Phage therapy

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 here!

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 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:

Craig Rouskey, MSc in Molecular Biology, Biochemistry, 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

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 . 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.

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.

During our pilot program, we plan to:

1. isolate N. gonorrhoeae from 10 patients
2. isolate bacteriophage from 10 clinical samples of ARNG
3. characterize bacteriophages from antibiotic-resistant, sexually-transmitted, Neisseria gonorrhoeae, and
4. conduct proof-of-concept experiments in which ARNG-derived phage cocktails are used to lyse cultures of ARNG in vitro.

This project is in its early stages. Our longer-term goals are 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 assays for the rapid detection of N. gonorrhoeae.
4. Develop methods by which bacteriophages can be cultured and used as prophylaxis and therapy for gonoccocal infections;
5. Develop a space that has the equipment (acquired or built) we need in which we can conduct our research;
6. Develop a community education program to bring science to underrepresented cultural groups.

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

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

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 facultative intracellular diplococci induce more serious illness such as pelvic inflammatory disease.

Antibiotic Resistant Gonorrhea

Until recently, gonorrhea treatment was simply a matter of picking the right antibiotics; however, that is quickly ceasing to be the case. Gonorrhea is currently one of the most common treatable STDs in the United States, but soon it may be just one of the most common STDs. The number of gonorrhea cases resistant to treatment with antibiotics has continued to rise, and scientists are quickly running out of options. Single-dose antibiotics for gonorrhea treatment are quickly becoming a thing of the past.

Gonorrhea, otherwise known as the clap, often seems like nothing more than a nuisance, particularly since it is so frequently asymptomatic, but that won't continue to be the case if we run out of antibiotics to treat it. Left untreated, gonorrhea can lead to serious problems. It is, for example, a major cause of pelvic inflammatory disease and infertility. Gonorrhea can also lead to an infection known as disseminated gonorrhea and cause problems in pregnant women and infants.

Because gonorrhea is so common, doctors would like to be able to treat it with a single, effective dose of medication. Single-dose gonorrhea antibiotics reduce problems with drug compliance that can increase the prevalence of antibiotic resistance, and also decrease the need for follow-up. Unfortunately, one-dose regimens may soon no longer be an option. The affordable antibiotics that have been widely used to treat gonorrhea in the past are losing effectiveness against a growing number of strains. Although it is still possible to find an antibiotic that can treat individual cases of gonorrhea, the choices are narrowing as multi-drug-resistant strains of the bacteria continue to appear. At this point, American doctors have been recommended to stop giving oral antibiotics as a primary treatment and switch over to an injectable cocktail.

The specific types of antibiotic-resistant gonorrhea strains seen in the U.S. and around the world vary from year to year, city to city, and population to population. Some scientists hope that by eliminating use of gonorrhea antibiotics that are becoming ineffective, strains that are resistant to those drugs will decrease in prevalence so that the drugs will become useful once again. Scientists have to hope, because they are quickly running out of drugs. In late 2012, the scientists reported that the last, effective oral antibiotic used to treat gonorrhea had begun to fail. In one clinic in Ontario, up to 7 percent of patients were not effectively treated with cephalosporins.

In a few years, gonorrhea treatment will cease to be a simple process. Kicking an infection may require course after course of antibiotics, followed by repeated testing to see which, if any, of the antibiotics have worked. At that point, your best option will be one that you also have right now -- consistently practicing safe sex to avoid getting infected in the first place.

Antibiotics That Are No Longer Recommended For Gonorrhea Treatment

Sulfonamides - Over a period of only 9 years, 30 percent of gonorrhea strains became resistant to treatment with sulfonamides. They stopped being used in the mid-1940s and were replaced by penicillin.

Penicillin - Although initially quite effective, required penicillin doses for gonorrhea treatment climbed significantly over time, until eventually, in the 1980s, U.S. doctors stopped using penicillin to treat gonorrhea.

Tetracycline - In the 1980s, tetracycline also ceased being a first-line treatment option due to the spread of treatment-resistant gonorrhea strains.

Fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin) - In 2007, the CDC changed their gonorrhea treatment guidelines to remove single-dose fluoroquinolones from the recommended list. Fluoroquinolone-resistant strains have been identified around the world, including in many areas of the U.S. The prevalence of fluoroquinolone-resistant gonorrhea in California went from less than one percent of infections in 1999 to over 20 percent in 2003.

Oral Cephalosporins (ceftriaxone, cefixime) - Cephalosporin-resistant gonorrhea strains were first identified in Asia and Australia and have been slowly becoming more common around the globe. As of August 2012, oral cephalosporins are no longer recommended for the treatment of gonorrhea in the United States. Between 2006 and 2011, the percentage of gonorrhea strains resistant to these drugs went up more than ten-fold in many areas of the U.S. Cefixime is no longer recommended for gonorrhea treatment at all, except in cases where ceftriaxone can not be used.

Antibiotics Currently Used to Treat Gonorrhea

Combination Treatment with Injectable Cephalosporins - As of August, 2012, the recommended treatment for gonorrhea is one injection of 250 mg ceftriaxone. This is combined with either a single oral dose of 1 g azithromycin or a week of taking 100 mg oral doxycycline twice a day. To date, few gonorrhea strains are resistant to both types of antibiotic. However, this will not be true forever. There are alternate treatment regimens available for people allergic to ceftriaxone, but they require patients to return for a second test to make certain they have been cured.

Bacteriophages

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.

History of Phage Therapy

The initial discovery bacteriophage has been subject to speculation to who was the first. In the western world, Ernest Hankin, a British bacteriologist, first reported observing unidentified antibacterial preventing the spread of cholera (Vibrio cholerae) in the rivers Ganges and Jumna in India in 1896 [mini]. These unidentified antibacterial remained of unknown origin until bacteriologist Frederick Twort hypothesized that the cause of inhibition of bacterial growth was from viruses [M13] in 1915. Twort would be unable to continue pursuing his findings due to various reasons.

D'Herelle first observed bacteriophages as 2-3mm "clear spots" which was a pathogenic agent to coccobacillus bacteria cultures studying locusts in South America and Africa in the early 1900s [M18]. He would use his observations of bacteriophages to perform one of the first phage therapy techniques on severe hemorrhagic dysentery outbreaks among French soldiers stationed at Maisons-Laffitte in the summer of 1915 [M18, Mini]. On September 15, 1917 Felix d'Herelle would present his findings in the Academy of Sciences naming this phenomena 'Bacteriophage' after the Greek words "bacteria" and "phagein", which means to devour [M18].

Continuing on the study of bacteriophages, d'Herelle starts researching on the effects of phage therapy on a 12-year-old boy with severe dysentery in 1919 at the Hôpital des Enfants-Malades in Paris, under the hospital's Chief of Pediatrics, Victor-Henri Hutinel. The patient's symptoms ceased after a single administration of d'Herelle's anti-dysentery phage, and the boy fully recovered within a few days [DHERELLE-BOOK]. Phage therapy was accept as the reason for cure after three more patients having bacterial dysentery were each treated with one dose of the preparation and started to recover within 24 hours of treatment.

Due to poor scientific experimentation, understanding of pathogenesis, phage-host interactions, and pharmacokinetics knowledge, bacteriophages soon lost interest in the western world as antibiotics and penicillin was discovered. However research continued in the eastern Europe in the Soviet era where two prominent research centers established, The Eliava Institute (EIBMV) in 1923 and The Hirszfeld Institute (HIIET) in 1952. Both continue to research bacteriophages presently. With new knowledge bacteriophages properties and due to increasingly drug resistant bacteria, bacteriophages has been brought to light again in the fight against disease.

Present Day Phage Therapy in Animals

Present day research on phage therapy in gram negative bacteria in animals have been very extensive and promising.

With a better understanding of phage therapy and more advanced equipment, modern day phage therapy can close the gaps in experimental accuracy and knowledge that was missing in the past. Using blood culture machines and better filtration methods, such as caesium chloride density centrifugation, have lead the better understanding of host-phage relationships such as longer-circulating strains of phage [nih11]. This has led to more efficient phage therapy techniques. With the knowledge of heat effects on phages, control groups observe differences in bacteriophage effect, therefore eliminating immunologic response variation in experiments. The National Institute of Health has done extensive controls on phage therapy on Vancomycin-Resistant Enterococcus faecium in mice, where single injection of phage administered 45 min after bacterium contact rescued 100% of mice compared to 100% fatality when no phage was administered [m10]. Numerous studies have shown substantial efficacy of phage on gram negative bacteria in calves, pigs, mice, and lamb[soothill01].

Extensive studies of phage therapy on gram negative bacteria have already been proved extremely effective [soothill, smith hw]. Experimentation of E. coli done on in vivo mice showed that bacteriophages were more efficient in vivo experimenation than in vitro. [59] Research on calves showed that one injection of specific phage strain 8 hours after contact with E coli protected the calves from death and diarrhoea, and again the in vitro experimentation underestimated the virulence of phage. Futhermore, different strains of phage were tested for efficiency in isolated does and cocktail formulas [60]. Some strains were proven more effective than leading antibiotics []. [soothill experiments needed].

Questions and Specific Aims

Pilot Project Aims

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 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.

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.

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.

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.

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

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

Protocol Documentation

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

Laboratory Needs

Your Financial Support

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.

Ultracentrifuge

SDS-PAGE gel electrophoresis equipment and reagents.

Autoclave

much much much more.

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 question. 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

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: 8/4/13 8:00pm PST/10:00pm CST

if you'd like to attend the conference call, please contact: Craig (moleculararts [at] riseup [dot] net)