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The Antimicrobial Potential of Natural Products

  1. Abstract

There are an ever growing number of microorganisms that are becoming resistant to current antimicrobials.  Microorganisms can become resistant for a number of reasons such as over prescription of antibiotics, patients not completing the full course or from random mutation.  This has lead to infections such as MRSA becoming virtually untreatable.  There is now a great need from the health industry for new antimicrobials and the answer may lie within natural products.  This experiment compared four natural products (chives, oyster mushroom, cherry and vanilla) to known antimicrobials (ethanol, garlic and antibiotics) in a hope that they would produce a significant effect and show antimicrobial potential.  The compounds were tested on two bacteria’s, one gram positive (S.aureus) and one gram negative (E.coli), this was to show how different bacteria’s behave.  All compounds were suspended in ethanol and a t test was used to show their significance.  The compound that worked best at the lowest concentration (0.1) was cherry on S.aureus, all of the other compounds produced significant results at the 0.2 concentration.  None of the natural compounds selected produced significant results on E.coli. On neither of the bacteria did any of the compounds produce a significant result at the highest dilutions (1 and 0.5).  In further study, compounds could be separated into single ingredients to determine which part of the product has the antimicrobial effect and this could then be used to treat resistant microorganisms.

  1. Introduction

Over the past few years the amount of antimicrobial resistant pathogens has sharply increased.  There is now a need to find new compounds that have antimicrobial properties in order to prevent this from rising further.

Bacteria have developed resistance at a fast rate due to a number of reasons…

  1. Doctors have overprescribed antibiotics for minor infections that present no danger. By overexposing bacteria to antibiotics they are more likely to develop resistance as the more they come into contact together the less effective the antibiotic becomes.
  2. Doctors have prescribed antibiotics for viral infections. As viruses live inside host human cells they have no metabolism of their own and therefore antibiotics cannot destroy a virus.  Hence it is pointless to prescribe antibiotics to a virus as it will not do anything.  However any bacteria that may be present in the patients system will be exposed to the antibiotic and so may develop resistance.
  3. Patients are not finishing the full course of antibiotics. When patients begin to feel better they tend to stop taking their prescription but there may be some bacteria left in their system, these will be the most resistant to the antibiotic.  By stopping the course the bacteria has not been killed and so will reproduce passing on the resistance gained through the exposure of the antibiotic.  This will also lead to the patient returning to the doctors and getting another prescription which causes more exposure to the bacteria.

Resistance can be caused by a chance mutation in a bacteria’s plasmid.  This new gene may be able to break down a particular antimicrobial before it affects its own metabolism.  This increases the pathogens chance of survival and so when they come to reproduce they pass on this allele to their offspring.  Plasmids are also interesting as they can pass from bacteria to bacteria without reproduction, this means resistance can spread very quickly throughout a colony and between different strains of bacteria.  This is one of the reasons why antimicrobial resistance has become such a large issue.

Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the most commonly known antimicrobial infections.  MRSA is a strain of Staphylococcus aureus that is no more infectious than the normal one but much harder to treat.  MRSA is known as a superbug as it is resistant to not only methicillin but also amoxicillin, penicillin, oxacillin and lots of other antibiotics.  MRSA causes mild skin infections such as sores and boils however can cause serious infections if it enters the bloodstream or urinary tract.  People most vulnerable to catching MRSA are those in hospital, due to the close contact of other patients on wards, their weakened immune systems and the poor hygiene from hospital staff.  This has led to a large change in the way patients are treated, all patients who are about to have surgery are screened beforehand for MRSA and if a patient is found to have MRSA they are isolated on wards with other patients who also have MRSA, this is to reduce the spread.

An antimicrobial agent is something that kills or inhibits the growth of microorganisms whilst causing little or no harm to the host.  An antimicrobial could be an antibiotic, antiviral or antifungal.  Traditional antimicrobials are becoming less and less effective because of the reasons explained above.  Therefore there is an ever demanding need for new antimicrobials to be found to treat these infections.

From this experiment it is hoped that some of the products tested will show some antimicrobial potential.  The products chosen were; Chives, Oyster Mushroom, Cherry and Vanilla.  Chives were chosen as they are a member of the allium family and this will be interesting to compare with garlic which is one of the positive controls.  Oyster mushroom was chosen because mushrooms are known to have lots of health benefits, but also because mushrooms are a fungus like Penicillin.  There were lots of fruits that would have been good to test and many are known to have lots of health benefits but there wasn’t much about cherries and that is why it was chosen.  Vanilla was a more random choice just to see what happened.

The success of this experiment will be determined from

  1. Did the compounds produce any inhibition
  2. Was this inhibition down to the compound or the ethanol suspension ( t test)
  3. What is the lowest concentration the compounds produce inhibition at, if any
  4. Are the natural products selected any better than the positive controls

Bacteria

The bacteria used in this experiment are Escherichia coli (E.coli) and Streptococcus aureus (S.aureus) which are both a category II hazard.  E.coli is a gram negative bacteria and S.aureus is a gram positive bacteria, this will allow a comparison as some compounds may only work on gram positive bacteria.  The difference between gram positive and gram negative is how the cell wall is structured, gram negative have a thin peptidoglycan layer and a high lipid content due to the outer membrane, whereas gram positive have a thick peptidoglycan layer which is what retains the crystal violet dye.  The differences can be seen through a gram stain.

Figure 1 – Gram stain of E.coli

gram stain ecoli

Figure 2 – Gram stain of S.aureus

gram stain saureus

Controls

The negative controls in this experiment are just bacteria and sterile water, this is to check that the spreads are even and the bugs are growing (no problems with incubation time/temperature-37°C). The positive controls are ethanol, garlic and antibiotics.  Both the ethanol and garlic will be diluted like the natural compounds so a direct comparison can be made.  All compounds will be suspended in ethanol (including garlic) so this will act as base to measure against in the statistical analysis.  The garlic came in a tablet form, which had an enteric coating, this may affect the results of the experiment as the garlic may not be able to metabolise properly with it.  If this was proved to be a problem then we would either use fresh garlic cloves or react the tablet with some hydrochloric acid as it would be in the stomach.  The antibiotics used were Ciprofloxacin 1µg (CIP) and Fusidic acid 10µg (FC).  CIP is a gram negative antibiotic and FC is a gram positive antibiotic.

CIP is a bactericidal which kills bacteria by inhibiting enzymes responsible for DNA replication, transcription, recombination and repair.  They do not normally cross-resist with other bacteria and mutations develop slowly which make them ideal for treating bacteria that are resistant to other antibiotics.  CIP can be used to treat both gram-positive and gram-negative bacterias.  FC is a bacteriostatic which inhibits protein synthesis (bacterial replication).  FC is only effective for gram positive bacteria.

Chives- Allium schoenoprasum

Chives are part of the Allium family of vegetable, which contains such foods as onions, garlic and leeks.  Onions and Garlic have known antimicrobial properties and this is one of the reasons why Garlic acts as one of the positive controls in this experiment. The active ingredient that causes there antioxidant effects is Allicin, a combination of alliin and the enzyme allinase which is a highly unstable and a very reactive compound (Kourounakis, 1991).  It is believed that alliin and allinase are kept separate during growth and only when cut/cooked do they mix, giving them their properties (Focke, 1990).  Chives also contain vitamin A, vitamin C, potassium and folic acid which have numerous benefits such as; lowering blood pressure, aid digestion, stimulate appetite and possess some antiseptic properties.

Figure 3 – Sftructural formula of Allicin (C6H10OS2)

structure alicin

Oyster Mushroom-  Pleurotus osteratus
Oyster mushrooms contain lots of active ingredients including; polypeptides, polysaccharides, alpha-glucans, pleuran, beta-glucans, lovastatin, natural statins, pelonic compounds and tannins.  Due to the high amount of natural statins, particually lovastatin which makes up about 2.8% of the mushrooms dry weight, oyster mushrooms have a large affect against reducing LDL cholesterol by binding to the enzyme, HMG-CoA reductase.  Recent in-vivo research has suggested that the extract may induce apoptosis (programmed cell death) in some cancer lines.  Oyster mushrooms are also known to have anti-inflammatory, antiviral and antibacterial properties and so often used in preventive medicine.

Figure 4: Structural fromula of Lovastain (C24H3605)

structure Lovastain

Cherry-  Prunus avium

Cherries contain lots of antioxidants which protect against free radicals produced in the body.  This is important as oxidation of the bodies’ molecules can cause arthritis, heart disease and ageing skin and many other chronic diseases.  Cherries contain anthocyanins (350-400mg per 100g) which may have antiviral properties and could improve vision, however these are not proven.  Melatonin regulates sleep patterns and cherries are a natural source of melatonin, supplements are used to treat jet lag and in the future reduce chronic cluster headaches and help people who work night shifts.

Figures 5 and 6: Strucural formula of Anthocyanin (C15H11O+) and Melatonin (C13H16N2O2)

structure Anthocyanin and Melatonin

Vanilla-  Vanilla planifolia

Vanilla is a phenolic aldehyde and contains the functional groups; aldehyde, hydroxyl and ether.  Vanilla’s active ingredient is called Vanillin and makes up approximately 2% of a vanilla pods dry weight.  At the moment Vanilla is not needed for any medicinal purposes other than pharmaceutical flavourings however a synthetically produced version of Vanillin, Ethyl Vanillin is used widely in the food industry.  Although it is more expensive the ethoxy group makes this compound much stronger than its natural version.

Figure 7: Structural formula of Vanillin (C8H8O3)

structure Vanillin

  1. Method

Prepare agar plates using aseptic techniques.

Serially dilute bacteria on a streak plate.  Take a single colony and make a bacteria emulsion.

Working aseptically, remove 100µl of bacterial emulsion into the centre of a fresh agar plate.  Using a spreader, cover the entire plate with the bacteria.  Starting in the middle, take care not to damage the surface of the agar.   Sterilise equipment between different strains of bacteria.

Using good aseptic techniques flame a cork borer (size 4) in ethanol, allow to cool briefly and punch 4 holes into the agar.  Gently remove with a sterile picking needle.

Using 0.5g of each compound, grind in a mortar and pestle to a pulp.  Pulse spin in a centrifuge, using the supernatant produce 4 dilutions as shown below.

Concentration (compared to original) Amount of compound/µl Amount of sterile water/µl
1 200
0.5 110 110
0.2 50 200
0.1 25 225

Pipette 100µl of each compound into the wells.  The order of concentrations is relevant for ethanol, garlic, chives, mushroom, cherry and vanilla.  Two plates will not have wells as the antibiotic compounds come in discs.

Repeat this so results are in triplicate.

Incubate overnight the right way up at 37°C.

Measure the diameter of each inhibition zone in cm using a ruler.  Calculate a mean and standard deviation for each concentration of the compounds. Carry out a t test, comparing all natural compounds results to ethanol.

  1. Results and Discussion

These are the hypotheses for the t test.

H0: no difference between ethanol and natural compounds

H1: some difference between ethanol and natural compounds

graph ecoli

These are the results for the E.coli bacteria.  The largest mean inhibition zone was the CIP antibiotic with 3.667cm.  A t test was carried out to see if the natural compounds had any effect on their own or if it was down to the ethanol.  None of the compounds with concentrations of 1 or 0.5 produced a significant result and so therefore the null hypothesis can be accepted.  Garlic with a concentration of 0.2 produced a significant result as ethanol 0.2 did not produce any inhibition, this means in this case the null hypothesis can be rejected (*** = p<0.001).

graph saureus

These are the results of the S.aureus experiment.  The mean inhibition zones for all compounds were much greater in this experiment than in the E.coli one.  The antibiotic FC gave the largest inhibition zone with 3.2333cm.  Like E.coli, none of the concentrations 1 or 0.5 were greater than the ethanol base, however all were significant at lower concentrations.  Cherry was the most significant at 0.2, closely followed by chives.  These were also the compounds that came closest to being significant at higher concentrations and were only narrowly under the critical values.  Cherry was also the only compound that gave a significant result at the lowest dilution (0.1) from both bacteria’s.

The diameters of inhibition zones for E.coli, decrease as concentration decreases which is what is expected.  However some of the results on S.aureus do not behave this way, these are cherry and chives between 0.5 and 0.2.  This could mean they are anomalous results and would have to be repeated in order to verify this.  These results could be down to the freshness of the ingredients used as cherry is a fruit it oxidises very quickly. In fact between the two days when we made the compound dilutions the cherry was a completely different colour, on the first day it was a very bright red colour and after it had been left for a day and oxidised the colour was a deep brown.  This supernatant was redone with fresher cherry to give more consistency throughout the experiment.  A similar explanation could be seen for chives as they began to dry out.

One of the main reasons for selecting chives was to compare it to the positive control garlic as they come from the same family.  From previous research it has been shown that the Allicin is weaker in chives than garlic.  The results from the E.coli experiment support this as garlic (0.2) produced a significant result and had much larger inhibition zones for all concentrations.  However on the S.aureus experiment this is not seen as chives (0.2) produced a significant result and garlic did not.  This may be down to the fact that a garlic tablet was used instead of a fresh clove, this may have skewed the results leading to this conclusion.  It is also interesting that chives (0.2) had a larger inhibition zone than chives (0.5) which may be anomalous and can only be proved through further repetitions of the experiment.

Figure 8and 9: Inhibition zones of garlic and chives on S.aureus

inhibition zones

Limitations

The reason the compounds worked better on S.aureus than E.coli may be to do with their structure. S.aureus is a gram positive bacteria and so doesn’t have a cell wall or extra outer membrane, this makes it easier to penetrate and affect the metabolism of the cell.  It is surprising that the chives had a larger affect than the garlic in the t test (particularly S.aureus), as Allicin is weaker in chives.  This may be explained by the fact that the garlic used was a tablet and not a natural clove.  The enteric coating on the garlic may have stopped the full reaction as ethanol may not have been able to dissolve it.  It would be better to repeat this part of the experiment with fresh garlic.

Although all compounds used had the same starting mass (0.5g) there were very different amounts of the active ingredient in each sample, due to the water content of each compound, for example there was very little cherry compared with the amount of chives.  Some of the active ingredients may have reacted with the ethanol and this could have affected the results, to see if this did affect the results, repeat with suspension in sterile water.  In all natural compounds there are a variety of active ingredients so without further experiment we do not know which were responsible for the inhibition, but could find this out by separating the compounds.  The plates had to be incubated right side up as otherwise the liquid would run out, this meant that condensation from incubation may drip onto the agar, possibly causing dilution.  Some plates may have had thinner agar than others, which may affect the results as less compound could fit in the wells and may diffuse quicker.  The spreading method of bacteria was quantitative as 100µl was used each time, however the amount of bacteria that remains on the spreader will be different and the spread may not be completely even over the agar.  The temperature of the lab at the time of the experiment was very hot and this may have evaporated some of the supernatant.  The garlic tablet and vanilla required 2ml of ethanol to form a liquid, this was twice the amount the other compounds needed and so they are effectively twice as diluted as the others.  The emulsions were made by different technicians.  The experiment was only repeated three times which is no really enough to draw conclusions from and so for some of the compounds that came very close to being significant more repeats would be needed to see if they did have an effect.  The compounds needed to be fresh when tested to keep results as similar as possible, this includes oxidisation of the cherry, as the supernatants of an oxidised and non-oxidised cherry looked very different and it is not known if this is to impact on its antimicrobial ability.

  1. Conclusion

The results of this experiment showed that none of the selected compounds significantly affected the growth of E.coli.  All four of the compounds had a significant effect on S.aureus at the 0.2 concentration and cherry was the only compound to have an effect at the lowest concentration, 0.1.  Cherry and chives were also very close to being significant at their higher concentrations and only narrowly missed the critical value.  This means that both cherry and chives could have antimicrobial properties and this could be verified with further research.  In future experiment the compounds could be separated and purified into singular active ingredients and repeating the test as above, with the singular compounds to see which had the effect on inhibition.  Also more compounds could be tested on a wider variety of not only bacteria but also viruses and fungi.  This in turn will provide the pharmaceutical industry with alternatives to traditional prescriptions and prevent further antimicrobial resistance developing.  Through this research it has been shown that natural compounds do have the potential to be used as antimicrobials, to treat infections that are currently untreatable.

  1. References

About Pleurotus Osteratus medicinal mushroom

Available at www.mycolivia.com/pleurotus-mushroom (Accessed 8th August 2014)

Anthocyanins and anthocyanidins

Available at www.food-info.net/uk (Accessed 9th August 2014)

Antimicrobials: An Introduction (2011)

Available at www.amrls.cvm.msu.edu (Accessed 14th August 2014)

Ciprofloxacin (13 April 2014)

Available at www.drugs.com/ciprofloxacin.html (Accessed 6th August 2014)

Focke, M., Feld, A., Lichtenthaler, K., February 1990, Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase.

Kourounakis, P.N., Rekka, E.A., November 1991, Effect on active oxygen species of alliin and Allium sativum (garlic) powder, Res Commum Chem Pathol Pharmacol, 74 (2): 249-252

Sleep disorders health centre: Melatonin-Overview (June 20, 2012)

Available at www.webmd.com/sleep-disorders (Accessed 9th August 2014)

  1. Appendix
Product E.coli
Expt 1/cm Expt 2/cm Expt 3/cm mean SD SEM UB LB significance
lawn 0 0 0 0 0 0 0 0
Sterile water 0 0 0 0 0 0 0 0
Ethanol 1 1.55 1.3 1.6 1.483333 0.160728 0.093 1.884 1.083
Ethanol 0.5 N/A 0 1.1 0.55 0.777817 0.55 2.917 -1.817
Ethanol 0.2 N/A 0 0 0 0 0 0 0
Ethanol 0.1 N/A 0 0 0 0 0 0 0
Garlic 1 2.4 1.3 2 1.9 0.556776 0.321 3.281 0.519 1.245
Garlic 0.5 1.3 0 1.4 0.9 0.781025 0.451 2.841 -1.041 -0.492
Garlic 0.2 1.3 0 1 0.766667 0.680686 0.393 2.458 -0.924  
Garlic 0.1 0 0 0 0 0 0 0 0
CIP 1µg 3.3 3.7 4 3.666667 0.351188 0.203 4.543 2.796
FC 10µg 0 0 0 0 0 0 0 0
Chives 1 0 1.7 1.3 1 0.888819 0.513 3.207 -1.207 0.926
Chives 0.5 0 0 1.1 0.366667 0.635085 0.367 1.946 -1.212 0.277
Chives 0.2 0 0 0 0 0 0 0 0
Chives 0.1 0 0 0 0 0 0 0 0
Mushroom 1 1.5 1.1 1.8 1.466667 0.351188 0.203 2.341 0.593 0.074
Mushroom 0.5 0 1 0 0.333333 0.57735 0.333 1.766 -1.1 0.337
Mushroom 0.2 0 0 0 0 0 0 0 0
Mushroom 0.1 0 0 0 0 0 0 0 0
Cherry 1 1.9 1.4 1 1.433333 0.450925 0.26 2.552 0.314 0.181
Cherry 0.5 0 0 0.9 0.3 0.519615 0.3 1.591 -0.991 0.399
Cherry 0.2 0 0 0 0 0 0 0 0
Cherry 0.1 0 0 0 0 0 0 0 0
Vanilla 1 2.5 1.6 2 2.033333 0.450925 0.26 3.152 0.914 -1.99
Vanilla 0.5 0 1.2 0 0.4 0.69282 0.4 2.121 -1.321 0.221
Vanilla 0.2 0 0 0 0 0 0 0 0
Vanilla 0.1 0 0 0 0 0 0 0 0

 

Product S.aureus
Expt 1/cm Expt 2/cm Expt 3/cm mean SD SEM UB LB significance
lawn 0 0 0 0 0 0 0 0
Sterile water 0 0 0 0 0 0 0 0
Ethanol 1 2.25 2.5 2.8 2.516667 0.275379 0.159 3.201 1.833
Ethanol 0.5 N/A 1.4 1.1 1.25 0.212132 0.15 1.895 0.605
Ethanol 0.2 N/A 0 0 0 0 0 0 0
Ethanol 0.1 N/A 0 0 0 0 0 0 0
Garlic 1 3.1 2.1 3.4 2.866667 0.680686 0.393 4.558 1.176 -0.826
Garlic 0.5 1.6 0 2.1 1.233333 1.096966 0.633 3.957 -1.491 0.025
Garlic 0.2 0 0 0 0 0 0 0 0
Garlic 0.1 0 0 0 0 0 0 0 0
CIP 1µg 1.9 3.1 3.2 2.733333 0.723418 0.418 4.532 0.934
FC 10µg 2.1 3.6 4 3.233333 1.001665 0.578 5.72 0.746
Chives 1 1.4 1.4 1.1 1.3 0.173205 0.1 1.73 0.87 3.919
Chives 0.5 0.8 0 0 0.266667 0.46188 0.267 1.416 -0.882 3.214
Chives 0.2 1.3 0 0 0.433333 0.750555 0.433 2.296 -1.43  
Chives 0.1 0 0 0 0 0 0 0 0
Mushroom 1 1.6 1.5 2.2 1.766667 0.378594 0.219 2.709 0.825 2.775
Mushroom 0.5 1.8 0 1.1 0.966667 0.907377 0.524 3.222 -1.288 0.52
Mushroom 0.2 1 0 0 0.333333 0.57735 0.333 1.766 -1.1  
Mushroom 0.1 0 0 0 0 0 0 0 0
Cherry 1 1.5 1.6 2.1 1.733333 0.321455 0.186 2.533 0.933 3.206
Cherry 0.5 0 0 1 0.333333 0.57735 0.333 1.766 -1.1 2.508
Cherry 0.2 1.4 0 0 0.466667 0.80829 0.467 2.477 -1.543  
Cherry 0.1 1.1 0 0 0.366667 0.635085 0.367 2.377 -1.643  
Vanilla 1 2.9 1.9 2 2.266667 0.550757 0.318 3.635 0.899 0.703
Vanilla 0.5 2.1 1.4 1.3 1.6 0.43589 0.252 2.684 0.516 -1.195
Vanilla 0.2 1.2 0 0 0.4 0.69282 0.4 2.121 -1.321  
Vanilla 0.1 0 0 0 0 0 0 0 0

Orange cells (ethanol) are the comparison in the t test.

Purple cells are compounds with overlapping bounderies to ethanol, so will be tested for their significance.

Green rows are significant to p<0.001 degree of accuracy.

Critical value- 4.303

 


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