For each day you will need to have a lab notebook entry.  Please use the template page provided.  If you do not have one component for a particular day, just explain that in your entry.  Include images of your plates, plaque, or soils for accurate record.

In America, “one-third of the major foodborne disease outbreaks, occurring in the U.S. between 2003 and 2007, were associated with contaminated fresh produce,” (volume 236) leaving scientists with a difficult problem; how do we combat dangerous bacterium that may be lurking on our food? Because of the fragility of most produce, harvested produce undergoes very minimal cleaning before being packaged and sent away for human consumption. An especially dangerous microorganism that can be found on produce is Escherichia coli, commonly referred to as E. Coli. A team of scientists from the University of Ohio came up with a possible solution, the use of macrophages in produce processing that will specifically target the dangerous strain of bacteria. However the question remains, is this possible and if so would the public be supportive of the idea that their produce is being washed in viral solutions?

Initially the scientists in the study decided to use E. Coli O157:H7 as the target bacterium. This bacterium can grow on the open surfaces of produce and the produce used in this study consisted of green pepper and spinach. The stain of E. Coli was first transferred to Luria Bertani Broth and incubated, spun, and incubated in a second LB broth for use in the study. Phage was isolated from cattle, sheep, and horse manure in addition to sewage from the city of Columbus Ohio. The phage was then isolated using the following procedure: samples where put into 90mL of broth and then shaken, the supernatants were then sterilized using a millipore size syringe-driven filter unit, next the samples were inoculated with the target bacterium at 35 degrees Celsius for 48 hours with shaking, lastly samples were centrifuged and filter-sterilized. The samples where then plated and individual plaques were plated a second time to isolate a single phage.

Each type of phage was tested to see which one was the most effective and the most effective phage was the one that caused the most inactivation of the target bacterium when plated in LB agar. Before the individual produce could be tested green pepper was put under an UV light for one hour on each side to kill any background microorganisms. Spinach was not put under the UV light because it was too fragile to withstand the UV treatment.

Actual testing involved dipping green pepper in crude lysate for five minutes and shaking. Spinach was also dipped in crude lysate for 2 minutes and shaken because it was too fragile to withstand the total shaking time. A control was also only dipped in E. Coli. The samples were then left to dry for a period of 4 hours at room temperature and then refrigerated for a period of 72 hours to simulate produce manufacturing. Pepper samples and spinach samples were then plated and calculations preformed. Because spinach could not be treated with UV light the bacterium used was altered to produce a green fluorescent light and the colonies were then counted.

Compared to just the rinse the use of phages to combat E. Coli O157:H7 was extremely successful showing major inhibition of bacterial growth.

Overall the experiment was a success. The use of phages to combat bacterium is not only possible, but extremely effective – reducing E. Coli populations by 2.0 Log. Thus the applications of phages in produce manufacturing may soon become a reality. One problem still remains, this was small scale, further research would have to be preformed industrially and currently the scientists from Ohio are working to create that opportunity. Lastly public education would be required before this treatment can be put into practice as the majority of the American and world population for that matter knows very little about phages and the benefits that they can also have. In conclusion the experiment was a success and in the coming years we may begin to see phages integrated into our sanitation systems worldwide.

The link to, “Developing and optimizing bacteriophage treatment to control enterohemorrhagic Escherichia Coli on fresh produce,” can be found here. The article, written by Abigail B. Snyder, Jennifer J. Perry and Ahmed E. Yousef from the University of Ohio, was found using the Scopus search engine. Key words and limits used to find the article include: bacteriophage, article (limit to), 2016 (limit to) and yielded a total of 587 articles.

Over the past few summers since the summer after my sophomore year in high school, I have been researching under a Cardiac Pathologist in Dallas, Texas. This is why I choose a topic on bacteriophages that have to do with the human heart. I went on Web of Science and searched for bacteriophage which resulted in over 30,000 different articles. To narrow the search results, I searched within the articles for cardiology at first. This didn’t work, so I went back and searched for heart instead. This resulted in only 49 articled which is much easier to deal with. I found an article titled “Phenoptosis: programmed death of an organism.” The article was written by V. P. Skulachev.

This article describes apoptosis (which purifies a tissue from cells that became useless or even harmful for the organism) and how bacteriophages may help induce this process in order to purify bacterial population. The figure below shows how apoptosis can be harmful to an organism.  

http://protein.bio.msu.ru/biokhimiya/contents/v64/pdf/bcm_1418.pdf

This article was the first written about apoptosis and the benefits that can have for getting rid of unwanted bacteria. If I am not mistaken, V. P. Skulachev was the first to discover the benefits of such processes. Apoptosis is an integral part of Biology. I have heard Cardiologists where I do research talk about cell processes, and how drugs affect them and essentially how some drugs work in helping our bodies.

Knowing that there are plenty of medical applications for bacteriophages, I was curious to discover why phage therapies are not as commonplace in the medical field today. Are there many practical advantages of using bacteriophages over other medical approaches? With healthcare costs on the rise, does phage therapy offer more accessible and financially sound treatment options? With these questions in mind, I started my quest for an article on Scopus by searching “bacteriophages in medicine” in order to get some general ideas about how bacteriophages can be used in the medical field. After narrowing my search to more recent articles (between 2015 and 2016) and adding the keyword “practical”, I came across the study: Phage Cocktails and the Future of Phage Therapy.

Written by authors Benjamin K Chan, Stephen T Abedon, and Catherine Loc-Carillo, this article published in July of 2013, explores the potential of phage cocktails, mixtures of a variety of therapeutic phages that can target a range of bacteria, as well as suggests methods for integrating phage therapies into clinical practice.

Compared to monophage therapies which can only offer one form of treatment, polyphage approaches, allow more room for development as different cocktail combinations create broader prospects for successful treatment. Although these therapies offer promising results, western commercial development stalls due to a lack of Phase III human clinical trials. In order to move on to the next steps of commercial development, this paper offers three different models of development that would help circumnavigate some of the blockages that currently exist. The first is the Western pharmaceutical model. This model provides a middle ground between specialized, yet expensive monophage treatments and large polyphage treatments that could impact non-target bacteria. It suggests simplifying cocktails so that while although there would still be a variety of phages in the cocktails, the treatment can be more targeted as well as more cost-efficient. The second model suggests the idea of a personalized phage bank where preformulated cocktails could be distributed and modified over time. This approach would be more labor intensive but provides personalized medicine and flexibility. The last approach this paper suggests is the Georgian Approach. Modeled after the phage therapy development in the Soviet Republic of Georgia, this phage cocktail development would create more streamlined cocktails that also have the potential to be modified to fit the needs of a diverse population.

Finally, this paper poses that despite certain obstacles, phage therapy can be expected to be more common practice within the next 5-10 years. It presumes that further research must be done in human clinical trials and become more mainstream among western researchers in order for development to truly begin, as there are still many questions surrounding the long term effects of phage therapy in humans that remain unanswered.

My initial thoughts lead me to search on Scopus if there was any up and coming research on the use of bacteriophages in the “omics” fields; however, when I searched up “Bacteriophage” and “Omics” only 8 results appeared and only two seemed to focus on bacteriophage use. Therefore, I decided to take a different approach and see if any new treatments or discoveries using bacteriophages have been made, so I restarted my search on scopus using the keywords: Bacteriophage AND medicine. With this search, I resulted in 601 different articles, which gave me much more variety. I chose the article,  “What Can We Learn from a Metagenomic Analysis of a Georgian Bacteriophage Cocktail?” by Zschach. H and Joensen. KG Viruses 2015,7(12), mainly because the title had intrigued me due to the involvement of the genome which was involved in my initial search and also because I wanted to learn the significance of the Georgian Bacteriophage Cocktail.

Phage therapy has become widespread in Eastern Europe; however its potential to combat antibiotic-resistant bacterial infections in Western medicine has not been unlocked. In order to help facilitate this process, the Eliava Institute in Georgia has chosen to use this specific study to identify and analyze the major components of the Intesti phage cocktail, which was developed in Paris at the Pasteur Institute and is a combination of sterile phage lysates active against Shigella, E. coli, Proteus vulgaris and mirabilis, Enterococcus, Staphylococcus aureus, and Pseudomonas aeruginosa.

To execute the research, scientists sequenced the complete cocktail as a metagenome, amplified the component phages on eight different hosts and isolated DNA from the resulting lysates. However, before scientists were able to sequence the samples, they had to prepare the samples with solutions in order to remove possible bacterial DNA leftovers. Phage clusters were then constructed using grouping contigs sorted by size and depth of coverage. Lastly, in order to verify the cocktail’s capability to cause lysis of the specified pathogens, five to ten strains were selected for each pathogen and tested for susceptibility towards the phage cocktail by streaking the bacteria onto an agar plate perpendicular to a streak of phage solution.

The table represents the exact number of strains tested and the fraction of strains found susceptible. The streaking tests confirmed that the cocktail was in principle able to cause lysis of strains of all seven pathogens specified by the producer, albeit with differing specificity for the different pathogens. The apparent low efficiency in lysis of Staphylococcus is due to the fact that only five of the ten tested isolates were S. aureus, of which all but one were susceptible.

The article allowed scientists to explore which types of sequence-based analysis are able to result in useful conclusions using phage cocktails.

Keywords: Bacteriophage, medicine, metagenomic

Hits: 601

As I was looking for an article, I was thinking about how Bacteriophages can help with genetics, so on Scopus I searched “Bacteriophage use in genetic research.” Since the use of bacteriophages in gene therapy is relatively new, I got 223 results right off the bat. The name of my article is “Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents,” and it is written by Amanda Donnelly, Teerapong Yata, Kaoutar Bentayebi, Keittisak Suwan, and Amin Hajitou.

The article begins by saying how although there have been many advances in the development of transfection reagents for gene transfer, it is still a challenge to ensure that they are efficient, safe, cheap, and reproducible. According to the article, the definition of gene transfection is that nucleic acids are delivered into cells so that they can be genetically modified. It talks about how bacteriophages have the potential to really be helpful with gene transfections for many different reasons such as a protein coat which is arranged in an alpha helical array, the protein coat can tolerate mutations, they can safely be given to humans in order to treat bacterial infections, and they can be easily produced for a low cost. However, there is a problem with the bacteriophages. They do not have the ability to enter and convert the cells, so the researchers had to genetically engineer the phage to be able to bind to the surface of the cell and eventually enter the cell. But they discovered that even the genetically engineered phages are poor delivery vectors when they are compared against the traditional viral vectors because they cannot easily bind to the mammalian cell. So something that would be a good topic for future research is looking into how phages can effectively bind and enter mammalian cells in order to have a more efficient gene transfer ability.

There were nine steps to this research project. It started off with genetically modifying bacteriophage vectors. Then they chemically modified the phages by testing three transfection reagents with phage vectors such as cationic lipids, cationic polymers, and calcium phosphate. Afterwards, they started growing a cell culture containing human MDF-7 breast cancer cells. Then they did an In Vitro cell transduction that was a vector derived from a phage. After that, they treated HEK-293 cells, which were in 48 different plates, with phage vectors that had the right ratio of transfection reagents. After all of the steps of their experiment, they did a statistical analysis to compare the results.

One result that the researchers discovered was that the integration of the transfection reagents and the phage vectors boosted the positive outcomes of gene transfer. They also notices that different transfection reagents affected the phage-mediated gene transfer in different ways. The calcium phosphate and PEI combinations had a higher increase in gene transfer than the lipid-based reagents.

When searching for articles, I was attempting to find an one that would be related to the research project being worked on during class. I began with the question “How have other individuals or groups been researching bacteriophages and soil?” Using Scopus, I searched “bacteriophage” and then searched for “soil” within the results; I also limited the search to articles that were published in 2016 because I wanted to learn about some recent discoveries. One of the articles displayed was “Phage-bacteria interaction network within human oral microbiome”. While this was not related to what I was initially looking for, I was interested in why this particular article was appearing with with limits and keywords I included.

The authors of the article are Jinfeng Wang, Yuan Gao, and Fangqing Zhao, and the journal the article was published in is Environmental Microbiology.

The purpose of this research is to explore bacteriophages in a novel microbial ecosystem. Because the phage-bacteria interaction in human oral cavities is less understood, the authors decided to research the relationship.

To complete the research, the authors collected samples of salivary or dental plaque specimens from individuals with varied periodontal statuses. After sequencing and data processing on software, the researchers came to the conclusion that the phage-bacteria interaction in the human oral microbiome was an idea that could be expanded upon in the future. Based on the results that periodontally healthy individuals have a richer phage communities, further exploration of the topic can be carried out.

Figure A depicts the phage contig characteristics after sequencing. Figure B is a heat map of the different types of samples the researchers collected. It shows the relative abundance of each phage as well as the sequence identity from the viral genome databases. Figure C compares the phages of the samples from the individuals with different periodontal health.

After reading the work the researchers completed, I noticed they focused on certain conditions and diseases. They looked to see how the phage-bacteria interacted in the oral cavities of those individuals, leading me to the question of “How would the results differ if they tested individuals exposed to different levels of environmental pollution?”

I wondered if bacteriophages could be medically beneficial to us. I searched “bacteriophage antibiotics” on the Web of Science database and limited my search to articles from newest to oldest. I wanted a more recent article that had something to do with bacteriophages as a new cure or a new discovery in which they help us solve a major problem or disease. The keywords that showed up was bacteriophages, antibiotics, bacteriophage adaptation, and antibiotic resistance. I finally came across the article “Bacteriophages as a Potential Treatment for Urinary Tract Infections” and the more I read, the more intrigued I was. The article was published under the terms of the Creative Commons Attribution License and written by Wilbert Sybesma, Reinhard Zbinden, Nino Chanishvili, Mzia Kutateladze, Archil Chkhotua, Aleksandre Ujmajuridze, Ulrich Mehnert, and Thomas M. Kessler.

The objective of these scientists was to examine the effect of lytic bacteriophages on E. coli and K. pneumoniae strains derived from the urine of UTI patients. A total of 50 urinary cultures were collected from 50 patients (41 E. coli and 9 K. pneumoniae strains) by sterile catheterization. There were different causes of their UTIs which increases randomness for a good experiment. The urine samples were cultivated on sheep agar blood and after the susceptibility testing was performed, the strains were stored in skimmed milk at -70 degrees Celsius. Then they were subcultured and transferred to Amies transport agar. Four commercial bacteriophage preparations were used in a bacterial cell lysis screening assay and were then titrated by Appelmans method. Next, screening was performed using the spot test method. The following plates show plaque morphology of E. coli of different strains in which all the results are positive.

E. Coli stain #33 – overgrown lysis of Pyo and Intesti bacteriophages – confluent lysis of Ses and Enko bacteriophages.

E. coli strain #16 – individual plaques of Pyo and Intesti bacteriophages – overgrown partial lysis of Ses and Enko bacteriophages.

In the following tables, the results show that bacteriophage and antibiotic resistance had no correlation to each other. In table 1, by using a antibiogram, the susceptibility deviates from sensitive to resistant to the antibiotics. Also the lytic activity differed in the strains. In table 2, by using spot tests, it can be shown that the highest lytic activity came from the bacteriophages. In table 3, in regards to K. pneumoniae strains, the bacteriophages showed promise. In particular, the v_BRKpS 10 could lyse all 9 strains.

Table 1 – 41 E. Coli strains and 9 K. pneumoniae strains

Table 2 – 41 E. Coli strains

Table 3 – 9 K. Pneumoniae strains

In conclusion, the outcome of this experiment shows that bacteriophage therapy is a possible option for treating UTIs.

My further questions include: Is this safe enough to be used to treat UTIs? What consequences can this have on the patient? Can this be what causes us to depend less on antibiotics? What are the next steps into making this an official, open, and widely used treatment?