Difference between revisions of "Phage therapy"
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:b. Harvest phage and perform proof-of-concept, in vitro experiments in which gonorrhea is lysed with preparations of bacteriophage.
:b. Harvest phage and perform proof-of-concept, in vitro experiments in which gonorrhea is lysed with preparations of bacteriophage.
== Anticipated Problems ==
== Anticipated Problems ==
== Experiments ==
== Experiments ==
Revision as of 00:31, 14 July 2013
The Gonorrhea Eradication Team and Integration Taskforce (GETit!)
We are a volunteer group of experienced citizen scientists working to foster healthy communities through innovative medical research. Our team includes PhD-, Master- and Bachelor-level microbiologists, immunologists and cellular biologists. Together, we are taking on our first major project: the eradication of antibiotic resistant gonorrhea! We would like to work with you to: (1) Implement an on-the-street Gonorrhea screening program; (2) Provide a consistent feed of data that is open to the public (open-access); and (3) develop novel, phage therapies for the treatment of antibiotic resistant Gonorrhea.
- Craig Rouskey, MSc in Molecular Biology, Biochemistry, Microbiology and Immunology (2005)
- Ami Knop Ullrich, MSc in Medical Microbiology and Immunology (2006)
- Nina DiPrimio, PhD in Pharmacology (2009)
- Louis Huang, BSc in Chemical Engineering and Economics (2012)
- Marc Juul, MSc in Biotechnology Engineering (2009)
- Jenny Ryan, MA in Anthropology, MA in Communication (2010)
Who We Are
- Throughout the course of this project, participants will co-create and foster an open access citizen science project that accomplishes specific scientific goals while educating and nurturing community and scientific creativity.
- Those participating in this project adhere to the following statements:
We are building egalitarian networks.:
- Hierarchy, privilege and discrimination have the potential to repress creative thought in science, deny access to science, and as a result, hold back global scientific development. As a collective of citizen scientists working toward a more complete understanding of molecular medicine, we commit ourselves to nurturing creative, positive voices within our community. We commit ourselves to listening to, hearing and acknowledging the diverse voices that make our community great!
We believe in open access and transparency:
- Everyone is invited to participate in and learn from this project. We will always make time for conversation and teaching opportunities. Online meetings, communications regarding this project, and all findings will be publicized on this fully-editable wiki with the stipulation that those participating adhere to a value system of mutual respect, compassion and safety; while accepting that these values will be enforced via community-based decision making.
We believe in consensus:
- We believe scientists should be open to discussion around research experiments, data interpretation, and project directions. To foster a spirit of openness and understanding, decision making is made by consensus. Proposals are brought to weekly meetings and offered to the group. The group develops and integrates proposals into organizational policy and scientific research aims.
We believe in asking (and answering) questions:
- Science is a question. Our social quest is the pursuit of knowledge. We desire to collectively answer questions and serve our communities. We believe that all knowledge must be free and accessible and will continue to work to maintain that openness.
- Neisseria gonorrhoeae has progressively developed resistance to the antibiotic drugs used to treat it. Since the late 1970's, Gonorrhea has shown signs of developing resistance to 3rd-generation, cephalosporin antibiotics which are ultimately the last line of defense against this bacterial pathogen . Antibiotic resistant Gonorrhea is therefore a growing public health concern. In the United States, this concern is exacerbated by the fact that primary treatments for gonorrheal infections are solely antibiotic-based. Currently, CDC STD treatment guidelines recommend dual therapy with the injectable cephalosporin ceftriaxone and either azithromycin or doxycycline to treat all uncomplicated gonococcal infections among adults and adolescents in the United States. Dual therapy is recommended to address the potential emergence of gonococcal cephalosporin resistance. Given the ability of N. gonorrhoeae to develop antibiotic resistance, it is critical to continuously monitor gonococcal antibiotic resistance and encourage research and development of new treatment regimens for gonorrhea .
- This project was established to (1) Provide surveillance insight into the emergence of antibiotic resistant gonorrhea, (2) better understand and characterize bacteriophages from antibiotic-resistant, sexually-transmitted, Neisseria gonorrhoeae, and (3) develop phage-based prophylaxis and therapy to stop prevent further emergence of antibiotic-resistant gonorrhea.
- This project is in its early stages. We are working to:
- Begin a free-Gonorrhea screening program on the streets of Oakland and San Francisco;
- Enhance the Center for Disease Control's Surveillance of antibiotic-resistant gonorrheal infections;
- Identify the first known bacteriophages from N. gonorrhoeae;
- Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections;
- Develop a space that has the equipment (acquired or built) we need in which we can conduct our research;
- If you would like to help with any of these project goals, please contact Craig (contact [at] craigrouskey [dot] com).
What is Phage Therapy
- Phage therapy is a biological therapy that uses bacteriophages (bacterial viruses) to infect and lyse bacterial pathogens.
Sexually Transmitted Bacterial Infections
- This project will begin by studying Neisseria gonorrhoeae the causative agent of gonorrhea.
- Neisseria gonorrhoeae is a facultative intracellular pathogen that is able to infect the eye, pharynx, anus/rectum, urogenital tract, and may be disseminated throughout the body in more complex cases. The Center for Disease Control reports that in 2011 there were an 321,849 new cases of gonorrhea reported in the U.S. of which about 50% are estimated to be reported ( for a total of 700,000 estimated new cases in 2011). The World Health Organization reports that there are between 65-105 million new cases of gonorrhea nationally each year. Of these, 0.5-3% of cases develop into disseminated, systemic infection where the falcutative intracellular diplococci induce more serious illness such as pelvic inflammatory disease.
Symptoms of Gonorrhea
Antibiotic Resistant Gonorrhea
- Until recently, gonorrhea treatment was simply a matter of picking the right antibiotics; however, that is quickly ceasing to be the case. Gonorrhea is currently one of the most common treatable STDs in the United States, but soon it may be just one of the most common STDs. The number of gonorrhea cases resistant to treatment with antibiotics has continued to rise, and scientists are quickly running out of options. Single-dose antibiotics for gonorrhea treatment are quickly becoming a thing of the past.
- Gonorrhea, otherwise known as the clap, often seems like nothing more than a nuisance, particularly since it is so frequently asymptomatic, but that won't continue to be the case if we run out of antibiotics to treat it. Left untreated, gonorrhea can lead to serious problems. It is, for example, a major cause of pelvic inflammatory disease and infertility. Gonorrhea can also lead to an infection known as disseminated gonorrhea and cause problems in pregnant women and infants.
- Because gonorrhea is so common, doctors would like to be able to treat it with a single, effective dose of medication. Single-dose gonorrhea antibiotics reduce problems with drug compliance that can increase the prevalence of antibiotic resistance, and also decrease the need for follow-up. Unfortunately, one-dose regimens may soon no longer be an option. The affordable antibiotics that have been widely used to treat gonorrhea in the past are losing effectiveness against a growing number of strains. Although it is still possible to find an antibiotic that can treat individual cases of gonorrhea, the choices are narrowing as multi-drug-resistant strains of the bacteria continue to appear. At this point, American doctors have been recommended to stop giving oral antibiotics as a primary treatment and switch over to an injectable cocktail.
- The specific types of antibiotic-resistant gonorrhea strains seen in the U.S. and around the world vary from year to year, city to city, and population to population. Some scientists hope that by eliminating use of gonorrhea antibiotics that are becoming ineffective, strains that are resistant to those drugs will decrease in prevalence so that the drugs will become useful once again. Scientists have to hope, because they are quickly running out of drugs. In late 2012, the scientists reported that the last, effective oral antibiotic used to treat gonorrhea had begun to fail. In one clinic in Ontario, up to 7 percent of patients were not effectively treated with cephalosporins.
- In a few years, gonorrhea treatment will cease to be a simple process. Kicking an infection may require course after course of antibiotics, followed by repeated testing to see which, if any, of the antibiotics have worked. At that point, your best option will be one that you also have right now -- consistently practicing safe sex to avoid getting infected in the first place.
Antibiotics That Are No Longer Recommended For Gonorrhea Treatment
- Sulfonamides - Over a period of only 9 years, 30 percent of gonorrhea strains became resistant to treatment with sulfonamides. They stopped being used in the mid-1940s and were replaced by penicillin.
- Penicillin - Although initially quite effective, required penicillin doses for gonorrhea treatment climbed significantly over time, until eventually, in the 1980s, U.S. doctors stopped using penicillin to treat gonorrhea.
- Tetracycline - In the 1980s, tetracycline also ceased being a first-line treatment option due to the spread of treatment-resistant gonorrhea strains.
- Fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin) - In 2007, the CDC changed their gonorrhea treatment guidelines to remove single-dose fluoroquinolones from the recommended list. Fluoroquinolone-resistant strains have been identified around the world, including in many areas of the U.S. The prevalence of fluoroquinolone-resistant gonorrhea in California went from less than one percent of infections in 1999 to over 20 percent in 2003.
- Oral Cephalosporins (ceftriaxone, cefixime) - Cephalosporin-resistant gonorrhea strains were first identified in Asia and Australia and have been slowly becoming more common around the globe. As of August 2012, oral cephalosporins are no longer recommended for the treatment of gonorrhea in the United States. Between 2006 and 2011, the percentage of gonorrhea strains resistant to these drugs went up more than ten-fold in many areas of the U.S. Cefixime is no longer recommended for gonorrhea treatment at all, except in cases where ceftriaxone can not be used.
Antibiotics Currently Used to Treat Gonorrhea
- Combination Treatment with Injectable Cephalosporins - As of August, 2012, the recommended treatment for gonorrhea is one injection of 250 mg ceftriaxone. This is combined with either a single oral dose of 1 g azithromycin or a week of taking 100 mg oral doxycycline twice a day. To date, few gonorrhea strains are resistant to both types of antibiotic. However, this will not be true forever. There are alternate treatment regimens available for people allergic to ceftriaxone, but they require patients to return for a second test to make certain they have been cured.
- Bacteriophage (phage) is a virus that infects bacteria host cells. Viruses are acellular microbes that are obligate intracellular pathogens; requiring living cell hosts to carry out metabolic and reproductive needs. Bacteriophages carry with them a protein coat called a capsid that surrounds a small amount of DNA genetic material. The size of the DNA can vary from 5 genes to over 100 genes (3). The majority of the genes on phage DNA code for capsid proteins, proteins to protect viral DNA from degradation, and proteins used in the release from the host cell (3, 4). Because phage cannot reproduce or undergo metabolism on their own, they must infect living bacteria cells in order to reproduce. As part of their reproductive cycle, phages kill the bacteria cell they are infecting. There are two main types of reproductive cycles that a phage can use: the lytic cycle and the lysogenic cycle. A typical phage lytic cycle consists of five main steps. The first step is attachment. The attachment occurs between the phage and a receptor or structure on the surface of the bacterial cell. Attachment is very specific for the bacteriophage, with each phage being able to only infect one species of bacteria. After attachment is entry and this is where the phage DNA enters into the cytoplasm of the bacteria cell. Once inside the bacteria cell, the phage takes over the metabolic machinery of the cell, degrades the bacteria DNA, and changes the cell into a phage producing factory. The viral DNA is translated and viral proteins are made in the synthesis part of the viral cycle. In addition to translation, viral DNA is also being replicated to produce more viral DNA. Once enough viral capsid proteins and viral DNA are synthesized, the assembly part of the cycle occurs. During assembly, the viral capsid proteins surround the viral DNA to build more bacteriophage. When enough bacteriophage particles have been assembled, the release phase occurs. During the release phase, the host cell lysis open, releasing numerous bacteriophage into the environment. The bacteriophage can then go and attach to another bacteria host cell to repeat the lytic cycle over and over again until no bacteria are available for attachment.
- Although the lytic cycle can occur with all bacteriophage, some phage can enter a dormant cycle called the lysogenic cycle. In the lysogenic cycle, attachment and entry still occur but the host cell DNA is not degraded upon entrance. Instead, the phage DNA incorporates into the host cell DNA to form a prophage. A prophage implies that a bacteriophage has infected the host cell and is in a dormant cycle. The length of this dormant cycle depends on a number of parameters such as, the specific bacteriophage, the host cell, and the stress of the environment. Most bacteria that enter this dormant stage never re-enter the lytic cycle. Each time the bacterial cell divides and replicates its DNA, the prophage DNA is also being replicated. Eventually induction occurs which is when the prophage excises out of the host DNA and re-enters the lytic cycle at the synthesis stage. During the synthesis phase, the host cell DNA is degraded and viral proteins are translated. The assembly and release phases will follow. Many things can trigger induction such as nutrient depletion, UV damage to host cell, or any change in environment temperature or pH (5).
- Bacteriophages provide a selective method for targeting and destroying specific bacteria. In addition, because bacteriophage cannot replicate without the presence of their host bacteria, once the bacteria have been eliminated, the viral particles will soon degrade and also be eliminated. For each bacteria that exists, there is at least one bacteriophage specifically able to attach and infect it. This makes bacteriophage the most abundant entity on earth an estimated 1x10^31 present on Earth (3). With such an abundance, this makes bacteriophage an excellent candidate for eliminating bacterial infections.
History of Phage Therapy
- The initial discovery bacteriophage has been subject to speculation to who was the first. In the western world, Ernest Hankin, a British bacteriologist, first reported observing unidentified antibacterial preventing the spread of cholera (Vibrio cholerae) in the rivers Ganges and Jumna in India in 1896 [mini]. These unidentified antibacterial remained of unknown origin until bacteriologist Frederick Twort hypothesized that the cause of inhibition of bacterial growth was from viruses [M13] in 1915. Twort would be unable to continue pursuing his findings due to various reasons, among them financial difficulties.
- D'Herelle first observed bacteriophages as 2-3mm "clear spots" which was a pathogenic agent to coccobacillus bacteria cultures studying locusts in South America and Africa in the early 1900s [M18]. He would use his observations of bacteriophages to perform one of the first phage therapy techniques on severe hemorrhagic dysentery outbreaks among French soldiers stationed at Maisons-Laffitte in the summer of 1915 [M18, Mini]. On September 15, 1917 Felix d'Herelle would present his findings in the Academy of Sciences naming this phenomena 'Bacteriophage' after the Greek words "bacteria" and "phagein", which means to devour [M18].
- Continuing on the study of bacteriophages, d'Herelle starts researching on the effects of phage therapy on a 12-year-old boy with severe dysentery in 1919 at the HÃ´pital des Enfants-Malades in Paris, under the hospital's Chief of Pediatrics, Victor-Henri Hutinel. The patient's symptoms ceased after a single administration of d'Herelle's anti-dysentery phage, and the boy fully recovered within a few days [DHERELLE-BOOK]. The efficacy of the phage preparation was "confirmed" shortly afterwards, when three additional patients having bacterial dysentery and treated with one dose of the preparation started to recover within 24 h of treatment.
- Bacteriophages soon lost interest in the western world as antibiotics and penicillin was discovered, however research continued in the eastern Europe in the Soviet era. Two prominent research centers established are The Eliava Institute (EIBMV) in 1923 and The Hirszfeld Institute (HIIET) in 1952. Both continue to research bacteriophages presently. Due to observation of increasingly drug resistant bacteria, bacteriophages has brought to light again in the fight against disease.
Questions and Specific Aims
- Specific Aims
1. Begin a long-term mobile screening program for antibiotic resistant gonorrhea on the streets of Oakland and San Francisco.
- a. Develop a website through which patients, after being provided a patient number can retrieve their results with a full analysis of their strain.
- b. Compose and deploy a mobile screening unit at various locations throughout Oakland and San Francisco.
2. Support the Center for Disease Control's efforts to monitor the emergence of antibiotic resistant gonorrhea.
- a. Compile data from the mobile screening program and report it (anonymously) to the CDC.
- b. Send antibiotic-resistant gonorrhea samples to the center for disease control according to the Instructions for Submitting Specimens to CDC Gonorrhea Laboratory for Confirmation Testing and/or Testing of Clinical Treatment Failures.
3. Develop a community laboratory space in Oakland, CA that has the equipment (acquired or built) we need in which we can conduct our research.
- a. Procure capital equipment and reagents needed for this project.
- b. Procure reagents and equipment to begin molecular microbiological research.
- c. Open space up to provide education for Oakland teens.
4. Isolate, identify and characterize the first known bacteriophages from N. gonorrhoeae.
- a. Swab patients, grow samples on Modified Thayer Martin medium, broth cultures grown in Trypticase Soy Broth.
- b. Supernatants harvested and filtered through a 0.2um filter and analyzed by SDS-PAGE.
- c. Characterize phage via PCR
5.Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections.
- a. Test and optimize growth conditions for strains of antibiotic resistant gonorrhea from which bacteriophage can be isolated.
- b. Harvest phage and perform proof-of-concept, in vitro experiments in which gonorrhea is lysed with preparations of bacteriophage.
Anticipated Problems and Solutions
- We plan to begin acquiring clinical samples in April, 2013. Samples will be grown on Modified Thayer Martin agar supplemented with antibiotics. Colonies will (1)be stored in glycerol at -80oC, (2)grown in liquid culture for the assessment of bacteriophage production, (3)grown in Maltose to ensure we have cultured N. gonorrhoeae, and (4)grown in Fastidious Broth with 3rd-generation antibiotics to assess antibiotic resistance.
- Following growth in FB, supernatants will be analyzed for the presence of bacteriophage using SDS-PAGE electrophoresis.
- If bacteriophage proteins are present in the supernatants, these cultures will be re-grown in bulk and bacteriophage-containing sups will be stored at -80oC.
- All clinical screening data will be available to patients online through our website. If patients are positive, they will be asked to fill out a form on their results page that will detail their risk level. This data is important in the surveillance of antibiotic-resistant N. gonorrhoeae emergence. All screening procedures and results are anonymous.
- CO2 incubator (we've built a prototype incubator but it has no CO2 component). We can grow these organisms in candle jars inside the incubator which means we need candle jars and candles.
- Benchtop butane torches.
- Sterilized toothpicks.
- MTM agar plates (ordered!)
- Fastidious Broth 
- Glass test-tubes.
- Maltose for differentiation of N. meningiditis vs. N. gonorrhoeae.
- Rayon Swabs and tongue depressors (ordered!)
- -80c Freezer to store glycerol stocks of clinical samples.
- SDS-PAGE gel electrophoresis equipment and reagents.
- much much much more.
Here are some videos to review
Meeting Announcements and Contact Information
Next Call: 3/24/13 8:00pm PST/10:00pm CST
- if you'd like to attend the conference call, please contact: Craig (moleculararts [at] riseup [dot] net)
- ↑ http://www.cdc.gov/std/Gonorrhea/arg/default.htm
- ↑ http://www.cdc.gov/std/Gonorrhea/arg/basic.htm
- ↑  Phage Treatment of Human Infections. Abedon, et al. 2011.
- ↑  Detailed STD Facts
- ↑ Cultivation of Neisseria gonorrhoeae in Liquid Media and Determination of Its In Vitro Susceptibilities to Quinolones. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1234085/