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Fisher Et Al-2009-Journal of Applied Microbiology
Fisher Et Al-2009-Journal of Applied Microbiology
ORIGINAL ARTICLE
Keywords
Enterococcus, citrus essential oils, mechanisms
of action.
Correspondence
Katie Fisher, University of Northampton,
School of Health, Park Campus, Boughton
Green Road, Northampton, NN2 7AL, UK.
E-mail: katie.fisher@northampton.ac.uk or
carol.phillips@northampton.ac.uk
Abstract
Aims: The aim was to explore the mechanisms by which a blend of orange
(Citrus sinensis) : bergamot (Citrus bergamia) (1 : 1 v v) EO (essential oil)
(2% v v) and its vapour (15 mg l)1 air) brings about its antimicrobial effect
against Enterococcus faecium and Enterococcus faecalis.
Methods and Results: Cells were exposed to the blend in oil or vapour form in
a sealed unit. Membrane permeability was measured using an NPN assay and
intra and extracellular ATP concentrations were assessed using luminescence.
Assays using 3,3-dipropylthiacarbocyanine and carboxyfluorescein diacetate
succinimidyl ester measured membrane potential and intracellular pH changes.
TEM images of treated cells indicate morphological differences and show the
possible uptake of the EO into the cell. After cells were exposed to EO or
vapour, cell permeability increased by 2 and 40 respectively. A decrease of
15 in intracellular pH, 20 a.u. in membrane potential and 18 pmol mg)1 protein of intracellular ATP occurred.
Conclusions: The EO blend affects the cell membrane and cell homeostasis
resulting in inhibition of growth or cell death.
Significance and Impact of the Study: Understanding the mechanisms by
which EOs bring about their antibacterial effect could lead to an alternative to
chemical-based bactericides for use against Enterococcus sp.
Introduction
In the last decade, Enterococcus was reported as the second most common cause of wound and urinary tract
infection and the third most common cause of bacteraemia (De Fatima Silva Lopes et al. 2005). In 2005, in
the UK, 28% of all cases were antibiotic-resistant. Of
these, 63% were due to Enterococcus faecalis and 28% to
Enterococcus faecium, both of which have increasing
numbers of antibiotic resistant isolates being reported
especially those resistant to vancomycin (Health Protection Agency, 2007). In the USA, there have been similar
increases in vancomycin resistant Enterococcus (VRE).
Many factors determine the virulence of Enterococcus,
for example, its ability to colonize the GI tract, to
adhere to a range of extracellular matrix proteins, urinary tract epithelial and human embryo kidney cells
(Franz et al. 1999). The capability for Enterococcus to
1343
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Results
(a)
Oil
12846 24 3083 8
13066 18 30996 5
50
45
40
35
30
25
20
15
10
5
0
50
(b)
Fluroscence (a.u)
Nigericin
Oil
Glucose
Glucose
100
150
Time (s)
Nigericin
200
250
300
200
250
300
Oil
40
35
30
25
20
15
10
5
0
0
50
100
150
Time (s)
Figure 1 Changes in membrane potential after the addition of bergamot : orange (1 : 1 v v) EO blend to (a) Ent. faecium and (b) Ent.
faecalis (n = 6).
Discussion
The findings of this investigation suggest that the antimicrobial action of a bergamot : orange (1 : 1 v v) EO on
Enterococcus spp. may be effected by the uptake of the EO
into the cell (Fig. 3). Vacuoles are observed within the
cells which were not present in the control cells. The lipophilic characteristic of the EOs will allow the preferential
partitioning from the aqueous phase into the membrane
structure of the cell (Cox et al. 2000) and thus could
result in the uptake of the EO into the cell. However, the
mechanism of action of the vapour is not clear and,
although it may work via the same mechanism, no resi-
Vapour
Fluorescence
value
Increase
24 01 419165 9 3263 02
237 02 524339 10 4013 03
1346
20
18
16
14
12
10
8
6
4
2
0
0
8
6
Time (min)
12
10
14
due of oil was noted when being examined by UV spectrophotometry unlike when the cells were treated with
EO when between 85 and 919 ll ml)1 remained in the
supernatant of the lysed Ent. faecalis and Ent. faecium
cells suspensions respectively. Previous studies using TEM
to assess cell damage from EOs have suggested that the
vacuoles observed when cells are treated with EOs may be
due to co-agulation of the membrane, for example, when
using cinnamon and oregano in combination with packaging against E. coli and Staph. aureus (Becerril et al.
2007 or oregano, cinnamon and savory against E. coli and
L. monocytogenes (Oussalah et al. 2006). The use of thyme
against L. monocytogenes also showed splitting of the cell
wall layers (Rasooli et al. 2006). The uptake of the EOs
into the cell to form vacuoles and thus having an
Ent. faecium
Before
Oil
Vapour
(a)
n
(c)
5 m
pH intracellular
D pH (pHinpHout)
pH intracellular
D pH (pHinpHout)
634
135
634
135
Ent. faecalis
After
024
021
024
021
546
0
532
0
Before
018
02
016
026
651
163
651
163
After
038
008
038
008
498
0
425
0
027
011
014
012
(b)
05 m
(d)
05 m
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Acknowledgements
We would like to thank AMPHORA for the supply of the
EOs and heat diffuser and J. Sheldon at Oxford Brookes
University for her expertise on the TEM experiments.
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