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Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Antibacterial activity of Thymus vulgaris essential
oil alone and in combination with cefotaxime
against blaESBL producing multidrug resistant
Enterobacteriaceae isolates
Qada Benameur, Teresa Gervasi, Vito Pellizzeri, Mária Pľuchtová, Hassiba
Tali-Maama, Farida Assaous, Badia Guettou, Kheira Rahal, Daniela Gruľová,
Giacomo Dugo, Andreana Marino & Meriem-Hind Ben-Mahdi
To cite this article: Qada Benameur, Teresa Gervasi, Vito Pellizzeri, Mária Pľuchtová, Hassiba
Tali-Maama, Farida Assaous, Badia Guettou, Kheira Rahal, Daniela Gruľová, Giacomo Dugo,
Andreana Marino & Meriem-Hind Ben-Mahdi (2018): Antibacterial activity of Thymus vulgaris
essential oil alone and in combination with cefotaxime against blaESBL producing multidrug resistant
Enterobacteriaceae isolates, Natural Product Research, DOI: 10.1080/14786419.2018.1466124
To link to this article: https://doi.org/10.1080/14786419.2018.1466124
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Published online: 04 May 2018.
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NATURAL PRODUCT RESEARCH, 2018
https://doi.org/10.1080/14786419.2018.1466124
Antibacterial activity of Thymus vulgaris essential oil alone
and in combination with cefotaxime against blaESBL producing
multidrug resistant Enterobacteriaceae isolates
QadaBenameura,b, TeresaGervasic, VitoPellizzeric, MáriaPľuchtovád,
HassibaTali-Maamae, FaridaAssaouse, BadiaGuettoue, KheiraRahale, DanielaGruľovád,
GiacomoDugoc,f, AndreanaMarinog and Meriem-HindBen-Mahdib,h
aFaculty of Natural Sciences and Life, Nursing Department, University of Abdelhamid Ibn Badis of Mostaganem,
Mostaganem, Algeria; bLaboratoire de Recherche «Santé et Production Animale», Ecole Nationale
Supérieure Vétérinaire d’El-Harrach, Algiers, Algeria; cDepartment of Biomedical and Dental Sciences and
Morphofunctional Imaging, University of Messina, Messina, Italy; dFaculty of Humanities and Natural Sciences,
Department of Ecology, University of Prešov, Prešov, Slovakia; eLaboratoire de Bactériologie Médicale, Institut
Pasteur d’Algérie, Algiers, Algeria; fScience4Life, Spin Off Company, University of Messina, Messina, Italy;
gDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
Messina, Italy; hEcole Supérieure des Sciences des Aliments et des Industries Agroalimentaires, Algiers, Algeria
ABSTRACT
The aim was to evaluate the susceptibility of blaESBL producing
Enterobacteriaceae to Slovakian Thymus vulgaris essential oil (TVEO)
alone and in combination with cefotaxime (CTX). TVEO composition
was determined by gas chromatograph-mass spectrometer (GC/MS).
Susceptibility to 21 antibiotics was determined by disc diusion assay.
Genes characterization for resistance to β-lactams was accomplished
by polymerase chain reaction (PCR). The antibacterial activity was
investigated by standard methods. The synergistic interaction was
determined by checkerboard test. Thymol (34.5%), p-cymene (22.27%)
and linalool (5.35%) were the major components present in the TVEO.
The identied strains were multi-drug resistant (MDR). TVEO showed
high activity against all MDR strains, including blaESBL producing
isolates, with inhibition zones and MIC values in the range of 24–
40mm/10μL and 2.87–11.5μg/mL, respectively. TVEO in combination
with CTX showed a synergistic action against blaSHV-12 producing
Escherichia coli (FICI 0.28) and an additive eect vs ESBL producing
Enterobacter cloacae (FICI 0.987).
© 2018 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Thymus vulgaris; essential
oil; antibacterial-resistance;
Enterobacteriaceae; ESBL;
synergism
ARTICLE HISTORY
Received 12 December 2017
Accepted13 April 2018
CONTACT Teresa Gervasi tgervasi@unime.it
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1466124.
2 Q. BENAMEUR ET AL.
1. Introduction
One of the main sources of antibiotic resistant bacteria is represented by food-producing
animals (Gervasi et al. 2014; Lambrecht et al. 2017).
β-lactam antibiotics including cephalosporins are one of the preferred therapeutic options
for treating Enterobacteriaceae infections. The persistent exposure of bacterial strains to a
multitude of β-lactams has induced dynamic and continuous production and mutation of
β-lactamases in these bacteria, expanding their activity even against the newly developed
β-lactam antibiotics. These enzymes are known as extended-spectrum β-lactamases (ESBLs)
(Pitout and Laupland 2008). Production of ESBLs is a signicant resistance-mechanism that
impedes the antimicrobial treatment of infections caused by Enterobacteriaceae and is a
serious threat to the currently available antibiotic armory (Shaikh et al. 2015). Herbal med-
icines have always been a rich source of drug discovery programs, and many plant derived
compounds have shown promising activity against multidrug resistant (MDR) pathogens
(Saeidi et al. 2015). Currently innovative and promising antimicrobial molecules have been
extensively studied (Marino et al. 2016). Potential synergy of some essential oils with anti-
biotics has also been previously postulated, with the aim to alleviate the burden of antimi-
crobial resistance to conventional antimicrobials (Yahiaoui et al. 2017). It has been showed
that Thymus vulgaris essential oil (TVEO) had the best antibacterial activity against some
Enterobacteriaceae species, especially Escherichia coli (Rota et al. 2008; Santurio et al. 2014).
However, little information is available on the antibacterial activity of TVEO against ESBL
producing Enterobacteriaceae strains (Anžlovar et al. 2014), and no study has been conducted
to investigate its synergistic eect with cefotaxime (CTX) against ESBL producing
Enterobacteriaceae strains. The aim of this study was to evaluate the susceptibility of blaESBL
producing Enterobacteriaceae strains to Slovakian origin TVEO and the synergistic interac-
tions between TVEO and CTX.
NATURAL PRODUCT RESEARCH 3
2. Results and discussion
2.1. Chemical composition of TVEO
The GC-MS analysis identied three major components in TVEO (Table 1). It was observed
that thymol (34.50%) was present in the highest concentration in TVEO followed by
p-cymene (22.27%) and linalool (5.35%). Signicant variation is observed in the chemical
composition of the TVEO. Mancini et al. (2015) found that the major component is thymol,
followed by carvacrol and caryophyllene oxide. However, Šegvić Klarić et al. (2007) and El
Hattabi et al. (2016) reported that the major constituent in this essential oil is carvacrol,
followed by p-cymene and E-caryophyllene. As previously reported the variation of the
chemical composition of the EOs may be due to many factors such as the geographical
origin, genetic factors, plant material and harvesting season (Dugo et al. 2014; Tuttolomondo
et al. 2015).
Chemical composition together with the contaminant amount investigation is very impor-
tant for foods characterization (Di Stefano et al. 2014; Mallamace et al. 2014; Naccari et al.
2014; Cicero et al. 2015; Cacciola et al. 2016; Pantano et al. 2016).
2.2. Antimicrobial susceptibility testing
The seven Enterobacteriaceae strains isolated from poultry were identied as E. coli (n = 2),
Klebsiella pneumoniae (n = 2), Enterobacter cloacae (n = 2) and Salmonella sp. (n = 1).
Susceptibility testing results showed that all the isolates were MDR to three or more dierent
antimicrobial agents belonging to dierent classes of antibiotics (Table 2). Among those
strains, four phenotypically conrmed ESBL producing Enterobacteriaceae isolates were
detected. PCR results showed the presence of blaCTX-M gene in E. coli S22/12 and blaSHV gene
in E. coli S77/15 (Table 2). According to the BLAST analysis, the blaCTX-M and blaSHV genes
harbored by E. coli isolates were blaCTX-M-1 (97% query coverage, 98% sequence similarity)
and blaSHV-12 (93% sequence coverage, 97% sequence identity).
Table 1.Thymus vulgaris essential oil composition.
Notes: RT: Retention time, %: percentage of identified compound.
N. Compound name RT RI % N. Compound name RT RI %
1α-pinene 8,45 939 2,8 15 endo-borneol 16.691 1166 1,1
2 camphene 8.943 951 1,2 16 α-terpineol 17.702 1190 1,5
3 β-pinene 9.894 974 0,9 17 γ-terpineol 17.931 1195 0,5
4 β-myrcene 10.387 992 1,2 18 thymol 21.346 1297 34,5
5 p-mentha-1(7).8-diene 10.829 1001 0,1 19 carvacrol 21.507 1307 1,1
6 3-carene 11.058 1007 Tr 20 4-terpinenyl acetate 21.796 1340 0,2
7 o-cymene 11.286 1020 0,1 21 (-)-β-bourbonene 24.217 1412 Tr
8 p-cymene 11.712 1026 22,3 22 caryophyllene 25.339 1417 1,9
9 D-limonene 11.78 1031 1,1 23 γ-muurolene 26.912 1474 0,1
10 1,8 cineole 11.857 1030 0,9 24 δ-cadinene 28.499 1519 0,1
11 γ-terpinene 12.80 1030 0,2 25 δ-cadinene 28.499 1519 0,1
12 Linalool 14.363 1100 5,3 26 caryophylleneoxide 30.309 1578 0,9
13 fenchol 14.813 1121 Tr 27
14 isoborneol 16.359 1146 0,6 28
Total identified 78,7
4 Q. BENAMEUR ET AL.
The occurrence and dissemination of ESBL producing Enterobacteriaceae strains, especially
E. coli, has been increasingly reported over the past few years, both in human (Fernandes et
al. 2014) and veterinary medicine (Belmahdi et al. 2016). β-lactamases are the primary cause
of resistance to β-lactams among members of the family Enterobacteriaceae. Based on pri-
mary structure (Ambler 1980), enzymatic properties and biochemical attributes (Bush et al.
1995), and the increasingly available amino acid sequences (Bush and Jacoby 2010) four
major classes (A, B, C, D) can be acknowledged. Serine β-lactamases belonging to class A
are the most abundant (Philippon et al. 2016), with more than 500 enzymes, including the
most clinically signicant extended spectrum β-lactamases (ESBL) variants, i.e. CTX-M-, TEM-,
and SHV-type enzymes (Bush and Fisher 2011). Among ESBLs, the CTX-M-1 group was dom-
inant in most African surveys on husbandry animals (Alonso et al. 2017). Bacteria that produce
CTX-Ms. are frequently resistant to other classes of antimicrobial agents (Woodford et al.
2004). The MDR bacteria can be transmitted to human through the food chain adding serious
burden to human health (Awad et al. 2016; Benameur et al. 2016).
Surveillance activities in healthy animals worldwide are generating a tremendous amount
of data on ESBL distribution. Majority of SHV variants in this reservoir belong to blaSHV-2,
blaSHV-2a, blaSHV-5, and blaSHV-12 (Liakopoulos et al. 2016). Although, SHV enzymes did not
undergo the explosive dissemination observed for CTX-M-type variants (Cantón et al. 2012),
in recent years they have been found in several Enterobacteriaceae (Liakopoulos et al. 2016).
2.3. Antibacterial activity of TVEO
The initial screening of the antibacterial activity of the TVEO showed its very strong activity
against all the tested strains, with growth inhibition zones ranging from 24 to 40 mm in
diameters (Table 3). The antimicrobial activity of EOs can be classied into three levels: no
Table 2.Characteristic of tested Enterobacteriaceae strains.
Notes: ESBL: extended-spectrum β-lactamase; NA, nalidixicacid; CIP, ciprofloxacin; LVX, levofloxacin; AM, amoxicillin; AMC,
amoxicillin+clavulanicacid; TIC, ticarcillin; PIP, piperacillin; CZ, cefazollin; CF, cephalothin; CTX, cefotaxime; SSS, sulfon-
amides, TMP, trimethoprim; SXT, trimethoprim-sulfamethoxazole, TE, tetracycline; C, chloramphenicol; N, neomycin;
ND: Not determined.
Strain Antimicrobial resistance prole Bla gene
Klebsiella pneumoniae S34/15 (ESBL) NA, CIP, LVX, AM, AMC, TIC, PIP, CZ, CF, CTX, SSS, TMP, SXT, TE, C, N ND
Klebsiella pneumoniae S42/15 AM, SSS, TMP, SXT, TE, C, N /
Escherichia coli S22/12 (ESBL) NA, CIP, LVX,AM, TIC, PIP, CZ, CF, CTX, SSS, TE CTX-M-1
Escherichia coli S77/15 (ESBL) NA, CIP, LVX, AM, TIC, PIP, CZ, CF, CTX, SSS, TMP, SXT, TE SHV-12
Enterobacter cloacae S63/15 (ESBL) NA, CIP, LVX, AM, AMC, TIC, PIP, CZ, CF, CTX, SSS, TMP, SXT, TE, N ND
Enterobacter cloaceae S29/15 AM, AMC, TIC, PIP, CZ, CF, TE, N /
Salmonella sp. S61/15 NA, CIP, LVX, AM, TIC, PIP /
Escherichia coli ATCC 25,922 / /
Table 3.Diameters of inhibition zones and MIC values of TVEO.
Notes: TVEO: Thymus vulgaris essential oil, ESBL: extended spectrum β-lactamase, SD: standard deviation.
Strains Diameter of inhibition zone (mm)±SD (n=3) MIC (μg/mL) of TVEO
Klebsiella pneumoniae S34/15 (ESBL) 39 ± 0.5 2.857
Klebsiella pneumoniae S42/15 35 ± 0.3 11.5
Escherichia coli S22/12 (blaCTX-M-1) 40 ± 0.3 2.857
Escherichia coli S77/15 (blaSHV-12) 30 ± 0.5 11.5
Enterobacter cloacae S63/15 (ESBL) 24 ± 0.7 2.87
Enterobacter cloaceae S29/15 35 ± 0.3 11.5
Salmonella sp. S61/15 35 ± 0.5 11.5
Escherichia coli ATCC 25,922 37 ± 0.6 11.5
NATURAL PRODUCT RESEARCH 5
activity (inhibition zone < 12 mm), moderate activity (<20–12 mm) and strong activity (inhi-
bition zone ≥ 20 mm) (Rota et al. 2008).
The MICs values conrmed the results obtained by the agar disk diusion method (Table 3).
The values ranged from 11.5–2.87 μg/mL against the tested strains. The highest antibacterial
activity of TVEO was observed against blaCTX-M-1 producing E. coli S22/12 and ESBL producing
Klebsiella pneumoniae S34/15 with MIC value of 2.87 μg/mL, with both methods. Moreover,
blaCTX-M-1 producing E. coli was more sensitive to TVEO than blaSHV-12 producing E. coli S77/15.
TVEO also exhibited signicant antibacterial activity against ESBL producing Klebsiella pneu -
moniae than non ESBL producing Klebsiella pneumoniae S42/15.
The FICI of TVEO combined with CTX were calculated to determine their possible inter-
actions against ESBL producing Enterobacteriaceae strains (Table 4). High MIC values for CTX
(32 μg/mL) were obtained against all strains. Checkerboard test results showed a synergistic
eect against blaSHV-12 producing E. coli (FICI 0.28), an additive action against ESBL producing
Enterobacter cloacae (FICI 0.987), and an antagonistic eect against blaCTX-M-1 producing E.
coli and ESBL producing Klebsiella pneumoniae (9.064).
These dose-dependent eects are in agreement with other studies (Rota et al. 2008;
Anžlovar et al. 2014). The antimicrobial properties of essential oils of genus Thymus against
a wide range of resistant microbial strains have been documented extensively (Nabavi et al.
2015). The antimicrobial proprieties of TVEO could be associated with the thymol content,
which has been tested previously and was found to have a signicant antibiotic activity
(Guarda et al. 2011). Also, the synergistic eect between the dierent oil’s compounds, i.e.,
thymol and carvacrol (Guarda et al. 2011) and p-cymene and carvacrol (Ultee et al. 2002)
has also been reported. The mechanism of action of EO and their constituents is not fully
elucidated. This is complicated by the fact that there are many phytochemicals in EO and its
antibacterial activity may not be attributable to one specic mechanism, but probably there
are dierent targets in the bacterial cell (Burt 2004).
There are few published data on combination between CTX and essential oils (Yahiaoui
et al. 2017). This is the rst report concerning the synergistic eects of TVEO in combination
with CTX against ESBL-producing bacteria. This synergistic activity can be attributed to thy-
mol, p-cymene, linalool and other phytochemicals present in minor concentrations in TVEO
and to their dierent targets in the bacterial cell that could potentiate the activity of CTX.
Table 4.Fractional inhibitory concentration index (FICI) of TVEO tested in combination with CTX.
Notes: ESBL: extended spectrum β-lactamase, TVEO: Thymus vulgaris essential oil, CTX: cefotaxime.
Strains
MIC (μg/mL)
cefotaxime
MIC (μg/mL) of
TVEO
MIC of TVEO
combined with
cefotaxime
MIC of cefotaxime
combined with
TVEO FICI
Klebsiella pneumo-
niae S34/15 (ESBL)
32 2.87 23 32 9.064
Escherichia coli
S22/12 (blaCTX-M-1)
32 2.87 23 32 9.064
Escherichia coli
S77/15 (blaSHV-12)
32 11.5 0.36 8 0.28
Enterobacter cloacae
S63/15 (ESBL)
32 2.87 1.38 16 0.987
6 Q. BENAMEUR ET AL.
3. Experimental
See Supplementary materials for source of materials, gas chromatography/mass spectrom-
etry (GC-MS) analysis and evaluation of antimicrobial activity.
4. Conclusion
Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been documented
in humans as well as in food-producing birds, including chickens. In this study, the TVEO
exhibited high activity against all studied MDR Enterobacteriaceae isolates, including ESBL
strains. Moreover, TVEO in combination with CTX showed a synergistic action against blaSHV-12
producing E. coli and an additive eect vs ESBL producing Enterobacter cloacae.
These data suggest essential oil from Slovakian origin Thymus vulgaris, alone or in com-
bination with CTX, has promising antibacterial activity against blaESBL producing MDR
Enterobacteriaceae isolates and it could be used in several elds such as food and pharma-
ceutics. However, further investigations are needed to understand the relation between
TVEO and antibacterial-resistance.
Disclosure statement
No potential conict of interest was reported by the authors.
ORCID
Giacomo Dugo http://orcid.org/0000-0003-0185-6591
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