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The anti-biofilm activity of lemongrass ( Cymbopogon flexuosus ) and grapefruit ( Citrus paradisi ) essential oils against five strains of Staphylococcus aureus

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To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment.
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ORIGINAL ARTICLE
The anti-biofilm activity of lemongrass (Cymbopogon
flexuosus) and grapefruit (Citrus paradisi) essential oils
against five strains of Staphylococcus aureus
E.C. Adukwu, S.C.H. Allen and C.A. Phillips
School of Health, The University of Northampton, Northampton, UK
Keywords
biofilms, microbial contamination,
staphylococci.
Correspondence
Carol A. Phillips, School of Health, The
University of Northampton, Boughton Green
Road, Northampton NN2 7AL, UK. E-mail:
carol.phillips@northampton.ac.uk
2012/0575: received 30 March 2012, revised
27 June 2012 and accepted 27 July 2012
doi:10.1111/j.1365-2672.2012.05418.x
Abstract
Aims: To determine the sensitivity of five strains of Staphylococcus aureus to
five essential oils (EOs) and to investigate the anti-biofilm activity of
lemongrass and grapefruit EOs.
Methods and Results: Antimicrobial susceptibility screening was carried out
using the disk diffusion method. All of the strains tested were susceptible to
lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition
varying from 285 to 860 cm although they were resistant to lemon EO.
Lemongrass EO inhibited biofilm formation at 0125% (v/v) as measured by
colorimetric assay and at 025% (v/v) no metabolic activity was observed as
determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm
activity. Following exposure to lemongrass EO extensive disruption to
Staph. aureus biofilms was shown under scanning electron microscopy.
Conclusions: In comparison to the other EOs tested, lemongrass exhibited the
most effective antimicrobial and anti-biofilm activity.
Significance and Impact of the Study: The effect of lemongrass EO highlights its
potential against antibiotic resistant Staph. aureus in the healthcare environment.
Introduction
Staphylococcus aureus is an important human pathogen
whose ability to acquire resistance mechanisms and other
pathogenic determinants has added to its emergence in
both acute and community healthcare settings (Said-
Salim et al. 2005; Zetola et al. 2005). Although the majority
of the Staph. aureus infections described in community
settings has been associated with methicillin susceptible
Staph. aureus (MSSA) strains (Said-Salim et al. 2005)
rather than methicillin resistant Staph. aureus (MRSA)
strains, infections caused by the latter are linked with
higher subsequent treatment costs than those caused by
MSSA strains (Gordon and Lowy 2008). Over 50% of
healthcare associated MRSA strains are known to be
resistant to both b-lactam and non-b-lactam antibiotics
while the community acquired (CA) Staph. aureus strains
are susceptible to non-b-lactam antibiotics (Fey et al.
2003; Weber 2005; King et al. 2006). The emergence of
CA infections especially CA MRSA strains is of particular
importance as they possess molecular characteristics absent
in hospital MRSA strains highlighted in a review by Cham-
bers and Deleo (2009). These characteristics include; a
staphylococcal chromosomal cassette mec (SCCmec) type
IV allele and genes which encode the virulence factor Pan-
ton Valentine-Leukocidin (PVL) (King et al. 2006). PVL is
a cytotoxin which causes leucocyte destruction and tissue
necrosis (Weber 2005; Loughrey et al. 2007), and both
MSSA and MRSA strains from community settings can
carry the PVL gene (Rasigade et al. 2010).
Biofilms have been defined by Donlan and Costerton
(2002), as immobile communities of organisms attached
to a substratum or to each other, embedded in a matrix
of extracellular polymeric substances and showing an
altered phenotype. Biofilm formation has been identified
in Pseudomonas sp. (Klausen et al. 2003; Ghafoor et al.
2011), Candida sp. (Ramage et al. 2002; Silva et al.
2011), Enterococcus sp. (Seno et al. 2005; Mohamed and
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 1
Journal of Applied Microbiology ISSN 1364-5072
Huang 2007) and Staph. aureus (Donlan and Costerton
2002; Knobloch et al. 2002; Yarwood et al. 2004). The
ability to form biofilms is also known to provide the
organisms with protection against antibiotics (Yarwood
et al. 2004).
Interest in natural antimicrobials has grown in recent
years and the most important and well researched of
these are plant products which have many medicinal and
antimicrobial properties (Bourne et al. 1999; Cowan
1999). Essential oils (EOs) extracted from plants have
been shown to possess antimicrobial activity in in vitro
assays against a range of bacteria including known antibi-
otic resistant strains (Fisher and Phillips 2006; Warnke
et al. 2009). EOs have been used as topical antimicrobials
(Barker and Altman 2010; Dai et al. 2010), as dental and
oral treatments (Jeon et al. 2011; Palombo 2011), and for
burns and wound healing (Edwards-Jones et al. 2004;
Thakur et al. 2011). Recently, the use of EOs in vapour
phase has also been shown to be anti-bacterial and
anti-fungal as reviewed by Laird and Phillips (2012).
In recent years, reports of studies on the anti-biofilm
activity of EOs have been increasing. For example, cinna-
mon EO against Candida sp. (Pires et al. 2011) a citrus EO
in vapour phase against Enterococcus faecium (Laird et al.
2012) and lemongrass EO against biofilm formation in
Listeria monocytogenes (De Oliveira et al. 2010). The resis-
tance of biofilm-associated organisms is estimated at
50500 times more than planktonic cells (Jabra-Rizk et al.
2006). The effect of different EOs on biofilms has been
investigated with studies showing sufficient eradication of
biofilms of Pseudomonas aeruginosa (Kavanaugh and
Ribbeck 2012) and Staph. aureus and Staphylococcus epide-
rmidis (Nuryastuti et al. 2009; Nostro et al. 2007) at similar
concentrations as corresponding planktonic cells. In other
in vitro studies, there was increased activity in biofilms
exposed to EOs when compared to their effect on cells in sus-
pension (Al-Shuneigat et al. 2005; Karpanen et al. 2008).
The aim of this present study was to determine the
anti-staphyloccocal activity of a range of EOs against five
strains which included those of CA origin using in vitro
screening assays and to further investigate the anti-bio-
film effect of the EOs found to be the most effective after
initial screening.
Materials and methods
EOs and components
The EOs used in this study were lemongrass (Cymbopo-
gon flexuosus), grapefruit (Citrus paradisi), lime (Citrus
aurantifolia), bergamot (Citrus bergamia) and lemon
(Citrus limon) obtained from Belmay Plc., Northampton,
UK. Two known EO components limonene (Sigma-Aldrich,
Dorset, UK) and citral (95%, natural; SAFC Supply
Solutions, St Louis, MO, USA) were also investigated.
Micro-organisms
The test micro-organisms used in this study included:
three MSSA strains (a hospital MSSA isolate, MSSA
NCTC 13297 and a CA PVL positive MSSA) and two
MRSA strains (one CA MRSA MW2, and a PVL positive
CA MRSA strain). MSSA NCTC 13297 was obtained
from Health Protection Agency, London, UK, while the
other strains were provided by Professor Mark Fielder at
Kingston University, UK. Stock cultures were stored on
beads at 80°C. Working cultures were maintained on
Brain Heart Infusion (BHI) agar, sub-culturing weekly
for a maximum of 3 weeks, to maintain viability and col-
ony characteristics. For inoculum preparation, single col-
onies were picked from a BHI agar plate into BHI broth
(CM225; Oxoid Ltd, Basingstoke, UK) and incubated
overnight at 37°C. Enumeration was on BHI agar
(CM0375; Oxoid).
Disc diffusion
The screening method was adapted from Prabuseenivasan
et al. (2006) and the British Society for Antimicrobial
Chemotherapy (BSAC) guidelines Version 10 (Andrews
and Howe 2011). Briefly, 100 ll of each of the EOs was
deposited onto sterile 2 cm diameter filter paper discs
placed on the surface of BHI plates previously spread
with 100 llofa10
8
CFU ml
1
overnight culture. The
plates were left to dry for 15 min in a sterile environ-
ment, inverted and incubated at 37°C for 24 h. The
diameters of zones of inhibition (ZOI) were measured
using Vernier calipers. The controls were bacterial
cultures without EO exposure.
Minimum inhibitory and minimum bactericidal
concentrations
The method used was adapted from Hammer et al.
(1998). An aliquot (20 ll) of a 10
8
CFU ml
1
overnight
culture was added to wells of a sterile 96-well micro-titre
plate. Each EO was diluted in BHI broth containing 05%
(v/v) Tween 20 and added to wells to give final EO con-
centrations of 003, 006, 012, 05, 1, 2 and 4% (v/v).
The positive control wells contained BHI broth and cells
without EOs while the negative control wells contained
BHI only. Optical density (OD) was measured at 595 nm
using a microplate reader (Bio-Rad 680XR, Hertfordshire,
UK) and again after incubation for 24 h at 37°C. The
Minimum Inhibitory Concentration (MIC) was deter-
mined as the lowest EO concentration at which the OD
©2012 The Authors
2Journal of Applied Microbiology ©2012 The Society for Applied Microbiology
Staph. aureus biofilms and EOs E.C. Adukwu et al.
at 24 h of the inoculum remained the same or reduced
compared with the initial reading.
For Minimum Bactericidal Concentration (MBC)
determination, 10 ll was taken from each well after incu-
bation and spot inoculated (Hammer et al. 1998) onto
BHI agar and incubated for 24 h at 37°C. The concentra-
tion at which no growth was observed on subculture was
determined as the MBC.
Minimum biofilm inhibitory concentration (MBIC)
Inhibition of biofilm formation was assessed using a
method adapted from Nostro et al. (2007). An aliquot
(100 ll) from an overnight culture diluted in BHI broth
supplemented with 1% (w/v) glucose to 10
8
CFU ml
1
was dispensed into each test well of a 96 well plate. In
all, 100 ll of the EO concentrations 0064% (v/v) for
lemongrass EO and 14% (v/v) for grapefruit EO were
added into the wells. The negative control was BHI
broth only whereas the positive control contained cell
cultures alone with no added EO. Following 24 h incu-
bation at 37°C, the contents of the wells were decanted
and each well gently rinsed twice with 300 ll of sterile
phosphate buffered saline (PBS) (pH: 73±03). The
plates were air dried for 30 min, stained with 01%
(w/v) crystal violet for 30 min at room temperature
(Wijman et al. 2007), washed three times with PBS
(200 ll per well) and dried. The crystal violet was then
solubilized using 10% (v/v) glacial acetic acid and the
OD measured at 595 nm using a Microplate reader
(Bio-Rad 680XR). The MBIC was determined as the EO
concentration at which the OD negative control
(Pettit et al. 2005; Sandoe et al. 2006). Each experiment
was performed in quadruplicate and performed on four
separate occasions.
Minimum biofilm eradication concentration (MBEC)
The method used was similar to that described by
Kwiecinski et al. (2009). After biofilm formation for
48 h, the medium was discarded and the wells gently
rinsed twice with PBS. A total of 200 ll of the EOs
(lemongrass or grapefruit) were serially diluted and
added into the wells ranging from 006 to 4% (v/v) for
lemongrass EO and 1 to 4% (v/v) for grapefruit EO.
The plates were then incubated for 24 h at 37°C after
which the wells were washed with PBS and stained
using the Crystal Violet (CV) staining method as
described previously. The positive control was biofilm
without EO. The concentration at which already estab-
lished biofilms were removed from the bottom of the
treated wells was determined as the MBEC (Muli and
Struthers 1998; Ceri et al. 1999).
Biofilm metabolism assay XTT reduction
This method is based on reduction of tetrazolium salt
XTT [2, 3-bis (2-methyloxy-4-nitro-5-sulphophenyl)-2H-
tetrazoluim-5-carboxanilalide] and was performed to
determine the metabolic activity of the biofilm formed
using methods described by Cerca et al. (2005) and Laird
et al. (2012). Stock solutions of XTT in PBS (5 mg ml
1
)
and menadione (1 mmol l
1
) were prepared. At the start
of each experiment, a fresh solution of XTT/menadione
was prepared at a ratio of 125/1 v/v. Biofilms were
formed for 48 h in the wells of 96 well plates and 200 ll
of the XTT/menadione mix was added into each test and
control well, incubated in the dark at 37°C for 24 h and
the OD measured at 450 nm.
Biofilm viability assay (CFU ml
1
)
Biofilm viability was measured using a method adapted
from Pettit et al. (2005). Following 24 h exposure of bio-
films to lemongrass or grapefruit EO, a sterile scraper
was used to dislodge each biofilm into the micro-titre
wells, and 100 ll of the well contents removed and
spread onto BHI agar. Plates were incubated for 24 h at
37°C before enumeration.
Gas chromatography mass spectrometry
GC/MS analysis of the EOs was performed using the Tur-
boMass (Perkin-Elmer, Buckinghamshire, UK) instru-
ment with column 1 stationary phase Rtx 1 column
(60 m 9025 mm i.d.; film thickness: 025 lm; Restek).
The oven temperature programme was: initial tempera-
ture of 50°C; increasing by3°C min
1
to 265°C and held
for 13 min. Helium was used as the carrier gas with a
1ll injector volume, an injector temperature of 285°C
and a split ratio 30 : 1. The MS was performed with an
EI+source and operated in scan mode, from 35 to
350 m/zat a detector temperature of 300°C. The com-
pounds were identified by comparing retention times and
mass spectra with those of standards or their retention
indices with published data and their mass spectra with
the National Institute of Standards and Technology
(NIST) library.
Scanning electron microscopy (SEM)
Following preliminary investigations of biofilm forma-
tion, PVL CA MSSA was selected for SEM observations
due to its higher biofilm OD values compared with the
other strains (data not shown). In all, 2 cm diameter
sterile stainless steel discs (Goodfellows Cambridge Ltd,
Huntingdon, UK) were immersed in six well plates (Nun-
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 3
E.C. Adukwu et al. Staph. aureus biofilms and EOs
clon Surface, Roskilde, Denmark) containing 5 ml of BHI
broth supplemented with 1% (w/v) glucose for 48 h. A
total of 100 llofa10
8
CFU ml
1
overnight culture was
then added and the plates incubated for 48 h in a shak-
ing incubator. After incubation the discs were removed
and gently rinsed with sterile PBS to remove loosely
attached cells and re-suspended in 0125, 05 and 1%
(v/v) lemongrass or 4% (v/v) grapefruit EO. After expo-
sure to the EOs, the discs were washed three times with
PBS and fixed with 25% (v/v) glutaraldehyde in PBS
solution for 2 h at 4°C, washed twice with PBS and dehy-
drated for 10 min using a graded ethanol series; 30, 50,
70, 90, 100% (v/v). The samples were then dried prior to
coating with gold and observed using a Hitachi S-3000
Scanning Electron Microscope (Hitachi High-Technolo-
gies Europe, Maidenhead, UK).
Statistical analysis
Statistical analysis was conducted using SPSS version 17.0
(IBM, Armonk, NY, USA). Significance levels was set at
P=005. After assumptions of normality and variances
of homogeneity were checked, one way analysis of
variance (ANOVA) was performed.
Results
Screening
No ZOIs were observed with either lemon EO or limo-
nene (Table 1) whereas lemongrass EO and citral, the
major component in lemongrass EO (Table 2), both
completely cleared the plate of bacterial growth with a
ZOI of >860 cm. Grapefruit, lime and bergamot EOs
produced ZOIs ranging from 285 to 463 cm (Table 1).
Consequentially, lemongrass and grapefruit EOs were
selected for determination of MICs and MBCs and for
anti-biofilm activity, as these were the most effective at
>860 and 348 cm respectively (Table 1). Although ZOI
produced by the latter was only marginally more effective
than lime EO (ZOI =347 cm), it was selected for fur-
ther studies as it has been shown to have anti-bacterial
activity and potential in other antimicrobial applications
(Williams et al. 2007; Uysal et al. 2011).
The MIC for lemongrass EO at 006% (v/v) for all
strains tested was lower than that for grapefruit EO at
05% (v/v) for MSSA NCTC 13297 and 1% (v/v) for the
other strains. Similarly, the MBC for lemongrass EO was
the same for all the strains tested (0125% v/v), while for
grapefruit the MBC was 2% (v/v) for the hospital MSSA
Table 1 Zones of inhibition (cm) ±SE measured after exposure to essential oils and components against Staphylococcus aureus strains
Lemongrass Grapefruit Lime Bergamot Citral Lemon Limonene
Hospital MSSA 86(±0) 396 (±017) 417 (±018) 336 (±007) 86(±0) 0 (±0) 0 (±0)
PVL CA-MSSA 86(±0) 323 (±006) 290 (±017) 289 (±007) 86(±0) 0 (±0) 0 (±0)
MSSA NCTC 13297 86(±0) 335 (±007) 327 (±005) 312 (±011) 86(±0) 0 (±0) 0 (±0)
CA-MRSA (MW2) 86(±0) 343 (±004) 357 (±012) 285 (±009) 86(±0) 0 (±0) 0 (±0)
PVL CA-MRSA 86(±0) 342 (±011) 358 (±008) 285 (±009) 86(±0) 0 (±0) 0 (±0)
Mean (cm) 860 348 347 301 860 000 000
CA, community acquired; MRSA, methicillin resistant Staph. aureus; MSSA, methicillin susceptible Staph. aureus; PVL, Panton Valentine-
Leukocidin.
Table 2 GC/MS analysis of the essential oils showing major
components
Components
Cymbopogon
flexuosus
Citrus
aurantifolia
Citrus
paradisi
Citrus
bergamia
Alpha Pinene 020 230 050 130
Methyl
heptenone
200 ––
Myrcene 100 232 100
Limonene 040 4730 9350 3850
Linalool 150 015 1140
Citronellal 100 ––
Neral* 3300 200 020
Geranial* 4700 290 040
Geranyl acetate 150 ––
Beta
carypohyllene
400 100 ––
Beta pinene 2270 040 720
Alpha
terpinene
025 ––
Para cymene 025 050
Gamma
terpinene
750 600
Trans alpha
bergamotene
110 ––
Beta bisabolene 270 ––
Decanal ––026
Nootkatone ––010
Alpha thujene ––030
Sabinene ––110
Terpinolene ––030
Linalyl acetate ––2790
*Neral is the Z-isomer of Citral (also known as Citral B) and geranial
is the E-Isomer of Citral (Citral A).
©2012 The Authors
4Journal of Applied Microbiology ©2012 The Society for Applied Microbiology
Staph. aureus biofilms and EOs E.C. Adukwu et al.
strain and MSSA NCTC 13297, and 4% (v/v) for the
three community strains; PVL CA MSSA, CA-MRSA
(MW2) and PVL CA MRSA (Table 3).
Lemongrass EO prevented biofilm formation at 006%
(v/v) for the hospital MSSA strain and 0125% (v/v) for
the other strains tested (Table 3) which, for four of the
five strains were the same concentration as the MBC.
However, lemongrass EO did not remove already formed
biofilms (MBEC) at any of the concentrations tested, i.e.,
0064% (v/v). Grapefruit EO did not either prevent bio-
film formation or remove already formed biofilms at
14% (v/v) (Table 3).
Inhibition of metabolic activity occurred in the pres-
ence of lemongrass EO after 24 h for all five
Staph. aureus strains at 0125 and 006% (v/v) with no
significant difference in the reduction brought about by
these two concentrations (Fig. 1a). At 025% (v/v), no
metabolic activity was observed (results not shown).
Grapefruit EO did not reduce the metabolic activity as
measured by the XTT assay after 24 h incubation. When
the effect of grapefruit EO was compared for four strains
(not including PVL CA MSSA), there was no statistical
significant difference as determined by the one-way
Anova for 2% (P=0390) and 1% (P=0259) EO,
although at 4% there was a statistical significant differ-
ence (P=0039) with PVL CA MRSA having a higher
metabolic activity than the other three strains.
There was a significant difference between the meta-
bolic activity of PVL CA MSSA and the other strains at
all the concentrations of grapefruit EO tested (at 4%,
P=0036; at 2%, P=0038; at 1%, P=0012) with PVL
CA MRSA having a metabolic activity approximately 27
times that of the control compared to the other four
strains (Fig. 1b).
Following lemongrass EO treatment for 24 h, biofilms
showed total loss of viability at concentrations of 0125
4% (v/v) dependent on strain (results not shown),
although at 006 (v/v) some viable cells were recovered
(Fig. 2a) there was no significant difference between the
treated and control viable counts (P=057) and with no
significant differences between the results for the five
strains (P=049). Grapefruit EO treated biofilms showed
no reduction in viability at any of the concentrations
tested (Fig. 2b).
When biofilms were treated with 2 and 1% grapefruit
EO, there was no statistically significant difference
between the viable counts obtained for all strains (Pvalue
at 2% =070; Pvalue at 1% =072). At 4%, there was a
statistical significant difference between the strains
(P=0049) which can be attributed to the larger varia-
tion observed for the hospital MSSA strain although there
was no statistical differences between the other four
strains (P=045).
Following biofilm quantification, the PVL CA MSSA
strain consistently showed increased biofilm formation
compared to the other strains tested, and therefore was
chosen for SEM. After 24 h exposure to lemongrass EO,
the control (Fig. 3a) showed intact biofilm structure, and
at 0125% (v/v) (Fig. 3b) it was observed that the integ-
rity of the biofilm structure was disrupted. At 05% (v/v)
lemongrass EO, there was evident damage on the biofilm
structure (Fig. 3c) and at 1% (v/v) of lemongrass EO
treatment, no biofilms were observed on the discs
although biofilm debris remained (Fig. 3d). When PVL
CA MSSA was treated with 4% (v/v) grapefruit EO, no
effect on biofilm formation and integrity was observed in
comparison to the control (results not shown).
Discussion
Lemongrass EO at low concentrations between 003 and
006% (v/v) was effective at inhibiting the growth of all
five Staph. aureus strains, and at 0125% (v/v) the effect
of lemongrass EO was bactericidal. The results presented
here are consistent with those of a previous study
(Barbosa et al. 2009) in which it was demonstrated that
lemongrass EO inhibited the growth of Gram positive
bacteria, including Staph. aureus at a concentration of
005% (v/v). In this present study the MIC for grapefruit
EO was higher than that for lemongrass EO for all the
Table 3 Minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration (MBEC) and minimum
biofilm eradication concentration (MBEC) (% v/v) for lemongrass essential oil (EO) and grapefruit EO against Staphylococcus aureus strains
Lemongrass EO Grapefruit EO
MIC (%) MBC (%) MBIC (%) MBEC (%) MIC (%) MBC (%) MBIC (%) MBEC (%)
Hospital MSSA 006 0125 006 >412>4>4
PVL CA-MSSA 006 0125 0125 >414>4>4
MSSA NCTC 13297 006 0125 0125 >4052 >4>4
MRSA MW2 006 0125 0125 >414>4>4
PVL CA-MRSA 006 0125 0125 >414>4>4
CA, community acquired; MRSA, methicillin resistant Staph. aureus; MSSA, methicillin susceptible Staph. aureus; PVL, Panton Valentine-
Leukocidin.
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 5
E.C. Adukwu et al. Staph. aureus biofilms and EOs
strains, i.e., between 05 and 2% (v/v), while bactericidal
activity was observed between 2 and 4% (v/v) EO. To
date, there are very few studies that have investigated the
antimicrobial activity of grapefruit EO although it has
been shown to possess both anti-fungal and anti-bacterial
activity (Viuda-Martos et al. 2008; Uysal et al. 2011).
When the effects of the components were compared to
the overall effect of the EO, contrasting results were
observed. First, citral, the major component in lemon-
grass EO also showed a ZOI of 86 cm (i.e., no growth
on plate) at screening and similar MIC profiles to lemon-
grass EO (data not shown) suggesting that citral may be
responsible for the majority of the anti-bacterial activity.
This high activity by citral has been previously reported
(Hayes and Markovic 2002; Da Silva et al. 2008; Aiems-
aard et al. 2011). Limonene is the major component in
the grapefruit EO used in this study, at approximately
94%, but it did not show any antimicrobial effect as
demonstrated by the screening results which has also
been observed in previous studies by Fisher and Phillips
(2006) and Inouye et al. (2001). In comparison to this
lack of activity by limonene, grapefruit EO produces
inhibition zones in the Staph. aureus strains between 323
and 396 cm which suggests that other components of
the grapefruit EO are involved in the anti-bacterial activ-
ity observed in this study. Onawunmi et al. (1984)
observed that myrcene showed no anti-bacterial activity
per se, but enhanced activity of other EO components
when in combination which suggests that the presence of
other components in small amounts could enhance the
EO antimicrobial activity. Although the individual com-
ponents of EOs are important, they act in a synergistic
manner so that the EO exhibits a greater anti-bacterial
activity than the sum of that brought about by its
components (Gill et al. 2002).
The results of this present study demonstrate that lem-
ongrass EO possesses anti-biofilm activity at low concen-
trations between 006 and 0125% (v/v) which has been
reported previously (Aiemsaard et al. 2011). As biofilm
formation is a survival mechanism but also contributes to
virulence and persistence (Vuong et al. 2004; Soto et al.
2006), it has been suggested that preventing biofilm
attachment is a way of dealing with the problem of bio-
films in the food industry (Sinde and Carballo 2000).
–0·2
0
0·2
0·4
0·6
0·8
1
1·2
1·4
1·6
Hospital MSSA PVL CA MSSA MSSA NCTC 13297 CA-MRSA MW2 PVL CA MRSA
Relative metabolic activity (OD450 nm)
0·00
0·50
1·00
1·50
2·00
2·50
3·00
3·50
Hospital MSSA PVL CA MSSA MSSA NCTC 13297 CA-MRSA MW2 PVL CA MRSA
Relative metabolic activity (OD450 nm)
(a)
(b)
Figure 1 Changes in metabolic activity
following 24 h exposure of biofilms of
Staphylococcus aureus strains to (a)
lemongrass essential oil (EO) (0125%,
006%, , control) and (b) grapefruit EO (
1%, 2%, 4%, control) as determined
by the XTT assay (control =biofilms not
exposed to EO; N=4 for each treatment and
for each strain).
©2012 The Authors
6Journal of Applied Microbiology ©2012 The Society for Applied Microbiology
Staph. aureus biofilms and EOs E.C. Adukwu et al.
Therefore considering the results presented here, there
may be a possible potential for lemongrass EO use in
food processing environments. The effect on the organo-
leptic properties of the foodstuff at the anti-biofilm con-
centrations would need to be determined, although
lemongrass per se is generally recognized as safe and is
used as a food ingredient world-wide.
To our knowledge, this is the first time the anti-biofilm
activity of grapefruit EO has been reported. The results
described here demonstrate that although grapefruit EO
is bactericidal at 24% (v/v) against the different
Staph. aureus strains tested, it has limited or no activity
against biofilm formation. This suggests that biofilm for-
mation could offer protection against EOs, or at least
against grapefruit EO. Previous studies have shown how-
ever that when grapefruit EO was combined with other
EOs against MRSA (not in biofilms), there was synergistic
activity and improved antimicrobial potential (Edwards-
Jones et al. 2004) hence, combining grapefruit EO with
other EOs or antimicrobial compounds might also
enhance its activity against biofilms. The synergistic
action of EOs against surface adhered cells has previously
been demonstrated by the results of a study by De Oliveira
et al. (2010) who reported a 100% log reduction of a
mature L. monocytogenes biofilm after 60 min contact time
with a combination of lemongrass and citronella EOs.
Both lemongrass and grapefruit EOs were unable to
eradicate already established biofilms (Table 2). The
inability of antimicrobial compounds to remove biofilm
deposits has been observed previously (Lin et al. 2011).
As biofilms develop, the pioneer cells undergo irreversible
attachment leading up to maturation (Mittelman 1998)
and at this point, removal of biofilms is said to be diffi-
cult and would require mechanical force or chemical dis-
ruption (De Oliveira et al. 2010). In addition, Pitts et al.
(2003), after an investigation of reductions in Ps. aerugin-
osa and Staph. epidermidis biofilms using a range of
chemical agents such as hydrogen peroxide and 1 mol l
1
sodium chloride, suggest that such reductions are micro-
organism and antimicrobial agent specific further
highlighting the difficulty with regard to biofilm removal.
For example, 1 mol l
1
sodium chloride significantly
reduces Ps. aeruginosa biofilms but not those of
Staph. epidermidis, while hydrogen peroxide was more
0
0·2
0·4
0·6
0·8
1
1·2
1·4
1·6
MSSA 110 PVL CA MSSA MRSA NCTC 13297 MRSA MW2 PVL CA MRSA
Relative biofilm viability (Log10 cfu/ml)
0
0·2
0·4
0·6
0·8
1
1·2
1·4
MSSA 110 PVL CA MSSA MRSA NCTC 13297 MRSA MW2 PVL CA MRSA
Relative biofilm viability (Log10 cfu/ml)
(a)
(b)
Figure 2 Effects of (a) lemongrass essential
oil (EO) (0125%, 006%, control)
and (b) grapefruit EO ( 1%, 2%, 4%,
control) on the relative biofilm viability of
Staphylococcus aureus strains following 24 h
exposure as determined by the CFU ml
1
assay (control =biofilms not exposed to EO;
N=4 for each treatment and for each
strain).
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 7
E.C. Adukwu et al. Staph. aureus biofilms and EOs
effective against the latter than it was against the former
(Pitts et al. 2003). It has been suggested (Kelly et al.
2012) that biofilm prevention is preferable to disruption
and removal due to factors such as the nature of biofilms,
adherence of staphylococci to surfaces and treatment
problems associated with biofilms.
Biofilms tolerate high amounts of antibiotic between
101000 fold when compared to planktonic cells
(Yarwood et al. 2004; Resch et al. 2006; Kelly et al.
2012), and in this study, lemongrass at twice the MIC
and same concentration as the MBC prevented biofilm
formation highlighting antimicrobial activity as well as its
potential as an anti-biofilm agent. Exposure of the bio-
films to grapefruit EO showed no reduction in metabolic
activity in four of the five Staph. aureus strains and an
increased metabolic activity in the PVL positive MSSA
strain. The reasons for such increase in metabolic activity
are unknown. However, a study by Kwiecinski et al.
(2009) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide] colorimetric assay
reported an increase in metabolism within Staph. aureus
biofilms when treated with tea tree oil (TTO) concentra-
tions lower than the MBEC which, it was suggested,
could be a result of a stress response. Whether this is the
case for the increase in metabolic activity in PVL positive
MSSA strain when treated with grapefruit EO remains to
be determined.
Lemongrass EO at the MBC and MBIC concentrations
disrupts PVL CA MSSA biofilms and at 05% (v/v), and
at 1% (v/v), total destruction of the biofilm was
observed. Kwiecinski et al. (2009) has suggested that
TTO treatment of Staph. aureus biofilms causes damage
to the extra cellular matrix and damage to the biofilm
structure was observed on treatment with 1% (v/v) lem-
ongrass EO suggesting a similar mode of action. With the
grapefruit EO, SEM analysis did not show any disruption
of the biofilm structure which further confirms the lack
of anti-biofilm activity and indicates the importance of
biofilm formation as a protective mechanism against
EOs.
This study is one of few that have investigated the
anti-biofilm properties of EOs especially the effect of lem-
ongrass EOs against Staph. aureus biofilms. Where previ-
ously (Bearden et al. 2008), investigated commercial
formulations containing EOs against CA MRSA, this is
first study that has demonstrated the anti-biofilm activity
of lemongrass EO against biofilms of CA MSSA and
MRSA including PVL positive strains. Further studies
would be required to evaluate any potentially toxic effect
of lemongrass EO; however, the antimicrobial and anti-
biofilm properties provide another option for future anti-
microbial therapeutic interventions in both clinical and
industrial applications.
Acknowledgements
E.C.A. acknowledges the financial support provided from
NHS Northamptonshire and The University of North-
ampton, UK through the Centre of Health and Wellbeing
Research, for a Postgraduate Studentship. The authors
(a) (b)
SE 11:21 WD12·6 mm 5·00 kV x5·0k 10 um
(c)
SE 12:03 WD14·1 mm 5·00 kV x5·0k 10 um
(d)
SE 12:59 WD12·8 mm 5·00 kV x5·0k 10 um
SE 11:45 WD12·8 mm 5·00 kV x5·0k 10 um
Figure 3 Scanning electron micrographs of
Panton Valentine-Leukocidin community
acquired methicillin susceptible
Staphylococcus aureus following treatment
with lemongrass essential oil at (a) 0%
(control), (b) 0125% (c) 05% and (d) 1%
(v/v) after 24 h exposure (magnification
95000, Scale 10 lm). Arrows indicate biofilm
formation (a, b), biofilm disruption (c) and
biofilm debris (d).
©2012 The Authors
8Journal of Applied Microbiology ©2012 The Society for Applied Microbiology
Staph. aureus biofilms and EOs E.C. Adukwu et al.
also thank Belmay Plc., Northampton, UK for supplying
the oils and for the GC/MS analysis and Professor Mark
Fielder, Kingston University UK for supply of the clinical
isolates.
References
Aiemsaard, J., Aiumlamai, S., Aromdee, C.,
Taweechaisupapong, S. and Khunkitti, W. (2011) The
effect of lemongrass oil and its major components on
clinical isolate mastitis pathogens and their mechanisms of
action on Staphylococcus aureus DMST 4745. Res Vet Sci
91, e31e37.
Al-Shuneigat, J., Cox, S.D. and Markham, J.L. (2005) Effects
of a topical essential oil-containing formulation on
biofilm-forming coagulase-negative staphylococci. Lett
Appl Microbiol 41,5255.
Andrews, J.M. and Howe, R.A. (2011) BSAC standardized disc
susceptibility testing method (version 10). J Antimicrob
Chemother 66, 27262757.
Barbosa, L.N., Rall, V.L., Fernandes, A.A., Ushimaru, P.I., Da
Silva Probst, I. and Fernandes, A. Jr (2009) Essential oils
against foodborne pathogens and spoilage bacteria in
minced meat. Foodborne Pathog Dis 6, 725728.
Barker, S.C. and Altman, P.M. (2010) A randomised, assessor
blind, parallel group comparative efficacy trial of three
products for the treatment of head lice in children-
melaleuca oil and lavender oil, pyrethrins and piperonyl
butoxide, and a “suffocation” product. BMC Dermatol 10,6.
Bearden, D.T., Allen, G.P. and Christensen, J.M. (2008)
Comparative in vitro activities of topical wound care
products against community-associated methicillin-
resistant Staphylococcus aureus.J Antimicrob Chemother 62,
769772.
Bourne, K.Z., Bourne, N., Reising, S.F. and Stanberry, L.R.
(1999) Plant products as topical microbicide candidates:
assessment of in vitro and in vivo activity against herpes
simplex virus type 2. Antiviral Res 42, 219226.
Cerca, N., Martins, S., Cerca, F., Jefferson, K.K., Pier, G.B.,
Oliveira, R. and Azeredo, J. (2005) Comparative
assessment of antibiotic susceptibility of coagulase-
negative staphylococci in biofilm versus planktonic culture
as assessed by bacterial enumeration or rapid XTT
colorimetry. J Antimicrob Chemother 56, 331336.
Ceri, H., Olson, M.E., Stremick, C., Read, R.R., Morck, D. and
Buret, A. (1999) The Calgary biofilm device: new
technology for rapid determination of antibiotic
susceptibilities of bacterial biofilms. J Clin Microbiol 37,
17711776.
Chambers, H.F. and Deleo, F.R. (2009) Waves of resistance:
Staphylococcus aureus in the antibiotic era. Nat Rev
Microbiol 7, 629641.
Cowan, M.M. (1999) Plant products as antimicrobial agents.
Clin Microbiol Rev 12, 564582.
Da Silva, C.D.B., Guterres, S.S., Weisheimer, V. and
Schapoval, E.E.S. (2008) Antifungal activity of the
lemongrass oil and citral against Candida spp. Braz J
Infect Dis 12,6366.
Dai, T., Huang, Y.Y., Sharma, S.K., Hashmi, J.T., Kurup, D.B.
and Hamblin, M.R. (2010) Topical antimicrobials for
burn wound infections. Recent Pat Antiinfect Drug Discov
5, 124151.
De Oliveira, M.M.M., Brugnera, D.F., Cardoso, M.D.G., Alves,
E. and Piccoli, R.H. (2010) Disinfectant action of
Cymbopogon sp. essential oils in different phases of biofilm
formation by Listeria monocytogenes on stainless steel
surface. Food Control 21, 549553.
Donlan, R.M. and Costerton, J.W. (2002) Biofilms: survival
mechanisms of clinically relevant microorganisms. Clin
Microbiol Rev 15, 167193.
Edwards-Jones, V., Buck, R., Shawcross, S.G., Dawson, M.M.
and Dunn, K. (2004) The effect of essential oils on
methicillin-resistant Staphylococcus aureus using a dressing
model. Burns 30, 772777.
Fey, P.D., Said-Salim, B., Rupp, M.E., Hinrichs, S.H., Boxrud,
D.J., Davis, C.C., Kreiswirth, B.N. and Schlievert, P.M.
(2003) Comparative molecular analysis of community- or
hospital-acquired methicillin-resistant Staphylococcus
aureus.Antimicrob Agents Chemother 47, 196203.
Fisher, K. and Phillips, C.A. (2006) The effect of lemon,
orange and bergamot essential oils and their components
on the survival of Campylobacter jejuni,Escherichia coli
O157, Listeria monocytogenes,Bacillus cereus and
Staphylococcus aureus in vitro and in food systems. J Appl
Microbiol 101, 12321240.
Ghafoor, A., Hay, I.D. and Rehm, B.H. (2011) Role of
exopolysaccharides in Pseudomonas aeruginosa biofilm
formation and architecture. Appl Environ Microbiol 77,
52385246.
Gill, A.O., Delaquis, P., Russo, P. and Holley, R.A. (2002)
Evaluation of antilisterial action of cilantro oil on vacuum
packed ham. Int J Food Microbiol 73,8392.
Gordon, R.J. and Lowy, F.D. (2008) Pathogenesis of
methicillin-resistant Staphylococcus aureus infection. Clin
Infect Dis 46(Suppl 5), S350S359.
Hammer, K.A., Carson, C.F. and Riley, T.V. (1998) In-vitro
activity of essential oils, in particular Melaleuca alternifolia
(tea tree) oil and tea tree oil products, against Candida
spp. J Antimicrob Chemother 42, 591595.
Hayes, A.J. and Markovic, B. (2002) Toxicity of Australian
essential oil Backhousia citriodora (Lemon myrtle). Part 1.
Antimicrobial activity and in vitro cytotoxicity. Food
Chem Toxicol 40, 535543.
Inouye, S., Takizawa, T. and Yamaguchi, H. (2001)
Antibacterial activity of essential oils and their major
constituents against respiratory tract pathogens by gaseous
contact. J Antimicrob Chemother 47, 565573.
Jabra-Rizk, M.A., Meiller, T.F., James, C.E. and Shirtliff, M.E.
(2006) Effect of farnesol on Staphylococcus aureus biofilm
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 9
E.C. Adukwu et al. Staph. aureus biofilms and EOs
formation and antimicrobial susceptibility. Antimicrob
Agents Chemother 50, 14631469.
Jeon, J.G., Rosalen, P.L., Falsetta, M.L. and Koo, H. (2011)
Natural products in caries research: current (limited)
knowledge, challenges and future perspective. Caries Res
45, 243263.
Karpanen, T.J., Worthington, T., Hendry, E.R., Conway, B.R.
and Lambert, P.A. (2008) Antimicrobial efficacy of
chlorhexidine digluconate alone and in combination with
eucalyptus oil, tea tree oil and thymol against planktonic
and biofilm cultures of Staphylococcus epidermidis.
J Antimicrob Chemother 62, 10311036.
Kavanaugh, N.L. and Ribbeck, K. (2012) Selected
antimicrobial essential oils eradicate Pseudomonas spp. and
Staphylococcus aureus biofilms. Appl Environ Microbiol 78,
40574061.
Kelly, D., McAuliffe, O., Ross, R.P. and Coffey, A. (2012)
Prevention of Staphylococcus aureus biofilm formation and
reduction in established biofilm density using a
combination of phage K and modified derivatives. Lett
Appl Microbiol 54, 286291.
King, M.D., Humphrey, B.J., Wang, Y.F., Kourbatova, E.V.,
Ray, S.M. and Blumberg, H.M. (2006) Emergence of
community-acquired methicillin-resistant Staphylococcus
aureus USA 300 clone as the predominant cause of skin
and soft-tissue infections. Ann Intern Med 144, 309317.
Klausen, M., Heydorn, A., Ragas, P., Lambertsen, L.,
Aaes-Jorgensen, A., Molin, S. and Tolker-Nielsen, T.
(2003) Biofilm formation by Pseudomonas aeruginosa wild
type, flagella and type IV pili mutants. Mol Microbiol 48,
15111524.
Knobloch, J.K., Horstkotte, M.A., Rohde, H. and Mack, D.
(2002) Evaluation of different detection methods of
biofilm formation in Staphylococcus aureus.Med Microbiol
Immunol 191, 101106.
Kwiecinski, J., Eick, S. and Wojcik, K. (2009) Effects of tea
tree (Melaleuca alternifolia) oil on Staphylococcus aureus in
biofilms and stationary growth phase. Int J Antimicrob
Agents 33, 343347.
Laird, K. and Phillips, C. (2012) Vapour phase: a potential
future use for essential oils as antimicrobials? Lett Appl
Microbiol 54, 169174.
Laird, K., Armitage, D. and Phillips, C. (2012) Reduction of
surface contamination and biofilms of Enterococcus sp. and
Staphylococcus aureus using a citrus-based vapour. J Hosp
Infect 80,6166.
Lin, S.M., Svoboda, K.K., Giletto, A., Seibert, J. and Puttaiah,
R. (2011) Effects of hydrogen peroxide on dental unit
biofilms and treatment water contamination. Eur J Dent 5,
4759.
Loughrey, A., Millar, B.C., Goldsmith, C.E., Rooney, P.J. and
Moore, J.E. (2007) Emergence of community-associated
MRSA (CA-MRSA) in Northern Ireland. Ulster Med J 76,
6871.
Mittelman, M.W. (1998) Structure and functional
characteristics of bacterial biofilms in fluid processing
operations. J Dairy Sci 81, 27602764.
Mohamed, J.A. and Huang, D.B. (2007) Biofilm formation by
enterococci. J Med Microbiol 56, 15811588.
Muli, F. and Struthers, J.K. (1998) Use of a continuous-
culture biofilm system to study the antimicrobial
susceptibilities of Gardnerella vaginalis and Lactobacillus
acidophilus.Antimicrob Agents Chemother 42, 14281432.
Nostro, A., Sudano Roccaro, A., Bisignano, G., Marino, A.,
Cannatelli, M.A., Pizzimenti, F.C., Cioni, P.L., Procopio,
F. et al. (2007) Effects of oregano, carvacrol and thymol
on Staphylococcus aureus and Staphylococcus epidermidis
biofilms. J Med Microbiol 56, 519523.
Nuryastuti, T., van der mei, H.C., Busscher, H.J., Iravati, S.,
Aman, A.T. and Krom, B.P. (2009) Effect of cinnamon oil
on icaA expression and biofilm formation by
Staphylococcus epidermidis.Appl Environ Microbiol 75,
68506855.
Onawunmi, G.O., Yisak, W.A. and Ogunlana, E.O. (1984)
Antibacterial constituents in the essential oil of
Cymbopogon citratus (DC.) Stapf. J Ethnopharmacol 12,
279286.
Palombo, E.A. (2011) Traditional medicinal plant extracts and
natural products with activity against oral bacteria:
potential application in the prevention and treatment of
oral diseases. Evid Based Complement Alternat Med 2011,
Article ID 680354. doi: 10.1093/ecam/nep067.
Pettit, R.K., Weber, C.A., Kean, M.J., Hoffmann, H., Pettit, G.
R., Tan, R., Franks, K.S. and Horton, M.L. (2005)
Microplate Alamar blue assay for Staphylococcus
epidermidis biofilm susceptibility testing. Antimicrob Agents
Chemother 49, 26122617.
Pires, R.H., Montanari, L.B., Martins, C.H., Zaia, J.E.,
Almeida, A.M., Matsumoto, M.T. and Mendes-Giannini,
M.J. (2011) Anticandidal efficacy of cinnamon oil against
planktonic and biofilm cultures of Candida parapsilosis
and Candida orthopsilosis.Mycopathologia 172, 453464.
Pitts, B., Hamilton, M.A., Zelver, N. and Stewart, P.S. (2003)
A microtiter-plate screening method for biofilm
disinfection and removal. J Microbiol Methods 54,
269276.
Prabuseenivasan, S., Jayakumar, M. and Ignacimuthu, S.
(2006) In vitro antibacterial activity of some plant essential
oils. BMC Complement Altern Med 6, 39.
Ramage, G., Bachmann, S., Patterson, T.F., Wickes, B.L. and
Lopez-Ribot, J.L. (2002) Investigation of multidrug efflux
pumps in relation to fluconazole resistance in Candida
albicans biofilms. J Antimicrob Chemother 49, 973980.
Rasigade, J.P., Laurent, F., Lina, G., Meugnier, H., Bes, M.,
Vandenesch, F., Etienne, J. and Tristan, A. (2010) Global
distribution and evolution of Panton-Valentine
leukocidin-positive methicillin-susceptible Staphylococcus
aureus, 19812007. J Infect Dis 201, 15891597.
©2012 The Authors
10 Journal of Applied Microbiology ©2012 The Society for Applied Microbiology
Staph. aureus biofilms and EOs E.C. Adukwu et al.
Resch, A., Leicht, S., Saric, M., Pasztor, L., Jakob, A., Gotz, F.
and Nordheim, A. (2006) Comparative proteome analysis
of Staphylococcus aureus biofilm and planktonic cells and
correlation with transcriptome profiling. Proteomics 6,
18671877.
Said-Salim, B., Mathema, B., Braughton, K., Davis, S.,
Sinsimer, D., Eisner, W., Likhoshvay, Y., Deleo, F.R. et al.
(2005) Differential distribution and expression of Panton-
Valentine leucocidin among community-acquired
methicillin-resistant Staphylococcus aureus strains. J Clin
Microbiol 43, 33733379.
Sandoe, J.A.T., Wysome, J., West, A.P., Heritage, J. and
Wilcox, M.H. (2006) Measurement of ampicillin,
vancomycin, linezolid and Gentamicin activity against
enterococcal biofilms. J Antimicrob Chemother 57,
767770.
Seno, Y., Kariyama, R., Mitsuhata, R., Monden, K. and
Kumon, H. (2005) Clinical implications of biofilm
formation by Enterococcus faecalis in the urinary tract.
Acta Med Okayama 59,7987.
Silva, S.N., Negri, M., Henriques, M., Oliveira, R.R., Williams,
D.W. and Azeredo, J. (2011) Adherence and biofilm
formation of non-Candida albicans Candida species.
Trends Microbiol 19, 241247.
Sinde, E. and Carballo, J. (2000) Attachment of Salmonella
spp. and Listeria monocytogenes to stainless steel, rubber
and polytetrafluoroethylene: the influence of free energy
and the effect of commercial sanitizers. Food Microbiol 17,
439447.
Soto, S.M., Smithson, A., Horcajada, J.P., Martinez, J.A.,
Mensa, J.P. and Vila, J. (2006) Implication of biofilm
formation in the persistence of urinary tract infection
caused by uropathogenic Escherichia coli.Clin Microbiol
Infect 12, 10341036.
Thakur, R., Jain, N., Pathak, R. and Sandhu, S.S. (2011)
Practices in wound healing studies of plants. Evid Based
Complement Alternat Med 00, 17.
Uysal, B., Sozmen, F., Aktas, O., Oksal, B.S. and Kose, E.O.
(2011) Essential oil composition and antibacterial activity
of the grapefruit (Citrus paradisi. L) peel essential oils
obtained by solvent-free microwave extraction:
comparison with hydrodistillation. Int J Food Sci Technol
46, 14551461.
Viuda-Martos, M., Ruiz-Navajas, Y., Fernandez-Lopez, J. and
Perez-Alvarez, J. (2008) Antifungal activity of lemon
(Citrus lemon L.), mandarin (Citrus reticulata L.),
grapefruit (Citrus paradisi L.) and orange (Citrus sinensis
L.) essential oils. Food Control 19, 11301138.
Vuong, C., Kocianova, S., Voyich, J.M., Yao, Y., Fischer, E.R.,
Deleo, F.R. and Otto, M. (2004) A crucial role for
exopolysaccharide modification in bacterial biofilm
formation, immune evasion, and virulence. J Biol Chem
279, 5488154886.
Warnke, P.H., Becker, S.T., Podschun, R., Sivananthan, S.,
Springer, I.N., Russo, P.A., Wiltfang, J., Fickenscher, H.
et al. (2009) The battle against multi-resistant strains:
renaissance of antimicrobial essential oils as a promising
force to fight hospital-acquired infections. J
Craniomaxillofac Surg 37, 392397.
Weber, J.T. (2005) Community-associated methicillin-resistant
Staphylococcus aureus.Clin Infect Dis 41, S269S272.
Wijman, J.G., de Leeuw, P.P., Moezelaar, R., Zwietering, M.H.
and Abee, T. (2007) Air-liquid interface biofilms of
Bacillus cereus: formation, sporulation, and dispersion.
Appl Environ Microbiol 73, 14811488.
Williams, G.J., Denyer, S.P., Hosein, I.K., Hill, D.W. and
Maillard, J.Y. (2007) The development of a new three-step
protocol to determine the efficacy of disinfectant wipes on
surfaces contaminated with Staphylococcus aureus.J Hosp
Infect 67, 329335.
Yarwood, J.M., Bartels, D.J., Volper, E.M. and Greenberg, E.P.
(2004) Quorum sensing in Staphylococcus aureus biofilms.
J Bacteriol 186, 18381850.
Zetola, N., Francis, J.S., Nuermberger, E.L. and Bishai, W.R.
(2005) Community-acquired meticillin-resistant
Staphylococcus aureus: an emerging threat. Lancet Infect
Dis 5, 275286.
©2012 The Authors
Journal of Applied Microbiology ©2012 The Society for Applied Microbiology 11
E.C. Adukwu et al. Staph. aureus biofilms and EOs
... Given this, the addition of emulsifiers is another good strategy for the EO dilution in the MIC determination. Adukwu et al. [76] added 0.5% (v/v) Tween 20 into the broth medium to achieve higher oil solubility in the MIC determination. Furthermore, sonication as an assistant method applied to the emulsion showed a reduction in droplet size and an increment in the emulsion's stability. ...
... Judging visible growth is vague; so, determining the absorbance at a certain wavelength using a spectrophotometer is performed. The wavelengths used are not uniform and multiple, such as 560 nm, [43] 590 nm, [45] 595 nm, [76] 600 nm, [75,81] and 620 nm, [27] which increases the difficulty in comparison. In addition, the inhibition degree of microbial growth is also different from the complete inhibition in the standard. ...
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Essential oils were extracted from two Vietnam local varieties of pomelo namely Citrus Grandis L. CV Nam Roi and Citrus Grandis L. CV Da Xanh. Storage methods by freezing or drying of young fruit peels affect the yield of extracted essential oils. The yield of essential oils obtained from Da Xanh variety was higher in Nam Roi variety. The extraction under ultrasound assisted condition increased the yield by two folds. The composition of volatile compounds in pomelo essential oils varied with different pomelo varieties and storage methods of the materials. The dominant compound in young peels essential oils Nam Roi, Da Xanh is limonene (55 – 90%). Nam Roi peels essential oils have antioxidant and antimicrobial activities (inhibition ability against Bacillus subtilis and Staphylococcus aureus ) higher than Da Xanh peels’ essential oils.
... At 37°C, plates were incubated for microbe and at 27°C for fungi for 24 hrs. In millimeters, the zone of inhibition was measured [27,28]. ...
... All examined free EOs and their active ingredients successfully inhibited planktonic bacterial cells [82,83]. However, the biofilms required significantly higher concentrations of Eos, or their active components, than their planktonic counterparts, to achieve comparable or zero microbial reductions [75,[84][85][86][87][88][89]. In several studies, even when EOs or their active components were applied at twice or even higher concentrations than their minimum inhibitory concentrations (MIC) against planktonic cells, the biofilms were not completely eradicated [85,88,[90][91][92][93][94][95][96]. ...
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Rosa damascena is mostly grown for its usage in the food, medical, and perfume industries, while it is also used as an attractive plant in parks, gardens, and homes. The use of R. damascena essential oil may yield new results in relation to the antimicrobial activity of essential oils and their use mainly in extending the shelf life of foods. This study investigates the chemical composition and antimicrobial properties of Rosa damascena essential oil (RDEO) using gas chromatography-mass spectrometry (GC-MS) and various bioassays to explore its potential applications in food preservation and microorganism growth control. The GC-MS analysis revealed that RDEO is predominantly composed of phenylethyl alcohol (70%), which is known for its antimicrobial and aromatic properties. Additionally, other significant constituents were identified, including nerol, citronellol, and geraniol, which may contribute to the EOs overall bioactivity. The antimicrobial activity was assessed through the minimal inhibition concentration against five Candida yeast strains, four Gram-positive, and four Gram-negative bacteria, including biofilm-forming Salmonella enterica. Determination of minimum inhibitory concentrations (MIC) revealed the strongest effects of RDEO's on Gram-negative species, with MIC 50 values as low as 0.250 mg/mL for S. enterica. Moreover, an in situ assessment utilizing Foods 2024, 13, 3579. https://doi.org/10.3390/foods13223579 https://www.mdpi.com/journal/foods Foods 2024, 13, 3579 2 of 30 fruit and vegetable models demonstrated that the vapor phase of RDEO significantly suppressed microbial growth, with the most substantial reductions observed on kiwi and banana models. As a result of our study, the antimicrobial effect of RDEO on the microbiota of sous vide processed eggplant was detected, as well as an inhibitory effect on S. enterica during storage. The insecticidal activity against Megabruchidius dorsalis Fahreus, 1839, was also studied in this work and the best insecticidal activity was found at the highest concentrations. These results suggest that RDEO has the potential to serve as a natural antimicrobial agent in food preservation and safety applications, providing an alternative to synthetic preservatives.
... 6,7 Other researchers reported the effectiveness of the essential oils extracted from the grapefruit peels against different strains of microorganisms, such as (Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsellia pneumonia, Proteus vulgaris, Pseudococcus spp., Salmonella typhimurium, Serratia marcescens, Shigella flexneri, Staphylococcus aureus, S. epidermidis. [8][9][10] On the other hand, grapefruit seed extracts were effective against a wide range of gram-negative bacteria. 11,12 It was also reported in 2008 that grapefruit juice and its furocoumarins are effective against the bacterial autoinducer signaling that are the main cause of pathogenicity in bacteria as well inhibited biofilm formation in E. coli, S. typhimurium and Pseudomonas aeruginosa. ...
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Introduction: Worldwide, grapefruit is known to have medicinal values with antimicrobial, anti-inflammatory, antioxidant, and astringent propensities. Only a few researchers studied its antibacterial effects underlining the efficacy of the flavonoids extracted from the grapefruit peels against gram-positive and negative bacteria. Aims: The purpose of this work was to evaluate the antibacterial activity of the grapefruit water and ethanol extracts and their furanocoumarins against three common infectious bacteria and their multi-drug resistant clinical isolates. Methods: Antimicrobial activity of 4 different grapefruit extracts (Red-Ethanol, Yellow-Ethanol, Red-n-Hexane, Yellow-n-Hexane) from 2 different varieties (Red & Yellow) along with 4 of its active materials (naringin X, Bergaptol, Bergapten, Bergamottin) have been evaluated in vitro against 7 different bacteria that are frequently incriminated in human infection four of which were ATCC (Salmonella paratyphi, E.coli, Staphylococcus aureus, Pseudomonas) and three clinical bacteria (Pseudomonas aeruginosa drugs sensitive, Klebsiellas pneumonia multi-drug resistant, Pseudomonas aeruginosa multi-drug resistant) by assessing the minimum inhibitory concentration (MIC), using the microdilution method to test the microorganisms. Results: Most of the grapefruit’s tested extracts showed great activity level, including the active materials (Furanocoumarins) of the grapefruit, which showed a strong level of antimicrobial activity against the tested microorganisms, especially against the gram-negatives. Conclusions: The results obtained from this study confirm that the grapefruit extracts had a significant antimicrobial effect on the tested microorganisms, especially the Red-Ethanol extract, which had more activity against gram-negative bacteria, including E.coli, Pseudomonas as well as Pseudomonas aeruginosa drugs sensitive.
... The viability of each microbial strains in the biofilm was evaluated via the XTT assay [39][40][41][42]. After incubation for 48 h, the plates were washed with phosphate-buffered saline (PBS, 0.01 M, pH 7.2). ...
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Biofilms are a severe problem for public health because of the contributing recurrence of infections. Therefore, combating biofilms is a critical issue. In our study, we loaded zinc oxide (ZnO), zinc oxide borax (ZnOBorax), zinc copper oxide (ZnCuO2) nanoparticles and borax into bacterial cellulose (BC) to impart anti-biofilm and wound healing activity. The prepared BC loaded with nanoparticles (BC–NPs) was analysed via scanning electron microscopy. The nanoparticles’ geometric structure and placement in BC fibres were observed. We evaluated the biofilm inhibition and biofilm degradation activities of the BC–NPs against some pathogens via a crystal violet (CV) assay and XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide) reduction assay. The effects of BC–NPs on cell proliferation and wound-healing ability were analysed in L929 cell line. BC–NPs exhibited better biofilm degradation activity than biofilm inhibition activity. According to the results of the CV assay, BC–ZnONPs, BC–Borax and BC–ZnOBoraxNPs inhibited 65.53%, 71.74% and 66.60% of biofilm formation of Staphylococcus aureus, respectively. BC–ZnCuO2NPs showed the most degradation activity on Pseudomonas aeruginosa and Listeria innocua biofilms. The XTT reduction assay results indicated a considerable reduction in the metabolic activity of the biofilms. Moreover, compared to the control group, BC loaded with borax and ZnO nanoparticle promoted cell migration without cytotoxicity.
... The VOCs of R. damascena was assessed for antibacterial activity against a range of opportunistic pathogenic bacterial strains using the agar well diffusion method (Balouiri et al., 2016) followed by the broth microdilution method, as detailed in the studies of (Adukwu et al., 2012;Elshikh et al., 2016;Rathore et al., 2023). The bacterial strains taken for this study included Micrococcus luteus MTCC 2470, Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC 96 (Gram-positive), and Pseudomonas aeruginosa MTCC 2453, Escherichia coli MTCC 43, and Klebsiella pneumoniae MTCC 109 (Gram-negative). ...
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The objective of this work was to study the effect of the essential oils of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) on the growth of moulds commonly associated with food spoilage: Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum and Penicillium verrucosum, using the agar dilution method. All the oils showed antifungal activity against all the moulds. Orange essential oil was the most effective against A. niger, mandarin essential oil was most effective at reducing the growth of Aspergillus flavus while grapefruit was the best inhibitor of the moulds P. chrysogenum and P. verrucosum. Citrus essential oils could be considered suitable alternatives to chemical additives for use in the food industry.