- 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;
- Develop an intern program for Oakland teens;
- Develop a space that has the equipment (acquired or built) we need in which we can conduct our research;
- Acquire culture materials, and clinical specimen from which bacteriophages can be harvested;
- 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;
- 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.
- 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.
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.
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.
- 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.
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!
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)
-  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/