2017/2018 Practical Project

School of Biological Sciences

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and analysis of antimicrobial and synergistic properties of Oregano oil with
ampicillin against Bacillus subtilis


Student name: Andrew Waddingham

Student number: 1544297

Supervisor: Gary Foster

Word count:
























Antibiotic resistance is an ever-growing problem, and
research has focused on finding new antibiotics as well as increasing the
effectiveness of those we have. In this investigation we built upon previous
work undertaken at the University of Bristol, which found antibiotic activity
and potential synergism of the monoterpene alcohol components of Oregano oil, thymol
and carvacrol, with the antibiotic pleuromutilin. A range of other essential oils
have been found to have antibiotic properties, including some synergistic
activity for specific combinations of bacteria and antibiotics. In this investigation
we first confirmed the findings of previous studies, as our results showed
Oregano oil and each of its component monoterpene alcohols showed significant antibiotic
activity when plated against Bacillus subtilis
on an agar plate, each producing a clearing zone. Synergism was tested between Pleuromutilin
and Oregano oil (and its component monoterpene alcohols individually), against the
bacteria Bacillus subtilis. The
pleuromutilin used was sourced from the fungus Clitopilus passeckerianus.
Though our tests did not show any synergistic activity between any test
substance and the antibiotic, we believe this to be the result of imperfect
methodology, and have made suggestions as to how further work could improve
this method.



The looming antibiotic resistance crisis is a huge threat to
many current basic medical procedures, and threatens a world where the smallest
cut can become fatal due to multi-resistant bacteria being untreatable by drugs
(O’Neil, 2014). 50,000 people die annually across
Europe and the United States from antimicrobial resistant bacteria, and this
number is expected to rise rapidly without significant advancements in this
field (O’Neil, 2014). The golden age of antibiotics of
the mid-twentieth century has come to an end and the future of antibiotics is
uncertain, although it is clear new antibiotics are a necessity for modern
medicine (Rossiter et al. 2017).

The extreme usage of antibiotics in the farming and
livestock industry has been responsible for very strong selection towards
resistance over the past century (Rosenblatt-Farrell,
2009). It has been proposed that natural products should be used as a
basis for engineering new and more potent antibiotics (Rossiter et al. 2017).
In an effort to help combat this crisis, the scientific community has moved to
researching naturally occurring essential oils, which have been historically
used as an antimicrobial treatment (Fournomiti et al. 2015). While reducing the usage of antibiotics may
be the most effective counter of resistance development, being able to use
essential oils as a supplement to antibiotics would help counter resistance
selection, without sacrificing the ability to use them within industry. This is
achieved by reducing the minimum inhibitory concentration of antibiotic
required, which is the lowest possible concentration of antibiotic which will
inhibit bacterial growth (Cirino
et al. 2014). Reducing this allows for a smaller amount of antibiotic to
be used when supplemented with synergistic substances, decreasing the potential
for resistance to develop (Cirino
et al. 2014). There have been other similar investigations into the uses of
carvacrol and thymol for both their antibiotic properties and potential
synergisms with current antibiotics (Cirino et al. 2014), however we
believe it is a novel experiment to test pleuromutilin for synergistic activity
with Oregano oil and its components.



Materials & Method

Preparation of Bacillus spores – In order to create a culture of Bacillus subtilis, a cultivation loop
with Bacillus subtilis spores was
introduced into a 50ml LB media flask (1.5g Tryptic Soy Broth, 50ml distilled
water, autoclaved). The flask was then incubated in a shaking incubator at 30?C
for 24h. 100ml TSB (3g Tryptic Soy Broth, 2g Agar powder and distilled water to
100ml) was made in a Thompson bottle, autoclaved and left to harden at a 45?
angle. The set TSB was inoculated using 1ml of the Bacillus subtilis culture; the liquid was swirled around the
surface of the TSB to ensure an even spread of the inoculums. The Thompson
bottle was then left at a 45? angle with a loosened lid in an incubator at 30?C
for 6 days. 100ml sterile distilled water was pipetted into the Thompson bottle
and water was washed over the surface of the TSB, collecting the bacterial
spores from the TSB surface. 15ml aliquots of this bacterial suspension were transferred
into universal tubes using a sterile pipette and vortexed to ensure an even
suspension. A 70?C water bath was used to heat the suspensions for 30 minutes
to activate the spores. The suspensions were removed, the tubes sealed with
parafilm and stored at 4?C for use throughout the experiment.

Bioassay of Oregano
oil and components- To test for antimicrobial activity, base media (TSAb)
was prepared from 15g Tryptic Soy Broth, 10g Agar powder and distilled water to
500ml; vortexed and then autoclaved. 20ml of TSAb was poured under sterile
laminar flow into 10cm petri dishes, then left to cool and harden.  Overpour media (TSAg) was prepared from 15g
Tryptic Soy Broth, 2.5g Agar powder, 5g Glucose, and distilled water to 1000ml,
autoclaved after mixing. A 4% TTC solution was made from 400mg TTC powder
(>99% TTC) dissolved in 10ml distilled water, vortexed, sealed and stored at
4?C. 20µl Bacillus subtilis Spore
suspension per 100ml was added to hot (50?C -70?C) TSAg, to heat shock the
spores. 750µl of 4% TTC solution was then added per 100ml TSAg. 5ml of this
TSAg solution was poured onto each plate of hardened TSAb under laminar flow,
and left to set.

A circular well, 8mm diameter, was created in the centre of
each plate using a sterilised borer tool. Each of the following test substances
was then added to these wells, in 40µl aliquots:

Oregano Oil was added at 100% concentration.

Distilled water was added at 100% concentration.

The ampicillin was in solution with distilled
water at 100µl/ml.

The carvacrol was in solution with distilled
water at 100µl/ml.

The thymol, provided in granular form, was
dissolved in distilled water at 100mg/ml, warmed and vortexed to ensure it was
fully dissolved immediately before it was added to the wells.

The ampicillin was used as a positive control for
antimicrobial activity and the distilled water was used as a negative control. The
plates were sealed with tape and incubated for 24h at 25?C immediately after
addition of the test substance. The radius of the clearing zones produced after
incubation was recorded. In cases of non-circular zones, an average of four
perpendicular measurements was used. Each substance was plated in triplicate,
then this was repeated to give a total of 6 plates for each test substance.

Bioassey of synergism
using Clitopilus passeckerianus – A
solution of Potato Dextrose Agar (PDA) was prepared from 24g potato dextrose
broth, agar powder 15g and distilled water to 1L. 20ml of PDA was poured into a
petri dish in laminar flow and allowed to set. Using a scalpel, a small (5mm x
5mm) square of Clitopilus
passeckerianus mycelium was cut from a fungal plate. This subculture was then placed
into the centre of the set PDA plate, sealed and incubated at 25?C for 5 days
to allow the fungus to grow. This plate was then used to generate all the
subcultures used in the synergism bioassay. 18 petri dishes of 20ml PDA each
were then poured under laminar flow and allowed to set. 5mm x 5mm squares were
then cut from the growth plate at the edges, and the mycelium cuttings were
added to the centre of each of the PDA plates. These plates were then sealed
and incubated at 25?C for 5 days. A well was then cut into the centre of the
fungal growth and 40ml of the test substances were then pipetted into the

Oregano Oil and distilled water were added at 100%
concentration. The ampicillin and carvacrol were in solution with distilled
water at 100µl/ml. The thymol, provided in granular form, was dissolved in
distilled water at 100mg/ml, warmed and vortexed to ensure it was fully
dissolved immediately before it was added to the wells. The ampicillin was used
as a positive control and the distilled water as the negative control. 20ml TSAg
{Overpour media} (30g Tryptic Soy Broth, 5g Agar, 10g Glucose, distilled water
to 1000ml, autoclaved) was poured over each plate. They were then sealed and
incubated for 24h at 25?C. The radii of the clearing zones were measured from
the well. In cases of non-circular zones, an average of four perpendicular
measurements was used Each substance was plated in triplicate, then this was repeated
to give a total of 6 plates for each test substance.



The ability of the test substances to inhibit growth of Bacillus subtilis on an agar plate is
recorded in Figure 1. The negative control sterile distilled water did not
produce a clearing zone. The positive control ampicillin produced the largest
clearing zone of 24.2mm radius. Pure Oregano oil, carvacrol and thymol each
produced a clearing zone, showing antibiotic activity.


Figure 1. Mean
clearing zone radius produced by agar well diffusion of test substances on Bacillus subtilis. Incubated at 35?C for
24h. Error bars show the standard deviation around the mean. A One-way ANOVA
was used to analyse the clearing zone radii (F=112.486; d.f.=4,25; p<0.001). A Tukey post-hoc revealed Ampicillin (p<0.001), Oregano oil (p=0.014), and Carvacrol (p<0.001) to be statistically significantly different from the negative control, therefore displaying antibiotic activity. Thymol (p=0.206) was not statistically significantly different from the negative control.   The synergistic ability of each test substance is recorded in Figure 2. To be able to say synergistic activity occurred, the test substances' mean clearing zone would have to be larger than the non-synergistic positive control, 10% Ampicillin. None of the three test substances exhibited any synergistic effect, as all test substances produced a mean clearing zone smaller than both the positive and negative controls.   Figure 2.  Mean clearing zone produced by test substances or controls when plated on Clitopilus passeckerianus over a Bacillus subtilis inoculated Potato Dextrose Agar plate, incubated for 5 days at 25?C. Sterile Distilled Water was used as the negative control. 10% Ampicillin used as positive control. All test substances used in same concentrations as in antibiotic bioassay. The negative control produced a mean clearing zone of 23.3mm. The positive control produced a mean clearing zone of 31.7mm, larger than the negative control as expected. A One-way ANOVA shows the positive control is statistically significantly larger (F=7.773; d.f.=1,16; p=0.013) than the negative control, allowing us to use it as a comparison for the rest of this investigation. The test substances all have smaller means than the negative control, which therefore shows no evidence of synergistic activity occurring, as this would produce a larger clearing zone radius. Discussion The antibiotic activity of three test substances was investigated; the results showed that 100% Oregano oil as obtained from a bottle of 'over-the-counter' capsules had an antibiotic effect on Bacillus subtilis when plated with a TSB agar (Fig. 1). This conclusion supports the findings of (K. Wareham. 2016)(G. Niblock. 2016), who also found that Oregano oil had antibiotic activity. This report was the basis of this investigation, and as such we followed a similar protocol for performing an antibiotic activity bioassay, which was a standard bacterial bioassay protocol provided by the University of Bristol Life Sciences Building laboratories. As our intention was to expand upon this work, and focus upon the possible synergistic activity of Oregano oil with pleuromutilin, we also tested the two monoterpene alcohols that make up Oregano oil: thymol and carvacrol. This allowed us to exclude any bulking agents as variables in causing antibiotic activity or synergistic activity. The monoterpene alcohols both produced a clearing zone, however only carvacrol and pure Oregano oil had a statistically significant difference from the negative control. This suggests that a repeat of these findings utilising a greater number of plates may find a statistically significant difference, and future work would benefit from greater investigation into these substances. This experiment utilised just six plates in total for each test substances for the bacterial bioassay, which may have been too few to be able to conclusively decide upon the antibiotic activity, or lack thereof, of thymol against Bacillus subtilis. There is also a need for future work involving testing multiple different species of bacteria against these test substances. Bacillus subtilis was used for a range of reasons. It is a Gram-positive, spore-forming and non-pathogenic bacterium (Hiraoka et al. 1992). These factors allow much easier laboratory growth and usage than other bacteria available. Bacillus subtilis is a model organism; it is highly studied and understood, making it ideal as a test bacterium (Hiraoka et al. 1992). Gram-positive bacteria were chosen because it has been reported that "Gram positive bacteria are more susceptible to the phenolic components of essential oils than Gram negative bacteria since they act as membrane permeabilisers" (Cacciatore et al. 2015). Our decision to test for potential synergistic activity within carvacrol and thymol was prompted by reports of synergistic relationships between these monoterpene alcohols and other antibiotics, such as tetracycline (Cirino et al. 2014). The findings of last year's project students (K. Wareham. 2016)(G. Niblock. 2016) had also previously suggested possible synergistic activity, as well as further literature (Cacciatore et al. 2015). Carvacrol and thymol constitute 70%-85% of pure Oregano oil, suggesting that any Oregano oil synergism found may be due to the presence of Carvacrol and Thymol (Ultee et al., 2002). They are also believed to be the cause of Oregano Oil's antimicrobial activity (Fournomiti et al., 2015). The method by which they cause an antibiotic effect is by causing damage to cell membrane integrity, affecting pH homeostasis and equilibrium of inorganic ions (Lambert et al., 2001). Our investigation failed to find any evidence of synergism. However, we believe this is likely due to the methodology we employed. As stated previously, we used the fungus Clitopilus passeckerianus, a natural producer of the antibiotic pleuromutilin, as the source of antibiotic for the synergism bioassay. Upon finding the results showed no synergism, we had to revaluate our methods and now believe the use of this fungus may be the cause of our findings not supporting previous findings. Carvacrol has fungicidal properties (Cacciatore et al. 2015). This means that the fungal growth and production of pleuromutilin was almost certainly negatively affected in comparison to the control substance. This would explain not only the lack of synergistic activity found, but the smaller mean clearing zone radius than the negative control. Thymol also has similar antifungal properties (Robledo et al. 2018), meaning we can assume the same to be true of the thymol results. Clearly for any further work into this area, a new methodology would need to be created to avoid the need for Clitopilus passeckerianus. This presents a challenge however, as pleuromutilin is a novel, semisynthetic antibiotic only recently being thoroughly investigated (Jacobsson et al. 2017) (Abbas et al. 2017). It was not readily available for use in this experiment, unlike ampicillin, and so we could not use it in its pure form. It was this inability to acquire the drug that led us to choose to use the fungal method.  Any further work would benefit from be able to procure pleuromutilin directly if they wish to test for synergism in a similar way to this investigation. However, this may be either very expensive and/or difficult, or need to be done once the availability of pleuromutilin has increased. It is also important that the antibiotic activity of Oregano oil and its components are tested against a wide range of both gram-positive and gram-negative bacteria, if there is to be any future use of the compounds for their antibiotic activity. Although using Bacillus subtilis due to its status as a model organism is beneficial, it cannot be assumed that the antibiotic activity would extend to other gram-positive bacteria without clinical trials. If there is hope for using Oregano oil in human medicine, tissue and eventually human trials are essential before it can be introduced to the public to ensure there are no unwanted side effects. The oil may be easily available to buy for the public, but until it has been shown to not have any negative effects it cannot be medically prescribed. Our conclusion from this investigation is that although there is evidence behind the claims of antibiotic activity of Oregano oil, as there is for many other essential oils, the claim of synergism with pleuromutilin has not been provable within this investigation. Carvacrol is the more antibiotically potent than thymol, and although we did not find significant evidence for thymol having antibiotic activity, we believe that further, more rigorous investigation may do. However, we can say that the antibiotic activity of carvacrol is far more potent than thymol. We did not find evidence of synergism, we believe due to our methodology, and have set out potential suggestions for further work which may be able to learn from and avoid our sources of error. References Abbas, M., M. Paul, and A. Huttner. 2017. "New and Improved? A Review of Novel Antibiotics for Gram-Positive Bacteria." Clinical Microbiology and Infection: The Official Publication of the European Society of Clinical Microbiology and Infectious Diseases 23 (10):697–703. Cacciatore, Ivana, Mara Di Giulio, Erika Fornasari, Antonio Di Stefano, Laura Serafina Cerasa, Lisa Marinelli, Hasan Turkez, et al. 2015. "Carvacrol Codrugs: A New Approach in the Antimicrobial Plan." PloS One 10 (4):e0120937. Cirino, Isis Caroline S., Suellen Maria P. Menezes-Silva, Helena Tainá D. Silva, Evandro Leite de Souza, and José P. Siqueira-Júnior. 2014. "The Essential Oil from Origanum Vulgare L. and Its Individual Constituents Carvacrol and Thymol Enhance the Effect of Tetracycline against Staphylococcus Aureus." Chemotherapy 60 (5-6):290–93. Fournomiti, Maria, Athanasios Kimbaris, Ioanna Mantzourani, Stavros Plessas, Irene Theodoridou, Virginia Papaemmanouil, Ioannis Kapsiotis, et al. 2015. "Antimicrobial Activity of Essential Oils of Cultivated Oregano (Origanum Vulgare), Sage (Salvia Officinalis), and Thyme (Thymus Vulgaris) against Clinical Isolates of Escherichia Coli, Klebsiella Oxytoca, and Klebsiella Pneumoniae." Microbial Ecology in Health and Disease 26 (April):23289. Hartley, Amanda J., Kate de Mattos-Shipley, Catherine M. Collins, Sreedhar Kilaru, Gary D. Foster, and Andy M. Bailey. 2009. "Investigating Pleuromutilin-producingClitopilusspecies and Related Basidiomycetes." FEMS Microbiology Letters 297 (1):24–30. Hiraoka, Hideji, Orie Asaka, Takashi Ano, and Makoto Shoda. 1992. 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Robledo, Nancy, Paola Vera, Luis López, Mehrdad Yazdani-Pedram, Cristian Tapia, and Lilian Abugoch. 2018. "Thymol Nanoemulsions Incorporated in Quinoa Protein/chitosan Edible Films; Antifungal Effect in Cherry Tomatoes." Food Chemistry 246 (April):211–19. Rosenblatt-Farrell, Noah. 2009. "The Landscape of Antibiotic Resistance." Environmental Health Perspectives 117 (6):A244–50. Rossiter, Sean E., Madison H. Fletcher, and William M. Wuest. 2017. "Natural Products as Platforms To Overcome Antibiotic Resistance." Chemical Reviews 117 (19):12415–74. Ultee, A., M. H. J. Bennik, and R. Moezelaar. 2002. "The Phenolic Hydroxyl Group of Carvacrol Is Essential for Action against the Food-Borne Pathogen Bacillus Cereus." Applied and Environmental Microbiology 68 (4):1561–68. Kezia Wareham, 2016: "An investigation into antibacterial properties of claimed natural antibiotics against Bacillus subtilis and the possibility of synergism between the test substances and the antibiotic pleuromutilin." 3rd year practical project, University of Bristol Life Sciences Building. Grace Niblock, 2016: "Analysis of the Antimicrobial Properties of Claimed Natural Antibiotics against Bacillus subtilis and Possible Synergism with the Antibiotic Pleuromutilin" 3rd year practical project, University of Bristol Life Sciences Building.

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