Difference between revisions of "Phage therapy"
→Questions and Specific Aims
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:'''Pilot Project Aims''' | :'''Pilot Project Aims''' | ||
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'''Isolate N. gonorrhoeae from 10 patients'''. 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. | '''Isolate N. gonorrhoeae from 10 patients'''. | ||
''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. | |||
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). | ''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). | ||
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 | ''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. | ||
'''Isolate ARNG-derived bacteriophage from 10 patients'''. | '''Isolate ARNG-derived bacteriophage from 10 patients'''. | ||
''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. | |||
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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. | |||
'''Characterize ARNG-derived bacteriophage from 10 patients'''. | '''Characterize ARNG-derived bacteriophage from 10 patients'''. | ||
''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). | |||
''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. | |||
''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'''. | '''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 == | ||