Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Antibacterial activity and mode of action of a commercial

citrus fruit extract


A. Alvarez-Ord
n
o ~ ez, A. Carvajal, H. Arguello, F.J. Mart
ınez-Lobo, G. Naharro and P. Rubio

n, Leo
Infectious Diseases and Epidemiology Unit, Department of Animal Health, Veterinary Faculty, University of Leo
n, Spain

Keywords Abstract
antimicrobial, Brachyspira hyodysenteriae,
citrus fruit, Escherichia coli, FTIR spectroscopy, Aims: This study addresses the antibacterial activity and mechanism of action
membrane integrity, Salmonella enterica. of BIOLL+â, a commercial extract obtained from citrus fruits.
Methods and Results: Strong activities with minimum inhibitory concentra-
Correspondence tions (MIC) ranging from 10 ppm (for some Brachyspira hyodysenteriae strains)


Avelino Alvarez-Ord
n
o ~ez, Infectious Diseases
to 80 ppm (for various Salmonella enterica and Escherichia coli strains) were
and Epidemiology Unit, Department of
observed. Membrane integrity tests and Fourier transform infrared (FT-IR)
Animal Health, Veterinary Faculty, University

n, Campus de Vegazana s/n,
of Leo spectroscopic analyses were performed to shed light on the effects caused on
24071-Leo
n, Spain. molecular structure and composition. Physical effects, with formation of pores
E-mail: aalvo@unileon.es and leakage of intracellular components, and chemical effects, which were
dependent on the bacterial species, were evident on cellular envelopes. Whereas
2013/0047: received 8 January 2013, revised for S. enterica and E. coli, changes were focused on the carboxylic group of
11 March 2013 and accepted 27 March 2013
membrane fatty acids, for B. hyodysenteriae, the main effects were found in
polysaccharides and carbohydrates of the cell wall.
doi:10.1111/jam.12216
Conclusions: The great antibacterial activity shown by BIOLL+â and its
proposed dual physico-chemical mode of action, with species-specific cellular
targets, show its attractiveness as an alternative to antibiotics.
Significance and impact of the study: Antibiotic resistance is becoming a
serious problem. Our study characterizes a novel antimicrobial extract, which
could represent an alternative to antibiotics for treatment or prevention of
bacterial infectious diseases.

Reichling et al. 2009; Sol
orzano-Santos and Miranda-

Introduction
Antibiotic-resistant bacteria represent a major concern
worldwide. The possibility of developing bacterial resis-
tant populations when using antibiotics as growth pro-
moters in animal production has led to the EU and USA
ban on the use of antibiotics as feed additives on farm
animals (Casewell et al. 2003; Gaggıa et al. 2010; Has-
hemi and Davoodi 2011). Therefore, intense research
efforts focused on the search for new alternatives to pre-
vent intestinal infectious diseases, including use of probi-
otics, prebiotics and feed additives, have been made in
the last decade. Special attention has been paid to the
antimicrobial activity of diverse plant oil extracts and
their components, which have been reported to show
great inhibitory effects against pathogenic bacteria, yeasts,
fungi and viruses (Burt 2004; Alviano and Alviano 2009;
Novales 2012). Among the potential candidates, citrus
fruit extracts are generating great interest because their
components have been reported to show not only antimi-
crobial effects (Nannapaneni et al. 2008; O’Bryan et al.
2008; Bevilacqua et al. 2010; Frassinetti et al. 2011;
Settani et al. 2012), but also other beneficial biological
activities – for example anti-inflammatory properties
(Manthey et al. 2001; Cushnie and Lamb 2005).
BIOLL+â is a natural extract obtained from Citrus
paradisi (grapefruit), Citrus reticulata (mandarin), Citrus
aurantium subsp. bergamia (bergamot) and Citrus sinensis
(sweet orange) and commercialized by Grupo Omega
(Spain) to supplement animal feed. Indeed, it is especially
recommended in swine and poultry production at dos-
ages of 100–500 g per ton of feed (granulated presenta-
tion) or 50–350 ml per 103 l of drinking water (liquid

50 Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology


A. Alvarez-Ord
n
o ~ez et al. Antibacterial activity of BIOLL+â

presentation) as a supplement indicated to increase pro-
ductivity and feed conversion and to control digestive
processes and gastrointestinal pathogenic bacteria. This
extract is rich in flavonoids (1
74 g/100 mg) and citric
acid (1
87 g/100 mg), compounds with known antibacte-
rial activity. Among those flavonoids with antibacterial
activity present in BIOLL+â, the most numerous are
quercetin (1972 ppm), naringenin (833 ppm) and narin-
gin (54 ppm). However, the extract also contains other
compounds with antibacterial activity, such as ascorbic
acid, hesperidin, rutin and saponins (Grupo Omega,
personal communication).
The antibacterial activity and mode of action of
BIOLL+â are not completely characterized yet. The aim
of this study was to test its efficacy to inactivate or to
inhibit the growth of several pathogenic bacteria of inter-
est for animal health, that is, S. enterica subsp. enterica,
Escherichia coli and Brachyspira hyodysenteriae. Various
strains of these important animal pathogens, most of
them isolated from swine finishing farms, were included
in the study. In addition, to elucidate its mechanisms of
action, membrane integrity tests and Fourier transform
infrared (FT-IR) spectroscopic analyses were performed
on treated cells to shed light on the effects caused by this
citrus fruit extract on the molecular structure and com-
position, with special focus on the cellular envelopes.
Materials and methods
Bacterial strains and culture conditions
Strains of S. enterica, E. coli and B. hyodysenteriae used in
this study, listed in Table 1, belonged to the Infectious
Diseases Unit Collection (IDUC) of the University of

Table 1 Bacterial strains and their susceptibility to BIOLL+â

IDUC Id. Strain Origin Source* Minimum inhibitory concentrations (ppm)

RS37 Salmonella Typhimurium CECT443 Spanish Type Culture Collection IDUC 40

RS38 S. Typhimurium CECT 883 Spanish Type Culture Collection IDUC 20
RS39 Salmonella Enteritidis CECT 4300 Spanish Type Culture Collection IDUC 40
RS40 Salmonella Infantis CECT 700 Spanish Type Culture Collection IDUC 80
RS41 Salmonella Cholerasuis CECT 915 Spanish Type Culture Collection IDUC 80
SP11 S. Typhimurium DT104 Swine Finishing farm isolate IDUC 80
SP28 Salmonella London Swine Finishing farm isolate IDUC 80
SP36 Salmonella Rissen Swine Finishing farm isolate IDUC 80
SP58 S. Typhimurium DT104 Swine Finishing farm isolate IDUC 80
SP62 Salmonella Derby Swine Finishing farm isolate IDUC 80
EC58 Escherichia coli (haemolytic strain) Swine Finishing farm isolate IDUC 80
EC59 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 80
EC60 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 40
EC61 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 80
EC62 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 40
EC63 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 40
EC64 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 40
EC65 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 80
EC66 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 40
EC67 E. coli (haemolytic strain) Swine Finishing farm isolate IDUC 80
B78 Brachyspira hyodysenteriae ATCC 27164 American Type Culture Collection IDUC 10
B204 B. hyodysenteriae ATCC 31212 American Type Culture Collection IDUC 40
H2 B. hyodysenteriae Swine Finishing farm isolate IDUC 20
H9 B. hyodysenteriae Swine Finishing farm isolate IDUC 40
H40 B. hyodysenteriae Swine Finishing farm isolate IDUC 20
H52 B. hyodysenteriae Swine Finishing farm isolate IDUC 20
H76 B. hyodysenteriae Swine Finishing farm isolate IDUC 20
H151 B. hyodysenteriae Swine Finishing farm isolate IDUC 10
H363 B. hyodysenteriae Swine Finishing farm isolate IDUC 10
H507 B. hyodysenteriae Swine Finishing farm isolate IDUC 20
H549 B. hyodysenteriae Swine Finishing farm isolate IDUC 40
H555 B. hyodysenteriae Swine Finishing farm isolate IDUC 40
H583 B. hyodysenteriae Swine Finishing farm isolate IDUC 40
H591 B. hyodysenteriae Swine Finishing farm isolate IDUC 10

n, Spain.
*All strains belonged to the Infectious Diseases Unit Collection (IDUC) of the University of Leo

Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology 51
Antibacterial activity of BIOLL+â
Le
on. Salmonella enterica and E. coli cultures were resus-
citated and maintained on tryptic soy agar (TSA) plates.
Test cultures were prepared by transferring an isolated
colony from a plate into a test tube containing 10 ml of
sterile brain heart infusion (BHI) followed by incubation
at 37°C for 24 h in a shaking incubator. Brachyspira hyo-
dysenteriae strains were grown on blood agar plates and
subsequently on fastidious anaerobe agar (FAA) at 42°C
in an anaerobic chamber. Test cultures were prepared by
transferring the bacterial load from a FAA plate into a
test tube containing 10 ml of sterile BHI supplemented
with 10% foetal bovine serum, followed by incubation at
42°C for 48 h under anaerobic atmosphere in a shaking
incubator. Salmonella enterica, E. coli and B. hyodysente-
riae test cultures were subsequently used for all experi-
ments performed.
Antibacterial activity of the commercial citrus fruit
extract
A stock suspension of BIOLL+â (Grupo Omega; liquid
presentation, Arganda del Rey, Spain) was prepared at
3200 ppm and used for all tests. Susceptibility testing was
performed by the broth dilution method using 48-well
tissue culture plates (Iwaki, Tokyo, Japan). Briefly, two-
fold serial dilutions of the citrus fruit extract, with con-
centrations ranging from 160 to 1
25 ppm, were carried
out in BHI (supplemented with 10% foetal bovine serum
in the case of B. hyodysenteriae). Afterwards, the 48-well
plate was prepared by dispensing into each well 250 ll
of the test growth medium and 250 ll of the test
bacterial cell suspension standardized with 0
5 McFarland
turbidity standard and diluted to a final level of
~5 9 105 cells ml 1. Positive control wells (containing
250 ll of bacterial suspension and 250 ll of BHI without
citrus fruit extract supplementation) and negative control
wells (containing 500 ll of BHI) were included. The min-
imum inhibitory concentration (MIC) was determined
for each strain as the lowest concentration which pre-
vented visible growth after incubation in a shaking incu-
bator at 37°C for 24 h (S. enterica and E. coli) or 42°C
for 96 h (B. hyodysenteriae). The absence of contamina-
tion was confirmed for B. hyodysenteriae by phase-con-
trast microscopy. Aliquots from wells not showing visible
growth were plated onto solid media to confirm whether
those concentrations were bactericidal. MIC50 and MIC90
values, defined as the lowest concentrations that inhibited
the growth of 50 and 90% of total isolates tested, were
estimated for each bacterial species as the median and the
90th percentile values, respectively, of the MICs observed
for all bacterial isolates tested.
To study the bactericidal activity of the citrus fruit
extract, inactivation kinetic experiments were performed
52 Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology


A. Alvarez-Ord
n
o ~ez et al.
for some strains of S. enterica and E. coli. In these experi-
ments, 1 ml of stationary-phase cultures was centrifuged
at 11 000 g for 3 min at 4°C. The supernatant liquid was
discarded, and the cellular pellet was resuspended in 1 ml
of BHI supplemented with different concentrations of the
citrus fruit extract. After incubation for 90 min at room
temperature, survival was monitored. Tenfold serial dilu-
tions were produced in sterile phosphate-buffered saline
(PBS), and suitable dilutions were plated in duplicate on
TSA. Viable cell counts were enumerated following incu-
bation of TSA plates at 37°C for 48 h (longer incubation
times did not show any influence on the count). Inactiva-
tion experiments were performed in triplicate using three
different fresh cultures.
Fourier transform infrared spectroscopic analysis
Control cells and cells exposed to different (sub)lethal
concentrations of the citrus fruit extract for 90 min were
harvested by centrifugation at 11 000 g for 3 min at 4°C
and suspended in 50 ll of PBS, placed (15 ll) in a ZeSn
window and stove-dried (15 min, 50°C). Infrared spectra
were obtained with a FT-IR spectroscope (Perkin-Elmer
2000 FT-IR, Waltham, MA, USA) equipped with a DTGS
detector. Measurements were recorded over the wave-
length range of 3500–700 cm 1 with an interval of
1 cm 1. The spectral resolution was 4 cm 1. The final
spectra were achieved averaging 20 scans. FT-IR experi-
ments were performed in triplicate.
A software application developed for the Perkin-Elmer
environment was used for transformation, including
normalization (0 setting with absorption at 1800 cm 1; 1
setting at maximal absorption, located around 1650
cm 1), smoothing and first derivative. After transforma-
tion, spectra were recorded in ASCII format and processed
(Maradona 1996; Mouwen et al. 2005).
The spectral data were subjected to multivariate statis-
tical methods [hierarchical cluster analysis (HCA) and
factor analysis (FA)] to separate spectra into different
classes. Pearson’s product moment correlation coefficient
was used to measure the similarity between spectra, and
strain clustering was achieved using Ward’s algorithm. All
of the analyses (calculation of coefficients, joining of vari-
ables, canonical analysis and graphical display) were car-
ried out with the Statistica for Windows, version 7.0,
program (Statsoft Inc., Tulsa, OK, USA).
Membrane integrity tests
Assessment of propidium iodide intake by flow cytometry
Bacterial cultures grown until stationary phase and
exposed to different concentrations of the citrus fruit
extract for 90 min were subsequently diluted in PBS to


A. Alvarez-Ord
n
o ~ez et al.
achieve a final cellular concentration of ~108 cells ml 1.
Afterwards, propidium iodide (PI) (Molecular Probes,
Life Technologies, Madrid, Spain; Invitrogen, Life Tech-
nologies) was added (final PI concentration of 0
1% v/v),
and the mix was incubated in the dark at room tempera-
ture for 10 min. Prior to the analysis of the samples by
flow cytometry, the cell suspension was centrifuged at
11 000 g for 3 min at 4°C, and the cellular pellet was
suspended in PBS. Flow cytometry experiments were car-
ried out using a CyAn-adp flow cytometer (Beckman
Coulter, Brea, CA, USA). Samples were excited using a
488-nm air-cooled argon-ion laser. The instrument was
set up with the following configuration: forward scatter
(FS), side scatter (SS) and red fluorescence (613/20 nm)
for PI. The results were collected on logarithmic scale.
The cell population was selected by gating in a FS vs SS
dot plot, which allowed for the exclusion of aggregates
and cell debris. Fluorescence histograms were represented
in single-parameter histograms. Data were analysed with
Summit version 3.1 software (Cytomation, Fort Collins,
CO, USA).
Measurement of cellular leakage
Aliquots of 3 ml of cell cultures exposed to different con-
centrations of the citrus fruit extract during 90 min were
filtered through a 25-mm-diameter, 0
22-lm-pore-size
Millex-GS syringe filter (Millex-GS, Millipore, Madrid,
Spain). The presence of nucleic acids in the filtrate was
checked by measuring the absorbance at 260 nm (Uvikon
810 spectrophotometer, Kontron analytical, Munich,
Germany).
Results
The activity of a commercial citrus fruit extract against
34 strains belonging to three bacterial species of interest
for animal health, S. enterica, E. coli and B. hyodysente-
riae, was assessed by means of the broth dilution method
(Table 1). The citrus fruit extract was able to inhibit bac-
terial growth at very low concentrations. Thus, the mini-
mum inhibitory concentrations (MICs) ranged from 20
to 80 ppm for S. enterica (MIC50 of 80 ppm; MIC90 of
80 ppm), from 40 to 80 ppm for E. coli (MIC50 of
60 ppm; MIC90 of 80 ppm) and from 10 to 40 ppm for
B. hyodysenteriae (MIC50 of 20 ppm; MIC90 of 40 ppm).
It is worth noting that for all strains studied, the minimal
inhibitory concentration resulted to be bactericidal (data
not shown).
To further investigate the antibacterial activity and
mode of action of the extract, three Salmonella Typhimu-
rium strains with different susceptibilities were selected
(strain RS38, with a MIC of 20 ppm, and strains SP11
and SP58, with a MIC of 80 ppm). Inactivation
Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology
Antibacterial activity of BIOLL+â
experiments were performed after exposure of the strains
to different concentrations of the citrus fruit extract
(5, 20, 80, 160 ppm) for 90 min (Fig. 1a). Whereas no
significant reductions in cell numbers were observed with
5 and 20 ppm, the exposure to 80 ppm gave rise to a
2
6-log-cycle reduction for strain RS38, the most sensitive
one. In the presence of 160 ppm, the log-cycle reductions
achieved for strains RS38, SP11 and SP58 were 4
4, 2
0
and 2
2, respectively.
To elucidate the effects caused by the citrus fruit
extract on the cellular structure and composition, bacteria
exposed to these (sub)lethal conditions were subsequently
analysed using FT-IR spectroscopy. The spectrum of
S. Typhimurium nontreated cells was visually similar to
the spectra reported in previous studies (Al-Qadiri et al.


2008; Alvarez-Ord
~ez and Prieto 2010; Alvarez-
on

Ord
~ez et al. 2010). Strong absorptions were detected in
on
the five spectral regions (w1, w2, w3, w4 and w5) that
characterize the major cellular constituents. The spectra
obtained for treated samples showed visible changes, even
without any spectral transformation, in the w3 spectral
region. With the aim of minimizing methodological vari-
ability and amplifying the chemically based spectral dif-
ferences, the spectra were further processed – that is,
normalization, smoothing and first-derivatization of spec-
tra were carried out. The signal transformation thus
achieved made differences between spectral features much
more prominent. Figure 1b shows the w3 region of repre-
sentative transformed spectra of strain RS38 after its
exposure to different concentrations of the citrus fruit
extract for 90 min. Concentrations exceeding the MIC
caused striking changes in this region of the infrared
spectrum, which were especially located around
1400 cm 1, frequency where vibrations from the carbox-
ylic functional group (mainly C–O symmetric stretching
of the deprotonated carboxylate group) of fatty acids are
reflected (Jiang et al. 2004). Changes in other regions of
the spectra were limited (data not shown). When strains
SP11 and SP58 were tested, similar changes in FT-IR
spectra were observed (data not shown). Multivariate
analysis of the first-derivative spectra revealed significant
differences among phenotypes within the spectral region
w3 (Fig. 1c). A FA of this spectral region allowed us to
discriminate between four groups of samples: (i) cells of
strain RS38 exposed to 160 ppm, (ii) cells of strain RS38
exposed to 80 ppm, (iii) cells of strain RS38 exposed to
20 ppm and cells of strains SP11 and SP58 exposed to
160 ppm (iv) and spectra belonging to strains RS38,
SP11 and SP58 exposed to lower concentrations of the
citrus fruit extract.
The leakage of intracellular material after exposure to
the different concentrations of the extract for 90 min was
assessed by measuring the optical density at 260 nm
53
Antibacterial activity of BIOLL+â

A. Alvarez-Ord
n
o ~ez et al.

(a) (b)
6 0·020
Reduction in cell number (log CFU/mL)
0·015
5
0·010

Absorbance (nm)
4
0·005

3 0·000

–0·005
2
–0·010
1
–0·015

0 –0·020
5ppm 20ppm 80ppm 160ppm 1500 1450 1400 1350 1300 1250 1200
Bioll® concentration Wavelength (cm–1)

(c) (d)
0·3
0·8
Other spectra
0·1

0·6
–0·1

ΔOD 260nm
RS38 20 ppm
Factor 2

SP11 160 ppm

SP58 160 ppm
0·4
–0·3

RS38 80 ppm
0·2
–0·5 RS38160 ppm

–0·7 0
–1·0 –0·9 –0·8 –0·7 5ppm 20ppm 80ppm 160ppm
Factor 1 Bioll® concentration

Figure 1 Antibacterial activity of BIOLL+â against Salmonella Typhimurium strains. (a) Reduction in cell numbers (in log CFU ml 1) for
S. Typhimurium strains RS38 (black), SP11 (grey) and SP58 (white) after exposure to different concentrations of BIOLL+â for 90 min. The averages
of three independent experiments  standard deviations are shown. (b) Transformed representative FT-IR spectra (1500–1200 cm 1) of
S. Typhimurium RS38 nontreated cells (grey continuous line) and S. Typhimurium RS38 cells exposed for 90 min to 5 ppm (grey broken line),
20 ppm (black continuous line), 80 ppm (black broken line) or 160 ppm (black dotted line) of BIOLL+â. (c) Scatter plot obtained from factor anal-
ysis of the w3 (1500–1200 cm 1) spectral region, showing the distribution of control and BIOLL+â-treated samples of S. Typhimurium strains
RS38, SP11 and SP58. Three replicates of each experimental condition are represented. (d) Increase in OD260 of cell-free filtrates of S. Typhimuri-
um strains RS38 (black), SP11 (grey) and SP58 (white) after exposure to different concentrations of BIOLL+â for 90 min. The averages of three
independent experiments  standard deviations are shown.

(OD260) of cell-free filtrates (Fig. 1d). An increase in trations equal to the MIC, two populations of cells were
OD260 is indicative of leakage of intracellular nucleic evident: a first population with an intact membrane and
acids and, consequently, reflects a loss in membrane a second population with damaged membrane (embrac-


integrity (Sampathkumar et al. 2003; Alvarez-Ord
on~ez ing 67–79% of total cells). With concentrations over the
and Prieto 2010). The increase in OD260 was dependent MIC, near 100% of cells were PI stained.
on the citrus fruit extract concentration and S. Typhimu- In a similar way, two E. coli strains (EC65, with a MIC
rium strain. Thus, it was more marked at high concentra- of 80 ppm, and EC66, with a MIC of 40 ppm) were fur-
tions of the extract, with strain RS38 showing in all cases ther studied after their exposure to different concentra-
the highest OD260 values. Membrane integrity was also tions of the citrus fruit extract (20, 40, 80, 160 ppm) for
assessed by measuring the intake of PI, dye that enters 90 min. Inactivation experiments showed that whereas at
the cell when membrane integrity is compromised and low concentrations (20 and 40 ppm), no significant
binds to intracellular nucleic acids (Fig. 2). For the three reductions in cell numbers were achieved, at higher con-
S. Typhimurium strains tested, <12% of cells were PI centrations similar reductions were observed for strains
positive when exposed to concentrations of the extract EC65 and EC66, with 0
3–0
6 and 3
1–3
5 log cycles
below the MIC (Table S1; Fig. 2). However, with concen- of inactivation after exposure to 80 and 160 ppm,

54 Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology


A. Alvarez-Ord
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o ~ez et al. Antibacterial activity of BIOLL+â

0 ppm 5 ppm 20 ppm 80 ppm 160 ppm

202 217 166 547 532

151 162 124 410 399

RS38

R3 R3 R3 R3 R3
Counts

Counts
Counts

Counts

Counts
101 R2 108 R2 83 R2 273 R2 266 R2

50 54 41 136 133

0 0 0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
PI Log PI Log PI Log PI Log PI Log
192 219 195 243 591

144 164 146 182 443

SP11

R3 R3 R3 R3 R3

Counts
Counts

Counts

Counts

Counts
96 R2 109 R2 97 R2 121 R2 295 R2

48 54 48 60 147

0 0 0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
PI Log PI Log PI Log PI Log PI Log

Figure 2 Flow cytometry histograms (red fluorescence) showing the uptake of propidium iodide (PI) by Salmonella Typhimurium strains RS38 and
SP11 after their exposure to different concentrations of BIOLL+â for 90 min. Undamaged cells are embraced in R3 gate. Damaged cells are
embraced in R2 gate.

(a) (b)
6 0·020
Reduction in cell number (log CFU/mL)

0·015
5
0·010
Absorbance (nm)

4
0·005

3 0·000

–0·005
2
–0·010
1
–0·015

0 –0·020
20ppm 40ppm 80ppm 160ppm 1500 1450 1400 1350 1300 1250 1200
Bioll® concentration Wavelength (cm–1)
(c) (d)
0·3 0·8
0·2
Other spectra
0·1
0·6
0·0
ΔOD 260nm

–0·1
Factor 2

–0·2 0·4
EC65 160 ppm
–0·3
–0·4 EC66 80 ppm
0·2
–0·5 EC66 160 ppm

–0·6
–0·7 0
–1·00 –0·95 –0·90 –0·85 –0·8 –0·75 –0·70 20ppm 40ppm 80ppm 160ppm
Factor 1 Bioll® concentration

Figure 3 Antibacterial activity of BIOLL+â against Escherichia coli strains. (a) Reduction in cell numbers (in log CFU ml 1) for E. coli strains EC65
(black) and EC66 (white) after exposure to different concentrations of BIOLL+â for 90 min. The averages of three independent experi-
ments  standard deviations are shown. (b) Transformed representative FT-IR spectra (1500–1200 cm 1) of E. coli EC65 nontreated cells (grey
continuous line) and E. coli EC65 cells exposed for 90 min to 20 ppm (grey broken line), 40 ppm (black continuous line), 80 ppm (black broken
line) or 160 ppm (black dotted line) of BIOLL+â. (c) Scatter plot obtained from factor analysis of the w3 (1500–1200 cm 1) spectral region, show-
ing the distribution of control and BIOLL+â-treated samples of E. coli strains EC65 and EC66. Three replicates of each experimental condition are
represented. (d) Increase in OD260 of cell-free filtrates of E. coli strains EC65 (black) and EC66 (white) after exposure to different concentrations
of BIOLL+â for 90 min. The averages of three independent experiments  standard deviations are shown.

Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology 55
Antibacterial activity of BIOLL+â

A. Alvarez-Ord
n
o ~ez et al.

respectively (Fig. 3a). FT-IR analyses indicated that unexposed to citrus fruit extract were already PI stained
changes caused by the citrus fruit extract in FT-IR spectra and showed high OD260 values after routine laboratorial
were mainly located in the w3 spectral region, as hap- manipulation (data not shown). Brachyspira hyodysente-
pened to S. Typhimurium strains. Figure 3b shows the riae strain B204 was selected for FT-IR spectroscopic
w3 region of representative transformed spectra of strain analyses. Exposure of this strain to various concentrations
EC65 after its exposure to different concentrations of the of the citrus fruit extract (10, 20, 40, 80 and 160 ppm)
extract for 90 min. As previously described for S. for 90 min gave rise to noticeable changes in the FT-IR
Typhimurium, the main spectral modifications were spectra. The characteristic effect above described for
found around 1400 cm 1. A similar effect was evident for S. Typhimurium and E. coli in the w3 region of
strain EC66 (data not shown). After a FA of the w3 spec- transformed spectra was less evident for treated B. hyody-
tral region, it was possible to discriminate four groups of senteriae samples (Fig. 5a). However, major spectral mod-
samples: (i) cells of strain EC66 exposed to 160 ppm, (ii) ifications occurred in the w4 region of samples exposed
cells of strain EC66 exposed to 80 ppm, (iii) cells of to concentrations exceeding the MIC (Fig. 5b). This
strain EC65 exposed to 160 ppm (iv) and spectra belong- region is informative mostly for the carbohydrates and
ing to strains EC65 and EC66 exposed to lower concen-

polysaccharides of the cell wall (Alvarez-Ord
~ez and Pri-
on
trations (Fig. 3c). Leakage of intracellular content was eto 2012). A HCA of the w4 region from transformed
also monitored for strains EC65 and EC66 (Fig. 3d). The spectra clearly discriminated the spectra obtained for
increase in OD260 of cell-free filtrates observed after expo- samples exposed to concentrations over the MIC from
sure to the citrus fruit extract was in all cases higher for those obtained at lower concentrations (Fig. 5c).
strain EC66 and was more marked at high concentrations
of BIOLL+â. When the intake of PI was evaluated, it was
Discussion
observed that at low concentrations (20 and 40 ppm),
<12% of cells were PI stained, while exposure to 80 ppm This study shows the great efficacy of BIOLL+â (a com-
gave rise to the presence of a numerous subpopulation mercial citrus fruit extract) in both inhibiting the growth
with damaged membrane, which embraces 51% (strain and killing three bacterial gastrointestinal pathogens of
EC65) and 76% (strain EC66) of total cells (Table S2; interest for animal health (S. enterica, E. coli and B. hyo-
Fig. 4). When strains were exposed to 160 ppm, near dysenteriae). MIC, defined as the lowest concentration of
100% of cells showed a damaged membrane. the citrus fruit extract that avoids visible growth, was
In the case of B. hyodysenteriae, no inactivation experi- determined for a collection of strains through the broth
ments were performed, because this bacterial species does dilution method. All the strains tested were very suscepti-
not form isolated colonies on agar plates, which hinders ble to the effect of the extract, with MIC ranging from
the enumeration of survivors. Nevertheless, for all strains 10 ppm (for some B. hyodysenteriae strains) to 80 ppm
assayed, the MIC was found to be bactericidal after plat- (for various S. enterica and E. coli strains), very low con-
ing in blood agar plates, as shown by the lack of haemol- centrations in comparison with those previously
ysis for samples exposed for long time periods to the described for several essential oils (Burt 2004). This
MIC (data not shown). Performance of membrane integ- strong activity may be associated with the presence within
rity tests was not possible either. The outer membrane of BIOLL+â of a wide range of compounds with antimicro-
B. hyodysenteriae is associated with the protoplasmic cyl- bial properties, that is, ascorbic acid, citric acid, naringin,
inder by unknown connections in a very loose manner, hesperidin, quercetin, rutin, naringenin and saponins
and therefore, the membrane is easily removed during (Grupo Omega, personal communication), which may
gentle laboratorial manipulative procedures, such as cen- show additive or synergic activities due to their action on
trifugation (Sellwood and Bland 1997). Thus, control cells different cellular targets. In addition, our experiments

0 ppm 20 ppm 40 ppm 80 ppm 160 ppm

174 195 183 226 504

130 146 137 169 378

EC65

R3 R3 R3 R3 R3
Counts
Counts

Counts
Counts

Counts

87 R2 97 R2 91 R2 113 R2 252 R2

43 48 45 56 126

0 0 0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
PI Log PI Log PI Log PI Log PI Log

Figure 4 Flow cytometry histograms (red fluorescence) showing the uptake of propidium iodide (PI) by Escherichia coli EC65 after its exposure to
different concentrations of BIOLL+â for 90 min. Undamaged cells are embraced in R3 gate. Damaged cells are embraced in R2 gate.

56 Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology


A. Alvarez-Ord
n
o ~ez et al. Antibacterial activity of BIOLL+â

(a) 0·020 (b) 0·020

0·015 0·015

0·010 0·010
Absorbance (nm)

Absorbance (nm)
0·005 0·005

0·000 0·000

–0·005 –0·005

–0·010 –0·010

–0·015 –0·015

–0·020 –0·020
1500 1450 1400 1350 1300 1250 1200 1500 1450 1400 1350 1300 1250 1200
Wavelength (cm–1) Wavelength (cm–1)

(c)
B204 0ppm
B204 10ppm
B204 10ppm
B204 0ppm
B204 40ppm
B204 40ppm
B204 0ppm
B204 40ppm
B204 20ppm
B204 20ppm
B204 10ppm
B204 20ppm
B204 80ppm
B204 80ppm
B204 160ppm
B204 160ppm
B204 80ppm
B204 160ppm

0·0 0·1 0·2 0·3 0·4 0·5 0·6 0·7 0·8

Linkage Distance

Figure 5 Antibacterial activity of BIOLL+â against Brachyspira hyodysenteriae strains. (a) Transformed representative FT-IR spectra (1500–
1200 cm 1) of B. hyodysenteriae B204 nontreated cells (grey continuous line) and B. hyodysenteriae B204 cells exposed for 90 min to 10 ppm
(grey broken line), 20 ppm (grey dotted line), 40 ppm (black continuous line), 80 ppm (black broken line) or 160 ppm (black dotted line) of
BIOLL+â. (b) Transformed representative FT-IR spectra (1200–900 cm 1) of B. hyodysenteriae B204 nontreated cells (grey continuous line) and
B. hyodysenteriae B204 cells exposed for 90 min to 10 ppm (grey broken line), 20 ppm (grey dotted line), 40 ppm (black continuous line),
80 ppm (black broken line) or 160 ppm (black dotted line) of BIOLL+â. (c) Dendrogram obtained from hierarchical cluster analysis of the w4
(1200–900 cm 1) spectral region of control and BIOLL+â-treated B. hyodysenteriae B204 cells. Cluster analysis was performed with the Pearson
product moment correlation coefficient and by the Ward’s algorithm method. Three replicates of each experimental condition are represented.

revealed that the citrus fruit extract acts at its MIC as a
bactericidal agent against S. enterica, E. coli and B. hyody-
senteriae. However, inactivation experiments evidenced
that short-term exposition (for up to 90 min) to the
MIC caused low or no reductions in cell numbers, which
shows that this bactericidal activity is time dependent.
Thus, concentrations of the extract exceeding the MIC
(29 or 49) or longer exposition times were required to
achieve significant reductions in bacterial populations.
Nevertheless, although there were no immediate effects of
the citrus fruit extract at its MIC on bacterial viability,
clear effects in membrane integrity and composition were
observed after 90-min exposures, as assessed by mem-
brane integrity tests and FT-IR spectroscopic analyses
(below described).
Although recent investigations have focused on the elu-
cidation of the mechanism of action of different natural
antimicrobials and essential oils, including citrus fruits
oils (Xu et al. 2008; Fisher and Phillips 2009; Bouhdid
et al. 2010; Devi et al. 2010; Lu et al. 2011; Lv et al.
2011; Silva et al. 2011; Muthaiyan et al. 2012), no defini-
tive conclusions have been drawn yet. Considering the
large number of different groups of chemical compounds
present in most essential oils and, especially, in BIOLL+â,
it is likely that their antibacterial activity was not due to
one only specific mechanism but that several targets in
the cell were affected (Skandamis et al. 2001; Carson
et al. 2002). However, it has been speculated that the
main cellular target is the membrane. Due to their
lipophilic/hydrophobic character, essential oils partition

Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology 57
Antibacterial activity of BIOLL+â
in the lipids of bacterial cell membranes, which results in
membrane disruption, membrane expansion, increased
membrane fluidity and permeability, inhibition of respi-
ration and alteration in ion transport processes (Tromb-
etta et al. 2005). Moreover, certain components of
essential oils also appear to act on cell proteins embedded
in the cytoplasmic membrane through distortion of the
lipid–protein interaction or through direct effects on
hydrophobic regions of proteins (Knobloch et al. 1989;
Juven et al. 1994; Sikkema et al. 1995).
In our study, the effects of the commercial citrus fruit
extract on whole cellular composition and membrane
integrity were monitored. For the first purpose, FT-IR
spectroscopy was used. FT-IR spectroscopy is a method-
ology able to provide nondestructive, rapid, relevant
information on microbial systematics, useful for classifi-
cation and identification. In addition, it has been recently
proposed as a useful technique for the study of the mech-
anisms of death induction after bacterial exposure to
different antimicrobial compounds and adverse environ-
mental conditions, with special focus on cytoplasmic


membrane composition and structure (Alvarez-Ord
~ez
on
et al. 2011). Our results evidence that FT-IR spectra of
S. Typhimurium, E. coli and B. hyodysenteriae are greatly
affected after exposition for 90 min to the citrus fruit
extract and that the changes observed are dependent on
the extract concentration. Thus, spectral modifications
were only evident at lethal concentrations equal to (for
some strains), or exceeding (in most cases), the MIC.
Another interesting finding was the fact that spectral
changes observed were species dependent. Whereas for
S. Typhimurium and E. coli, the main effects on FT-IR
spectra were located in the w3 spectral region
(1500–1200 cm 1), for B. hyodysenteriae the w4 region
(1200–900 cm 1) was more seriously affected. This sug-
gests that the citrus fruit extract shows a broad-spectrum
antibacterial activity based on the action of several of its
constituents on different cellular targets. The spectra
obtained for S. Typhimurium and E. coli treated cells evi-
dence that for these bacterial species, the more striking
alterations were observed around 1400 cm 1, frequency
where vibrations from carboxylic functional groups
(mainly C–O symmetric stretching of the deprotonated
carboxylate group) are reflected (Jiang et al. 2004). These
changes suggest that the citrus fruit extract alters the
macromolecular structure of bacterial membranes, mainly
affecting carboxylic groups of membrane fatty acids. On
the other hand, alterations in this region of the spectra
were minimal for B. hyodysenteriae, which showed major
changes in the w4 spectral region, where ring vibrations
of functional groups C–O–C and C–O from polysaccha-
rides and carbohydrates of the cell wall are recorded


(Alvarez-Ord
~ez and Prieto 2012), which suggests the
on
58 Journal of Applied Microbiology 115, 50--60 © 2013 The Society for Applied Microbiology


A. Alvarez-Ord
n
o ~ez et al.
presence of damage or conformational/compositional
alterations in some or all of the components of the cell
wall.
The ability of the commercial citrus fruit extract
to damage the bacterial membrane was evaluated for
S. Typhimurium and E. coli cells by measuring the release
of intracellular contents and the uptake of PI, dye that
enters the cell when membrane integrity is compromised
and binds to intracellular nucleic acids. The exposure to
concentrations equal to or higher than the MIC gave rise
to the leakage of nucleic acids, as shown by the increase
in OD260 of cell-free filtrates, which was more marked at
high extract concentrations. This leakage of intracellular
components suggests that the commercial citrus fruit
extract may cause formation of pores in the cytoplasmic
membrane. Regarding PI uptake experiments, in general,
samples exposed to the MIC showed a relevant subpopu-
lation of cells with damaged membrane, while part of the
population remained undamaged. On the other hand,
near 100% of cells showed a damaged membrane when
concentrations higher than the MIC were used. Thus,
flow cytometric results also evidence the capability of the
citrus fruit extract to disrupt the cell membrane, render-
ing it more permeable. These findings are in agreement
with previous studies that demonstrated that different
essential oils are able to disturb the bacterial membrane,
leading to a loss of cytoplasmic material (Moosavy et al.
2008; Fisher and Phillips 2009; Bouhdid et al. 2010; Devi
et al. 2010; Lv et al. 2011).
On the basis of our findings, it can be postulated that
BIOLL+â has a dual mode of action. First, it shows a
physical effect on bacterial membranes, with the forma-
tion of pores and a consequent leakage of intracellular
components. This physical effect is already evident in
most cases after short-time exposures to the MIC, when
no reduction in bacterial numbers has been achieved yet,
and therefore, it is a reversible damage that can be
repaired after culturing under optimal conditions. How-
ever, bacterial exposition to concentrations exceeding the
MIC causes a chemical effect on the cellular envelopes,
which is dependent on the bacterial species, as shown
by FT-IR spectroscopic results. Thus, whereas for S.
Typhimurium and E. coli, the changes are focused on the
carboxylic group of membrane fatty acids, for B. hyody-
senteriae the main effects are found in polysaccharides
and carbohydrates of the cell wall. Exposition to the MIC
for longer time periods is likely to cause similar effects
on FT-IR spectra. This chemical effect is already linked to
a reduction in bacterial numbers, which suggests that the
damage caused is at this point irreversible. The proposed
mode of action, with dual physico-chemical effects on
bacterial envelopes and species-specific cellular targets,
may be associated with a decreased likelihood of bacteria


A. Alvarez-Ord
n
o ~ez et al.
developing resistances. This fact, added to the great anti-
bacterial activity showed by BIOLL+â, with MIC values
 80 ppm, shows the attractiveness of this commercial
citrus fruit extract as an alternative to antibiotics for its
inclusion as a feed additive in animal nutrition. Never-
theless, further research in clinical trials under field con-
ditions is necessary to verify its efficacy without
detrimental side effects.
Acknowledgements
This work was funded by ‘Ministerio de Econom
ıa y
Competitividad’ (project AGL 2010-18804). We acknowl-
edge the contribution of Grupo Omega. The authors
declare no conflict of interests.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1 Percentage of PI-stained cells evaluated
by flow cytometry for control and BIOLL+â-treated
S. Typhimurium strains RS38, SP11 and SP58.
Table S2 Percentage of PI-stained cells evaluated by flow
cytometry for control and BIOLL+â-treated E. coli strains
EC65 and EC66.