Difference between revisions of "Phage therapy"

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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 ==
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== Sexually Transmitted Bacterial Infections ==  
== Sexually Transmitted Bacterial Infections ==  
: 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.
: 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.


== Symptoms of Gonorrhea ==
== Symptoms of Gonorrhea ==
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== Questions and Specific Aims ==
== Questions and Specific Aims ==
:
[[Experiments]]
:
:'''Pilot Project Aims'''
:
:'''Project Aims'''
<br />
<br />
1. Monitor the emergence of antibiotic resistant gonorrhea.<br />
:'''Isolate N. gonorrhoeae from 10 patients'''. 
:a. Compose and deploy a mobile screening unit at various locations throughout Oakland and San Francisco.
:''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. Compile data from the mobile screening program and integrate it with data from other public health organizations.
 
:''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. Develop a community laboratory space in Oakland, CA that has the equipment we need in which we can conduct our research.
:'''Isolate ARNG-derived bacteriophage from 10 patients'''.  
:a. Procure capital equipment and reagents needed for this project.   
:''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. Procure reagents and equipment to begin molecular microbiological research.
<br />
<br />
3. Isolate, identify and characterize the first known bacteriophages from '''N. gonorrhoeae.'''
: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. Harvest samples from our mobile screening program.
:b. Perform Genomic and Proteomic Analyses on Bacteriophage samples.
<br />
<br />
4.Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections.
:'''Characterize ARNG-derived bacteriophage from 10 patients'''.
: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.
:''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).
<br />
 
5. Provide educational outreach opportunties.
:''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.
:a. Provide information on prevention and treatment of gonorrhea.
 
:b. Provide in-the-lab training and experience for those with the desire to pursue citizen science.
:''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.  
 
:'''Conduct proof-of-concept lysis experiments'''.
:''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).


== Anticipated Challenges to Our Approach ==
== Anticipated Challenges to Our Approach ==
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#Addressing previous citizen science failures and conducting research with a greater adherence to quality.
#Addressing previous citizen science failures and conducting research with a greater adherence to quality.
#Preventing Lysogeny.
#Preventing Lysogeny.
== Experiments ==
: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.'''''


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


== Laboratory Needs ==
== Laboratory Needs ==
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== References ==
== References ==
<references />
<references />
[[Category: Health]]
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