Difference between revisions of "Phage therapy"

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==Contribute To Our Pilot Program!==
If you have time or resources to contribute to this project, we urge you to attend our bi-weekly meetings or donate to our Pilot Program WePay Campaign [https://wepay.com/donations/gonorrhea-eradication-team here!]
==The Gonorrhea Eradication Team and Integration Taskforce (GETit!) ==
==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 are working with you to: (1) Implement an on-the-street Gonorrhea screening program through which emergence data are acquired and integrated with current data via strategic collaboration with public health organizations, (2) Provide a consistent feed of data that is open to the public (open-access), (3) conduct research on gonorrhea-associated bacterial viruses (bacteriophage) and, (4) develop novel, phage therapies for the treatment of antibiotic resistant gonorrhea.
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 scientists. Together, we are taking on our first major project: the eradication of antibiotic resistant gonorrhea using Phage Therapy! We are asking for support in launching our Pilot Program which aims to: (1) Isolate and Characterize Bacteriophage from 10 patients, and (2) create a proof-of-concept bacteriophage cocktail to be tested ''in vitro''.


''Our team:''
''Our team:''
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:'''Craig Rouskey''', MSc in Molecular Biology, Biochemistry, Microbiology and Immunology
:'''Craig Rouskey''', MSc in Molecular Biology, Biochemistry, Microbiology and Immunology
:'''Ami Knop Ullrich''', MSc in Medical Microbiology and Immunology
:'''Ami Knop Ullrich''', MSc in Medical Microbiology and Immunology
:'''Nina DiPrimio''', PhD in Pharmacology
 
:'''Louis Huang''', BSc in Chemical Engineering and Economics
:'''Marc Juul''', MSc in Biotechnology Engineering
:'''Jenny Ryan''', MA in Anthropology, MA in Communication
:'''Tess Westbrook''', BSc Biology, MBA
:'''Ianto Lin Xi''', BSc Molecular Biology


== Introduction ==
== Introduction ==
: ''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 <ref>http://www.cdc.gov/std/Gonorrhea/arg/default.htm</ref>. 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 <ref>http://www.cdc.gov/std/Gonorrhea/arg/basic.htm</ref>.
Neisseria gonorrhoeae, the sexually transmitted pathogen that causes Gonorrhea, has progressively developed resistance to the antibiotic drugs used to treat it. Within the past 40 years, this pathogen has developed resistance to 3rd-generation, cephalosporin antibiotics which are ultimately the last line of defense against this bacterial pathogen <ref>http://www.cdc.gov/std/Gonorrhea/arg/default.htm</ref>. Antibiotic resistant Neisseria gonorrhoeae (ARNG) 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. These treatments have been mostly successful, but given the ability of ARNG to develop antibiotic resistance, it is critical to continuously research and develop new treatment regimens for gonorrhea<ref>http://www.cdc.gov/std/Gonorrhea/arg/basic.htm</ref>.  


: 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.
To meet the needs of a rapidly changing public health field in which traditional antibiotic-based therapies are becoming ineffective, the Gonorrhea Eradication Team and Integration Task-force (GETit) was created. This organization was created to conduct research into bacteriophage associated with Neisseria gonorrhoeae. Bacteriophage are viruses that infect bacterial hosts and can induce lysis upon infection. Ultimately, our goal is to identify and characterize bacteriophage from wild type ARNG and use a composite cocktail of ARNG-associated phage as therapy to treat hosts infected with this pathogen.
:<br />
 
: This project is in its early stages. We are working to:
'''During our pilot program, we plan to''':
# isolate ''N. gonorrhoeae'' from 10 patients
# isolate bacteriophage from 10 clinical samples of ARNG
# characterize bacteriophages from antibiotic-resistant, sexually-transmitted, ''Neisseria gonorrhoeae'', and  
# conduct proof-of-concept experiments in which ARNG-derived phage cocktails are used to lyse cultures of ARNG ''in vitro''.
:
<br />
: '''This project is in its early stages. Our longer-term goals are to''':
# Begin a free-Gonorrhea screening program on the streets of Oakland and San Francisco;
# 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;
# Enhance the Center for Disease Control's Surveillance of antibiotic-resistant gonorrheal infections;
# Identify the first known bacteriophages from ''N. gonorrhoeae;''
# Develop assays for the rapid detection of ''N. gonorrhoeae.''
# Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections;
# 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;
# Develop a space that has the equipment (acquired or built) we need in which we can conduct our research;
# Develop a community education program to bring science to underrepresented cultural groups.


:
:
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: Phage therapy is a biological therapy that uses bacteriophages (bacterial viruses) to infect and lyse bacterial pathogens.<ref>[http://www.landesbioscience.com/journals/bacteriophage/AbedonBP1-2.pdf] Phage Treatment of Human Infections. Abedon, et al. 2011.</ref>  
: Phage therapy is a biological therapy that uses bacteriophages (bacterial viruses) to infect and lyse bacterial pathogens.<ref>[http://www.landesbioscience.com/journals/bacteriophage/AbedonBP1-2.pdf] Phage Treatment of Human Infections. Abedon, et al. 2011.</ref>  


== Sexually Transmitted Bacterial Infections ==
== 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.<ref>[http://www.cdc.gov/std/gonorrhea/STDFact-gonorrhea-detailed.htm] Detailed STD Facts </ref> 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 facultative intracellular diplococci induce more serious illness such as pelvic inflammatory disease.
: 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.<ref>[http://www.cdc.gov/std/gonorrhea/STDFact-gonorrhea-detailed.htm] Detailed STD Facts </ref> 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 ==
== Symptoms of Gonorrhea ==
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== Questions and Specific Aims ==
== Questions and Specific Aims ==
:
[[Experiments]]
:
:'''Pilot Project Aims'''
:
:'''Specific Aims'''
<br />
<br />
1. Begin a long-term mobile screening program for antibiotic resistant gonorrhea on the streets of Oakland and San Francisco.<br />
:'''Isolate N. gonorrhoeae from 10 patients'''. 
:a. Develop a website through which patients, after being provided a patient number can retrieve their results with a full analysis of their strain.
:''Acquiring samples''. We are currently in negotiations with local clinics (ie. Berkeley Free Clinic, Magnet, City Clinic :of San Francisco and St. James Infirmary) to enroll patients in our pilot study. Briefly, patients who are screened by :these clinics and test positive for oropharyngeal N. gonorrhoeae will be provided with information on our study. Within :the informational packet, patients will find their assigned Patient ID and use this number to schedule an in-house visit. :The Patient ID allows the patient to remain anonymous during our screening process. During their primary visit, patients :will provide their consent, sign liability waivers, fill out our surveillance survey, and provide us with an :oropharyngeal specimen. Patients can then use their Patient ID to track the results from their specimen using our :anonymous web tracking system.
:b. Compose and deploy a mobile screening unit at various locations throughout Oakland and San Francisco.
 
:''Primary isolation of N. gonorrhoeae''. Primary gonococcal cultures will be grown on Modified Thayer-Martin (MTM) medium containing chocolate agar (5% SRBC), Vancomycin, Colistin, Nystatin and SXT. This medium allows selective growth of Neisseria species. Primary cultures will be propagated in tryptic soy broth (TSB) according to the standards set forth by the Centers for Disease Control (REF).  
 
:''Confirmation of N. gonorrhoeae''. Upon primary culture and propagation, N. gonorrhoeae samples will be confirmed using nucleic acid testing. A panel of polymerase chain reactions will be performed on DNA isolated from bacterial cultures. These panels will be performed to (1) identify genes responsible for 1st-, 2nd- and 3rd-generation antibiotic resistance, (2) differentiate N. gonorrhoeae from other Neisseria species. The differentiation PCR will be confirmed using CTA sugar growth testing as described by the CDC (REF). Briefly, isolates will be assessed for their ability to grow in Glucose, Maltose, Lactose and Sucrose. N. gonorrhoeae is positive for glucose metabolism, but not maltose or lactose metablism.
<br />
<br />
2. Support the Center for Disease Control's efforts to monitor the emergence of antibiotic resistant gonorrhea.
:'''Isolate ARNG-derived bacteriophage from 10 patients'''.  
:a. Compile data from the mobile screening program and report it (anonymously) to the CDC.
:''Bacteriophage Isolation''. Bacteriophage will be isolated from 10 clinical samples as previously described (REF). Briefly,  isolated colonies will be selected and cultured in  5 mL TPY broth (trypticase, phytone, and yeast extract) and cultued for 24 hours at 37oC in 5% CO2.
: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.
<br />
<br />
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.
:After culture, 1500 µL of broth will be transferred to Trypticase Soy Agar (TSA) plates in triplicate (500uL each). One plate will be used for harvesting bacteriophage, a second plate will be used for propagating bacteriophage, and a third plate used for storing cultures. The remaining culture (3500uL) will be centrifuged for 10min at 2500 RPM. The supernatant will be removed and filtered through a 0.22 µm syringe filter. The syringe filters removes all cellular debris resulting in a pure bacteriophage culture. Bacteriopahge cultures will then be added to a bacterial lawns using 10-fold titrations to determine titers. Positive titers will result in plaque formation.
: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.
<br />
<br />
4. Isolate, identify and characterize the first known bacteriophages from '''N. gonorrhoeae.'''
:'''Characterize ARNG-derived bacteriophage from 10 patients'''.
: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.
:''Genomic Molecular Weight Analysis''. Bacteriophage genomes will be extracted as previously described (REF). The genomes :will then be analyzed via gel-electrophoresis to determine approximate molecular weight(REF).
:c. Characterize phage via PCR
<br />
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 ==
:''SDS-PAGE''. Purified bacteriophage lysates will be analyzed by denaturing SDS-PAGE as previously described. The SDS-:PAGE characterization assays will determine the molecular weight of proteins and assess their relative abundance. All :samples will be compared to positive control T4-phage panels.


Key issues that need to be addressed. Table _ shown is a list of problems and solutions that are needed to address our research into phage therapy.
:''Host Range Studies''. Host range studies will be performed as previously described (REF). The purpose of this assay is :to determine the ability of ARNG-associated phage to infect different Neisseria species, bacterial from different genii :(I.e gram positive, and coliforms), and alternate clinical N. gonorrhoeae samples. Briefly, bacterial lawns of varying :host bacteria will be created and phage will be drop-tested and assessed for their ability to produce plaques in :different genii, species, or isolates cultures. We also propose a novel broth-culture assay in which OD readings are :taken before and after bacteriophage infection. Should our bacteriophage isolates maintain their capacity for lysis, OD :culture readings should drop significantly.  


#rapid clearance of phages
:'''Conduct proof-of-concept lysis experiments'''.
#patient safety
:''Infection Assay''. Following testing of each individual phage, a 10-sample phage cocktail will be generated and tested :for its ability to lyse clinical samples of N. gonorrhoeae. These methods are similar to "Host Range Studies," the only :difference being that instead of using single-phage samples, the proof-of-concept treatment will be a 10-phage cocktail :in which all bacteriophage samples are combined at (a) the same titer, and (b) in the same proportions. These assays are :previously described (REF).
#bacterium targeting and optimization
#bacterial strains resistance/ anti-phage antibodies
#spread of bacterial resistance
#narrow range of hosts
#phage purity and purification technique (while administering directly may be hazardous if not bacteria is removed)
#poor stability of phage
#poor scientific style/methods of past research
#lysogeny


== Experiments ==
== Anticipated Challenges to Our Approach ==
: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.'''''


#rapid clearance of phages by human host and bacteria: bacterial strain resistance / anti-phage antibodies.
#patient safety: altering phage to prevent lysis of gram negative species.
#bacterium targeting and optimization: altering the binding kinetics of Phage adherence.
#narrow range of hosts
#achieving phage purity: while administering directly may be hazardous if bacteria is not removed.
#poor stability of phage: how do we preserve phage ex vivo and ensure that is viable upon administration?
#Addressing previous citizen science failures and conducting research with a greater adherence to quality.
#Preventing Lysogeny.


===Protocol Documentation===
===Protocol Documentation===
http://aegia.nu/folder/getitproject/130721_prephage-protocols.txt


== Laboratory Needs ==
== Laboratory Needs ==
#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.
:[https://www.wepay.com/donations/gonorrhea-eradication-team Your Financial Support]
#Benchtop butane torches.
: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.
#Sterilized toothpicks.
:Benchtop butane torches.
#MTM agar plates '''(ordered!)'''
:Sterilized toothpicks.
#Fastidious Broth <ref>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/</ref>
:MTM agar plates '''(ordered!)'''
#Glass test-tubes.
:Fastidious Broth <ref>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/</ref>
#Maltose for differentiation of N. meningiditis vs. N. gonorrhoeae.
:Glass test-tubes.
#Rayon Swabs and tongue depressors ('''ordered!''')
:Maltose for differentiation of N. meningiditis vs. N. gonorrhoeae.
#-80c Freezer to store glycerol stocks of clinical samples.  
:Rayon Swabs and tongue depressors ('''ordered!''')
#Ultracentrifuge
:-80c Freezer to store glycerol stocks of clinical samples.  
#SDS-PAGE gel electrophoresis equipment and reagents.
:Ultracentrifuge
#Autoclave
:SDS-PAGE gel electrophoresis equipment and reagents.
:Autoclave
:
:
: '''''much much much more'''''.
: '''''much much much more'''''.
<br />
==Guiding Principles==
:
''We believe in 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 ''quest''ion'''. 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.
''We believe in equal access to healthcare.''


== Relevance ==
== Relevance ==
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== References ==
== References ==
<references />
<references />
[[Category: Health]]
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