2017/2018 Practical ProjectSchool of Biological Sciences Investigationand analysis of antimicrobial and synergistic properties of Oregano oil withampicillin against Bacillus subtilis  Student name: Andrew WaddinghamStudent number: 1544297Supervisor: Gary FosterWord count:                      Abstract Antibiotic resistance is an ever-growing problem, andresearch has focused on finding new antibiotics as well as increasing theeffectiveness of those we have. In this investigation we built upon previouswork undertaken at the University of Bristol, which found antibiotic activityand potential synergism of the monoterpene alcohol components of Oregano oil, thymoland carvacrol, with the antibiotic pleuromutilin. A range of other essential oilshave been found to have antibiotic properties, including some synergisticactivity for specific combinations of bacteria and antibiotics. In this investigationwe first confirmed the findings of previous studies, as our results showedOregano oil and each of its component monoterpene alcohols showed significant antibioticactivity when plated against Bacillus subtilison an agar plate, each producing a clearing zone. Synergism was tested between Pleuromutilinand Oregano oil (and its component monoterpene alcohols individually), against thebacteria Bacillus subtilis. Thepleuromutilin used was sourced from the fungus Clitopilus passeckerianus.Though our tests did not show any synergistic activity between any testsubstance and the antibiotic, we believe this to be the result of imperfectmethodology, and have made suggestions as to how further work could improvethis method.

 IntroductionThe looming antibiotic resistance crisis is a huge threat tomany current basic medical procedures, and threatens a world where the smallestcut can become fatal due to multi-resistant bacteria being untreatable by drugs(O’Neil, 2014). 50,000 people die annually acrossEurope and the United States from antimicrobial resistant bacteria, and thisnumber is expected to rise rapidly without significant advancements in thisfield (O’Neil, 2014). The golden age of antibiotics ofthe mid-twentieth century has come to an end and the future of antibiotics isuncertain, although it is clear new antibiotics are a necessity for modernmedicine (Rossiter et al. 2017). The extreme usage of antibiotics in the farming andlivestock industry has been responsible for very strong selection towardsresistance over the past century (Rosenblatt-Farrell,2009). It has been proposed that natural products should be used as abasis for engineering new and more potent antibiotics (Rossiter et al.

2017).In an effort to help combat this crisis, the scientific community has moved toresearching naturally occurring essential oils, which have been historicallyused as an antimicrobial treatment (Fournomiti et al. 2015). While reducing the usage of antibiotics maybe the most effective counter of resistance development, being able to useessential oils as a supplement to antibiotics would help counter resistanceselection, without sacrificing the ability to use them within industry. This isachieved by reducing the minimum inhibitory concentration of antibioticrequired, which is the lowest possible concentration of antibiotic which willinhibit bacterial growth (Cirinoet al. 2014).

Reducing this allows for a smaller amount of antibiotic tobe used when supplemented with synergistic substances, decreasing the potentialfor resistance to develop (Cirinoet al. 2014). There have been other similar investigations into the uses ofcarvacrol and thymol for both their antibiotic properties and potentialsynergisms with current antibiotics (Cirino et al. 2014), however webelieve it is a novel experiment to test pleuromutilin for synergistic activitywith Oregano oil and its components.   Materials & MethodPreparation of Bacillus spores – In order to create a culture of Bacillus subtilis, a cultivation loopwith Bacillus subtilis spores wasintroduced into a 50ml LB media flask (1.

5g Tryptic Soy Broth, 50ml distilledwater, autoclaved). The flask was then incubated in a shaking incubator at 30?Cfor 24h. 100ml TSB (3g Tryptic Soy Broth, 2g Agar powder and distilled water to100ml) 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 thesurface of the TSB to ensure an even spread of the inoculums. The Thompsonbottle was then left at a 45? angle with a loosened lid in an incubator at 30?Cfor 6 days. 100ml sterile distilled water was pipetted into the Thompson bottleand water was washed over the surface of the TSB, collecting the bacterialspores from the TSB surface. 15ml aliquots of this bacterial suspension were transferredinto universal tubes using a sterile pipette and vortexed to ensure an evensuspension.

A 70?C water bath was used to heat the suspensions for 30 minutesto activate the spores. The suspensions were removed, the tubes sealed withparafilm and stored at 4?C for use throughout the experiment.Bioassay of Oreganooil and components- To test for antimicrobial activity, base media (TSAb)was prepared from 15g Tryptic Soy Broth, 10g Agar powder and distilled water to500ml; vortexed and then autoclaved. 20ml of TSAb was poured under sterilelaminar flow into 10cm petri dishes, then left to cool and harden.  Overpour media (TSAg) was prepared from 15gTryptic 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 at4?C. 20µl Bacillus subtilis Sporesuspension per 100ml was added to hot (50?C -70?C) TSAg, to heat shock thespores.

750µl of 4% TTC solution was then added per 100ml TSAg. 5ml of thisTSAg 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 ofeach plate using a sterilised borer tool. Each of the following test substanceswas 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 distilledwater at 100µl/ml.

·       The carvacrol was in solution with distilledwater at 100µl/ml. ·       The thymol, provided in granular form, wasdissolved in distilled water at 100mg/ml, warmed and vortexed to ensure it wasfully dissolved immediately before it was added to the wells. The ampicillin was used as a positive control forantimicrobial activity and the distilled water was used as a negative control.

Theplates were sealed with tape and incubated for 24h at 25?C immediately afteraddition of the test substance. The radius of the clearing zones produced afterincubation was recorded. In cases of non-circular zones, an average of fourperpendicular 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 synergismusing Clitopilus passeckerianus – Asolution of Potato Dextrose Agar (PDA) was prepared from 24g potato dextrosebroth, agar powder 15g and distilled water to 1L. 20ml of PDA was poured into apetri dish in laminar flow and allowed to set.

Using a scalpel, a small (5mm x5mm) square of Clitopiluspasseckerianus mycelium was cut from a fungal plate. This subculture was then placedinto the centre of the set PDA plate, sealed and incubated at 25?C for 5 daysto allow the fungus to grow. This plate was then used to generate all thesubcultures used in the synergism bioassay. 18 petri dishes of 20ml PDA eachwere then poured under laminar flow and allowed to set. 5mm x 5mm squares werethen cut from the growth plate at the edges, and the mycelium cuttings wereadded to the centre of each of the PDA plates. These plates were then sealedand incubated at 25?C for 5 days. A well was then cut into the centre of thefungal growth and 40ml of the test substances were then pipetted into thewells. Oregano Oil and distilled water were added at 100%concentration.

The ampicillin and carvacrol were in solution with distilledwater at 100µl/ml. The thymol, provided in granular form, was dissolved indistilled water at 100mg/ml, warmed and vortexed to ensure it was fullydissolved immediately before it was added to the wells. The ampicillin was usedas a positive control and the distilled water as the negative control. 20ml TSAg{Overpour media} (30g Tryptic Soy Broth, 5g Agar, 10g Glucose, distilled waterto 1000ml, autoclaved) was poured over each plate.

They were then sealed andincubated for 24h at 25?C. The radii of the clearing zones were measured fromthe well. In cases of non-circular zones, an average of four perpendicularmeasurements was used Each substance was plated in triplicate, then this was repeatedto give a total of 6 plates for each test substance. ResultsThe ability of the test substances to inhibit growth of Bacillus subtilis on an agar plate isrecorded in Figure 1. The negative control sterile distilled water did notproduce a clearing zone. The positive control ampicillin produced the largestclearing zone of 24.2mm radius. Pure Oregano oil, carvacrol and thymol eachproduced a clearing zone, showing antibiotic activity.

 Figure 1. Meanclearing zone radius produced by agar well diffusion of test substances on Bacillus subtilis. Incubated at 35?C for24h. Error bars show the standard deviation around the mean. A One-way ANOVAwas 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), andCarvacrol (p<0.001) to be statistically significantly different from thenegative 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 recordedin Figure 2. To be able to say synergistic activity occurred, the testsubstances’ mean clearing zone would have to be larger than the non-synergisticpositive control, 10% Ampicillin. None of the three test substances exhibitedany synergistic effect, as all test substances produced a mean clearing zonesmaller than both the positive and negative controls.  Figure 2.  Mean clearing zone produced by testsubstances or controls when plated on Clitopiluspasseckerianus over a Bacillus subtilisinoculated Potato Dextrose Agar plate, incubated for 5 days at 25?C. SterileDistilled Water was used as the negative control. 10% Ampicillin used aspositive control. All test substances used in same concentrations as inantibiotic bioassay.

The negative control produced a mean clearing zone of23.3mm. The positive control produced a mean clearing zone of 31.7mm, largerthan the negative control as expected. A One-way ANOVA shows the positivecontrol 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 restof this investigation. The test substances all have smaller means than thenegative control, which therefore shows no evidence of synergistic activityoccurring, as this would produce a larger clearing zone radius.

Discussion The antibiotic activity of three test substances wasinvestigated; the results showed that 100% Oregano oil as obtained from abottle of ‘over-the-counter’ capsules had an antibiotic effect on Bacillus subtilis when plated with a TSBagar (Fig. 1). This conclusion supports the findings of (K. Wareham.

2016)(G.Niblock. 2016), who also found that Oregano oil had antibiotic activity. Thisreport was the basis of this investigation, and as such we followed a similarprotocol for performing an antibiotic activity bioassay, which was a standardbacterial bioassay protocol provided by the University of Bristol Life SciencesBuilding laboratories. As our intention was to expand upon this work, and focusupon the possible synergistic activity of Oregano oil with pleuromutilin, wealso tested the two monoterpene alcohols that make up Oregano oil: thymol and carvacrol.This allowed us to exclude any bulking agents as variables in causingantibiotic activity or synergistic activity. The monoterpene alcohols both produceda clearing zone, however only carvacrol and pure Oregano oil had astatistically significant difference from the negative control.

This suggeststhat a repeat of these findings utilising a greater number of plates may find astatistically significant difference, and future work would benefit fromgreater investigation into these substances. This experiment utilised just sixplates in total for each test substances for the bacterial bioassay, which mayhave been too few to be able to conclusively decide upon the antibioticactivity, or lack thereof, of thymol against Bacillus subtilis.There is also a need for future work involving testingmultiple different species of bacteria against these test substances. Bacillus subtilis was used for a range ofreasons. It is a Gram-positive, spore-forming and non-pathogenic bacterium (Hiraokaet al. 1992).

These factors allow much easier laboratory growth and usage thanother bacteria available. Bacillus subtilisis a model organism; it is highly studied and understood, making it ideal as atest bacterium (Hiraoka et al. 1992). Gram-positive bacteria were chosenbecause it has been reported that “Grampositive bacteria are more susceptible to the phenolic components of essentialoils than Gram negative bacteria since they act as membrane permeabilisers” (Cacciatoreet al. 2015).Our decision to test for potential synergistic activitywithin carvacrol and thymol was prompted by reports of synergisticrelationships between these monoterpene alcohols and other antibiotics, such astetracycline (Cirino et al. 2014).

The findings of last year’s project students (K.Wareham. 2016)(G.

Niblock. 2016) had also previously suggested possiblesynergistic activity, as well as further literature (Cacciatore et al. 2015). Carvacroland thymol constitute 70%-85% of pure Oregano oil, suggesting that any Oreganooil synergism found may be due to the presence of Carvacrol and Thymol (Ultee et al., 2002). They are alsobelieved to be the cause of Oregano Oil’s antimicrobial activity (Fournomiti etal.

, 2015). The method by which they cause an antibiotic effect is bycausing damage to cell membrane integrity, affecting pH homeostasis andequilibrium 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. Asstated previously, we used the fungus Clitopiluspasseckerianus, a natural producer of the antibiotic pleuromutilin, as thesource of antibiotic for the synergism bioassay. Upon finding the resultsshowed no synergism, we had to revaluate our methods and now believe the use ofthis fungus may be the cause of our findings not supporting previous findings.Carvacrol has fungicidal properties (Cacciatore et al.

2015). This means thatthe fungal growth and production of pleuromutilin was almost certainlynegatively affected in comparison to the control substance. This would explainnot only the lack of synergistic activity found, but the smaller mean clearingzone radius than the negative control. Thymol also has similar antifungalproperties (Robledo et al.

2018), meaning we can assume the same to be true ofthe thymol results. Clearly for any further work into this area, a newmethodology would need to be created to avoid the need for Clitopilus passeckerianus. This presents a challenge however, aspleuromutilin is a novel, semisynthetic antibiotic only recently beingthoroughly investigated (Jacobsson et al.

2017) (Abbas et al. 2017).It was not readily available for use in this experiment, unlike ampicillin, andso we could not use it in its pure form. It was this inability to acquire thedrug that led us to choose to use the fungal method. Any further workwould benefit from be able to procure pleuromutilin directly if they wish totest for synergism in a similar way to this investigation.

However, this may beeither very expensive and/or difficult, or need to be done once theavailability of pleuromutilin has increased. It is also important that theantibiotic activity of Oregano oil and its components are tested against a widerange of both gram-positive and gram-negative bacteria, if there is to be anyfuture use of the compounds for their antibiotic activity. Although using Bacillus subtilis due to its status as amodel organism is beneficial, it cannot be assumed that the antibiotic activitywould extend to other gram-positive bacteria without clinical trials. If thereis hope for using Oregano oil in human medicine, tissue and eventually humantrials are essential before it can be introduced to the public to ensure thereare no unwanted side effects.

The oil may be easily available to buy for thepublic, but until it has been shown to not have any negative effects it cannotbe medically prescribed. Our conclusion from this investigation is that althoughthere is evidence behind the claims of antibiotic activity of Oregano oil, asthere is for many other essential oils, the claim of synergism withpleuromutilin has not been provable within this investigation. Carvacrol is themore antibiotically potent than thymol, and although we did not find significantevidence for thymol having antibiotic activity, we believe that further, more rigorousinvestigation may do. However, we can say that the antibiotic activity of carvacrolis far more potent than thymol.

We did not find evidence of synergism, webelieve due to our methodology, and have set out potential suggestions forfurther work which may be able to learn from and avoid our sources of error. ReferencesAbbas, M., M. Paul, and A. Huttner.

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