Monday, May 24, 2010

Bacteriophage Research

PHAGE DRUG: It was March, 2004, when I decided on a course of action that might work. I had been thinking for a long time during my studies at Texas Tech University about the possibilities of bacteriophage therapy or phage therapy. Phage is a fancy word for virus and the idea is to use virus against harmful bacteria. For every type of bacteria in the world there are many virus that predate or feed on it. The idea is to find the viruses which are most efficient at killing the specific bacteria you want to control. It is an idea perfected by Felix d’Herrelle and it is a technique older than antibiotics. These virus do not attack eukaryotic cells (human tissue) but invade and lyse (kill) prokaryotic cells (bacteria). These are two very distinct types of cells and the human cells are not at all a target of bacteriophage, they are immune from harm. The former USSR had learned that hard way that they could not keep up with the US in the arms race by matching us nuclear bomb for nuclear bomb. It was just too expensive so they came up with a different strategy. After an initial nuclear exchange with the US in WWIII they planned to use missiles to rain down anthrax and bubonic plague germs on our cities and break our will to fight. It was called project Bonfire. The USSR calculated that they would have about two weeks or more prior warning that WWIII was about to begin so they would begin massive production of many thousands of tons of germ weapons and transfer these onto refrigerated warheads on top of ICBM’s. The USSR devoted billions of rubles and hundreds of thousands of scientists, technicians, and workers to this task for decades. In the end they evolved the mature capability to do just that which they planned. Anthrax cannot be spread from person to person but the plague bioweapon can be spread from lung to lung and would theoretically be capable of killing much of the population of the US if they decided to use it. In the Middle Ages certain varieties of the plague (septicemic) killed over 90% of the people exposed.
I began to research bacteriophage therapy and which phage worked on the germ that causes bubonic plague (Yersinia pestis). I read every paper I could find and spent every waking hour trying to find the perfect phage. In a matter of weeks I had found the exact phage I was looking for. In laboratory tests it had lysed or killed nearly every single strain of plague out of the 2,000 strains they had tested it on. I created a website ( and a company (Leonardo Concepts Inc.). The website was designed to show the PhD’s, the NIH and people like USAMRID that I was for real when I asked for information and later money. I emailed and called all the major companies in the world that made phage and tried to get them interested in partnering with me to make the drug. I found two business partners with decades of experience in biodefense who read my website and immediately contacted me and agreed to go into business together on the project. I worked 12 hours a day or more every day and finished the application and mailed it off to the NIH. All the forms and documents fit into a box nearly two feet tall. It was like mailing off one of your children in more ways than one. In the mean time I knew I had to get a skill set working with bacteriophage in a lab to give me the expertise I needed. I found just the place and with the recommendation of my old biology professor I was accepted to a school in Olympia, Washington. The T4 lab at The Evergreen State College was one of the few places in the world where an undergrad like me could get my foot in the door and start working on making phage drugs.
The world of bacteriophage research is very rarefied air where only a few scientists in a dozen or more labs work on discovering new phage and making new drugs to control pathogenic bacteria. I had a plan and the hope that when my grant application was evaluated I would be one of the 15% that were funded. The grant application results would come when I had gotten the skill set of working with phage in the lab. If I got the grant the next step would be to make a small quantity of the drug and have the animal tests done at the Battelle labs. The scientist I was working with there was very responsive to my questions and very enthusiastic about the project. He was an expert at measuring the amount of aerosol pathogen that was to be transferred to the experimental animals. The experiment consisted of exposing 200 mice to an aerosol of bubonic plague in a sealed container that resembled an oversized microwave oven. Half of the exposed mice would be given the phage drug at different stages to test the efficacy of the drug. If it worked and saved the lives of the exposed population that were treated 24 hours after the deadly germs entered their lungs then I would receive two million dollars to make the phage drug in large quantities at a production facility in Maryland. The experiment on the mice alone would cost $820,000 and was the make or break step. It was a unique experiment that had never been done before but I had read all the literature and was confident that the phage would save the mice and if necessary human beings. I don’t condone the superfluous testing of drugs on animals but this drug could save millions of lives in the event of a national emergency so the lives of 200 specially bred mice were not being wasted. It was a very dangerous experiment that could only be done in a very few places in the world. If the scientist I was working with accidently broke the seal on his glove during the experiment he could very well lose his life. At least he would have plenty of the phage drug handy. Whatever they were paying him he was earning every bit of it. The lab in Olympia where I would be working was a small room full of incubators, centrifuges of all sizes, chemicals, measuring equipment, supplies, glass ware, an air hood, and a -80 degree centigrade walk in freezer. There was a room next door for sterilizing and storing equipment and analyzing DNA and RNA sequences of phage. There was another room next to that which was not used heavily because it had been contaminated with radioactive isotopes but some of us used it out of necessity. Two scientists would direct about twelve students in their research projects in the small lab but there were rarely more than eight people in the lab. The main focus of research by the two professors was E. coli bacteria and the phage that killed them. We also worked with different kinds of staph and strep and other pathogens that were becoming resistant to antibiotics. Every year about 70,000 people who visit a hospital incur an infection and lose their lives to bacterial infections that can no longer be controlled with antibiotics. Excessive use of antibiotics in humans and the animals that make up our food supply make the resistance more prevalent and the deaths greater in number every year. Bacteria pass plasmids (genes) to each other that confer resistance to antibiotics. Our work was important because at some point most antibiotics will no longer control bacterial infections and humanity will suffer millions of deaths. The E. coli strains we worked with were the “McDonalds strain” that had been responsible for several deaths but these bacteria had the pathogenic genes removed so that we could work with them without becoming ill and dying. The methodology was to grow the E. coli or other bacteria in a flask in the warm water bath and then place the bacteriophage in the flask and do a time experiment to measure how much bacteria the phage killed over what period of time. The phage in relation to the bacteria is about the size of a BB to a basketball. The phage enters the liquid of the flask and cannot swim but randomly bumps into the walls of the bacteria. The first phase is called attachment when the phage “sticks” to the outside of the bacteria. The phage often looks like the lunar module as it goes through attachment and the proteins irreversibly adhere to the bacteria and inject their packet of DNA or RNA into the interior of the bacteria. The phage hijacks the proteins and other machinery that the bacteria uses to live and begins to manufacture exact copies of itself in parts like a Buick automobile that might enter a factory dedicated to making semi trucks and suddenly it is converted to making all the separate parts that make up a Buick. The following stage is called assembly when all the separate pieces of the phage reassemble into hundreds of complete virus (phage) particles while still inside the bacteria. The final stage is lysis when the phage slice through the bacterial wall from the inside and release the hundreds of copies of themselves at the same time killing the bacteria. The whole process might take about 40 minutes. The hundreds of new copies of phage then enter the liquid medium and randomly bump into the bacterial walls and repeat the process again. It is a delicate and complex dance that varies at every stage and might work or fail at any stage depending on the phage used and the conditions of the experiment. At different time points the experimenter removes portions of the liquid medium and pours it onto a petri dish that is incubated over night and shows how well the phage is killing the bacteria. Science is very precise and very hard because there are many things that can go wrong. Some people in the lab had worked for two years or more trying to write a thesis but had to admit their darkest fears that all of that blood, sweat, and tears might in the end lead nowhere. The stakes were enormous and the work was not easy. The two scientists we were working for were very professional and dedicated people who gave us support and encouragement. It was a heady atmosphere because we were potentially going to change the world but could just as easily fail. The people around me were young, highly intelligent, and idealistic and I felt like that I was in a special place. In the same way that the devout might enter the Vatican or the Dome of the Rock I entered the lab every morning and began my experiments.
I have never studied how to make organisms to be used to injure or kill people. I have no specialized knowledge about how to make biological weapons or organisms that could be used against specific individuals or races or nations. The entire focus of my research has been to make medicines that could save millions of lives in the event of a national emergency. I am not now nor have I ever been a threat to national security, and in fact the entire thrust of my career has been to enhance the health and safety of humanity. A careful study of my career in microbiology and interviews with my teachers and fellow students and researchers will confirm this fact beyond a shadow of a doubt. Marshall Gregory Thomas

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