Natural Antimicrobials against Bacteria

[yvetteds]

Hi - I hope you don't mind a comment/ question from me - I've been following all the detailed advice from Connie - And how great it is for you to have such a knowledgable resource! I've taught high school biology for 22 years and as soon as I saw your last question, I couldn't help but ask -What do you mean by DEVELOP the resistance?
The reason I am asking is because in my experience teaching evolution, classic examples are bacteria populations that have 'developed' resistance to certain antibiotics over repeated use of the antibiotic. But when we teach this concept, we are talking about the POPULTiON - meaning that one individual bacteria cell is not changing in response to the antibiotic - but that those bacteria cells that already have a mutation / resistance gene / will survive and those that did not have this resistance die - so the ones left on the plate according to Connie's directions survived because of natural selection. Is this what you are trying to find out? It's not the essential oil that changed the bacteria - they already had the resistance gene , so they survived- right?

[deleted-160001]

Oh I see, so it's not that the bacteria change, but the genes that they have are already mutated? I was wondering if I repeated the exposure of natural antibiotics to the same bacteria, even though the first time they were susceptible, the second or third time, since they already were exposed to it, they would be resistant, but I see what you mean. Thank you yvette.

[yvetteds]

Connie - is this correct? I don't want to confuse what he is trying to do- I just wanted to understand his purpose and what he thinks DEVELOP means. My teaching experience has shown me that the hardest concept in teaching evolution is that populations change as a result of selection of the most fit survive due to the environment - in this case the presence of the antibiotic.

[deleted-132180]

Hi both,

According to my understanding, what you said is right yvetteds. In a population of bacteria out in nature, many genes are present and each of these bacteria may express these genes in different ways. When exposed to an antibiotic or antimicrobial substance, this population of bacteria should die off if they are susceptible. However, it is possible that a small percentage of the population may survive due to expressing some gene in a different manner that confers resistance, or there may have been natural mutations that have occurred that (fortunately for those bacteria) confers resistance. Since the "resistant" bacteria are the ones that survive, they are the only ones that will reproduce and eventually all their descendants will have that same resistance.

However, if bacteria are repeatedly exposed to antibiotics, this stress could cause the bacteria to start mutating like crazy in hopes that certain mutations will allow them to cope with and survive this stress. This is potentially how many antibiotic resistant bacteria have arisen in clinical settings because overuse of antibiotics (especially in situations where they are not necessary) can greatly hasten the development of resistance. If you are interested in this topic more, here are some good articles to look at:

1) http://www.sciencedaily.com/articles/a/ ... stance.htm
2) http://www.icr.org/article/do-bacteria- ... tibiotics/

Other microbiologists in this forum... I hope I have explained this correctly. If not, feel free to share your opinion or correct anything.

According to my understanding, yvetteds, I think Anthony is saying that he is trying to test whether the bacteria will eventually "develop" resistance against the antimicrobials if you continually expose them to the antimicrobial. So Anthony, that prompts me to ask this question: What is the main question that you are trying to test? From our earlier discussions, it seems like you want to see whether the essential oils you've chosen have antimicrobial activity. In this case, the procedures that you've proposed are sufficient to answer the question of whether your compounds will kill/inhibit growth of your bacteria at whichever concentrations you have chosen to test. However, if your main question is if the bacteria can develop resistance to these antimicrobial compounds after repeated exposure, that will be a more difficult question to answer. First of all, you have to determine their susceptibility to these compounds. If they're already resistant to these compounds, that means that either these compounds don't have antimicrobial activity, or the bacteria are naturally resistant to them. If the bacteria are susceptible to these compounds, you would then have to repeatedly expose these bacteria to the antimicrobial and see if you eventually select for growth of resistant clones. I would suggest to not do this because it can potentially be dangerous if you select for a clone that is highly resistant to your antimicrobial compound because not only can the compound no longer kill your bacteria, but there is no way of knowing how this will affect the bacteria's response to other antimicrobials and antibiotics. However, if other experts have a different opinion, please share. There are also other parameters you have to consider because: 1) You don't know how long you have to expose the bacteria to the antimicrobial to see resistance show up, if it ever even shows up. 2) If all your bacteria died after the first exposure, there will be no other bacteria for you to continually expose the compound to.

And Anthony, one last thing about the culturing without the shaking, you can take the culture out after 24 hours and see if the broth has become more turbid--if it has, that indicates that the bacteria have grown. You should probably just put in a tube with broth but no bacteria inoculated into it as a negative control for growth. Your negative control broth should still remain clear after 24 hours in the incubator, whereas your broth with bacteria inoculated into it should become more turbid if the bacteria did grow!

I think the project you have proposed on looking at the antimicrobial activity of essential oils is already quite interesting and I think it will give some cool results! As for the question about whether you can make antimicrobial resistant clones, I would suggest to not do that due to potential health hazards, BUT... if other experts have any ideas to conduct these experiments safely or think that it's not a huge problem selecting for resistant clones, please share your ideas because I am sure that Anthony would benefit from them! Let me know if I didn't explain some parts clearly, Anthony, or if you have more questions. It is very important for these science fair projects that you clearly understand how these experiments are testing your question/hypothesis!

Best,
Connie

[yvetteds]

Hi again - Thanks Connie for all that information! Yes, exactly what we teach in high school biology - evolution - all about diversity in the genetic make up and eventually natural selection - so I was assuming this would be why certain bacteria would survive the antibiotic treatments.. microevolution -
but from that first article :

A new line of research has produced tantalizing results. Evidently, when stressed, some microbes go into a mutation mode, rapidly producing a variety of strains, thereby increasing the odds that some will survive the stress. This has produced some interesting areas for speculation by creationists, but it still mitigates against evolution. There is a tremendous scope of genetic potential already present in a cell, but E. coli bacteria before stress and mutation remain E. coli. Minor change has taken place, but not true evolution.

-- gives us some food for thought! but i'm not sure how you would know which process was happening? is it natural resistance due to a genetic variation that was there all along? or is it mutations because of repeated stress? As a high school biology teacher, our goal was to get kids to stop thinking that the individual could change 'because' of the environment - which this seems to be - Very interesting!

As you mentioned, it's really important to isolate the hypothesis - what is it exactly that this experiment is testing? This is really interesting - and such a good lesson that even when we think we know what we are looking for, more questions come up - forcing us to be even more specific and careful in our experiment design.

[deleted-160001]

Thank you connie, the idea about the negative control was very helpful! Sci B told me about the magnetic stir plate, so i think the culture will grow just fine. And thanks for ur warning about the mutations, and thank you yvetteds for explaining that. I will start the experiment next week, I'll tell you guys how it goes!

[deleted-132180]

Hi both,

I'm glad that you found this topic interesting. Microbes are fascinating organisms.

Good luck with your experiments! I'm excited to hear about your results.

Best,
Connie