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Jennifer Jung

1/15/16
Annotated Source List
Allen, H. K., Trachsel, J., Looft, T., & Casey, T. A. (2014). Finding alternatives to antibiotics.
Annals of the New York Academy of Sciences. Retrieved from
http://www.ncbi.nlm.nih.gov/nlmcatalog?term=0077-8923%5BISSN%5D
This actually compares both phage lysins and bacteriocins side by side.
Becker, S. C., Foster-Frey, J., & Donovan, D. M. (2008). The phage K lytic enzyme LysK and
lysostaphin act synergistically to kill MRSA. Federation of European Microbiological
Societies. Retrieved from http://femsle.oxfordjournals.org/content/287/2/185.long
Its about the use of antibiotics in synergy with lysins.
Biochemical Society. (n.d.). Resistance is futile by Margaret Riley [Video file]. Retrieved from
https://www.youtube.com/watch?v=NfqyVtvoqFU
Summary: At a conference devoted to bacteriocins and their use against drug-resistant
bacteria. Margaret Riley presented an article about bacteriocins through a webcast. In
addition to growing antibiotic resistance, broad spectrum antibiotics are generally not a
good option for defeating bacteria because they are not found commonly in nature and
target wide ranges of bacteria, wiping out the healthy flora already in the body. However,
bacteriocins have a multitude of benefits. For one, they have very low MICs. MICs are
the lowest concentration of a drug that will visibly inhibit the growth of bacteria
overnight. Bacteriocins perform at the same levels as antibiotics when compared side-byside. There are two reasons why they have lower resistance rates. Firstly, because they
target specific bacteria, they will not be used as often so there is less unnecessary
exposure to the drug. Next, it is very simple to create bacteriocin cocktails, which
consist of similar strains of the same bacteriocins all used to treat infection. They can be
genetically modified very easily and remain sturdy, so it functions the same way but the
bacteria has to mutate two or three times to fully achieve resistance to all three variations,
slowing down the process and making it more cumbersome. So far, there have been no
immune reactions to bacteriocins, and they are abundant in nature. After all, bacteria
make the bacteriocins themselves so they would know the best way to eliminate
competitors.
Application to Research: Margaret Riley also wrote a more in-depth article about the
benefits of bacteriocins, so I will read that. Also from the same conference there are many
other sources about bacteriocins and recent advances. It was held in 2012 so the
information is still recent.
BioWorld News & Views. (2014, June 27). Avidbiotics taps nature to tackle the toughest
infectious diseases [Video file]. Retrieved from https://www.youtube.com/watch?
v=8px8hgsO5AY

Summary: This is a video clip of an interview with the CEO of Avidbiotics, a company
that focuses on bacterial diseases and trying to influence the management of bacterial
infection both by treatment and prevention, though they focus more on the prevention
aspect because that requires less money and time spent in going through the long drug
development process. The company utilizes R-type bacteriocins, nanomachines that
bacteria can produce when stressed. It attaches to the cell envelope of another cell and
pokes a hole in it, killing the bacteria. They have retargeted these proteins to attack
pathogenic bacteria rather than the species that they would naturally kill. Avidocins are
for human use and puricins are used in everything else such as food safety and for
animals. In particular, the company has developed a bacteriocin that kills only C. difficile,
the number one hospital based infection. They have already tested this on mice and are in
preclinical trials. Normally E. coli are a minor part of the gut, where they cause no issue,
but in the urinary tract they are harmful. By killing the bacteria in the gut and in other
places with the use of a bacteriocin, it is possible to prevent a UTI by eliminating it in its
reservoirs.
Application to Research: Before conducting my interview, I had only heard of the
Pheromonicin institute and assumed that it was the only company of its kind out there.
Here, this company also focuses on their use in areas other than for humans, and even in
prevention of future disease rather than treatment, which I had previously not considered.
Briers, Y., Walmagh, M., & Lavigne, R. (2014). Engineered endolysin-based artilysins
to combat multidrug-resistant gram-negative pathogens. mBio. Retrieved from
http://mbio.asm.org/content/5/4/e01379-14.long
Summary: This journal article is about artilysins, endolysins fused with a protein that
allows them to get past the lipopolysaccharide layer around the peptidoglycan cell
membrane. It essentially works against gram-negative bacteria. Time-lapse microscopy
revealed that the endolysin hybrid passed through the outer membrane to degrade the
peptidoglycan layer and cause cell lysis. Since the first discovery of lysins for use as
antimicrobials, gram-negative bacteria has been largely ignored. Even though the fusion
of the lysin with a protein took away some of its capabilities to work as an enzyme, it still
effectively killed bacteria. The proteins are biodegradable, so they will not last very long,
decreasing the chances for resistance developing in bacteria. On the other hand,
antibiotics remain in the environment for longer periods of time, increasing the chances
that a bacteria develop resistance and pass it on. Other methods of combining lysins to
fight gram-negative bacteria are not as efficient as this one. For example, depending on
what membrane protein the antimicrobial binds to, there will be differences in the speed
resistance can develop.
Application to research: Many of the sources cited in the study could be useful to gain
more information. In addition, I could reach out to the authors of the article to see if they
would be willing to be part of an interview as I begin my data collection process in third
quarter. As far as I know, Im not aware as to how these hybrid lysins compare to
bacteriocins in terms of killing gram-negative bacteria.

Brumfitt, W., Salton, M., & Hamilton-Miller, J. (2002). Nisin, alone and combined with
peptidoglycan-modulating antibiotics: Activity against methicillin-resistant
Staphylococcus aureus and vancomycin-resistant enterococci. Journal of Antimicrobial
Chemotherapy. Retrieved from http://jac.oxfordjournals.org/content/50/5/731.full.pdf
Summary: This is a journal article. Nisin is a bacteriocin produced by a bacteria, but it
varies from others of its type in that it has a broad spectrum of antibiotic activity.
Considered a lantibiotic, it can both form pores in the cell membrane of a bacterium and
inhibit the growth of the peptidoglycan layer. Although it proves effective against some
gram-positive bacteria, some bacterial species have already developed resistance. When
tested with various antibiotics, nisin either antagonized it or participated in a two way
synergy with it to kill MRSA and VRE, two different bacterial species. Nisin acts very
quickly in comparison to the antibiotics it works with. This is not an example of an en
vivo experiment because everything was limited to only the two variables being tested.
Therefore, it is possible that this is not applicable in the human body, only in these trials.
Scientists are unsure whether nisin causes lysis of the cell membrane or stops lipid II,
meaning two different pathways for the lantibiotic, but the second reason appears more
plausible.
Application to Research: This is the first I have heard of lantibiotics and their possible
use in medicine, not just in the food industry. I found this article from one of the sources
mentioned in the previous one. I am not quite sure what the difference is between this and
bacteriocins in general. This article was written in 2002, so a lot of the questions that
arose in this experiment have been answered since. However, it is a good example of how
far research has come in just over 10 years.
Campbell, N. A., & Reece, J. B. (2002). Biology (6th ed.). San Francisco, CA: Benjamin
Cummings
Summary: This is an excerpt from a biology textbook. Antibiotic resistance develops in
previously susceptible bacteria through plasmids, double stranded rings of DNA. They
are separate from bacterial DNA. Scientists have noticed that in drug-resistant bacteria R
plasmids are in abundance, which stand for Resistance plasmids. They can replicate and
pass on their genetic information to other bacteria through conjugation. Some even carry
multiple resistance genes, using transposons. Transposons are fragments of DNA that can
move from one place in the genome to another. Sometimes they will jump from place
to place or they will replicate and then move. The simplest transposons are insertion
sequences, which only contain the DNA absolutely necessary to make it a transposon.
The actual gene codes for transposase, an enzyme that moves the gene to a different
location. On either end of the gene are inverted repeats. They contain the same pairs of
nucleotides but are inverted on the other side. Transposase uses them as boundary lines to
make sure other DNA is not removed. These insertion sequences will cause mutation if
they land in the coding sequence of a gene, but they do not do much benefit or harm to
the cell. Composite transposons are what cause expression of antibiotic resistance genes.
It only means that additional genes are added with the one coding for transposase

between the inverted repeats. The amount of these genes only grow as cells divide or
conjugation occurs.
Application to Research: I found this intro to antibiotic resistance and how exactly it
occurs was helpful to me. I looked through the index and marked all of the pages that
may be helpful to my research, so I will read those as well.
Cavera, V. L., Arthur, T. D., Kashtanov, D., & Chikindas, M. L. (2015). Bacteriocins and
their position in the next wave of conventional antibiotics [PDF]. International Journal
of Antimicrobial Agents.
Summary: This journal article reviews the capabilities of bacteriocins for use as
antibiotics. They are convenient because they can be manufactured synthetically with a
simple process, so they would be easy to produce. Traditional antibiotics utilize five
different types of pathways to destroy a bacterium, and bacteriocins four of the five, as
well as some other ones not applicable to antibiotics, such as bacteriocins that cause holes
or pores in the cell membrane. Scientists have manipulated bacteriocins to make them
more stable and efficient in various cases, especially concerning nisin because it has
already been approved by the FDA. Scientists have also created hybrids of bacteriocins
that had lower MICs (smaller amount necessary to halt the growth of an organism) than
the original ones. Using antibiotics in synergy with bacteriocins has also been considered
when doctors face an insufficient infection therapy. In some cases, this proved to be true.
In others, they antagonized each other and were unable to kill the bacteria. Scientists
tested nisin with antibiotics to fight Salmonella with positive results. However, resistance
can develop against bacteriocins just as they did against antibiotics. The method for
getting them into the body to fight pathogenic bacteria is also an issue because there are
difficulties to them being absorbed orally.
Application to Research: This article provided the idea that possibly bacteriocins and
antibiotics could work together to fight disease. I could also look at the studies mentioned
as evidence. The authors of the article were ones that I had not heard of before so I will
email them.
Cunningham, A. (2006, Sept 9). Genes as pollutants: Tracking drug-resistant DNA in the
environment. Science News, 170.11, 163. Retrieved from http://goo.gl/eTifrd
Summary: This article studies the way antibiotic-resistance genes move and exist in the
world. They are almost omnipresent, even existing in treated water. Scientists consider
them pollutants in the environment and are looking for ways to stop their spread. Farm
animals especially are given antibiotics in excess even when they are not necessary,
speeding up the evolution of bacteria to defend themselves. Then they spread throughout
various animals and eventually humans. Also, antibiotics from farm animals can end up
in the soil and water, giving the bacteria living there the chance to evolve. Researchers
studied how the resistant genes traveled through an environment by taking samples from
both rural and urban areas, which were then tested for four known antibiotic resistant
genes. The results concluded that the DNA travels through waterways as its main form of

transportation. The largest concentration of resistant genes were present in lagoons near
farms, and then they traveled through waterways to more densely populated areas. Two of
the four genes were even present in treated drinking water and wastewater, showing the
insufficiency of current water treatment plants.
Application to Research: Solutions to the issue of antibiotic resistance do not always rely
in a new medicine. Other devices can be used to slow the domination of drug-resistant
bacteria. There are many benefits to this. Firstly, there is not as much of an issue of
bacteria developing a resistance to these mostly physical blocks placed on them. Also,
this option is less costly, because not as much money has to be spent laboriously
experimenting with different drugs to make sure they are safe.
Das, M. C., Paul, S., Gupta, P., Tribedi, P., Sarkar, S., Manna, D. and Bhattacharjee, S. (2016), 3Amino-4-Aminoximidofurazan derivatives: small molecules possessing antimicrobial and
antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa. J Appl
Microbiol. Accepted Author Manuscript. doi:10.1111/jam.13063
Dr. Vince Fischetti - bacterial biofilms & phage lytic enzymes [Video file]. (2010, April 4).
Retrieved from https://www.youtube.com/watch?v=lUcMGktSc7c
Summary: This video clip is an interview with Professor Vincent Fischetti at Rockefeller
University. He discusses biofilms and possible treatments using phage lysins. Biofilms
are a colony containing only one species or multiple species. The secrete a
polysaccharide slime so they form an intricate set of pathways and communication. They
are very harmful in the human body and are not easily destroyed by antibiotics because
they do not grow very fast. Many antibiotics prohibit the growth of the cell membrane
along with the cell, causing the internal pressure to become too much and the cell will
explode. However, they are useless against biofilms. However, a phage lysin is an
enzyme that chews through the cell membrane. Because it is not an organism, there is no
way the bacteria can develop resistance. to it. In order to get through the cell membrane,
the lysin binds to a polysaccharide capsule on the cell membrane of gram-positive
bacteria. These capsules are made up of mostly water so it is easy for the enzyme to
diffuse through it and get to the peptidoglycan, cell wall layer. From there it will make a
hole in the membrane and cause the inner bits of the cell to leak out, causing cell death.
Fischetti also mentions the importance of vaccines in the medical world. There is a
definite need for fresh vaccines every year, because they are more economically
beneficial (families do not have to pay lots of money for treatment) and they also will
prevent unnecessary heartbreak of families.
Application to Research: I wrote to this professor, but this is the first time I have seen an
interview of him. The video raised some questions about why lysins do this or that in
comparison to bacteriocins, but I am not sure if I should ask these to professionals since
there are definite answers hidden somewhere.

Fischetti, V. A., Nelson, D., & Schuch, R. (2006). Reinventing phage therapy: Are the parts
greater than the sum? [PDF]. Nature Biotechnology. Retrieved from
http://www.nature.com/nbt/journal/v24/n12/full/nbt1206-1508.html
Summary: This journal article looks at bacteriophages and their antibacterial properties.
One downside to phages however is because they are so common, it would be difficult to
procure a patent and profit from them, which is the goal of pharmaceuticals. Lysisdeficient phages can infect a cell as well as replicate within it, killing the bacterium but
they are unable to get out of the cell. Macrophages create a hole in the cell membrane so
the phages can escape. Phages have also been considered for use in delivering fatal genes
to bacteria rather than them actually doing the killing. Possible uses for vaccination with
phages has been discussed as well. There is no need for adjuvants along with the antigen
to be delivered because phages themselves trigger an immune response. Cell wall
hydrolases are called lysins. They are enzymes used towards the end of the infection
cycle after the phage has multiplied within the bacterium. The chew through the
peptidoglycan layer on the inside the cell. They will also react the same way when
binding to the outer peptidoglycan. However, some bacteria have an extra outer layer.
Lysins are ineffectual against those gram-negative bacteria. They bond tightly to the
membrane, attaching to carbohydrates which cannot be structurally changed very easily,
preventing bacterial resistance. Using carbohydrates as binding sites was probably done
to make sure the phages can get out easily. Because they bind so tightly, they have a onetime use.
Application to Research: There are three authors of this article, two of which I have sent
out my business letter. Perhaps now I can look into the other scientist for more
information.
Grush, L. (2014, September 24). Editing the genes of superbugs to turn off antibiotic resistance.
Retrieved September 10, 2015, from Popular Science website:
http://www.popsci.com/article/science/editing-genes-superbugs-turn-antibiotic-resistance
Summary: This is an online article. As drug resistant bacteria becomes more of a
concern, so has finding a cure for them. Researchers at MIT have discovered a protein in
CRISPR, a set of proteins that make up the bacterias immune system. Called Cas9, it
acts as a scissor, cutting specific sequences of DNA. By modifying the RNA address, the
researchers can make the protein cut the genes that make the bacteria able to cause
disease in the first place, rendering it harmless. In order to get it into the superbugs in the
first place, the researchers are considering bacteriophages, essentially the viruses of
bacteria. They will transfer their DNA into their host, including the modified CRISPR.
Another option is to genetically engineer bacteria with this modification to pass this gene
on to other bacteria, making them traitors. However, this specific method takes time.
Depending on how Cas9 is altered, CRISPR will either remove genes that make the
bacteria resistant to antibiotics, boosting the medicines effectiveness, or it will cut the
genes that make it harmful. Clinical trials have proven this solution to be overwhelmingly
successful.

Application to Research: My question concerns cures for drug resistant bacteria, and this
article provides an overview of one possible solution in detail. From here, I can look
more into this method to see if it is the most suitable alternative to antibiotics. There were
also other links to help clarify certain words or concepts mentioned which led to other
sources of new information.
James, R. (2015, October 12). Bacteriocin research [E-mail to the author].
Summary: At a conference devoted to bacteriocins and their use against drug-resistant
bacteria. Margaret Riley presented an article about bacteriocins through a webcast. In
addition to growing antibiotic resistance, broad spectrum antibiotics are generally not a
good option for defeating bacteria because they are not found commonly in nature and
target wide ranges of bacteria, wiping out the healthy flora already in the body. However,
bacteriocins have a multitude of benefits. For one, they have very low MICs. MICs are
the lowest concentration of a drug that will visibly inhibit the growth of bacteria
overnight. Bacteriocins perform at the same levels as antibiotics when compared side-byside. There are two reasons why they have lower resistance rates. Firstly, because they
target specific bacteria, they will not be used as often so there is less unnecessary
exposure to the drug. Next, it is very simple to create bacteriocin cocktails, which
consist of similar strains of the same bacteriocins all used to treat infection. They can be
genetically modified very easily and remain sturdy, so it functions the same way but the
bacteria has to mutate two or three times to fully achieve resistance to all three variations,
slowing down the process and making it more cumbersome. So far, there have been no
immune reactions to bacteriocins, and they are abundant in nature. After all, bacteria
make the bacteriocins themselves so they would know the best way to eliminate
competitors.
Application to Research: Margaret Riley also wrote a more in-depth article about the
benefits of bacteriocins, so I will read that. Also from the same conference there are many
other sources about bacteriocins and recent advances. It was held in 2012 so the
information is still recent.
Joerger, J. D. (2003). Alternatives to antibiotics: Bacteriocins, antimicrobial peptides and
bacteriophages. Poultry Science. Retrieved from
http://ps.oxfordjournals.org/content/82/4/640.full.pdf
Summary: This is an article from Poultry Science magazine. Research about antibiotics
has been conducted concerning their effects on poultry and other livestock, and generally
they will promote their growth, so today, they are routinely fed to animals all over the
country. However, with the threat of limitations on drug use by the FDA are causing
livestock producers to look elsewhere for antibiotic alternatives. Bacteriocins are
considered more natural as well since bacteria produce them and they are in some edible
foods. Adding the bacteria that produce bacteriocins into animal feed may be more cost
effective, but it is impossible to know for sure exactly how much of the protein will be
created and used. Resistance to this antibiotic alternative is in the form of a change to the
cell membrane because a bacteriocin has a certain anchor that it will latch onto, so if that

is changed on the cell, the protein can no longer attach to the membrane. Sometimes
bacteriocin-resistant bacterial strains will also be resistant to traditional antibiotics as
well. Antimicrobial peptides (proteins that fight microbes) exist in many other organisms
as well, not just bacteria. It is very difficult for resistance to develop against these
peptides, though not much research has been conducted.
Application to Research: In addition to disease in humans, antibiotics are used in
livestock as well, which again relates to many because most people consume meats from
a food industry that uses antibiotics. However, told from the industrys perspective, they
do not see the overuse of antibiotics as anything bad and are only looking for alternatives
to get around rumored bans on antibiotics.
Lukacik, P., Barnard, T. J., & Buchanan, S. K. (2012). Using a bacteriocin structure to engineer a
phage lysin that targets Yersinia pestis. Biochemical Society Transactions, 40(6), 15031506. Retrieved from http://www.biochemsoctrans.org/content/40/6/1503
Summary: In this online journal article, the three researchers created a hybrid of lysins
and bacteriocins because there are advantages and disadvantages to both. The researchers
used the bacteria Yersinia pestis for their experiments. Lysins are very efficient in
destroying the peptidoglycan cell membrane but are useless against gram-negative
bacteria. Bacteriocins (Pesticin), on the other hand, are created by bacteria to attack other
strains of their own kind. Pesticin targets a transporter protein on the outer membrane
(FyuA, which transports iron) and uses it to hitch a ride to the peptidoglycan layer, in
which it pokes holes. However, certain strains (mainly the ones that create the
bacteriocins) have the cure that provides immunity to the bacteriocin (Pim), This is
why lysins, unaffected by this immunity protein, are necessary. The researchers created a
bacterial-phage hybrid to make a phage lysin that targets gram-negative bacteria. The
hybrid lysin was then tested next to Pesticin in clinical trials with various strains of
Yersinia. If the strain expressed the protein FyuA without Pim, then both proteins were
able to kill the bacteria. However, if it expressed FyuA and Pim, then only the hybrid
lysin was able to get through. Pesticin poked smaller holes in the membrane, letting it
leak out slowly, but the hybrid lysin chewed a big hole in the peptidoglycan membrane,
causing a faster cell death. However, the Pesticin was more effective even with a slower
cell death, most likely because it was able to move through the outermost layer faster
than the hybrid lysin.
Application to Research: Even on top of my two contending solutions to antibiotic
resistance, there is a third option, which is a hybrid between the two. This potentially
takes out the disadvantages of the other two alternatives, but as mentioned, there are other
cons to this method.
Majeed, H., Lampert, A., Ghazaryan, L., & Gillor, O. (n.d.). The weak shall inherit:
Bacteriocin-mediated interactions in bacterial populations. PLoS ONE. Retrieved from
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063837

Summary: This peer-reviewed journal article explains why weaker bacteriocins are
better at killing bacteria rather than more powerful ones, even though it seems unlikely.
In lethal doses, strong bacteriocins are able to effectively remove other bacteria.
However, in sublethal doses, the bacteriocin is a trigger for a strong immune reaction,
causing the bacteria to send out bacteriocins of its own. Through analysis, researchers
have concluded that weaker bacteriocins are more effective in killing bacteria, leading to
possible implementations when developing new drugs, especially because weaker drugs
will have less of a negative impact on the body. This could explain the abundance of
weaker bacteriocins in the world. Colicins are bacteriocins secreted by the various strains
of E. coli, and they all work differently, either by targeting the cell membrane or degrade
the DNA/RNA. The bacteria that produce them are safe because the gene is tightly linked
to a resistance gene. However, the production of a colicin requires the cell to die through
lysis and the resulting cytoplasmic depletion. Therefore, very few bacteria go through this
process, only if an SOS response is triggered by DNA blockage. This high-cost process
works well in the environment where it would be very complicated if there were many
bacteriocins going around killing cells.
Application to Research: I was looking for more information on bacteriocins and so far it
has provided valuable background knowledge on how they work. This source has an
extensive bibliography and Ive already found another source from it.
Majeed, H., Gillor, O., Kerr, B., & Riley, M. (n.d.). Competitive interactions in
Escherichia coli populations: The role of bacteriocins. Multidisciplinary Journal of
Microbial Ecology. Retrieved from
http://www.nature.com/ismej/journal/v5/n1/full/ismej201090a.html
Summary: Bacteriocins have an effect on bacterial populations in numerous ways as
stated in this journal article. For one, there are immune responses in almost all
prokaryotes, generally concerning the creation of its own bacteriocin or something of that
nature. The producing bacteria will always be immune to its bacteriocin, though it suffers
a large cost (it dies during bacteriocin production). In addition, there are some bacteria
without any kind of protection from the pathogen. There is a third species that is immune
to the bacteriocin but does not actually produce one itself. The researchers assume that
there is also a cost to this immunity, though less than that of the bacteriocin producing
bacteria. Therefore, a rock-paper-scissors situation occurs, with the sensitive strain
beating the immune one because it does not have a cost to immunity, the producing strain
beating the sensitive one because the bacteriocin kills it, and the immune strain beating
the producing one because it is successfully able to repel the pathogen. Originally
scientists believed toxin production was used to compete against other strains of bacteria
in low nutrient environments, but the opposite is true. They work better in high nutrient
environments, discrediting this theory. Bacteriocins are better in stopping the invasion of
a new species into the environment. In fact, when an unfamiliar strain appears in the
niche, there is enhanced production of bacteriocins from whatever producers happen to
be there already to get rid of it.

Application to Research: Two of the authors from the last article collaborated on this one
as well. I remember sending a business letter to one but not the other, so I will do that. I
still need a complete overview of bacteriocins so I have enough information when
interviewing professionals.
Nelson, D. (2015, December 23). [Telephone interview by the author]
Summary: This was a telephone interview done by the author. Daniel Nelson conducts
research on phage lysins in his lab. Phage lysins are foreign proteins, and when injected
into the bloodstream/used systemically they could cause an immune response from the
body, and they have. When lysins were injected into rabbits and then studied, scientists
found that antibodies attached to the lysin but did not neutralize it, so the enzyme was
able to continue working. Also, it takes quite long for the body to develop antibodies and
neutralize the foreign proteins while the lysins are able to kill the target bacteria in a
matter of seconds when they contact. However, because of this possible toxicity issue,
only external uses for lysins are plausible as of this moment. This means treatments like
nose sprays, creams, etc. Phage lysins only work when in direct contact with the
peptidoglycan layer, so they are ineffective against gram negative bacteria. Some ways to
get around this include artilysins, that are a lysin fused to a peptide able to punch through
the outer membrane, and when reaching the cell membrane the lysin can finish lysing the
cell. Additionally, pharmaceutical companies do not like lysins very much because they
are very species specific. Therefore, the company has to go through the very expensive
drug development every single time it wants a new antibiotic for a different species of
bacteria. In the meantime, there are a number of other applications of phage lysins in
disinfecting materials (especially in hospitals) and for animals.
Application to Research: Before this, I had considered bacteriocins more superior to
phage lysins, but now I think that lysins do have some advantages as well. The interview
was incredibly helpful for me and I have already used it a lot in formulating my outline.
OMahony, J., Fenton, M., Henry, M., Sleator, R. D., & Coffey, A. (2011). Lysins to kill
[PDF]. Bioengineered Bugs, 2(6). Retrieved from
http://www.tandfonline.com/doi/abs/10.4161/bbug.2.6.16804#aHR0cDovL3d3dy50YW5
kZm9ubGluZS5jb20vZG9pL3BkZi8xMC40MTYxL2JidWcuMi42LjE2ODA0QEBAMA
==
Summary; This article details the history of bacteriophages and their use for antibacterial
purposes. A British scientist first noticed in 1896 that certain filtrates in two Indian rivers
were able to halt cholera. Then, in 1915, Felix DHerelle discovered phages in the feces
of locusts and humans. Scientists took interest and bacteriophage therapy became a hot
topic in the western world until the discovery of penicillin and antibiotics. However, in
the country of Georgia, research has continued to this day. With the rise of antibiotic
resistant bacteria, many other countries have turned to the research institute in Georgia
for alternatives using bacteriophages which have been proven to work. Companies began
to start trying to manufacture and cell phages. The FDA gave approval to sell a food
additive to present bacterial contamination. Phages can be used to treat disease generally

in two different ways. There can be a cocktail of similar phages to treat infection or to
use the lysin from inside the bacteriophage. This would be implemented by cloning the
gene or protein that relates to the lysin, so gene technology could be applied to this. It
would be relatively easy to tweak the gene to better fight bacteria/resistance.
Application to Research: I actually think I will select bacteriocins for the better
alternative to antibiotics, mostly because they are not hindered by gram-negative bacteria.
However, this overview of the history of bacteriophages is useful in highlighting the
various pros and cons of them shown throughout history.
Phage lysins. (n.d.). Retrieved from The Rockefeller University website:
http://www.rockefeller.edu/vaf/phage.php
Summary: This online article looks more deeply into lysins, discussed in the last article
towards the end. On the Rockefeller University website there is a whole page dedicated
to lysin research. The enzymes are actually used when the phage has finished replicating
itself inside the bacteria and needs a way out. The phages use lysins, refined over millions
of years, to force the bacterial cell wall to unravel. In the peptidoglycan, a mesh-like layer
on the outer cell, the lysin targets one its five major bonds. Bacteriophages have been
used for much of the 20th century, but only recently have the lysins been extracted and
tested to use as a drug against bacterial infections. However, they only work on grampositive bacteria because gram-negative bacteria have an extra outer membrane that
prevents the lysin from penetrating the cell. The university has already developed lysins
that target a large range of bacteria that are gram-positive.
Application to Research: Vincent Fischetti is a PhD in biology and is a professional in the
field of lysin research for antibacterial drug use. I found his email on the website, so I
could interview him as I get more immersed in my research. Even though some of the
terminology is a bit over my head, the article provides a clear explanation of what a lysin
is and what it does. There are also links to publications by Fischetti that I can use for
more in depth research. This is definitely a possible solution to drug resistant bacteria.
Pheromonicins, an ecologically sound family of bacteriocin-based antibiotics for use in
the age of the microbiome [PDF]. (n.d.). Future Microbiology.
Summary: This article is about a company in Beijing. Pheromonicins are antibiotics
based off of bacteriocins. They utilize colicin Ia, a bacteriocin of E. Coli. Colicin Ia uses
biophysical means to create a channel in the cell membrane through which the
cytoplasmic material inside the cell can leak out depending on whether the
transmembrane potential is reached. Using pheromones, this colicin can be applied to
virtually any bacteria and cause cell death. The modified colicin PMC-SA for use against
methicillin-resistant S. aureus was tested in mice. All of the mice injected with the colicin
survived without any lasting damage while 76% of the ones injected with oxacillin, the
antibiotic used instead of methicillin, died in the six-day experiment. Similar experiments
have also been carried out with other pheromonicins. However, even though speciesspecific drugs are better than broad spectrum, this is not very plausible until disease-

detecting methods become more precise. Therefore, scientists have developed


pheromonicins to target a wide range of bacterial pathogens by linking an antibody to a
colicin. The antibody binds to many gram-negative bacteria such as E. Coli. However,
they do not affect a number of other types of bacteria so they will be less likely to
decimate the gut flora already in the body. Pheromonicins also have potential for use in
fungi, seeing that they as well produce pheromones, though less species-specific.
Therefore, they can be used to prevent and treat infection in crops.
Application to Research: Rather than hybrid lysins that dont have much evidence for
them, I think that bacteriocins seem the more plausible for use against bacterial pathogens
resistant to traditional antibiotics. Here there is actual evidence of the benefits of
bacteriocins.
Summary: The article also talks about how pheromonicins also have potential for use in
the treatment of cancer. Therefore, there are many possible uses for this new platform of
drugs because the only thing that needs to be done to change the target of the drug is to
just attach a different pheromone. They have also been shown for having potential for
cancer treatment. However, pheromonicins are still susceptible to antibiotic resistance
because all bacteria evolve and adapt to their surroundings based on what will help them
survive the most. However, there are ways to slow this process down significantly. For
one, cocktails of slightly different pheromonicins can be deployed in the body at once,
both destroying the same pathogenic bacterial species but by attaching to a different
surface antigen. This means that the cell must mutate multiple times in order for it to fully
develop resistance, slowing the process down significantly. However, this use of a colicin
with channel-forming tendencies will probably cause slower resistance rates because it is
a biophysical occurrence, not a biochemical one. In order to promote the discovery and
use for different antibiotics, some measures need to be taken by the government to
incentivize this process along with compliance from regulatory organizations such as the
FDA in the US. This is necessary because pheromonicins are a break from the
conventional antibiotic discovery process seeing that it uses bacteriocins rather than the
screening of many compounds.
Application to Research: This section of the file details the plausibility of pheromonicins
for use in the real world. Although they are depicted as overwhelmingly positive, the fact
that trials have been conducted on this new drug is very promising. If I could possibly
find some of these experiments or use the works cited page of this document I can find
other sources.
Qiu, X.-Q., & Wu, G. Y. (2009). A novel strategy to target lethal peptides against
antibiotic resistant bacteria. Discovery Medicine. Retrieved from
http://www.discoverymedicine.com/Xiao-qing-Qiu/2009/06/18/a-novel-strategy-totarget-lethal-peptides-against-antibiotic-resistant-bacteria/
Summary: This journal article is about new antibiotics that are actually variations of the
bacteriocin colicin Ia from E. coli that has channel forming properties. In order to make
the peptide narrow spectrum, the author utilized pheromones, bacterial peptides made and

bound by membrane receptors that are specific to a species or strain. Therefore, the
colicin could be adjusted by using a pheromone to attack almost any type of bacteria. The
bacteria Streptococcus pneumoniae was used for the experiment, with various strains
resistant to certain antibiotics tested against fusion peptides. They were successful in
stopping the growth of bacteria and when further tested showed that they did not just
inhibit growth. They managed to exterminate the bacteria as well. However, these trials
with very controlled variables are considered artificial and there is no sure evidence that
this will be the case in the body, or in vivo. Therefore, the researchers conducted
experiments with mice. All of the mice injected with the pheromonicin survived the six
day experimental period. It was injected directly into the bloodstream and was able to
enter tissues to cure the mice of lethal infection, showing that bacteriocins do work in
vivo. Another test on the toxicity of these pheromonicins involved their incubation with
human liver and kidney cells. There were no differences in growth in the tissues.
Additionally, organs from animals treated with the pheromonicin (PMC) showed no
inflammation or damage to them.
Application to Research: I felt like I hit a dead end in sources just before I found this
article, but now I have plenty more to look at. This is exactly the study I was looking for,
something that observes pheromonicins, the closest we have to a drug for treating
antibiotic-resistant disease.
Rayner-Nottingham, E. (2012, June 8). E.Colis weapon in bug-on-bug warfare. Retrieved from
Futurity website: http://www.futurity.org/e-colis-weapon-in-bug-on-bug-warfare/
Summary: This article is from Futurity, which summarized a larger, more in-depth study
from the University of Nottingham featured on the Journal of Biochemistry. Various
researchers from the university studied the bacteria E. Coli. In particular, a bacteriocin
called colicin A. Bacteriocins are proteins produced by bacteria (in this case E. Coli) to
eliminate other similar bacterial strains to reduce competition. If these proteins were
directed to destroy TolA, a membrane protein on E. Coli, then the colicin could attack the
very bacteria that created it. This has obvious antibacterial uses, and could possibly be
developed into a drug as an alternative to antibiotics. Also, these bacteriocins can attack
any bacteria, regardless of whether it is gram-positive or negative. They have also been
considered to use for cancer treatments as well as food security. Because colicin A latches
onto TolA, scientists can create various substances and compounds to attach to it, which
will block the functions of the protein on the cell membrane.
Application to Research: Bacteriocins are another possible solution to antibiotic
resistance, as well as lysins. Colicin has another large advantage over lysins: it does not
matter whether the bacteria is gram-positive or negative. Lysins will only work with
gram-positive bacteria. The next step from here is to find out more about this new
antibiotic alternative, as well as the various positives and negatives to it in comparison to
lysins.

Riley leads campus group at Beijing drug conference. (2015, November 4). Retrieved
from University of Massachusetts Amherst website:
http://www.umass.edu/newsoffice/article/riley-leads-umass-amherst-group-beijing
Summary: This is a news article from the University of Massachusetts website.There
was a conference in Beijing from October 19-21 in 2015 to discuss bacteriocin research.
30 scientists were from China and 30 scientists were international. Margaret Riley was
the leader of this group. They met to share the science behind pheromonicins, a new
family of drugs based on bacteriocins. Riley organized the conference with the creator of
pheromonicins, Xiao-Qing Qiu and met with the deputy mayor in Beijing whose office
donated $200 million to support a pheromonicin institute in Beijing. Riley plans to craft a
sister institute back in Amherst as well as to hold another conference for next year. Riley
discussed the use of pheromonicins as antibiotics against tuberculosis that is resistant
against multiple drugs as well as other applications, such as treating infection in bats.
There is a lack of funding in the United States, which is why Riley is collaborating with
Qiu to develop new drugs that fight resistance in bacteria that are very quick to develop
resistance. Targeted bacteriocins are also better to use instead of broad-spectrum
antibiotics that will also wipe out the beneficial bacteria in the body.
Application to Research: Of course there has been research into phage lysins and
bacteriocins, but this is the first step I have seen to actively try and implement them into
daily life for human use. Pheromonicins seem promising and I will make sure to keep an
eye out for any other information regarding this. I also read a few names in the article
such as Alex Gerson, Rolf Karlstrom, and Larry Schwartz. I can contact these people for
more information of pheromonicins, since I have not gotten a reply from Dr. Riley.
The rise of superbugs. (n.d.). Retrieved September 7, 2015, from Consumer Reports website:
http://www.consumerreports.org/cro/health/the-rise-of-superbugs/index.htm?
utm_source=dlvr.it&utm_medium=twitter
Summary: This online article is about how incorrect use of antibiotics has lead to the rise
of drug-resistant bacteria dubbed superbugs. In some cases, unnecessary antibiotics wipe
out the beneficial bacteria within the body, allowing more harmful, drug resistant bacteria
to multiply. In others, the overuse of antibiotics has lead to the emergence of bacteria
resistant to them. Sometimes, they will even share their resistant traits with other
organisms. Because they cannot be easily treated, death or lifelong health impacts can
occur when someone encounters this bacteria. Most antibiotics actually go to livestock,
so the food supply could be in danger. Some misuses of antibiotics are when they are
prescribed too early, to viral diseases, or just to satisfy the patient. Broad spectrum
antibiotics, used to eradicate a variety of bacteria, used too often and they both wipe out
helpful bacteria as well as advocate resistance to the drug.
Application to Research: In terms of background information on superbugs, this article
does a good job of clearly explaining how they work and why they have come about.
However, it does not go much deeper. It merely states facts known about the bacteria,

which is both a good and a bad thing. There is no speculation or question about what
could possibly come next, but there is also no authors bias to worry about.
Schofield, C. B. (n.d.). The anarchy of antibiotic resistance: Mechanisms of bacterial resistance.
Medical Laboratory Observer. Retrieved from http://www2.mloonline.com/features/201105/cover-story/the-anarchy-of-antibiotic-resistance-themechanisms-of-bacterial-resistance.aspx
Summary: This peer-reviewed journal article details the rise of antibiotic resistance in
gram-negative bacteria, Using plasmids, independent rings of DNA separate from the
chromosome, bacteria can both create enzymes that digest antibiotics, and they can also
replicate and transport the plasmids to other cells very easily. Issues with resistance began
in the 1970s and has continued to raise public concern. More and more increasingly,
doctors are forced to use stronger carbapenem antibiotics that wipe out all bacteria it
touches, including beneficial bacteria, providing opportunity for opportunistic infection.
In fact, there are even bacteria resistant to carbapenems as well. Sometimes the bacteria is
inherently unbothered by the antibiotic (intrinsic resistance) and other times it develops it
(acquired resistance). There are three modes of acquired resistance: Mutation of cellular
genes, which is a mutation of one nucleotide in the DNA, acquisition of resistance genes,
which involves the separation of genes from the chromosome to create plasmids, and
mutation of acquired genes, which is when DNA is transferred to the bacteria. There are
various ways to prevent the spread of these antibiotic-resistant genes, mostly by limiting
antibiotic use in people as well as plants and animals.
Application to Research: The other article provided information on antibiotics in general,
and this one is about developing antibiotic resistance. Because I have very little
background in biology, I need more knowledge on how antibiotic resistance really works
so possible future resources could include AP biology textbooks and teachers familiar
with the information.
Scholl, D., Cooley, M., Williams, S. R., Gebhart, D., Martin, D., Bates, A., & Mandrell,
R. (2009). An engineered R-type Pyocin is a highly specific and sensitive bactericidal
agent for the food-borne pathogen Escherichia coli O157:H7. Antimicrobial Agents and
Chemotherapy. Retrieved from http://aac.asm.org/content/53/7/3074.long
Summary: This journal article was released from a company that also develops
bacteriocins into drugs called AvidBiotics, so Pherominicins Inc. is not the only company
looking into bacteriocins as antimicrobials. Rather than a colicin, researchers took a
bacteriocin produced by Pseudomonas aeruginosa with a tail-like protein complex
resembling those of bacteriophage and attached a tail spike protein to it from a
bacteriophage specific to a certain strain of E. coli. The resulting hybrid, AVR2-V10,
worked well against the specific strain but not against any other strains tested. Pyocin has
a hollow core, and when inserted into the cell membrane, will cause depolarization of the
cytoplasm and eventually cell death. Producers of the bacteriocin are unaffected by it
because of a gene cluster coding for resistance, but since the cell must lyse to release the
pyocin, this mechanism is meant to protect kin strains rather than protect that specific

bacterium. Theoretically, one bacteriocin can kill a cell. This gives it potential for use as a
food preservative, since antibiotic resistance has developed in the food industry. In
addition, the E. coli killed by AVR2-V10 did not release Shiga toxin to defend itself,
eliminating the issue of toxicity when used in humans. Other antibiotics often cause
Shiga toxin to be released into cows which are unaffected hosts of the bacteria. However,
in contaminated beef given to humans, it will cause the food-borne illness colitis.
Application to Research: There are seven authors to this article. I could email some of
them to see if they wouldnt mind being interviewed to aid my data collection to come in
3rd quarter.
Sixsem. (2012, September 15). ShareBiotech Colin Hill, UCC microbial food safety bacteriocins
[Video file]. Retrieved from https://www.youtube.com/watch?
v=EB2HH9nI4a8&index=3&list=PL1WjRwg9HJKcRXSr0Gd0j9cFlUi5GmmhP
Stark, C. J., Hoopes, J. T., Nelson, D. C., & Bonocora, R. P. (2010). Bacteriophage lytic
enzymes as antimicrobials. In Bacteriophages in the control of food- and waterborne
pathogens. Retrieved from
http://www.asmscience.org/content/book/10.1128/9781555816629
Summary: In this chapter of a book the authors discuss cell wall hydrolase, which is just
a term for a wide range of enzymes that break down peptidoglycan. Some are produced
by bacteria, called autolysins, to repair the cell membrane. In the bacteriophage, the
endolysin and holin work together to pierce the cell membrane from the inside out
towards the end of the infection cycle. The cell membrane in gram-negative bacteria are
relatively uniform, almost all composed of peptidoglycan. Therefore, their lysins only
have one domain/active site to bind to. Gram-positive cell membranes are riddled with a
variety of proteins and carbohydrates. Their lysins may have multiple domains. Because
of this, they are very specific to a certain species. Professor Vincent Fischetti and his
group were the first to consider using lysins to treat illness, coining it enzybiotics. In
their experiments, mice were injected with bacteria and four days later given doses of a
lysin. Within two hours, all of the bacteria were gone from the swabs. Two days later,
only one mouse still had the bacteria in its body. This is most likely because the dosage
was not enough or was not applied enough to completely wipe out the illness. Other
experiments did the same thing on a larger scale. About 40% of the mice treated with the
lysin survived in comparison to the 100% death rate when untreated. This is not the best
statistic, but lysin development is still in its early stages.
Application to Research: This chapter is actually 50 some pages, so I can use some of the
other information for future research. I can also contact the other authors who wrote this
chapter for more human sources.
Svoboda, E. (2009, March 31). The Next Phage. Retrieved from Popular Science website:
http://www.popsci.com/scitech/article/2009-03/next-phage

Summary: This is an online article. Even though it was written in 2009 it gives insight
into earlier solutions people found to drug resistant bacteria, which have been an issue
since around the start of the 21st century. One doctor found that bacteriophages can be
used to fight superbugs, because they use them as hosts, similar to how viruses act
towards human cells. It would be difficult for the bacteria to evolve resistance against
them because the phages themselves are living and therefore can change as well.
However, that property makes it difficult for them to get approval from U.S. regulatory
authorities. Therefore, it is not the most realistic solution to superbugs. Rockefeller
University biologist Vincent Fischetti suggests that instead of using the whole
bacteriophage to kill bacteria, extracting the lysin, an enzyme in the phage that dissolves
bacterial cell walls, is a much better option. However, this comes with its own
drawbacks. Some bacteria like E. Coli have outer membranes that prevent the lysin from
penetrating the cell.
Application to Research: Even though bacteriophages are not a very viable solution to the
rise of drug resistant bacteria, more research can be conducted from there. Because lysins
can be formulated into a more realistic antibacterial drug, studies must have been
conducted on them and their various properties.
Walker-Monash, E. (2012, July 24). To kill infection, try warhead virus. Retrieved from Futurity
website: http://www.futurity.org/to-kill-infection-try-%E2%80%98warhead
%E2%80%99-virus/
Summary: This online article is a summary of another, larger study conducted
concerning lysins. The research of bacteriophages against bacterial infection has started
in the beginning of the twentieth century, but was largely forgotten as antibiotics were
discovered. The bacteriophage lysin PlyC will attack pneumonia, streptococcal toxic
shock syndrome, and the bacteria responsible for sore throats. Scientists have studied the
structure of PlyC for six years to see why and how it fights bacteria and have found its
structure is similar to a flying saucer, made up of 9 protein segments. The lysin holds
itself to the bacterial cell wall with eight points of contact, while two warheads pierce
through it to kill the cell. The information gathered in this study will pave the way to
creating new antibacterial drugs instead of antibiotics. As for its history, PlyC was
recognized in the 1920s when phage therapy was a topic of interest in the scientific
community, but until now its structure was not known. This lysin is the most effective at
eliminating gram-positive bacteria compared to any others that have been discovered so
far.
Application to Research: The article I found was a simpler version of a full study of lysin
structure and function. However, although the full study has much more information, it is
also longer and much harder to understand. I will work through the full study more
slowly, and through it I can gain background knowledge on lysins that I can use when
reading other studies.
What is an antibiotic? (n.d.). Retrieved from
http://learn.genetics.utah.edu/content/microbiome/antibiotics/

Summary: This article gives a basic overview of how antibiotics work. There are actually
different types of antibiotics, grouped based on how they kill bacteria. Beta-Lactam
antibiotics target bacterial cell walls by prohibiting the new creation of peptidoglycan,
which is what it is primarily made up of. Because the cell keeps getting larger and its cell
wall does not, the pressure will eventually make it burst open. Penicillin is a common
example in this family. Macrolides stop protein synthesis by disturbing the ribosomes that
connect the amino acids together to form the protein. Because all cells are hugely
dependent on proteins to function, without new ones, they cannot survive. Quinolones
such as ciprofloxacin and levofloxacin (used to treat bronchitis and pneumonia) damage
the DNA by causing breaks and then preventing them from mending, so when it comes
time to replicate, they are unable to. Also, without intact DNA, the cell will die. Even
though antibiotics sound very beneficial, they also kill the good bacteria already living
in the human body.
Application to Research: I realized that in my hurry to find antibiotic alternatives, I never
stopped and considered familiarizing myself with antibiotics in general and how they kill
bacteria. Now I need to look at drug-resistant bacteria and predict if the same thing will
happen to the other bacterial cures I have found. Also, there have been recent findings of
new antibiotics that can supposedly destroy drug-resistant bacteria, so I will look into that
as well.
Yang, H., Wang, M., Yu, J., & Wei, H. (2015). Antibacterial activity of a novel peptidemodified lysin against Acinetobacter baumannii and Pseudomonas aeruginosa. Frontiers
in Microbiology. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4686776
Summary: This is a peer-reviewed journal article about a new peptide-modified lysin
that can kill gram-negative bacteria. When tested against bacteria, it worked well in cell
stages of growth and reproduction but was much less active in cells that were in the
stationary phase, not growing much. This is possibly because the difference in structure
of the outer cell membranes and/or the peptidoglycan layer during the two different
stages. The results in this situation match up with those of similar studies. Lysins have a
low possibility for bacteria to develop resistance because of the unique mechanisms they
utilize to function and lyse the cell. However, when faced with gram-negative bacteria,
they are largely useless because they cannot get past the outer layer. There have been
some other techniques to get around this barrier such as physical pressure and the
addition of a chemical permeabilizer like citric acid, but these can only be used for
topical infections or non-human applications like food conservation. Although in vitro
situations ended with the modified lysin successful in killing bacteria, in more
complicated environments such as pasteurized milk it did not participate in any
antibacterial activity. This indicates that it is not difficult to inactivate the lysin, PlyA, so
it can only be applied to special or simple situations.

Application to Research: Originally I thought that there was no way around the issue that
lysins are ineffective against gram-negative bacteria, but this research shows that it is not
the case at all. However, bacteriocins still do have a bit of an edge because they need less
modification, making it easier on the producers.