MINT V.S. BACTERIA, WILL BACTERIA RESIST ?

MINT V.S. BACTERIA,

WILL BACTERIA RESIST ?

Arriving in Petnica, I had the idea of making a project linked with medicine and drugs research, as we had the opportunity to do a full microbiology project. When we did the field trip, one of the searcher talked about biodiversity of plants in the region, and he mentioned some ‘antibacterial properties of mint. Having done a bibliography project about antibiotic resistance, this idea hooked me up and I’ve done some research about it.

Several studies showed the antibacterial properties of peppermint and other varieties of mint. The more often, searchers use peppermint oil extract (POE - I need to do an abbreviation because I will talk A LOT about it). This extract concentrates chemical compounds such as menthol or menthone that are dangerous for bacteria. I wanted to do the same kind of study, and compare the impact of POE on both the growth and the survival of bacteria, and compare the results of two different species, one gram negative () and one gram positive (). So I came up with this question : What is the impact of POE on bacterial growth and survival of E. coli and B. subtilis ? The goal was to determine if POE was an efficient antibiotic for these strains, and if yes, at which concentration.

The protocol to study if it was bacteriocide (kills the bacteria) was a protocol based on the CFU (if you don’t know what this is, I advise you to check this website which is really comprehensive, or take a biology course). Basically, I would expose bacteria to POE, at different concentrations, and perform a classical CFU afterwards. Unfortunately, technical issues gave me unrelevant data to use for any conclusions.

The other protocol, to study if POE was bacteriostatic (make bacteria unable to grow or divide) consisted in spreading bacteria over a plate, and then disposing a paper disc soaked with POE at different concentrations on this same plate. After incubation, I would measure the surface where bacteria didn’t grow, called “inhibition zone”.

My data plotted looked like this :

On this graph, you can see the different surfaces of inhibition zones (in cm²), according to the concentration of POE. It presents the results for both B. subtilis and E. coli. The concentration 1 for B. subtilis is not shown on the graph because no bacteria grown in this case. The two types of points represents the two species as you can see on the legend. For each concentration on each species, I’ve made three replicates (measures in the same conditions), this is why there is always three times the same kind of point for a single concentration.

As we can see, controls show a clear difference of values between them (C=0 and C=1), and it seems that the values of surface of inhibition are getting higher with the concentration.This tendencies cannot be shown statistically, as I lack of data (but you always lack of data, right ?)

I can conclude from these results that POE does have a negative impact on bacterial growth, for both E. coli and B. subtilis. It seems that the more concentrated is POE exposure, the more it inhibits the growth of both species. However we can’t see a clear difference between gram + and gram - results. Finally, as further experiments, I would expand my range of bacterial strains to some multi-resistant strains, and compare efficiency of POE with a classical antibiotic. Could we use in the future, antibiotic based on POE for some infections ? Why not ? It’s cheap, easy to grow and it smells quite good !

If you want to know more…

Get an insight on research which is done in this field :

-Basheer A. Al-Sum, Abdullah A. Al-Arfaj. Antimicrobial Activity of the Aqueous Extract of Mint  Plant. Science Journal of Clinical Medicin. Vol. 2, No. 3, 2013, pp. 110-113. doi: 10.11648/j.sjcm.20130203.19

-McKay, Diane L., and Jeffrey B. Blumberg. "A review of the bioactivity and potential health benefits of peppermint tea (Mentha piperita L.)." Phytotherapy Research 20.8 (2006): 619-633

-Singh, Rajinder, Muftah AM Shushni, and Asma Belkheir. "Antibacterial and antioxidant activities of Mentha piperita L." Arabian Journal of Chemistry 8.3 (2015): 322-328

-Thomson Healthcare (2007). PDR for Herbal Medicines (4th ed.). p. 640

Are Grapefruit Seed Extract real antimicrobial components

Are Grapefruit Seed Extract real antimicrobial components ?

(Comparison of the Gentamicin and  Grapefruit Seeds Extract antimicrobial effects on Escherichia coli.)

 

The all story started from a compost. The rumors told that Jacob Harich, (1919-1996) a german researcher in physics but also a passionate gardener, would have observed one day a grapefruit without any kind of microbial rot around it, in his compost. From this observation started the idea that Grapefruit Seeds could be antimicrobial, preventing it from decomposition. This is the beginning of the great and controversial adventure of the Grapefruit Seed Extract. Let's call it GSE.

Today, GSE had been commercialized all over the world and can be bought in internet to treat microbial and fungal infections.

GSE is composed of Grapefruit (Citrus paradisius) Seeds grinded, dissolved with ethanol or water and added to glycerin.

But since more than twenty years, the scientific community are in disagreement about the possible antimicrobial capacity of GSE. Why ?  

In 1999, a German scientific team observed an antimicrobial activity in all the commercially available  GSE they tested but not in their several self-made GSE. Moreover, they found preservatives known to be bactericidal such as benzethonium chloride, triclosan and methyl paraben in the commercialized GSE, letting them to suppose that the antimicrobial capacity of GSE would be merely due to these synthetic agents.  (Woedtke T, Schlüter B, Pflegel P, Lindequist U, Jülich WD (June 1999). "Aspects of the antimicrobial efficacy of grapefruit seed extract and its relation to preservative substances contained". Pharmazie. 54 (6): 452–6. PMID 10399191).) From this moment until today, some studies support this conclusion while other still demonstrate the real antimicrobial capacity of GSE.

    “If you can’t agree each other, I will attempt to resolve this mystery on my own ! (modestly)”

    What was my experimentation ? 

I made my own GSE extract :

I decided to study the impact of GSE on  the growth of Escherichia coli, a bacteria very used in microbiology. To determine the growth rate, which is the number of colony forming units per ml according to the time, I used a colorimetric method, using spectrophotometer measures at 620 nm.  Indeed, while bacteria are growing and cloning, the opacity of a solution is increasing. To detect Absorbance of a solution according to time can let us determine the growth rate.  The main idea was to compare the E coli growth rate in different condition : E coli with bacteria / E coli with gentamicin ( a well known antibiotic ) / E coli with GSE. 

Each conditions has a different growth rate which is the slope of the curves.  The 6th measurement in all the differents conditions and replicates seems to be  a technical error of the spectrophotometer so we will not taking it in account.

The test with only E coli is a positive control, which allow us to compare this growth rate with the growth rate of the two other conditions. We observe that the Absorbance according to the time in the solution (5) composed of Gentamicin+E coli and LB is approximately constant. There is an insignificant growth rate, meaning that the bacteria were killed or couldn’t divide since the first contact of the gentamicin.

We did the average of the 6 replicates GSE+E coli +LB (6)  coli growth rate  and it was equal to  3.81×10⁻4 while the growth rate average of the 6 replicates E coli+ LB was equal to 1.97*10⁻4.

This two different growth rate are statistically different, by using a bilateral average comparator.

What is striking : E coli alone grows slower than E coli in contact with GSE !

It means the growth of E coli isn’t inhibited by the GSE, and is even faster ! What could be the different reason leading to this result ?

1. The “home made” Grapefruit Seeds Extract without any kind of preservatives has no  antimicrobial activities.
2. The GSE concentration in this experimentation was to low. I couldn't determine the concentration I used. Maybe I could redo the experimentation using a way for my GSE to be at the  Minimum Inhibitory Concentration determined by this paper : (“Antimicrobial activity of grapefruit seed and pulp ethanolic extract”, ZDENKA CVETNI, SANDA VLADIMIR-KNE@EVI, Acta Pharm. 54 (2004) 243–250, 2004”. )
3. The “home-made” GSE get more opaque over time ?

The next step of my experimentation would be to redo the spectrophotometric measures and determination of the growth rate but with a commercial GSE in order to compare it with the “natural” GSE+ E coli growth rate. If this growth rate would be lower than the control one,  this study would be really in favour of all the researcher thinking that the natural  Grapefruit  Seeds extract is not antimicrobial, and that this capacity is merely due to synthetic preservatives agents.

Acknowledgement : Petnica Center, Centre de Recherche Interdisciplinaire, Fondation Bettencourt Schueller, licence Frontière du Vivant, my wonderful tutor Gaspard, the assistant team, Bore promotion…

If you want to know more about this subject :

Reference :

-1(Woedtke T, Schlüter B, Pflegel P, Lindequist U, Jülich WD (June 1999). "Aspects of the antimicrobial efficacy of grapefruit seed extract and its relation to preservative substances contained". Pharmazie. 54 (6): 452–6. PMID 10399191).)

-  2 “Antimicrobial activity of grapefruit seed and pulp ethanolic extract”, ZDENKA CVETNI, SANDA VLADIMIR-KNE@EVI, Acta Pharm. 54 (2004) 243–250, 2004”

- “EFFECTS OF OREGANO ESSENTIAL OIL, GRAPEFRUIT SEED

EXTRACT AND THEIR COMBINATION ON THE GROWTH

AND SURVIVAL OF Salmonella Typhimurium AND Listeria

monocytogenes IN POULTRY FILLETS UNDER MODIFIED ATMOSPHERE PACKAGING

Tolga Kahraman 1 , Ghassan Issa 4 , Sema Sandikci Altunatmaz 2 , Beren Basaran Kahraman 3 , Filiz Aksu 2 , Ali Aydin 1 *, Harun Aksu ,

-Armando C, Maythe S, Beatriz NP (December 1997). "Antioxidant activity of grapefruit seed extract on vegetable oils". Journal of the Science of Food and Agriculture. 77 (4): 463–7.

-Takeoka G, Dao L, Wong RY, Lundin R, Mahoney N (July 2001). "Identification of benzethonium chloride in commercial grapefruit seed extracts". J. Agric. Food Chem. 49 (7): 3316–20. doi:10.1021/jf010222w. PMID 11453769.

-Cvetnić Z, Vladimir-Knezević S (September 2004). "Antimicrobial activity of grapefruit seed and pulp ethanolic extract". Acta Pharm. 54 (3): 243–50. PMID 15610620

-Wikipédia : https://en.wikipedia.org/wiki/Grapefruit_seed_extract

- Takeoka GR, Dao LT, Wong RY, Harden LA (September 2005). "Identification of benzalkonium chloride in commercial grapefruit seed extracts". J. Agric. Food Chem. 53 (19): 7630–6.

-Food and Bioprocess Technology,pp 1–13, Grapefruit Seed Extract and Lemon Essential Oil as Active Agents in Corn Starch–Chitosan Blend Films, M. J. Bof, A. Jiménez, D. E. Locaso, M. A. García, A. Chiralt, 13 August 2016, DOI: 10.1007/s11947-016-1789-8

- Why I Don’t Use Grapefruit SeedExtract :

http://www.mommypotamus.com/the-ugly-truth-about-grapefruit-seed-extract/

Is there any competition between two microbes in food-restricted environment?

Is there any competition between two microbes

in food-restricted environment?

Competition in biology refers to an interaction between organisms or species which often leads to conflict between them due to resources supply, space, or many other factors … This competition can occur as between members of the same specie as between members of two different species

For whatever reason, competition is one of the most important interaction occurring in nature and take such a large importance in ecology since it affects community structures and ecosystems.

Several types of competition subsist in ecology such as “exploitative competition” often referred as food competition but also “interference competition” in which individuals of one specie interacts directly with individuals of another specie with more aggressive behaviour in the aim of becoming predominant towards the other.

This study is very interesting since it allows us to focus on competition between two simple micro-organisms from the same type of environment (digestive system, human and rodent one) which are Escherichia coli (white colour) and Serratia marcescens (red colour) and try to see what append in this situation, if competition there is and which type.

In order to answer this question, I managed to prepare two different initial bacteria cultures in order to let them grow individually. Then, I prepared different environments with five different food quantities (under the form of glucose, a sugar) but still limited where I put either the two microbes or each microbes individually, these latter enabling to see how microbes react individually in the same environment conditions (we call this “negative control”). After a while in this environment suitable for competition, I diluted solutions of bacteria cells to keep enough quantity of cells but not too much that will enable me to observe them on “plates” where they will form colonies (a large amount of cells with the same genetic information, as we can see on the first picture above). As for quantification, I decided to focus on number of colonies (in mixed bacteria environment) and their size (between mixed and individual bacteria environment).

In order to answer this question, I managed to prepare two different initial bacteria cultures in order to let them grow individually. Then, I prepared different environments with five different food quantities (under the form of glucose, a sugar) but still limited where I put either the two microbes or each microbes individually, these latter enabling to see how microbes react individually in the same environment conditions (we call this “negative control”). After a while in this environment suitable for competition, I diluted solutions of bacteria cells to keep enough quantity of cells but not too much that will enable me to observe them on “plates” where they will form colonies (a large amount of cells with the same genetic information, as we can see on the first picture above). As for quantification, I decided to focus on number of colonies (in mixed bacteria environment) and their size (between mixed and individual bacteria environment).

This graph is part of my results and represents ratios of A/B numbers colonies in mixed bacteria plates according to the glucose concentration (A being E. coli and B being S. marcescens). We can see that there is a considerable number of S. marcescens colonies towards E. coli ones since the ratio is clearly lower than 1 except for the least concentration of glucose where this value is more important (due to error manipulation).

Other results concerning surface areas of colonies tend to show that E. coli colonies seem to be bigger when they’re alone unlike when they’re confronted to S. marcescens whereas S. marcescens colonies tend to be bigger when they’re confronted to E. coli unlike when they’re alone.

Thus, we can say that predominance of S. marcescens is shown in mixed plates and for all concentrations of glucose, gaining colonies surface area while exposed to E. coli, whose colonies are conversely bigger in negative controls.  Nevertheless, we have to take into account the lack of replicates (when we repeat the experiment) due manipulation problems that certainly impacts the reliability of my results. Furthermore, we have to be clear that I can’t prove any competition between microbes but maybe assume that there are at least interactions that led to the possible predominance that I just spoke before.

So, what would be good to do next is first and overall to repeat the experiment with replicates to have more reliability of the results. Secondly, it would be very interesting to work at the microscopic scale to have more criteria to quantify that will bring enough information about competition of microbes.

If you want to know more, here are some bibliography links to have more details about key aspects of my project and information that helped me to do it:

- Utida, S. (1953). Interspecific competition between two species of bean weevil. Ecology, 34(2), 301-307.

- Competition in biology information on Wikipedia

https://en.wikipedia.org/wiki/Competition_(biology)

v   

             - Interspecific competition information on Wikipedia

https://en.wikipedia.org/wiki/Interspecific_competition

  

             - Competition exclusive principle information on Wikipedia

https://en.wikipedia.org/wiki/Competitive_exclusion_principle

v            - Scramble competition information on Wikipedia

https://en.wikipedia.org/wiki/Scramble_competition

v           - Yoon, H., Klinzing, G., & Blanch, H. W. (1977). Competition for mixed substrates by microbial populations. Biotechnology and Bioengineering, 19(8), 1193-1210.

v            - E. coli information on Wikipedia

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

v            - S. marcescens information on Wikipedia

https://en.wikipedia.org/wiki/Serratia_marcescens

v                  - Chramostova, J., Mošnová, R., Lisova, I., Pešek, E., Drbohlav, J., & Němečková, I. (2014). Influence of Cultivation Conditions on the Growth of Lactobacillus acidophilus, Bifidobacterium sp., and Streptococcus thermophilus, and on the Production of Organic Acids in Fermented Milks. Czech J. Food Sci, 32(5), 422-429.

v            - Thayer, D. W., Muller, W. S., Buchanan, R. L., & Phillips, J. G. (1987). Effect of NaCl, pH, temperature, and atmosphere on growth of Salmonella typhimurium in glucose-mineral salts medium. Applied and environmental microbiology, 53(6), 1311-1315.

Here, you will find the poster of my project in the framework of Petnica research projects week in Serbia with Paris Descartes university, CRI Paris and Schueller Bettencourt Fondation :  

Acknowledgements

All the teaching team of Petnica, all Bore students.

T. Milosevic for all advices, support, pedagogy and experience in Petnica Science Center.

G. Baudrin for his support and follow all along research projects as tutor.

Petnica Science Center for having given all these opportunities during the stay

Corentin Mathé - - Deletang
L2 FDV Bachelor
Paris Descartes & CRI PARIS