteriophage plaques. So, the dirt sample from

the house could have E. coli bacteriophage.

E. coli is a type of bacteria that lives in the environment, food, and people. Usually, they

are harmless, but some strains are harmful to people. There are many food poisoning cases

where people get diarrhea, urinary tract infections, respiratory illness and pneumonia, and other

illnesses from E. coli exposure. Theoretically, if the two plaques are indeed E. coli

bacteriophage, then they can be used in the treatment of those with specific E. coli strains to

those used in the experiment.

This discovery could go further with running more tests with sample five, and growing

more bacteria. If more plaques were to form, then the bacteriophage can be isolated, and its

genome can later be sequenced in order to figure out more specifically the type of bacteriophage.

There are technological aspects to identifying and sequencing a phage. The process of finding a

phage is considered a wet lab part of research. Sequencing and identifying the phage is a dry lab

that is all computer-based and can be done online with different programs and tools. However,

with the time allowed, the process of finding samples, filtering the samples, mixing the samples

with pathogenic bacteria, and possibly discovering a phage has been successful. This experiment
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has helped gain knowledge to repeat the process of finding other phage. In the future, repeating

the process of finding phage, but in a more specific method where the chances of finding phage

are higher will be possible. This experiment proves how phage can be isolated from the

environment in order to find bacteriophage specific to bacteria that can cause infections harmful

to humans.

Environmental Samples 18 Dishes (6 samples for 3 bacteria)

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E-coli sample with two possible phage plaques (lower right side)

Conclusions

In the battle against antibiotic resistant drugs, bacteriophage therapy is the alternative

answer in fighting the superbugs. Bacteriophage are everywhere, and can be isolated from

environmental samples such as dirt, soil, and water. If the experiment with the E. coli bacteria

was continued, the discovery of a new kind of phage against E. coli is a possibility. The possible

phage plaques were found from a house dirt sample. Therefore, it is definitely possible for

anyone to go out and discover new phage to battle the superbugs with the right procedures,

equipment, and amount of time. If bacteriophage gains back attention, the issue with antibiotic

resistant bacteria can be lowered dramatically. Since bacteriophage are found in nature, they

pose an all-natural solution to fighting bacteria in agriculture as well. Due to phage, consumers

do not have to worry about eating food containing harmful chemicals and steroids.

Bacteriophage can benefit the community in many ways including ways in medicine and

agriculture. The phage therapy has limitations, but can become a more common practice in the

future. The use of bacteriophage can fight bacterial disease or prevent infection.
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References

Biswas, B., Adhya, S., Washart, P., Paul, B., Trostel, A. N., Powell, B., . . . Merril, C. R.
(2002, January). Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate
of Vancomycin-Resistant Enterococcus faecium. Retrieved November/December, 2017,
from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC127648/

Clokie, M. R., Millard, A. D., Letarov, A. V., & Heaphy, S. (2011, January 1). Phages in
nature. Retrieved November/December, 2017, from
https://www.tandfonline.com/doi/pdf/10.4161/bact.1.1.14942

Debarbieux, L., Leduc, D., Maura, D., Morello, E., Criscuolo, A., Grossi, O., . . . Touqui,
L. (2010, April 1). Bacteriophages Can Treat and Prevent Pseudomonas aeruginosa Lung
Infections. Retrieved November/December, 2017, from
https://www.ncbi.nlm.nih.gov/pubmed/20196657

El-Arabi, T. F., Villegas, A., Lingohr, E. J., & Kropinski, A. M. (2013, February 07).
Genome sequence and analysis of a broad-host range lytic bacteriophage that infects the
Bacillus cereus group. Retrieved November/December, 2017, from
https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-48

Ganz, H. H., Law, C., Schmuki, M., Eichenseher, F., Calendar, R., Loessner, M. J., . . .
Klumpp, J. (2014, January 27). Novel Giant Siphovirus from Bacillus anthracis Features
Unusual Genome Characteristics. Retrieved November/December, 2017, from
https://doi.org/10.1371/journal.pone.0085972

Goodridge, L. D., & Bisha, B. (2011, May 1). Phage-based biocontrol strategies to reduce
foodborne pathogens in foods. Retrieved November/December, 2017, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225777/

Haq, I. U., Chaudhry, W. N., Akhtar, M. N., Andleeb, S., & Qadri, I. (2012, January 10).
Bacteriophages and their implications on future biotechnology: A review. Retrieved
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November/December, 2017, from

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Madhusoodanan, J. (2016, January 1). Viral Soldiers. Retrieved November/December,

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8029e

Pires, D. P., Boas, D. V., & Sillankorva, S. (2015, August 01). Phage Therapy: A Step
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Rigvava, S., Tchgkonia, I., Jgenti, D., Dvalidze, T., Carpino, J., & Goderdzishvili, M.
(2013, January). Comparative analysis of the biological and physical properties of
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54c35

Russell, D. A., & Hatfull, G. F. (2016, December 6). PhagesDB: The actinobacteriophage
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