2017/2018 Practical Project

School of Biological Sciences

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

 

Investigation
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:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Abstract

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.

 

Introduction

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
wells.

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.

 

Results

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

Written by
admin
x

Hi!
I'm Colleen!

Would you like to get a custom essay? How about receiving a customized one?

Check it out