Origin of Milk Bacteria

A VERY HOT RESEARCH PROJECT FOR HIGH SCHOOL STUDENTS and TEACHERS

The Transport of "Dairy Bacteria"
within the NORMAL Lactating Mammal
 (A serious topic: if this didn't happen, none of us would be here today!)
...

... I have returned from Chicago's meeting of the American Society for Microbiology where I presented the work of several high school teachers and students. While I stood with the poster, which 314 people read in the space of 90 minutes, I had three visitors - division heads of the U.S. Food and Drug Administration. These three brought five others over to look at the poster also - these five were representatives of five major pharmaceutical firms who are competing in the market of synthetic infant formula.

... What struck these particular visitors were two aspects of our presentation on the origins of the bacteria in our intestines. The first was a wondering comment: "Isn't it interesting how little we actually know about ourselves?" The second was that they saw a little piece at the end of our poster which hinted at a preview of good things to come - that next year we might deal with the bacteria in milk. All nursing mothers produce milk that has a huge population of "dairy" bacteria in it. These eight stood around for a long time discussing something obviously important as the manufacturers were dickering with their regulators at our site. The gist of their conversation was that the manufacturers were thinking of adding bacteria to their artificial milk formulas so as to provide the babies with one more important ingredient that would hopefully prevent the diarrhea that so many formula-fed babies have. The buzz-word at the ASM meeting was "pro-biotics." Got any idea what that means?

... Nevertheless we wise ones, who have been thinking about our next year's projects, know that the FDA and the company people have only a view of HALF the scene. While they consider the benefits of the "dairy bacteria" for the infant, they overlook the wonders that are going on inside the mother. Are these bacteria of any direct benefit to her? (It is possible that these bacteria are her guard-dogs keeping other environmental microbes from infecting her milk production equipment that are producing a nutritious juice that many microbes should relish and in which they should thrive.) How is it that she can have so many of these bacteria in her and yet show no signs of massive infection? Afterall, fresh milk contains more than 100,000 bacteria per milliliter (or more that 45 million per pint!). How is it that these bacteria get inside of her milk ducts? Why only these dozen or so different species of "dairy bacteria" and normally no others? So many questions. Answer a few of these questions this coming year, and I believe we can be assured of not only being once again accepted for presentation but also of being chosen as one of a few groups to be highlighted in the international press room in Los Angeles, where the meeting will be held in year 2000.

... Thus I want to STRONGLY encourage all of you to consider this as your project for this coming year. The whole project is much too large for any single scientist to do in one year. So it is imperative that you form teams - especially geographically distributed teams where each team has something unique to contribute - such as milk goats, UV-equipment for making mutants, and continuous flow centrifuges.

... What are the rewards for doing such a project? Several. For high school students, it will open doors to colleges. Afterall, why apply to college when you can get invited! For high school teachers, while co-authorship gives very little continuing education credit (ASM is working on that), such an authorship credential may allow you to be a teacher in an community or regular college. Of course, the main reward will be that you have personally discovered something basic to being a human or a mammal, something that will be soon found in textbooks. You will have discovered something that no one in the history of the world knew before. Now that is exciting to the soul!

The General Steps in the Experiment

  1. Need to obtain a NORMAL, lactating female (thus, obviously a mammal). This female must be one that will be producing milk for the next three months. It would be good to have at least three such milk providers in the event mastitis or other reason causes a cessation of production.
  2. Three or more types of the major bacteria found in the milk must be isolated. A way to both count the number of bacteria in the milk AND to isolate the bacteria is a method called plate counts.
  3. From those bacteria mutants must be isolated such that each mutant is now resistant to two different antibiotics that the "parent" strain was once susceptible to. Each bacterium should have a different combination of antibiotic resistance mutations for later easy and rapid species identification.
  4. Two harmless strains of environmental bacteria should also be mutagenized. Suggested are a benign strain of E. coli and perhaps the common soil bacterium Bacillus subtilis.
  5. These five strains of mutants should then be grown up in large batches - perhaps as much as 5 gallons and then centrifuged down to a concentrated paste.
  6. The various pastes would be mixed together.
  7. The paste-mix would be fed to the lactating mothers.
  8. Immediately, and every 15 minutes, a few drops of milk would be collected from the mother and refrigerated. A small portion would be plated on a series of petri plates - each with a different combination of antibiotics in them. This allows for rapid and easy identification of any of the species fed to the mother and now being passed out in her milk.
  9. Once a particular bacterial species is seen to be coming out in the milk, the remainder of the sample can be plate-counted.
  10. The data can all be plotted for each of the strains. A wonderful graph out to result in answering a number of questions such as
    • how long does it take for the mother to begin "passing" that particular type of bacterium?
    • what levels of selectivity are seen? Are only the "dairy bacteria" transported and the environmental bacteria are screened out.
    • is there evidence of any preferential selection of different species of 'dairy bacteria'?

--Are These Guard-Dogs with Teeth?--

... On a very different front - one which is trying to answer the question of the benefit these bacteria might have for the mother, we need to test the guard-dog-with-teeth hypothesis. At first this can be tested using raw cheeses and milk. Later, purified cultures of the dairy bacteria should be used to see if they can kill environmental bacteria.

  1. E. coli and B. subtilis can be mixed with various cheeses. Soft cheeses such as yogurt and fine curd cottage cheese might be good starts.
  2. Plate counts of the mixtures must be made over a period of several days.
  3. Meanwhile some of the major bacteria from the cheeses should be isolated and grown up in medium to which the same environmental bacteria are added to see if the killing exists with these bacteria.

Now to approach some of the logistical problems in getting this project done.

  1. What sort of mammalian mother are we going to use?
  2. Which teams of students and teachers have special expertise in doing various aspects of this project?
  3. How do we get samples back and forth between the groups?
  4. Deadline: December 1, 1999 is when the abstract must be submitted for the May 2000 meeting of the ASM. By Dec. 1, a lot of good data must be coming in and indicating a direction of the overall project.
  5. Communication between task groups is easy by e-mail and shipment by FEDex.

... Two types of nursing mothers seem most easy to use - goats and dogs. The reason for this choice is that these two will "eat almost anything" including a favorite food laced with lots of the mutant bacteria.

... I have a pretty good idea of the various expertises of groups with which I have been working this past year. However, there are certain general methods that all would have to be able to do well: good aseptic technique; sterilizing media; pouring petri plates, and a few other more minor activities. On the whole the project will be a very good bacteriological experience.

Expertises of various groups are required. Choose yours:

I hope dozens of you are interested! Let me know soon at ecoligist@yahoo.com.

Dr. V

P.S.: For all you budding M.D.'s, one of the things we've noticed at these microbiology meetings is that hundreds of presentations are made about diseases such as diarrhea and mastitis, yet NOTHING much is known about the NORMAL condition other than "it doesn't hurt" or "it isn't giving any problems." But as even young scientists know, the normal is like the control. Only when something goes wrong with the normal does disease step in. Alas, the medical community knows precious little about the normal. Allow me to give you two blatant examples:

  1. Neonatal sepsis - infectious disease in newborns is still thought that some barrier was breached in the mother so that a pathogen was able to gain a foothold in the fetus. Medical textbooks state plainly that in the womb, the fetus is sterile and has no bacteria. And that the intestinal bacteria are gained during the birthing process by a mechanism called reflux, where fluids from the outside world rise up the birth canal to inoculate the baby. Babies born via caesarian should thus not have any bacteria. They do! At the instant that almost all babies are born, they already have about a hundred trillion bacteria in their intestines. That number would look like peanut butter. Reflux? Naw: notta chance!
  2. Many nursing women contract mastitis, which is a bacterial infection of their milk ducts by some environmental bacterium. Often it is so painful, and often the milk becomes so putrid that nursing stops. If such infections were the general rule, our cave-ancestors would have not been able to raise their young and we wouldn't be here today. Doctors usually tell the mothers that they are not keeping themselves clean enough, and more care should be taken. But we have pigs today, and I have never seen a mud-wallowing sow pay any attention to keeping herself clean - nor our cave-ancestors for that matter either! Guard-dogs? Maybe that's the answer!
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