Phage therapy

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 . 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:

1. Begin a free-Gonorrhea screening program on the streets of Oakland and San Francisco;
2. Enhance the Center for Disease Control's Surveillance of antibiotic-resistant gonorrheal infections;
3. Develop an intern program for Oakland teens;
4. Develop a space that has the equipment (acquired or built) we need in which we can conduct our research;
5. Acquire culture materials, and clinical specimen from which bacteriophages can be harvested;
6. Identify the first known bacteriophages from N. gonorrhoeae;
7. Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections;

If you would like to help with any of these project goals, please contact Craig (moleculararts [at] riseup [dot] net).

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.

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.

5.Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections.

Experiments and Anticipated Problems

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.

Laboratory Needs

1. 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.
2. Benchtop butane torches.
3. Sterilized toothpicks.
4. MTM agar plates (ordered!)
5. Fastidious Broth
6. Glass test-tubes.
7. Maltose for differentiation of N. meningiditis vs. N. gonorrhoeae.
8. Rayon Swabs and tongue depressors (ordered!)
9. -80c Freezer to store glycerol stocks of clinical samples.
10. Ultracentrifuge
11. SDS-PAGE gel electrophoresis equipment and reagents.
12. Autoclave

much much much more.

Social Project Goals and Community Agreements

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 Non-hierarchical:

We recognize the outcomes of hierarchy. Hierarchy represses creative thought in science, denies access to science, and holds 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 hearing everyone and refuse to acknowledge hierarchy based on: sex and gender, race, age, accreditation, education, class, or access to resources. We stand in solidarity against oppression and hierarchy.

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 full 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, no decision will be made without 100% consensus.

We believe in asking (and answering) questions:

Science is a question. Our social quest is the pursuit of knowledge. We are not approaching this project with the hope of making money. We want to collectively answer questions and serve our communities. We will stand in solidarity against anyone who chooses to co-opt or privatize any portion of this project. All knowledge must be free and accessible!

Relevance

http://www.smithsonianmag.com/science-nature/phenom_oct00.html?c=y&page=1

Resources

Here are some videos to review

http://www.youtube.com/watch?v=LiPZq2K_Tos&NR=1&feature=endscreen

http://www.youtube.com/watch?v=ehbZpo8oXSs

http://www.youtube.com/watch?v=3VjE1zddXWk

http://www.youtube.com/watch?v=ZENpYdQg-z4

http://www.youtube.com/watch?v=egY-Br_oTHE

http://www.youtube.com/watch?v=Oms5SA_4-kE

http://www.youtube.com/watch?v=3eQhNyX7DRQ

http://www.youtube.com/watch?v=lUcMGktSc7c&NR=1&feature=endscreen

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)