Caffeinated E. coli

by Lorien E. Menhennett

I don’t think I’ve ever been so excited to go to school on a Monday as I was on April 11. Yes, I was actually excited about a Monday. Because at 11:30 a.m., when I walked into my research seminar class, I was going to find out whether my E. coli bacteria had mutated in the presence of caffeine and become antibiotic resistant.

OK, that might not sound like anything worth getting worked up about. So let me back up. My classmates, professor, and I were studying antibiotic resistance in bacteria. This is a poignant research topic, given the rampant rate at which antibiotic-resistant infections such as MRSA spread in hospitals. But we weren’t looking to decrease antibiotic resistance; we were looking to increase it. The goal was to learn about how mutations in DNA can allow bacteria to develop antibiotic resistance.

DNA is a sort of “language” that tells cells how to build their proteins. If that “language” is altered, even by a single letter, then the protein structure can also be altered. And that can affect an antibiotic’s ability to chemically bind to a bacteria and either destroy it or prevent it from replicating.

Mutations – changes in DNA – occur naturally, at a very low rate. So that’s what we looked at first. We grew cultures of bacteria in small glass tubes overnight in a shaking incubator, providing them with a liquid medium that would keep them happy, fed, and replicating. Using what’s called spectrophotometry, we were able to determine the concentration of bacteria cells. In essence, we used a machine to measure the amount of light that was absorbed by a sample of the cells in a small cuvette (a fancy name for a plastic tube). Then we distributed 100 microliters (a really tiny squirt) of the cells onto petri dishes which had a jelly-like medium for the cells to grow on.

Half of the petri dishes contained an antibiotic called carbenicillin, but this wouldn’t kill the bacteria, because they were engineered with a gene that gave them resistance to this specific antibiotic. These “carb” plates were our controls. They should exhibit explosive growth, but only growth of our desired bacteria – any bacteria from outside that lacked that carbenicillin-resistance gene would die. The other half of the plates contained carbenicillin plus another antibiotic called rifampicin. Rifampicin is a broad-spectrum antibiotic, meaning it kills lots of things. It is now only used to treat tuberculosis as part of a multi-drug cocktail, though, because it has a tendency to promote antibiotic resistance. (Perfect for our purposes.) The rifampicin should kill everything, with the exception of any bacteria that had mutated and developed some way of resisting its effects. Those were the colonies we were after.

We let everything grow over the weekend, and returned to find “lawn” – a very scientific term for prolific – growth on the carbenicillin plates, as we expected. We also found a handful of colonies, which looked a little like mold growing, on the rifampicin plates. Success! We scooped out the colonies, and through a series of processes, amplified and isolated one specific gene of their DNA sequence – a gene that has been implicated in mutations associated with rifampicin resistance. We then had the gene sequenced by an outside company. And sure enough, after a computer program analysis, we found DNA mutations (when compared to the “normal” E. coli gene sequence).

But it was time to take things a step further. So my professor, Dr. Kreher, asked each of us to come up with some substance – any substance – that we thought might induce additional mutations in the bacteria and increase the rifampicin-resistance rate. In other words, a substance that would cause more colonies to grow on the rifampicin plates. Everyone chose something different, from cigarettes to deodorant to caffeine (mine).  I found a number of research papers on PubMed (research article heaven) that indicated caffeine is indeed a mutagen at high concentrations. So I grew more bacteria, and then prepared a solution using water and anhydrous (powdered) caffeine, and mixed it in with the jelly-like medium that goes into the plastic petri dishes. I used two concentrations of caffeine to see whether there was a difference related to dose. When all was said and done, I had 48 dishes to plate. Being a rather novice microbiologist, it took me the better part of my Friday afternoon to squirt all those cells onto the plates and spread them around. But I got it done, and Dr. Kreher and I loaded the plates into an incubator.

Come Monday, all 48 plates were stacked up at my lab bench. I started combing through them, looking for colonies … and found only two. Far fewer colonies than we had found without using caffeine, meaning I had a very low resistance rate. But I found something else interesting: the higher concentration of caffeine actually killed most of the bacteria on my control plates (the carbenicillin plates) – which should have had that “lawn” growth. So our bacteria definitely did not like caffeine.

I’m still working out my specific conclusions from the experiment. And while I know those are important, what I found even more valuable was just the whole process – this was the first experiment I had really designed and carried out on my own. I wouldn’t have known the first place to begin on such a thing when I started out the semester. I have come a long way as a … scientist.

(Scientist: I’m trying that word on to see how it fits, and I like it.)

Oh, and a note for you coffee drinkers (of whom I am one): have no fear. The caffeine in coffee (or soda) will not cause your cells to mutate. The concentrations of caffeine studied in the papers I read would require a person to drink more than 100 cups of coffee, basically at once, which one author noted would be toxic. So you can have your coffee and drink it too.

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