Science Buddies: "Ask an Expert"

Hi
I have a microbiology project and I need some help.
my project is based on the danger of antibiotics to the normal flora (esp. the normal flora of gut).
we all know that when we use antibiotics not only the pathogens but also the normal flora are destroyed. how good life could be if only the pathogens were destroyed by antibiotics. we can make bacteria of normal flora resistant to antibiotics (at present, only one bacteria, and only one antibiotic), and then add that resistant bacteria to the normal flora. but there is a problem here and that is conjugation. for example; we want to make E.coli resistant to an antibiotic. then if E-coli 0157:H7 enters our body, the E.coli of our normal flora conjugates with E-coli 0157:H7 and it becomes resistant to that kind of antibiotic too.

what I thought I could do to solve this problem was:
1) cutting down the conjugative pili.
2) finding a bacteria in normal flora that doesn’t conjugate.
3) finding a bacteria in normal flora that doesn’t have a pathogen family.
are these ways possible or what are the other ways that I can do?

Hi,

If you have not read this project writeup I think you will find it very useful. Pay particular attention to the bibliography links:

https://www.sciencebuddies.org/science- ... p013.shtml

You may also find these sites informative:

http://www.bio-medicine.org/biology-def ... njugation/

http://www.bookrags.com/Bacterial_conjugation

http://www.mun.ca/biochem/courses/3107/ ... ation.html

http://en.wikipedia.org/wiki/Bacterial_conjugation

http://www.slic2.wsu.edu:82/hurlbert/mi ... lab14.html

http://mbe.oxfordjournals.org/cgi/reprint/15/4/385.pdf

I am not an expert in this field, so I hope others will answer your more specific questions.
However I think these resources will get you started on thinking through your experimental plan.

Good luck! It sounds like a fascinating project!!!

Best regards,

Barrett L. Tomlinson

Hi,

This paper on interspecies transfer of plasmids should be of great interest to you:

http://mbe.oxfordjournals.org/cgi/reprint/15/4/385.pdf

In considering your project question you really need to be aware of three different mechanisms of gene exchange: transformation, transduction, and conjugation:

http://www.emunix.emich.edu/~rwinning/g ... actrec.htm

http://www.biologyreference.com/Ar-Bi/B ... etics.html

The more I have looked into the questions you posed in your post the more interesting it looks.
I think you have the seeds of ideas for really interesting project. I wish you the best of luck with it!

Best regards,

Barrett L. Tomlinson

Hi,

Your post asked about possible ways to avoid antibiotic resistance in pathogens while immunizing beneficial bacteria against the antibiotics by using conjugation. My take on your question is that the essence of your idea is combating antibiotic resistance in pathogens - the protections of beneficial bacteria is desirable,but of secondary importance. The idea of modifying or eliminating pilli from the beneficial bacteria sounds like a long shot to me, as many resources I have seen and cited to you inicate pilli are essentially involved in conjugation, and interspecies conjugation appears widespread. I would suggest for your creative thinking you consider ways to reverse antibiotic resistance, perhaps by trying to use conjugation to insert an antisense RNA gene to the antibiotic resistance gene into an F+ plasmid in the beneficial bacteria. This might enable the beneficial bacteria to disarm the pathogens by conjugation.

Some backgound on antisense RNA in this area:
Read this first article if you read nothing else. It is a good overview of the use of antisense RNAs to fight antibiotic resistance!

http://jac.oxfordjournals.org/cgi/content/full/dkn467v1

This paper targets your interest almost exactly, as I understand it:

http://www.ncbi.nlm.nih.gov/pmc/article ... 1-0636.pdf

http://jb.asm.org/cgi/content/abstract/177/7/1655

http://jb.asm.org/cgi/content/abstract/178/1/306

http://jb.asm.org/cgi/content/abstract/175/4/1026

http://aac.asm.org/cgi/content/full/47/11/3485


http://jb.asm.org/cgi/content/full/182/ ... alsMethods

http://www.ibiblio.org/ecolandtech/Soil ... 00838.html

http://www.bioline.org.br/request?oc03093


I think this book could be very useful to you:

http://books.google.com/books?id=9Hw-ms ... ce&f=false

Please note that the above site will help you locate the book in a library close to you!

Thank you for asking the question you did. I have had a lot of fun researching this subject, and learned a lot myself. I think you have found an outstandingly interesting subject to study. I wish you a very successful project!!!!!

Best regards,
Barrett L. Tomlinson

Hi
Thank you very much.
The links in your first post were really useful (because I have not read your new links yet)
I found my answer in that articles. But it has a problem. The solution : We can only put f- bacteria in the gut ,because the f- bacteria is not donor and its only recipient and the connection between male and female is only one way connection (from male to female). But the problem is that when another bacteria comes into the gut and do a conjugation with the normal flora bacteria they send their f-factors and then our female bacteria changes into the male one.
And I think that you misinformed about a part of my project I don’t want to make bacteria resistant to an antibiotic by using conjugation. I want to select some resisted bacteria from the culture and conjugation is a disease in my project. At present I want to find out a way to stop conjugation or not letting the bacteria be conjugated.
thanks kamran

Hi Kamran,

I haven't had a ton of experience with bacterial conjugation, but the question you raise is an interesting ones.

First, a few potential roadblocks that I spot. It seems to me that you are mostly focusing on gastrointestinal conjugation (several groups of bacteria will never conjugate with gut flora), and as mentioned above, in such cases where strains like O157:H7 are present, antibiotics to clear out the virulent bacteria are generally preferred even though they are still toxic to the normal flora. O157:H7, along with a few other strains, is particularly nasty because it produces Shigella-like toxins that selectively target the larger ribosomal subunit of our eukaryotic cells (interestingly enough, this is the reverse of several of our antibiotics--many antibiotics work by blocking the prokaryotic 30S ribosomal subunit, preventing the bacteria from producing proteins and ultimately leading to cell death; imaging the 30S subunit was actually just recently awarded a Nobel prize).

It further seems that your project would be limited even in the types of gastrointestinal bacteria it deals with. Certain bacteria like cholera actually produce toxins that trigger the body to wash out the normal flora, and in such a case, antibiotics would definitely produce more good than harm.

Nevertheless, this actually makes it easier for you to do a project at school since it seems like the simplest bacteria to work with would be E. coli. This article posted above (http://www.ncbi.nlm.nih.gov/pmc/article ... 1-0636.pdf) seems promising in blocking conjugation--be careful though, this seems to mostly be dealing with the donor bacteria, not the recipients that you want to target, but is still a start in terms of understanding the genes involved in conjugation.

Another route to blocking conjugation may be to get the bacteria to produce and secrete an enzyme that catalyzes the F factor (I'm not sure this is known, I'm looking into it, but imaginably such an enzyme exists).

Also, another thought off of the top of my head is the maping of E. coli genome. I know that to do so, they used bacteria that were especially selected for high frequency conjugation to initiate conguation, and they then interrupted it via a blender or some kind of force. Obviously that would not be a valid strategy inside of the gut, but looking into the specific mechanism of interruption (eg force, disruption of certain enzymes) may provide some clues as to more ways to interrupt conjugation.

Hope this helps,
Aaron Lin

Hi Aaron
When I exampled E.coli of normal flora and O157:H7 I knew that they must be from a same family (e.g. both are E.coli)
We have 2 kinds of antibiotics: 1.specialty antibiotics 2. Non specialty Antibiotics. The first one doesn’t have any disadvantages but the second one not only kills the pathogens but also it kills the bacteria of normal flora , I want to make antibiotics more beneficial and less harmful. (Sort of making a 2nd type antibiotic, half specialty)
Thanks kamran

Hi everyone
Thanks for your links they are very useful for such a project and any one who wants to have project in this case must read that articles.
Now 1.I know how to stop conjugation in a culture. And 2.I know how to stop f+ bacteria from conjugating. Actually I can use the first way only in lab (not in the gut). And going throw the second way can’t help me because there are always some bacteria that come into the gut from outside and I can’t stop their conjugation with our normal flora , What I should do is stopping f- bacteria from being conjugated by others. Any Idea?
Thanks kamran banan

Hi,

Here is a report of an inhibitor to bacterial conjugation:

http://mic.sgmjournals.org/cgi/content/ ... 51/11/3517

Here is a patent on the subject:

http://www.freshpatents.com/-dt20090903 ... escription

And here are papers on the same idea:

http://virtualgenomeproject.blogspot.co ... tance.html

http://www.pnas.org/content/104/30/12282.full.pdf+html

Hope this is what you needed.

Good luck!

Barrett L. Tomlinson

barretttomlinson wrote: Here is a report of an inhibitor to bacterial conjugation:

http://mic.sgmjournals.org/cgi/content/ ... 51/11/3517

Hi
I really appreciate you guys. that links were very useful(mostly the link above). now sth. about 80% of theoric part of my project is finished.
best wishes
kamran banan

Hi everyone
my mentor told me that I can't stop conjugation in gut by using fatty acids.
(the link in the post above)
I didn’t understood his reason. and he wants to change my project a bit but I strongly believe in my own project. How can I find a university professor to ask my question from him? and how should I start being in contact with them?
thanks
kamran banan

WhY my reply has been deleted????!!!!!
Anyway, http://www.ncbi.nlm.nih.gov/sites/entrez
check this page, it could be help you.

Hi
my mentor says that you can’t use fatty acids for stopping the conjugation in the gut.
Why?

http://mic.sgmjournals.org/cgi/content/ ... 51/11/3517

because in our country the rate of blood triglyceride is high in most of people.
and I want to know does only fatty acids (triglyceride-glycerol) could change the rate of
blood triglyceride?
thanks kamran

Hi,

If you are looking for agents that lowet pipids and blood triglycerides, look in the the statins:

http://en.wikipedia.org/wiki/Statin

Most triglycerides are synthesized in the liver, not gotten from food.

There were two methods of inhibiting conjugation in my answer of Nov r, 2009.. The second method is described in this patent and paper:

http://www.freshpatents.com/-dt20090903 ... escription

http://www.pnas.org/content/104/30/12282.full.pdf+html

Note that the paper identifies two drugs already on the market as being effective.
I hope this answers the question.

I hope this answers the question.

Best regards,

Barrett L. Tomlinson

Hi
the founded the real reason of "why I can't use fatty acids" because by doing so 1% we helped the person to live better and if only 1% of people become ill by doing so our project doesn’t have good effect on people life and it will become useless.
somebody told me that gr+ bacteria doesn’t have pili. if this piece of information be correct then gr+ bacteria doesn’t conjugate. then my project is going to finish.
are these piece of information correct?
thanks
kamran

Hi,

I don’t think your information is correct. Gr+ is the donor of the pair and has the pili. Gr- is the recipient and has no pili until transformed by conjugation into gr+:

http://en.wikipedia.org/wiki/Pilus

I have pointed this paper out to you twice, but I cannot emphasize its importance to your question enough:

http://www.pnas.org/content/104/30/12282.full.pdf+html

It may be a bit hard to read, but it seems well worth the effort to understand it thoroughly as I understand your question. I strongly recommend that you dissect it carefully, and get help if required to understand it!

Best regards,

Barrett L. Tomlinson

I have tried to read that article. it is really hard only thing that i understood from that article is that the enzyme DNA relaxas is something like DNA Polymerase and if I could find out the inhibitor of that enzyme that is made up of protein I can inject the gene into the bacteria as a plasmid (by transformation) then our cell will make inhibitor for its own enzyme. now my problem is that what is that inhibitor?
thanks,
kamran banan

Hi Kamran,

So I took a quick glance at the article, and it seems to suggest that relaxase actually has a much different role than DNA pol. Wikipedia has a pretty decent explanation too (http://en.wikipedia.org/wiki/Relaxase), but relaxase functions in the initiation step of DNA transfer. It starts by making a very specific cut in the single strand of DNA (ssDNA) to be transferred, modifies the ssDNA to make it more stable (since ssDNA is unstable on its own), allows another signaling pathway to pull the ssDNA into the recipient cell, and then ends by reversing the modification it made earlier.

But aside from that, the article already lists two potential inhibitors of relaxase, clodronate and etidronate, which are already FDA-approved drugs to treat a different condition. You wouldn't need to transform the bacteria in this case since these inhibitors could probably be purchased for research purposes, so you could just treat bacteria with the drugs directly. Good luck!

Hope this helps!
Aaron Lin

Hi
thanks for your helps
because in near future this drug is going to be used by human been the inhibitor must be made of amino acids and it must be suicide inhibitor. those inhibitors are made of phosphate and they cant help me.
there are some other ways to inhibit conjugation but there are as hard as this one.
thanks,
kamran banan

Hi Kamran,

I'm not sure quite what the problem is; perhaps you could clarify for me? The inhibitors are bisphosphonates (http://en.wikipedia.org/wiki/Bisphosphonate), so you are right in that they are not made of amino acids. However, the aforementioned paper demonstrates them to be effective inhibitors of the relaxase complex by interacting with two metal-containing active sites on the complex (metal is particularly good at binding with phosphates due to charge interactions). Moreover, it seems to selectively affect bacterial cells since human cells do not contain the relaxase enzyme (at least I don't think that they do). But I would suggest looking more into it first before abandoning the idea entirely.

Hope this helps!
Aaron Lin

Hi Aaron
The problem appears when a pathogenic bacteria from outside of body comes into the body and if this bacteria conjugates with our bacteria then the pathogenic bacteria become resistant to that specific antibiotic. so I must stop conjugation.
now think that I used amount of that inhibitor and think that after adding this amount all of enzymes were inhibited and because inhibitors are irreversible after reaction they become useless but bacteria has DNA and ribosomes and it will reproduce the enzyme. now we have enzyme but no amount of inhibitors. so the inhibitor must reproduce by bacteria too.
thanks,
Kamran Banan

Hi Kamran,

I do see what you're saying now, in terms of the problem with getting long-term inhibition. At this point though, I don't think you need to worry about long-term inhibition. Though an actual drug version would take that into account, most of the work you want to do is likely more exploratory, testing the efficacy of the drug before worrying about the details. After all, actual drugs often are taken on a scheduled basis, so the actual mechanics of drug delivery and timing are to be worked out later. For now, I feel that showing that the bisphosphate inhibitors were selectively effective in stopping conjugation would be very much a science fair worthy project.

However, if you are really concerned with long-term inhibition, try considering coupling this drug inhibition with RNAi to knock down gene expression of the relaxase enzyme. I have no idea how well (or if at all) the bacteria will take up miRNAs, but it would be worth a shot.

Hope this helps!
Aaron Lin

Hello,

Gram positive bacteria can conjugate, however, the systems they use are different from Gram negative bacteria. This process is less well understood in Gram positives. It is believed that Gram positive conjugation involves pheromones – see the following links.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC156469/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC176677/

As for pili in Gram positive bacteria, again this is less well understood than in Gram negatives, but is appears they do have pili, see the following links. Also, if you google “Gram positive pili” links to several articles come up.

http://aetiology.blogspot.com/2005/10/p ... -gram.html
http://www.nature.com/nrmicro/journal/v ... o1443.html

I hope this is helpful.

Mary