Similarities Between E-coli K-12 & Propionibacterium

[matthewjbrewer4]

So I am currently working on a science project titled "Effectiveness of Acne Vulgaris Treatments Using Escherichia Coli K-12."

I used this idea on Sciencebuddies: http://www.sciencebuddies.org/science-f ... p019.shtml

However, I did change up the experimental design quite a bit, but I used the same method of the disks to determine the zones of inhibition. I've been working on it for a while now; basically I have 4 medications: Benzoyl Peroxide 10%, Salicylic Acid 2%, Adapalene .1%, and a homemade solution consisiting of 2000 microliters apple cider vinegar, 1000 microliters tea tree oil, and 500 microliters distilled water (to keep things constant, I also added 500 microliters of water to the 3 other medications). My partner and I looked to see if there was any Propionibacterium that our teacher could order in the catalog, however we were unable to find any. However, based on the experiment previously conducted (explained in the link above), we can use E-coli bacteria in place of Propionibacterium. I was told today in class that the judges will ask questions about certain things and so my partner and I realized that we really have to know EVERYTHING. My partner and I have been researching for a while, but we were having trouble finding out the similarties between E-coli and Propionibacterium. We are using E-coli because the experiment previously conducted said that it is okay, but we wanted to figure out how they are similar, and why it is okay to use E-coli. I've been looking on google with no luck. Anyone out there who might be able to help?

Thank you very much, I appreciate it!
-Matthew (12th Grade)

[matthewjbrewer4]

I should also probably add, I did some "pre-testing" to test which concentrations of the apple cider vinegar and tea tree oil would work best. So I tried 2000 microliters apple cider vinegar to 1000 microliters tea tree oil (2 to 1 ratio), 2000 microliters apple cider vinegar to 500 microliters tea tree oil, 4000 microliters apple cider vinegar to 1000 microliters tea tree oil, 4000 microliters apple cider vinegar to 500 microliters tea tree oil, 6000 microliters apple cider vinegar to 1000 microliters tea tree oil, 6000 microliters apple cider vinegar to 500 microliters tea tree oil, 8000 microliters apple cider vinegar to 1000 microliters tea tree oil, and finally 8000 microliters apple cider vinegar to 500 microliters tea tree oil. And then I had of course the controls, containing just distilled water, just apple cider vinegar, etc. Through experimentation, the 2000 microliter apple cider vinegar to 1000 microliters tea tree oil (2 to 1 ratio) solution/mix had the highest average zone of inhibition. This is why I am comparing this solution (which is a fully natural solution) to the other 3 experimental groups (the benzoyl peroxide, salicylic acid and adapalane). I'm curious to see if the over-the-counter medications that you can buy at stores such as target, longs, etc. (the salicylic acid and benzoyl peroxide) are just as effective as a prescription, such as adapalene .1%. I am also curious to see if a "home" remedy is effective. I hope this all makes sense! Thanks again! -Matthew

[donnahardy2]

Hi,

Welcome to Science Buddies! This is a really great project.

Proprionibacterium acnes is an anaerobic, Gram-positive bacterium that lives on fatty acids and produces propionic acid and a number of enzymes that can degrade skin. It is in the bacteria kingdom and Actinobacteria phylum.

http://mic.sgmjournals.org/content/129/5/1301.full.pdf

Escherichia coli is a Gram-negative, facultative anaerobic, motile bacterium that is found as normal flora in the intestinal tract that lives on sugars and produces lactic acid, succinic acid, ethanol acetate, and carbon dioxide. It is in the bacteria kingdom and Proteobacteria phylum.

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

So both of these organisms are bacteria and they both live on humans, but their structure and physiology are very different. It is likely that your results might not apply to P acnes unless you were able to do the experiment with P. acnes. However, you were correct to use E. coli instead of the pathogenic bacterium if you did not have access to a regular microbiology laboratory and your experimental design sounds very good. I think it is always a good idea to do a pilot experiment to find out what will happen so you will get good results on your final experiment.

Do you have any results yet?


Donna Hardy

[matthewjbrewer4]

Hello Donna,

Thank you so much for the response!

So I've been working on this project for a while now, and I'm very interested in it myself being an 18 year old who has suffered from acne, just like most if not all other teens/young adults have in their lives. However, I haven't been able to compare the homemade remedy to the adapalene .1%, benzoyl peroxide 10% and salicylic acid 2%. Due to some setbacks (For example, one of my controls showed no bacteria and we were not sure if that meant that the control containing the tea tree oil alone killed all the bacteria, or the bacteria just didn't grow for whatever reason), it took us a while to do the pretesting (where we tested different mixes of the apple cider vinegar and tea tree oil, where we found the 2000 microliters apple cider vinegar and 1000 microliters tea tree oil mix to be most effective (I had 3 disks per plate, and this had the largest average zone of inhibition (in mm). So the only results we have gotten so far is the zones of inhibitions for the different mixes of apple cider vinegar/tea tree oil. This upcoming week we will be doing trial 1 (we started it this week, but had some complications).

So long story short, no not results involving the 3 other medications. For next week, what we will be doing is placing the 2000 microliters apple cider vinegar and 1000 microliters tea tree oil in a beaker and weighing it, and then we can make sure that the adapalene/benzoyl peroxide/salicylic acid are all of the same weight as the homemade solution (to calibrate the experiment).

So, that is what I am not so sure about. So they are both bacteria that live on humans, however they are also very different. So how can I justify (specificly to the judges) using E-coli instead of Propionibacteria. From what I read, E-coli is much more safer (BSL 1), and it's also much more easier to use (grows much faster; after adding the e-coli and medicated disks to the plates, the very next day showed results). However, is that enough? I'm afraid the judges will ask me "So, the actual causing agent of acne vulgaris is propionibacteria, or p. acnes, however you are using E-coli. How is this going to show which medication is the most effective in fighting acne?" And yes, I am working in my biotechnology classroom.

Thank you very much for all your time!

-Matthew

[donnahardy2]

Hi Matthew,

Thanks for the additional details on your experiment; it is really helpful for me to understand exactly what you are doing. The lack of growth on your control was unfortunate, but you are correct; you can’t make any conclusions without a control.

I have another idea that should help justify your project. Here is an abstract of a paper that investigated the mechanism of antimicrobial action of tree tea oil against a Gram-positive organism, Staphylococcus aureus. The authors concluded that the active ingredients in tree tea oil (1,8-cineole, terpinen-4-ol, and alpha-terpineol) caused damage to the cell membrane that made the bacteria more sensitive to autolysis.

http://aac.asm.org/content/46/6/1914.short

The good news is that both Propionibacterium and E. coli have cytoplasmic membranes with similar structures. The cytoplasmic membrane t is composed of a phospholipid bilayer and may have other components such as fatty acids included. There is a paragraph on the cytoplasmic membrane in the Wikipedia article with a little more information.

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

So if the tree tea oil/vinegar combination is effective against E. coli, it should have the same activity against Propionibacterium. Antimicrobial agents that have activity against a wide variety of organisms are described as “broad spectrum” agents. Your idea of mixing tree tea oil with vinegar was brilliant; the tree tea oil should damage the cell membrane and allow the vinegar to lyze the cells. Your experiment should verify your hypothesis.

I recommend that you do a Google search and try to find more information about E. coli and Propionibacterium cell membranes, studies of antimicrobial activity of tree tea oil, vinegar, benzoyl peroxide, and salicylic acid. Including information from the scientific literature will improve your project.

Good luck on your next experiment. Let me know about your results.

Donna Hardy

[matthewjbrewer4]

Thank you very much, again, Donna!

So that news is awesome! Honestly I chose tea tree oil and apple cider vinegar because of their antibacterial properties, but that, in my opinion, is a perfect justification. So thank you very much!

So that justifies the tea tree oil/apple cider vinegar "home" remedy, so I've been google searching some information about salicylic acid/benzoyl peroxide/adapalene .1%

So I found a pretty helpful website: http://www.healthcareveda.com/post/Does ... teria.aspx

"Unlike the over-the-counter ingredient benzoyl peroxide, salicylic acid penetrates into pores and exfoliate the walls of the pores to eliminate the clogging residue that causes acne. For this reason, it is particularly effective against comedonal acne, or blackheads. The main difference between Benzoyl peroxide and salicylic acid is that benzoyl peroxide kills bacteria on the skin's surface, whereas salicylic acid removes the clogging residue. Benzoyl peroxide can be used in conjunction with salicylic acid for a comprehensive over-the-counter acne regimen. Salicylic acid products must be used continuously to prevent acne on acne-prone skin. Use the lowest possible concentration to avoid irritation."

"This ingredient works on acne by actually helping to get rid of the dead skin cells that are on the skin. When the dead skin cells are not shed, they can often fall into the pores or build up on the skin. If the dead cells get into the pores and mix with sebum and bacteria, this can cause acne breakouts to occur. Of course, not only does the salicylic acid help to get rid of the dead skin cells, but it can also help to break down whiteheads and blackheads as well. Salicylic acid also helps the top layer of skin to peel off as well, which helps get rid of acne. While salicylic acid does many things, one thing it cannot do is kill the bacteria on your skin and in the pores."

So benzoyl peroxide somehow KILLS the bacteria, whereas salicylic acid essentially cleans out the pores and clogs in your skin. I am unsure how benzoyl peroxide kills the bacteria, as in if it just lyses it like apple cider vinegar, or if it has its own way of killing the bacteria, however based on this information above, it seems as though the benzoyl peroxide 10% should show some form of results, whereas the salicylic acid might not show anything, because it doesn't actually kill the bacteria according to this article. I hope that makes sense!

So I need to figure out how benzoyl peroxide kills the bacterial cell, and also about adapalene and how it treats acne specificly. Once I have this information, then I can hopefully justify the future results. Thanks so much for all the help!

[matthewjbrewer4]

Okay, so I found a website that might explain how the benzoyl peroxide kills the bacteria, but it's still a bit unclear:

"Benzoyl peroxide works by destroying p.acnes, the bacteria that contribute to the acne condition. Benzoyl peroxide acts as an antiseptic (killing bacteria) and oxidizing agent. By providing oxygen to the follicles it kills p.acne bacteria, which need an oxygen free environment to survive."
http://www.acnetreatmentsguide.com/rodanfields/

So it is an antiseptic, however does it have the same affect on all bacteria or just p.acnes, I am not sure.

And then I found this website on adapalene:
http://www.dailyglow.com/does-differin- ... -zits.html
"You can get a tube of Differin in a 35- or 45-gram tube, but both tubes are 0.1-percent adapalene. This ingredient successfully sloughs off dead skin, just like Retin-A. It speeds up cell turnover so that pores get cleaned out quickly. As you know, clogged pores turn into unsightly zits, blackheads and whiteheads."

So, once again, I don't see where it says that Adapalene .1% directly kills the bacteria, because instead it says that it speeds up "cell turnover."

I was unsure of the definition of cell turnover, so I looked it up online and the resulting definition was as follows: "In skin care, the term used to describe the constant shedding of dead skin cells and subsequent replacement with younger cells." http://acne.about.com/od/acneinformatio ... rnover.htm

Furthermore, I found another website, http://www.myacnetreatmentreview.com/fi ... -bacteria/, and it says "Sold under the trade name Differin and available as a cream, gel, or pledget (small compress), adapalene opens pore and counteracts the inflammation caused by acne bacteria. The bacteria are removed, however, as pores open and they can drain to the surface. Adapalene has little or no direct antibacterial action," which further proves that adapalene does not directly kill the bacteria. So that might be something important to know.

Another website, http://www.acnetreatments-help.com/3530 ... treatment/, claims that Benzoyl Peroxide is a "Broad Spectrum" antibacterial agent, and that it " it is a strong bactericidal agent, killing the p. acnes bacteria responsible for acne directly." That still doesn't show that it'll have the same effects on E-coli as it will on P.acnes, however hopefully that means something.

Once again, thank you for taking your time to read this and help me out. It's much appreciated!
-Matthew

[donnahardy2]

Hi Matthew,

You’ve gotten a lot of good information that will help you understand the science behind your project. Here's some more:

Here is a scientific article on the mechanism of adapalene.

http://www.ncbi.nlm.nih.gov/pubmed/17518990

This drug apparently works because it decreases the epidermal inflammatory response of the human. It apparently does not have direct antimicrobial activity. Specifically, adapalene increases CD1d expression and decreased IL-10 and TLR-2 expression by keratinocytes. The combination apparently makes the environment less hospitable to P. acnes growth.

http://www.ncbi.nlm.nih.gov/pubmed/17518990

CD1D is a glycoprotein that is expressed on the surface of certain cells that activate a special class of lymphocyte T cells called Natural killer T (NKT) cells.

IL-10 or interleukin 10 is a human cytokine that inhibits inflammation.

http://www.bio.davidson.edu/courses/imm ... in-10.html

TLR-2 is a human protein that helps the immune system by helping to recognize pathogens and activates the immune system.

http://en.wikipedia.org/wiki/TLR_2

Since this drug works by affecting the human immune response, you might not see any directly effect of the drug on E. coli in a Petri dish.

Salicylic acid works by removing the skin and also has an anti-inflammatory effect. . Since the P acnes grows on the lipids in the skin, this drug must work by reducing the food source for the bacteria.

http://dailymed.nlm.nih.gov/dailymed/ar ... veid=24328

Benzoyl peroxide is an oxidizing agent. It breaks down to form oxygen and benzoic acid. Since Proprionibacterium acnes is an anaerobic organism, the oxidizing properties of this drug are probably directly toxic to the bacterium.

http://www.ncbi.nlm.nih.gov/pubmed/9142553

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


E. coli is a facultative anaerobic bacterium, which means that it can grow with and without oxygen, so the benzoyl peroxide would probably not be as toxic for the E. coli as it is for P. acnes.

Everything you have chosen for this project works by a different mechanism to inhibit P. acnes. Your results should be very interesting.

Donna Hardy

[matthewjbrewer4]

Once again, thank you so much Donna.

So it seems like both adapalene .1% and salicylic acid should not show much results, and chances are the results of the benzoyl peroxide 10% would not be as strong on the E-coli is it would on Propionibacterium, but it could still show results. I was thinking, based on the information above, it might be better that I use something else instead of the salicylic acid and adapalene, because I'm not actually going to be testing these medications on others' faces, but rather only on the petri dishes. I looked online, and I read some stuff about natural honey and it's strong antibacterial effects:

http://www.aschoonerofscience.com/recen ... -bacteria/

I'm not sure if this website talks about honey in general or a specific type of honey, however based on what I have read from this site, there is a protein called bee defensin-1, which works as an antibiotic. I've also heard that honey has general antibacterial properties, and it's acidity is also beneficial I believe. So maybe I could replace that with the adapalene .1%/salicylic acid 2% with the honey. And honey also adds a second "all natural" remedy (along with the tea tree oil/apple cider vinegar).

Thanks again for all the help and support!
-Matthew

[donnahardy2]

Hi Matthew,

You are welcome! I’m always happy to offer advice for science projects.

Since honey is supposed to have broad spectrum antibacterial activity and acts directly against the bacteria, you could use it instead of the adapalene or salicylic acid. Or you could also do more dilutions of the tea tree oil and acetic acid to show a dosage effect. If your control works, you will have a good project either way, with good data for discussion.

I finally found information on vinegar. Here is a paper that measured the antibacterial activity of vinegar against a variety of bacteria, including a pathogenic strain of E. coli.

http://www.unc.edu/depts/spice/dis/ICHE ... an-p33.pdf

Here is an article that investigated the antibacterial activity of chitosan and chitosan plus different concentrations of acetic acid against E. coli. The experimental design is an example of the suggestion I had about investigating dosage or varying the concentration of the substances you are testing.

http://www.mwit.ac.th/~teppode/th_1_1.pdf

Both of the articles above verify that acetic acid does have antibacterial activity, but I could not find an article that described the molecular mechanism. Is it the low pH, or is there something about the chemical structure of acetic acid that makes an effective antibacterial agent?

Donna Hardy

[matthewjbrewer4]

Thanks again, Donna!

I was reading through http://en.wikipedia.org/wiki/Acetic_acid, and I did not find the answer, however it did say, "In the clinical laboratory dilute acetic acid lyse red blood cells in order to facilitate microscopic examination," so it in some way lyses the cell, as you stated in an earlier post. I have read a couple of times now that there was a study that took place that showed that vinegar "kills 99 percent of bacteria, 82 percent of mold, and 80 percent of germs (viruses)."

http://www.care2.com/greenliving/vinega ... z1itRizYUY

It also says on wikipedia that, " A 1.0 M solution (about the concentration of domestic vinegar) has a pH of 2.4."

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

So I do not see anywhere where it says directly that its acidity is one reason why it's such an effective antibacterial agent, and also looking at it's structure:

In solid acetic acid, the molecules form pairs (dimers), being connected by hydrogen bonds.[17] The dimers can also be detected in the vapour at 120 °C (248 °F). Dimers also occur in the liquid phase in dilute solutions in non-hydrogen-bonding solvents, and a certain extent in pure acetic acid,[18] but are disrupted by hydrogen-bonding solvents. The dissociation enthalpy of the dimer is estimated at 65.0–66.0 kJ/mol, and the dissociation entropy at 154–157 J mol−1 K−1.

I do not see any direct effects on bacteria. On your idea about the dilutions, I will look into that and definitely consider that. Thank you very much!

-Matthew

[donnahardy2]

Hi Matthew,

This is interesting. You found some good information, but, like me, did not\ find exactly what we were looking for. Someone must have done this study and published the results, but we can't find it. You do have plenty of background information on the science behind your experiment without this information, and in the event a science fair judge asks you about this, you can say that you looked.

I see that your project is due in about 4 weeks. Will you be able to repeat your experiment with a working control and have a week or two to write up the board?

Donna Hardy

[matthewjbrewer4]

Hey Donna,

I am not exactly quite sure of the due date, because it was going to be due earlier but our teacher pushed it back. But I have been in the classroom after school working on it for a while now, and she knows that because she has been in the classroom with me and my partner, so I'm sure she is understanding.

But yeah, basically I think I'm going to just stuck with the honey. So I'll have a control containing just tea tree oil, a control containing just apple cider vinegar, then 3 experimental groups: one with just raw wildflower honey, one of the benzoyl peroxide 10%, and one of the 2000 microliters apple cider vinegar/1000 microliters tea tree oil (I will be weighing this, and then making sure that the benzoyl peroxide and honey come out to be the same weight, to calibrate the experiment). And I will do 3 trials of this, and it takes about 1 week per trial (because I have to make the agar plates, put the disks in the solutions and let them soak over night, place the disks and e-coli cultured in luria broth in the plates, etc.), so assuming everything goes okay, it should be about 3 more weeks until we finish. So I think everything will work out.

Thank you!
Matthew

[donnahardy2]

Hi Matthew,

I sounds like you have a good plan to complete your project and your experiment should give you lots of data to analyze. Three separate trials of the same experiment will be impressive. Please do let me know about your results.


Donna

[matthewjbrewer4]

Okay, I'll make sure to keep you updated and see how the results go. Thank you!

-Matthew