Advances in Microbiology, 2014, 4, 73-80
Published Online January 2014 (
Grape Phenolic Extract Potentially Useful in the Control of
Antibiotic Resistant Strains of Campylobacter
Elisa Mingo1, Alfonso V. Carrascosa1, Sonia de Pascual-Teresa2,
Adolfo J. Martinez-Rodriguez1*
1Insti tuto de In vestigación en Cienci as de la Alimentación (CIAL), CS IC-UAM, Departamento de Biotecnología y Microbiología.
C/Nico lás Cabrera, 9. Cantoblanco Campus, Universidad Autónoma de Madrid, Madrid, Spain
2Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), CSIC, Departamento de Metabolismo
y Nutrición-C/Jose Antonio Novais, Madrid, Spain
Email: *
Received November 7, 2013; revised December 7, 2013; accepted December 14, 2013
Copyright © 2014 Elisa Mingo et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual
property Elisa Mingo et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.
In this work, a grape phenolic extract obtained by methanol extraction has been demonstrat ed to be effect ive in in-
hibiting the growth of different strains and species of Camp ylo bacter, one of the most important bacterial food-
borne pathogens causing gastroenteritis worldwide. Noteworthily, it was particularly effective against several
strains presenting multiple antibiotic resistances. In all cases, the minimu m inhibitory concentration (MIC) was
lower than 300 mg GAE/L, being of 60 mg GAE/L for one of the most resistant strains (C. coli LP2), while the
others were between 120 mg GAE/L and 180 mg GAE/L. The analytical study of the main phenolic compounds
in the grape extract revealed that it was mainly constituted by catechins (85.7%) and phenolic acids (13.7%).
However, experiments developed using pure standards demonstrate that phenolic acids (such as gallic, p-hi-
droxibenzoic, vanillic, and homovanillic acids) were the most active, provoking a Campylobacter gr owt h decrease
between 6.7 and 7.6 log, while epic atechin w as the only catechin w ith activ ity as pure c ompound (1 log of g rowth
Campylobacter; Food-Borne Pathogen; Antibiotic Resistance; Grape Phenolic Extract; Phenolic Acids; Flavanols
1. Introduction
Campylobacter species are the leading causes of bacterial
food-borne gastroenteritis worldwide and the species
Campylobacter jejuni (C. jejuni) and Campylobacter coli
(C. coli ) cause more than 95 % of the infectio ns attributed
to this genus [1]. Campylobacter infections in humans
are usually characterized by self-limiting diarrhea, abdo-
minal cramps, nausea and fever, but severe neurological
sequelae, bacteremia, and other extraintestinal complica-
tions may develop less frequently [2]. Several sources of
Campylobacter infection in huma ns have bee n suggest ed,
but the most common is mainly associated with the con-
sumption and/or handling of poultry meat, especially
fresh broiler meat [3]. Although most infections are re-
sol ved without specific treatment, antimicrobial therapy
can be critical in invasive or severe infections. Fluoroqui-
nolone agents, like ciprofloxacin and macrolides such as
erythromycin, are commo nly us ed for the treatment of in-
fections caused by Campylobacter [4]. However, the rise
in the incidence of infections caused by antibiotic-re-
sistant strains of Campylobacter makes this illness in-
creasingly difficult to treat [5]. Moreover, since a large
proportion of the European Union (EU) chicken produc-
tion is contami nated with the pathogen [6] and give n the
recent ban b y the E uropean Union o n the use of a ntibio-
tics in animal feed to promote growth [7], it is essential
to search for new, natural and sustainable strategies to
reduce the incidence of Campylobacter in the food chain,
especially in its main host. Consumer concerns about the
Corresponding author.
safety of food have increased, and in this regard, there is
a growing interest in the use of natural antibacterial com-
pounds, like plant extracts rich in phenolic compounds,
as food preservatives. In the last years, different works
about the antimicrobial properties of wine and grape phe-
nolic compounds have been published, and several stu-
dies have shown that these compounds could inhibit the
growth of different food-borne bacteria [8,9]. Concerning
Campylobacter, other researchers have reported that
some phenolic co mpounds from grape leaves can have an-
timicrobial activity against this pathogen [10], contribut-
ing to mo dulat ing the resistance to macrolide antibiotics
[11]. We have previously reported the antimicrobial ac-
tivity of a commercial extract of GSE against Campylo -
bacter, identifying the main phenolic compounds related
with the behavior observed [12]. In this work, we have
obtained several grape extracts using two solvents (me-
thanol and water). The most active extract has been se-
lected to study its antibacterial activity against different
species and s trains of Campylobacter, ide nti f yi n g t he ma i n
compounds responsib le for the antibacter ial activity. The
bactericidal effect was compared to that of 10 different
antibiotics, with the purpose of establishi ng the potential
of grape phenolic compounds in the control of Campylo-
2. Materials and Methods
2.1. Bacterial Strains, Growth Media, and
Culture Conditions
The microorganisms used in this study included 12 dif-
ferent strains: 9 of C. jejuni and 3 of C . coli. Stra in s pe-
cification and origin of the specimen is provided in
Table 1.
Table 1 . Source of Campylobacter spe cies obt ained fr om ve-
terinary, clinical and coll ection librari es. La Paz and Carlo s
III are hospital s of Madri d, Spain.
Spec ie Strain Origin Source
Veteri n ary
Veteri n ary
Veteri n ary
La Paz
Carlos III
Carlos III
Veteri n ary
La Paz
aBacteri al cultures obtaine d from the Nationa l Collection of Types Cu ltures
(NCTC), UK, bCollection from Instituto de Investigación en Ciencias de la
Alimentación, (CIAL), cSpanish Collection of Type Cultures (CECT).
All strains were stored at 80˚C. Liquid growth me-
dium for Campylobacter strains consisted of Brucella
Broth (BB) (Becton, Dickinson, & Company, New Jer-
sey, USA). The agar plating medium consisted of Müel-
ler-Hinton agar supplemented with 5% defibrinated sheep
blood (MHB) (Becton, Dickinson, & Company). The fro-
zen strains were reactivated by inoculation in MHB and
incubation under microaerophilic conditions (85% N2,
10% CO2, 5% O2) using a Variable Atmosphere Incuba-
tor (VAIN) (MACS-VA500) (Don Whitley Scientific,
Shipley, UK) at 42˚C for 48 h. Isolated colonies were
inoculated into 50 ml of BB and incubated under stirring
at 130 rpm on an orbital shaker (Sha ker S3) (Elmi, Riga,
Latvia) at 42˚C for 24 h in microaerophilic conditions in
the VAIN. These bacterial inocula cultures (~1 × 108
CFU/ml) were used for the antibacterial activity assays.
2.2. Antibio tic S us ceptib i l ity Te st in g
The antibiotic susceptibility was assessed for each strain
following the Kirby-Bauer disc diffusion method based
on the performance standards for antimicrobial disk sus-
ceptibility test [13]. The bacterial inoculum in BB was
spread onto MHB using sterile cotton-tipped swabs. An-
timicrobial discs (Oxoid, Basingstoke, UK) were placed
on the inoculated MHB plates and they were incubated in
the VAIN for 48 h. The following antimicrobial discs
were used: aztreonam, tetracycline, gentamicin, cephalo-
thin, amoxicilli n-clavula nic acid, nalidixic acid , chlora m-
phenic o l, erythr omyci n (a ll 30 µ g), st re p to myci n (2 5 µg) ,
and ciprofloxacin (5 µg). Interpretation of the result s wa s
performed using the resistance breakpoint for Campylo-
bacters described by others [14,15]. When no breakpoints
were available for Campylobacters, the resistance break-
point describ ed by CLSI for Enterobacteriaceae was used.
The breakpoints are shown in Table 2.
2.3. Grape Extracts Preparation
Phenolic extracts were prepared from three varieties of
Vitis vinifiera grapes: Tempranillo, Garnacha and Caber-
net Sauvignon. Extraction and concentration of the phe-
nolic fraction was carried out by the procedure describ-
ed by Pallauf et al. [16]. Two different solvents were us-
ed in the extraction process: methanol, as non-polar sol-
vent, and wate r (polar solvent).
Briefly, fresh grapes were homogenized using an Ul-
tra-Turrax T25 (IKA-WERKE GmbH & Co., Staufen,
Germany) for 2 - 3 min. to obtain 100 g of grape’s homo-
genated. 100 ml of methanol 100% (Sigma-Aldrich, Mis-
souri, USA) or water was added to the homogenate and
mixed for 15 min. Afterwards, it was centrifuged for 10
min. at 4500 rpm and the supernatant was collected. The
extraction process was repeated twice more.
Table 2. Bre akpoints for Campylobacter spp.
Antibiotic Pote ncy R I S
Gentamicin** 10 µg 12 13 - 14 15
Cephalotin*** 30 µ g 14 15 - 17 18
Streptomycin*** 10 µg 10 11 - 12 15
Tetracyclin** 30 µg 14 15 - 18 19
Clorampheni col* 30 µg 11 12 - 22 23
Eritromycin* 15 µg 15 16 - 18 19
Aztreo na m*** 30 µg 15 16 - 21 22
Nalidix ic Acid** 30 µg 13 14 - 18 19
Ciprofloxacin* 20/10 µg 13 14 - 17 18
Clavulanic Acid*** 5 µg 18 19 - 23 24
*Miflin et al. (2007), **Luangtongkum et al. (2007), ***Clinical Laboratory
Standards Institute (CLSI) (2007) .
The extracts were then combined, filtered through a
Büch ner fu nne ll and concentrated by evaporation at 30˚C
(Rotavapor® 210 R-210 BÜCHI) (Labortechnik AG, Fla-
wil, 211 Switzerland). The extract obtained in each case
was suspended in 100 ml of water. The resulting aqueous
extracts were then lyophilized and the powder stored at
2.4. Antibacterial Activity of the Grape Extract
A first screening was performed to analyze the antibac-
terial activity of the different grape extracts (Tempranillo,
Garna cha a nd Cabe rnet Sau vigno n) agai nst C.jejuni LP1.
The extract which showed the strongest antibacterial ac-
tivity was then selected for the following experiments
with antibiotic-resistant strains. In all cases we used the
following quantitative procedure: 1 ml of sample was
transferred into different flasks containing 4 ml of BB.
Bacterial inocula (50 µl with 1x108 colony forming units
x mililiter (CFU/ml) were then inoculated into the flasks
under aseptic conditions. All cultures were prepared in
triplicate and incubated microaerobically at 42˚C for 24 h
(130 rpm) in the VAIN. Positive growth controls were
prepared by transferring 1 ml of saline solution (NaCl
0.9%) to 4 ml of BB and 50 µl of bacterial inocula. After
incubation, serial decimal dilutions of mixtures were
prepared in saline solution and they were plated (10 µl)
onto fresh MHB agar and incubated microaerobically at
42˚C in the VAIN. The number of CFU was assessed
after 48 h of incubation. Results were expressed as log
CFU/ml. The minimal inhibitory concentration (MIC)
and the minimal bactericidal concentration (MBC) were
determined following the procedure described above and
by using each grape extract diluted in sterile water to
obtain the desired final concentration. MIC was defined
as the lowest concentration of sample that provokes a
statistically significant decrease in viability with respect
to the control growth after 24 h of treatment. MBC was
defined as the lowest concentration of sample where no
growth was observed after 24 h of treatment.
2.5. Assess ment o f Total Phen olic Cont ent (TPC)
The total phenolic content (TPC) in the different grape
extracts was determined in accordance with the Folin-
Ciocalteu micro method as previously described by Sch-
midt et al. [17]. Briefly, samples (10 µl) were added to a
96-well microtiter plate (Sarstedt, Nümbrecht, Germany)
at an adequate d ilution in triplicate. To star t the reaction,
150 µl of aqueous Folin-Ciocalteu (Sigma -Aldrich) solu-
tion (14 ml water to 1 ml o f F olin-Ciocalte u rea gent) was
added to each well. After 3 minutes, 50 µl of NaHCO3
solution (2 ml of saturated NaHCO3 to 3 ml of water)
was added to each well and the plate was placed in the
dark at room temperature for 2 h. Absorbance was mea-
sured at 725 nm using a BioTek Synergy HT Multi-Mode
microplate reader (BioTek Instruments Inc., Vermont,
USA), and the data were acquired and processed using
BioTek’s Gen5TM software (BioTek Instruments Inc.).
Gallic acid (Sigma-Aldrich) was used as the standard for
a calibration curve. TPC was expressed as milligrams of
gallic acid equivalents per liter (mg G AE/L).
2.6. Determination of Individual Phenolic
Compounds of the Extracts by HPLC and
Mass Spectrometry Detection
All HPLC analyses were carried out on a Hewlett-Pack-
ard Agilent 1200 Series liquid chromatography system
equipped with a quaternary pump and a photodiode array
detector (DAD) (Agilent Technologies, Waldrom, Ger-
many). The column used was a Phenomenex Luna C18
column (4.6 × 150 mm, 5 μm) (Phenomenex, California,
USA) which was set thermostatically at 25˚C. Chroma-
tographic data were acquired and processed using an
Agilent Chemstation for LC 3D system (Rev. B.04.01)
(Agilent Technologies). The HPLC method conditions
were as described by Avila et al. [18]. Briefl y, t he bina r y
mobile phase used for analyses were aqueous 4.5% for-
mic acid (A) and HPLC-grade acetonitrile (B) at a flow
rate of 0.5 ml/min. The elution started with 10% B, and
the gradient was 20% B from 0 to 20 min, 25% B from
20 to 30 min, and 35% B from 30 to 50 min. Detection
wavelengths were 280, 320, 440 and 520 nm and samples
were analyzed in triplicate. Peaks were identified by
comparing their retention time and UV-vis spectra with
the reference compounds, and the data were quantified
using the corresponding curves of the reference com-
pounds as standards. In order to confirm the identity of
the recorded compounds, additional analyses were per-
formed by using HPLC with mass spectrometry detection
(HP LC -MS). For mass spectrometry an Agilent 1100
series liq uid c hromatogr aph/ mass-selective detector eq uip-
ped with a quadrupole (G1946D) mass spectrometer
(Agil ent Te chnolo gies) was u sed. Separation was achiev-
ed with an O RB AX Eclip se X DB -C18, (4.6 × 150 mm, 5
µm) ( Agi lent Te chnol ogie s). Elut ion was p erfo r med with
a gradient between 2.5% acetic acid in Milli-Q water (so-
lution A), a mixture of 2.5% acetic acid in Milli-Q and
water-acetonitrile (90:10) (solution B), and pure acetoni-
trile (solution C) at a flow rate of 0.5 ml/min, and an in-
jection volume of 20 µl. The elution programme consist-
ing of the following: from 100% A to 100% B in 3 min,
from 100% to 93% B in 5 min, from 7% to 10% C in 7
min, from 10% to 15% C in 5 min, from 15% to 50% C in
5 min and isocratic 50% C and B for another 5 min.
Electrospray ionisation in the positive mode was used.
The electrospra y capillary voltage was set to 2500 V, wit h
a nebulising gas flow rate of 12 liters/min and a drying
gas temperature of 150˚C.
2.7. Antimicrobial Activity Assay of Pure
Phenolic Compounds
The phenolic compounds identified in the most active
extract were tested against C. jejuni LP1 as pure com-
pound s. The assayed compounds (quercetin, quercetin 3-
glucoside, homovanillic acid, vanillic acid, gallic acid,
protocatechuic acid, clorogenic acid, p-hidroxibenzoic
acid, sinapic acid, catechin, epicatechin, and procyani-
dins B1, B2 were purchased from Sigma-Aldrich). The
procedure used has been described above. CFU was as-
sessed after 48 h of incubation. Results were expressed
as log CFU/ml.
2.8. Statistical Analysis
Analysis of variance (ANOVA) was performed by SPSS
19.0 for Windows, version 19.0.0 (Dec. 2011).
3. Results and Discussion
3.1. Antibio tic Susceptibi l ity Test for
Campylobacter spp.
The results of the antibiotic susceptibility test are shown
in Table 3. The sensitivity to antibiotics was dependent
on the Cam pylobacter strain. Strains from international
culture collections (11168, 11351, 7572 and 7571) were
the most sensitive, presenting from null to 2 antibiotic
resistances. On the other hand, recent isolates from clin-
ical (LP2) and veterinary origin (CN1 and CNL4) were
resistant to five antibiotics. Among antibiotics, the high
percentage of resistances were found in ciprofloxacin
(66.6%) and nalidixic acid (58.3%), both of them of the
fluoroquinolones group, whereas all strains tested were
sensitive to a minogl ucosides (strepto mycin and gentami-
cin), chloramphenicol and amoxicillin/clavulanic acid. It
is known that bacteria often lose virulence by growth in
vitro, and that the genetics basis for virulence may be
expressed completely only during growth in vivo. This
fact has been observed for Campylobacters, where the
comparation of the transcriptional profile of the original
clonal isolate of C. jejuni 11168 and the genome-se-
quenced clone of C. jejuni 11168 showed important dif-
ferences in gene expression [19]. Also, the use of re-
peatedly subcultured strains of Helicobacter pylori (H.
pylori), a close-related microorganism, in virulence ex-
periments, has shown the loss of several virulence prop-
erties respect to the original strain [20]. These results
demonstrate the effect of laboratory culture and storage
on virulence properties, suggesting the importance to use
strains with low subcultures in studies of virulence and/
or se nsitivi ty to dr ugs. T he behavio r agai nst fluo roq uino-
lones has confirmed the striking increase in resistance of
Campylobacter against these drugs in the last years, ren-
dering now in a limited use of them in the treatment of
campilobacteriosis in many regions [2]. Only C. coli ( LP2 )
was resistant to erythromycin, one of the first choices in
the antibiotic treatment of campilobacteriosis due to its
low resistance rates (0% to 12%) , alt hough i t is gene rall y
higher in C. coli, ranging from 0% to 50% [21]. Even if
the majority of C. jejuni and C. coli are resistant to β-
lactam agents, amoxicillin plus clavulanic acid has al-
ready been reported as effective [22], in accordance with
the results obtained in the present work. Other drugs such
as tetracycline and chloramphenicol can be alternative
antibiotics, but up to 60% of strains may be resistant to
tetracycline [23].
In the last years, it has been found that some com-
pounds derived from plants can be active against antibi-
otic-resistant pathogens associated with foods, possibly
by using different mechanisms of action [24]. For this
reason, in the present work we evaluate the effect of
three different grape extracts against Campylobacter in
order to cla r ify its possible use a s an antimicrobial.
3.2. Antibacterial Effect of Grape Extracts on
Campylobacter spp.
The results of the antibacterial activity of the different
grape extracts against C. jejuni LP 1 showed t hat p he no l ic
extra ctio n usi ng metha no l as s olve nt (ave ra ge 4 .56 log of
growth inhibition) was more effective than the extrac-
tion with water (average 1.91 log of growth inhibition)
(Table 4). This is consiste nt with the total p henolic co n-
tent (TPC) determined for each extract, which showed
that the amount of phenolic compounds extracted using
methanol was higher than the one obtained with water
Table 3. Antimicrobial susceptibility profile for the Campylobacter spp. strains by Kirby-Ba uer disc diffusion method.
Antibiotic LP1 1168 118 11351 CIII CN1 CNL1 CNL2 7571 LP2 CN L4 7572
Gentamicin S S S S S S S S S S S S
Cephalotin S R R R R R R R S R R R
Streptomycin S S S S S S S S S S S S
Tetracyclin I S S S I R R R S I R S
Chlorampheni col S S S S S S S S S S S S
Eritromycin S S S S S S S S S R S S
Aztreo na m S S S R R R S S S R R R
Nalidix ic Acid R S R S S R R R S R R S
Ciprofloxacin R S R S R R R R S R R S
Amoxicillin-Clavu lanic Acid S S S S S S S S S S S S
S: susceptible; R: resist ant; I: intermediate.
Table 4. Antibacterial activity of three different grape extracts against C. jejuni LP1. Three varieties of grapes (Cabernet
Sauvignon, Garnacha and Tempranillo) were tested, and two different extractions were obtained (methanolic or water) for
each grape variety. T he results are express ed in Log CFU/ml ± standard deviation (SD ) (n = 3).
(mg G AE/L )
Median log CFU/ml ± SD (log reduction) (n = 3)
Strain Grape extract Control Ext ract Log Inhibition
C. jeju ni
Cab ernet S. (M) 224.67 8.49 ± 0.1 4.78 ± 1.1a 3.71
Cab ernet S. (W) 103.53 8.2 ± 0.1 5.14 ± 0.8a 3.06
Garnacha (M) 218.43 8.7 ± 0.1 3.92 ± 0.9a 4.78
Garnacha (W) 103.53 8.24 ± 0.2 8.28 ± 0.6 0.00
Tempra nillo ( M) 2 24.09 8.5 ± 0.1 3.32 ± 0.7a 5.18
Tempra nillo ( W) 126.31 8.2 ± 0.1 6.67 ± 1.0a 2.13
Cab ernet S. (M) 224.67 8.49 ± 0.1 4.78 ± 1.1a 3.71
coinciding with the reported by others [25]. However, no
differences were observed in the individual phenolic
compounds extracted, which were the same indepen-
dently of the extraction method used (data not shown).
Among the methanolic extracts, the most effective was
the Tempranillo extract. Thus, we selected it for next
series of experiments.
The results of the antibacterial activity of the selected
grape extract against different Campylobacter strains are
presented in Table 5. For this assay, the five most resis-
tant strains (with 4 or 5 antibiotic resistances) were used
and the clinical isolate LP1 (with 2 antibiotic r esistances)
was also included. With the purpose to determine the
lower concentration of the grape extract able to inhibit
Campylobacter growth we calculated the MIC and MBC
for each strain. The results obtained showed a relation-
ship between strain and sensitivity. In all cases, the MIC
was lower than 300 mg GAE /L, being the lowest value of
60 mg GAE/L for C. jejuni LP1 (two antibiotic resis-
tances) and C. coli LP2 (five antibiotic resistances). The
MIC for the other strains was 120 mg GAE/L (C. jejuni
CN1, CNL1, and CNL2) and 180 mg GAE/L for C. coli
CNL4. The MBC was between 240 mg GAE/L and 120
mg GAE/L, showing a similar behavior as MIC. In gen-
eral terms the extract showed a strong capacity to inhibit
Campylobacter growth regardless of the Campylobacter
species (C. jejuni or C. coli) or the origin of the strain
(human or veterinary). These MIC and MBC values seem
relevant, taking into account that they are below 100
mg/L [26].
3.3. Phenolic Composition of the Grape Extract
In Table 6 are sho wn the mai n individ ual phe nolic com-
pounds identified in the Tempranillo grape extract. The
main group of p henolic co mpounds i n the extract consi s t-
ed of catechins (85.7%) and phenolic acids (13.7%).
Table 5. MIC and M BC of the Tempranil lo grape ex tract against differe nt antibiotic resistant strai ns of Campylobacter spp.
The results are expressed in Log UFC/ml ± standard deviation (SD) (n = 2).
Median log CFU/ml ± SD (log reduction) (n = 2)
Concentration o f the grape
phenolic extract (mg GAE/L) Campylobacter strain
C. jejun i LP1 C. jejuni CNL1 C. jejuni C N1 C. jejuni CNL2 C. coli LP2 C. coli CNL4
Control 7.89 ± 0.2 8.04 ± 0.2 8.45 ± 0.2 8.39 ± 0.1 7.54 ± 0.2 8.01 ± 0.2
300 >1.48a* ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0
240 >1.48a ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0 >1.48a ± 0.0*** >1.48a ± 0.0 >1.48a ± 0.0**
180 >1.48a ± 0.0 >1.48a ± 0.0*** >1.48a ± 0.0 *** 4.90a ± 1.4 >1.48a ± 0.0 4.60a ± 0.2**
120 >1.48a ± 0.0*** 5.38a ± 0.0** 6.45a ± 0.7** 6.75a ± 0.1** >1.48a ± 0.0*** 7.70 ± 0.1
60 2.77a ± 1.2** 8.40 ± 0.8 8.40 ± 0.1 8.49 ± 0.7 7.13a ± 1.0** 7.95 ± 0.2
30 8.58 ± 0.2 8.77 ± 0.2 8.70 ± 0.0 8.29 ± 1.1 8.65 ± 0.2 8.17 ± 0.0
*Calculated log of detection limit (30 CFU per plate). **Minima l Inhib it ory Conce ntr ation (MIC) , ***Minim al Bacte ric idal Con cent rati on (MBC), a significantly
differ ent with respect to the growth control (p 0.05).
Table 6 . Individual phenolic composition (mg/L) of the Tem-
pranillo grape extract (mean ± standard deviation) (n = 3)
determined by HP LC-MS.
Family Compound Concentration (mg/L)
Flavonols Querc eti n -3-glucoside 4 ± 0.0
Querc et in 7 ± 0.0
Phenolic Acids Gallic Acid 37 ± 0. 1
Homogentisic Acid 21 ± 0.0
Protocatechuic Acid 27 ± 7. 0
Chlorogenic Acid 6 ± 0.1
Homovanillic Acid 72 ± 4.6
Vanillic Acid 41 ± 2.0
p-cumaric Acid 12 ± 0.0
p-Hidroxibenzoi c Acid 21 ± 1.6
Sinapic Acid 31 ± 1.0
Catechins Catechin (cat) 680 ± 4.4
Epi catechin (ec) 806 ± 8.1
B1 (cat-ec ) 66 ± 1. 3
B2 (ec-ec) 130 ± 23 . 1
TOTAL 300* ± 0.0
*Total phenolic content (mg GAE/L).
Epicatechin and catechin were the major identified com-
pounds, while homovanill ic, vanillic a nd gallic acid were
the most abundant within phenolic acids. The total phe-
nolic compounds in the grape extract were quantified in
300 mg GAE/L. Vanillic and gallic acid moieties have
been associated before with a loss of cytoplasmic mem-
brane integrity, with the resultant loss of ion gradients,
pH homeostasis and inhibition o f respira tory activit y [27].
On the other hand, the mechanisms of action proposed
for the antibacterial activity of catechins have been main-
ly attributed to c ytoplasmic membrane damage, although
other mechanisms could be involved [28].
With the purpose to evaluate the impact of the phenol-
ic compounds presented in the extract in the observed
behavior, the pure phenolic compounds identified as part
of the most active extract (homovanillic acid, vanillic
acid, gallic acid, protocatechuic acid, clorogenic acid, p-
hidroxibenzoic acid, catechin, epicatechin, and procya-
nidin dimmers B1 and B2) were tested against C. jejuni
LP1 and results are shown in Table 7. Phenolics acids
were the most active of the assayed compounds, provok-
ing a growth decrease between 6.7 and 7.6 log, while epi-
catechin was the only flavanol with activity as pure com-
pound (1 lo g). T hese resul ts sh ow the cont rib ution o f phe -
nolic acids to the inhibition of Campylobacter gro wth, al-
though as part of the extract, additive and/or synergistic
effec ts could be invo lved i n the beha vior ob served in t he
case of the grape extract. This fact was previously de-
scribed by us for the GSE extract [12] and others are ob-
served a similar behavior for some catechins [28] and for
phenolic acids such as gallic acid [ 29].
4. Conclusion
In summary, the results obtained in this work indicate
that grape extracts could be an important source of phe-
nolic compounds potentially active against Campylo-
bacter. The grape extract assayed has been demonstrated
to be useful against Campylobacter strains with multiple an-
tibiotic resistances. Phenolic acids have been identified as
Table 7. Effect of the standard phenolic compounds on the
growth of C jejuni LP1. All the compounds w ere tested at 1
mg/ml, except B1 and B2, which activity was assayed at 0.5
mg/ml. The results are expressed in Log UFC/mL ± stan-
dard deviation (SD) (n = 2).
Median log CFU/ml ± SD (log reduction) (n = 2)
Family Compound Contr ol Compound
Activity Log of
Flavonols Querc eti n -3
-glucoside 8.28 ± 0.0 8.28 ± 0.0 NI**
Quercet in 8.28 ± 0.0 8.28 ± 0.0 NI
Acids Homovanillic
Acid 8.36 ± 0.1 >1.48a* ± 0.0 6.88
Vanillic Acid 9.10 ± 0.1 >1.48a ± 0.0 7.62
Gallic Acid 8.15 ± 0.0 >1.48a ± 0.0 6.70
Acid 8.78 ± 0.0 8.77a ± 0.1 NI
Chlorogenic Acid
8.20 ± 0.1 8.17a ± 0.1 NI
Acid 8.15 ± 0.1 >1.48a ± 0.0 6.67
Sinapic Acid 8.40 ± 0.0 8.42 ± 0.2 NI
Catechins Catechin (cat) 8.31 ± 0.1 8.84 ± 0.1 NI
Epicat echi n ( ec) 8.31 ± 0.1 7.27a ± 0.0 1.04
B1 (cat-ec ) 8.21 ± 0.1 8.61 ± 0.1 NI
B2 (ec-ec) 8.21 ± 0.1 8.20 ± 0.1 NI
*Calculated log of detection limit (30 CFU per plate ). **NI: no growth inh i-
bition. a Signific antly different with respect to the growth control (p 0.05).
the main compounds related with the behavior observed,
and they are usually the main phenolics in grape and
grape-derived products [30], constituting an important
metabolite derived from the metabolism of more complex
phenolic compounds, such as anthocyanins [31]. This fact
could contribute to standardizing the production process
of grape extracts to inhibit Campylobacter growth.
Ackno wledgements
This work was founded through Projects AGL 2009-
07894 from the MICINN, CSD2007-00063FUN-C-FOOD
[1] M. Ganan, J . M. Silvan, A. V. Carr ascosa and A. J. Marti-
nez-Rodriguez, Alternative Strategies to Use Antibiotics
or Chemical Products for Controlling Campylobacter in
the Food Chain,” Food Contr ol, Vol. 24, 2012, pp. 6-14.
[2] C. Fitzgerald, J. Whichard and I. Nachamkin, Diagnosis
and Antimicrobial Susceptibility of Campylobacter Spe-
cies,” In: I. Nach amkin, C. M. S zymanski and M. J. Blas-
er, Eds., Campylobacter, 3rd Edition, Amer ican Society
for Microbiology, Washington DC, 2008, pp. 227-243.
[3] T. Humphrey, S. O’Brien and M. Madsen , Campylobac-
ters as Zoonotic Pathogens: A Food Production Perspec-
tive,” International Journal of Food Microbiology, Vol.
117, 2007, pp. 237-257.
[4] C. K. Olson, S. Ethelberg, W. van Pelt and R. V. Tauxe,
Epidemiology of Campylobacter jejuni Infections in In-
dustrialized Nations Campylobacter,” In: I. Nachamkin,
C. M. Szyman ski and M. J. Bl aser, Eds., Campylobacter,
3rd Edition, American Society for Microbiology, Wash-
ington DC, 2008, pp. 163-189.
[5] Q. Zhang and P. J. Plummer, Mechanisms of Antibiotic
Resistance in Campylobacter,” In: I. Nachamkin, C. M.
Szymanski and M. J. Blaser, Eds., Campylobacter, 3rd
Edition, American S ociety for Micr obiolo gy, Washington
DC, 2008, pp. 263-266.
[6] EFSA, “The Community Summary Report on Trends and
Sources of Zoonoses, Zoonotic Agents and Food-Borne
Outbreaks in the European Union in 2008,” EFSA Jour-
nal, Vol. 8, 2010, p. 1496.
[7] European Commission, “Regulation (EC) No 1831/2003
on Additives for Use in Animal Nutrition,” Official Jour-
nal of the European Union, Vol. L268, 2003, pp. 29-43.
[8] M. Anastasiadi, N. G. Chorianopoulos, G. J. E. Nychas
and S. A. Haroutounian, Antilisterial Activities of Poly-
phenolic-Rich Extracts of Grapes and Vinification Bypro-
ducts,” Journal of Agricultural and Food Chemistry, Vol .
57, 2009, pp. 457-463.
[9] J. D. Adamez, E. G. Samino, E. V. Sanchez and D. Gon -
zalez-Gomez, In Vitro Estimation of the Antibacterial
Activity and Antioxidant Capacity of Aqueous Extracts
from Grape-Seed s (Vitis vinifera L.),” Food Control, Vol.
24, 2012, pp. 136-141.
[10] V. Katalinic, S. Možina, I. Gener al iz, D . S kro za, I. Ljuben-
kov and A. Klančnik, Phenolic Profile, Antioxidant Ca-
pacity, and Antimicrobial Activity of Leaf Extracts from
Six Vitis vinifera L. Varieties,” International Journal of
Food Properties, Vol. 16, 2013, pp. 45-60.
[11] M. Kurinčič, A. Klančnik and S. Možina, “Epigallocate-
chin Gallate as a Modulator of Campylobacter Resistance
to Macrolide Antibiotics,” International Journal of Anti-
microbial Agents, Vol. 40, 2012 , pp. 467-471.
[12] J. M. Silvan, E. Mingo, M. Hida lgo, S. de Pascual-Teresa,
A. V. Carrascosa and A. J. Martinez-Rodriguez, “Antiba-
cterial Activity of a Grape Seed Extract and Its Fractions
against Campylobacter spp,” Food Control, Vol. 29, 2013,
pp. 25-31.
[13] Clinical and Laboratoy Standars Institute (CLSI, formerly
NCCLS), “Perfor mance St andards for Antimicrobial Sus-
ceptibility Testing,” Seventeenth Informational Supple-
ment, Vol. 27, No. 1, 2007, pp. M100-S17.
[14] T. Luangtongkum, T. Y. Morishita, A. B. El-Tayeb, A. J.
Ison and Q. Zhang, “Comparison of Antimicrobial Suscep-
tibility Testing of Campylobacter spp. by the Agar Dilu-
tion and the Agar Disk Diffusion Methods,” Journal of
Clinical Microbiology, Vol. 45, 2007, pp. 590-594.
[15] J. K. Miflin, J. M. Templeton and P. J. Blackall, “Antibi-
otic Resistance in Campylobacter Jejuni and Campylo-
bacter coli Isolated from Poultry in the South-East Queens-
land Region,” Journal of Antimicrobial Chemotherapy,
Vol. 59, 2007, pp. 775-778.
[16] K. Pall auf, J. C. Rivas-Gonzalo, M. D. del Castillo, M. P.
Cano and S. de Pascual-Teresa, Characterization of the
Antioxidant Composition of Strawberry Tree (Arbutus
unedo L.) Fruits,” Journal of Food Composition and
Analysis, Vol. 21, 2008, pp. 273-281.
[17] B. M. Sch midt , J. W. E r dma n and M. A. Lila, Effects o f
Food Processing on Blueberry Antiproliferation and An-
tioxidant Activity,” Journal of Food Science, Vol. 70,
2005, pp . S389-S394.
[18] M. Avila, M. Hidalgo, C. Sánchez-Mo reno, C. P elaez, T.
Requena and S. de Pascual-Teresa, Bioconversion of An-
thocyanin Glycosides by Bifidobacteria and Lactobacil-
lus,” Food Research International, Vol. 42, 2009, pp.
[19] E. C. Gaynor, S. Cawthraw, G. Manning, J. K. MacKi-
chan, S. Falkow and D. G. Newell, The Genome-Se-
Quenced Variant of Campylobacter jejuni NCTC 11168
and the Original Clonal Clinical Isolate Differ Markedly
in Colonization, Gene Expression, and Virulence-Asso-
ciated Phenotypes,” Journal of Bacteriology, Vol. 186,
2004, pp . 503-517.
[20] S. S. Kim, H. S. Lee, Y. S. Cho, Y. S. Lee, C. S. Bhang,
H. S. Chae, S. W. Han, I. S. Chung and D. H. Park, The
Effect o f the Repeat ed Subcultures of Helicob acter p ylori
on Adhesion, Motility, Cytotoxicity, and Gastric Inflam-
mation,” Journal of Korean Medical Science, Vol. 17,
2002, pp . 302-306.
[21] A. Gibreel and D. E. Taylor, Macrolide Resistance in
Campylobacter jejuni and Campylobacter coli,” Journal
of Antimicrobial Chemotherapy, Vol. 58, 2006, pp. 243-
[22] A. J. Hakanen , M. Lehtopolku, A. Siitonen, P. Huovinen,
Kotilainen, “Multidrug Resistance in Campylobacter jeju-
ni Strains Collected from Finnish Patients during 1995-
2000,” Journal of Antimicrobial Chemotherapy, Vol. 2,
2003, pp . 1035-1039.
[23] M. J. Blaser and J. Engbe rg, Clini cal Aspects o f Campy-
lobacter jejuni and Campylobacter coli Infections,” In: I.
Nachamkin, C. M. Szymanski and M. J. Blaser, Eds.,
Campylobacter, 3r d Edit io n, Amer ican Society for Micro-
biology, Washington DC, 2008, pp . 99-121.
[24] M. Friedman, P. R. Henika, C. E. Levin, R. E. Mandrell
and N. Kozukue, Antimicrobial Activities of Tea Cate-
chins and Theaflavins and Tea Extracts against Bacillus
cereus,” Journal of Food Protection, Vol. 69, 2006, pp.
[25] H. Nawaz, J. Shi, G. Mittal and Y. Kakuda, “Extraction
of Polyphenols from Grape Seeds and Concentration by
Ultrafiltration,” Separation and Purification Technology,
Vol. 48, 2006, pp. 176-181.
[26] J. L. Ríos and M. C. Recio, Medicinal Plants and Anti-
microbial Activity,” Journal of Ethnopharmacology, Vol.
100, 2005, pp. 80-84.
[27] M. Ravichandran, N. S. Hettiarachchy, V. Ganesh, S. C.
Ricke and S. Singh, Enhancement of Antimicrobial Ac-
tivities of Naturally Occurring Phenolic Compounds by
Nanoscale Delivery against Listeria monocytogenes, Es-
cherichia coli O157:H7 and Salmonella typhimurium in
Broth and C hi cken M eat Syste m,” Journal of Food Safety,
Vo l. 31, 2011, pp. 462-471.
[28] T. P. T. Cushnie and A. J. Lamb, Recent Advances in
Understanding the Antibacterial Properties of Flavono-
ids,” International Journal of Antimicrobial Agents, Vol.
38, 2011, pp. 99-107.
[29] M. J. R. Vaquero , P. A. A. Fernandez, M. C. M. de Nadra and A.
M. S. de S aad, Phenolic Compound Combinations on Es-
cherichia coli Viability in a Meat System,” Journal of
Agricultural and Food Chemistry, Vol. 58, 2010, pp. 6048-
[30] M. Monagas, B . Bartolome and C. Go m ez -Cordoves, Up-
dated Knowledge about the Presence of Phenolic Com-
pounds in Wine,” Critical Reviews in Food Science and
Nutrition, Vol. 45, 2005, pp. 85-118.
[31] T. Nurmi, J. Mursu, M. Heinonen, A. Nurmi, R. Hiltunen
and S. Voutilainen, Metabolism of Berry Anthocyanins
to Phenolic Acids in Humans,” Journal of Agricultural
and Food Ch e m istry , Vol. 57, 2009, pp . 2274-2281.