Les was nice enough to take some time out of his busy schedule as a micro-biologist to share some insights into his latest study regarding the response of the gut bacteria to repeated antibiotic use.
In few words, Les just completed a study which examined human’s gut bacteria communities. The humans from the study were all given two rounds of the antibiotic Ciprofloxacin (Cipro). The goal of the study was to determine if the original gut bacteria communities of each person were affected by the antibiotics.
There are several videos, that will be introduced right away when they are complete which should definitely explain some questions that everyone has regarding this subject.
When I met with Les, he showed me around his lab, and a few other rooms within his research building. Here is a transcription of part of the video above. Towards the end of this video, Les was explaining how he was able to identify the different bacteria that he found during the study. If you are anything like me, quite a bit of this topic is completely foreign, so I thought it might be helpful to type out some of the recorded conversation so everyone can re-read or re-view if you need another go-round before you “get it”.
Les: At the molecular level this agarose is a big whole mesh of openings, with the chains of the gel inbetween, if its a small molecule it can squeeze through these holes quickly, if its a big molecule it keeps getting hung up. So this is a way to separate the small molecules that move quickly and further down the gel, the big molecules will slowly and not move so far. So after a while, you can see patterns of chunks of DNA. Its a complicated way to make something that’s invisible, visible to us, which is to say we have this whole mix of different DNA molecules but we know the one we want is a particular size. This lets us filter the DNA. We can separate the small ones from the big ones and everything in-between. When we locate the DNA we are searching for, we can actually slice it out of the gel and purify it and examine it further. So now, instead of having a whole mix of DNA, we can now use just the DNA we want.
Adam: Did you use the method above for the Gut Bacteria Study?
Les: I wound up using this as part of that study.
Adam: How did you use this process?
Les: So the steps that I went through started with stool samples from people who agreed to take the antibiotic, I extracted DNA from whatever was present in the poop sample there, and mostly that’s bacterial DNA, there will be some human cells in there as well, but bacteria are abundant in health stool. I used chemical technique and heat(things were boiling hot), basically the cell walls and membranes just bust open. So now everything that was inside the cell is now leaking out all over the place. Then, I used chemical methods to separate deoxyribonucleic acid (DNA) the stuff our genes are made of, to pull the DNA out and wash the rest away. So now, instead of having a bunch of bacterial cells mixed in with the stool sample, now I’ve got the DNA from a whole bunch of different bacteria mixed up in a test-tube.
Somewhere along that DNA, each bacteria has a 16S gene. That is the identity card we use. It just turns out because all bacteria have this. The sequence is similar enough that we can always recognize it and find this, but it will be different enough that it will be different from one bacterium to another. It works just like a standard government ID card.
I know this might take a moment or two to get this, but the electrophoresis you saw from the video along with some other tools and chemicals and micro-biologist smarts is what Les used to determine which bacteria were present before during and after the subjects took the antibiotics.
I’ve got about an hour and a half of video with Les and will be putting it all together ASAP so the rest of the meeting can be out there for everyone.
Once again, thank you so very much to Les for sharing his time with me and for helping the ulcerative colitis community understand more about what is happening in our gut.(more videos to come soon!)