Original Article

In vitro activity of hybrid lavender essential oils against multidrug resistant

strains of Pseudomonas aeruginosa

Matthew Donadu1, Donatella Usai1, Antonio Pinna2, Tiziana Porcu2, Vittorio Mazzarello1, Maura
Fiamma1,5, Mauro Marchetti3, Sara Cannas1, Giovanni Delogu4, Stefania Zanetti1, Paola Molicotti1
1 Department of Biomedical Sciences, University of Sassari, Sassari, Italy
2 Department of Surgery, Microsurgery and Medical Sciences, Ophthalmology Unit, University of Sassari, Sassari,
Italy
3 CNR, Institute of Biomolecular Chemistry, Li Punti, Sassari, Italy
4 Institute of Microbiology, Sacro Cuore Catholic University, Rome, Italy
5 Analysis Laboratory, Hospital "San Francesco", Nuoro, Italy

Abstract
Introduction: Lavender is an evergreen shrub native to Northern Africa and other mountainous Mediterranean regions. It grows throughout
Southern Europe, the United States, and Australia. Lavender essential oil has been used since ancient times and is known for its anti-
inflammatory, antidepressant, antiseptic, antifungal and antimicrobial properties.
Methodology: in this study, the antimicrobial activity of two Lavender essential oils (Lavanda sumian and Lavanda grosso) against 16
multidrug-resistant P. aeruginosa strains from clinical ocular samples taken from migrant patients has been investigated. The in vitro cytotoxic
activity on human Wong-Kilbourne derivative (WKD) conjunctiva cells from healthy patients and nitric oxide synthase (NOS) activity on
murine macrophage (J774.1A) were also evaluated.
Results: L. sumian showed lower antimicrobial activity when compared to L. grosso. Both lavender oils tested had no cytotoxic effect at very
low concentrations, mostly L. grosso. The essential oils extracted from L. sumian and L. grosso significantly reduced NOS in a cell model.
Conclusion: Increase in drug resistance and lack of new antibiotics may encourage the development of natural antimicrobial treatments.

Key words: antimicrobials; biopharmaceuticals; cytotoxicity; infection; Pseudomonads.

J Infect Dev Ctries 2018; 12(1):009-014. doi:10.3855/jidc.9920

(Received 08 November 2017 – Accepted 18 January 2018)

Copyright © 2018 Donadu 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.

Introduction composition of EOs, which contain a striking number

Lavender belongs to the Lamiaceae family, which of different compounds.
includes several evergreen species able to grow in Typology and quantity of components determine
different climate conditions. It is characterized by and characterize EOs properties. Variety and richness
intensely fragrant flowers used in the production of of compounds contribute to peculiar features of each
perfumes, as well as for the extraction of essential oils EO [1], which can contain one predominant constituent
(EOs) known for their beneficial effects on human or consist of many components in equilibrated
health. For centuries, traditional medicine and concentrations. Even traces of some compounds can
aromatherapy have taken advantage of EOs properties significantly influence EOs biological activity [2]. As
and aromatic molecules are still appreciated nowadays for secondary metabolites, the chemical composition of
as therapeutic agents. These natural compounds exert EOs is strongly modulated by the environment [1], with
significant biological and pharmacological effects, particular importance given to climate and seasons
mainly due to their lipophilic nature. [1,3,4]. Phytotherapy has been set aside with the
The therapeutic potential of lavender EOs has not development of chemistry and the first synthetic drugs,
fully been estimated yet, leaving many features then rediscovered during recent decades as either an
regarding their pharmacological aspects to be integrative method associated with conventional
discovered, even though numerous medicinal herbs medicine or as an alternative therapy.
have been used since ancient times. The reason for the Following our previous investigations performed
current knowledge gap is due to the complex on Pseudomonas aeruginosa ocular isolates from
Donadu et al. – Lavender essential oils against P. aeruginosa J Infect Dev Ctries 2018; 12(1):009-014.

migrant patients affected by keratitis and with a 60 m × 0.25 mm, thickness 0.25 μm AT-5 fused
endophthalmitis [5-7], the present study has focused on SiO2 capillary column (Alltech, Nicholasville, USA).
EOs antimicrobial activity. Injection port and detector temperature were 280° C.
Thus the minimum bactericidal concentration The column temperature was programmed from 50 to
(MBC) of EOs derived from Lavanda sumian and 135° C at 5°C/minute (1 minute), 5 °C/minute to 225
Lavanda grosso has been evaluated against multidrug- °C (5 minutes), 5°C/minute to 260°C, and held for 10
resistant ocular isolates of P. aeruginosa. Moreover, minutes. The samples (0.1 μL each), generally analyzed
cytotoxicity on human conjunctival Wong-Kilbourne without dilution (using 2,6-dimethylphenol as internal
derivative (WKD) cells and nitric oxide synthase standard), were injected using a split/splitless automatic
(NOS) activity on murine macrophage (J774.1A) cells injector HP 7673 and using He as carrier gas. The
were also assessed, in view of a possible ocular quantification of each compound was expressed as
application. absolute weight percentage using internal standard and
response factors. The detector response factors (RFs)
Methodology were determined for key components relative to 2,6-
Sixteen bacterial isolates of P. aeruginosa were dimethylphenol and assigned to other components on
used in the present study. All the bacterial strains were the basis of functional group and/or structural
isolated by ocular swab from migrant patients of similarity.
different nationality whith corneal infections resistant MS analyses were carried out with a Gas
to standard antibiotic therapies from Ophthalmology Chromatograph Agilent Technologies model 7820A
Unit, Department of Surgery, Microsurgery and connected with a MS detector 5977E MSD (Agilent,
Medical Sciences, Sassari, Italy. Santa Clara, USA), by using the same conditions and
L. sumian and L. grosso essential oils used in the column described above. The column was connected to
present study were produced, standardized and the ion source of the mass spectrometer. Mass units
distributed by Exentiae (Exentiae s.r.l., Catania, Italy). were monitored from 10 to 900 at 70 eV. The
In order to identify and perform the antimicrobial identification of compounds was based on the
susceptibility test on the 16 P. aeruginosa strains comparison of their retention times with those of
assessed, the MicroScan (Beckman Coulter. Inc., Brea, authentic samples and/or by comparison of their mass
USA) MIC/Combo Panel was used. The Breakpoint spectra with those of published data (Nist Library Mass
Combo Panels use concentrations equivalent to the spectra) or on the interpretation of the EI-fragmentation
Breakpoint of Clinical and Laboratory Standards of the molecules.
Institute (CLSI) following the European Committee on In a second experiment, EOs cytotoxicity was
Antimicrobial Susceptibility Testing (EUCAST) analyzed on WKD cells. WKD cells were maintained in
interpretive criteria [8-9]. Roswell Park Memorial Institute-1640 (RPMI) medium
Minimum Bactericidal Concentration (MBC) (Sigma-Aldrich, Saint Louis, USA) supplemented with
values of the EOs on 16 multidrug-resistant P. 10% foetal bovine serum and 100 U/mL
aeruginosa strains was analyzed. penicillin/streptomycin and incubated at 37° C in 5%
Samples were diluted in Luria Broth (LB) added CO2 air atmosphere. 1,5×105/mL cells were further
with 0.5% Tween 80 at concentrations ranging from seeded in 96-well plates and incubated overnight at
16% to 0.0005% (v/v). The bacterial inoculum was 37°C, 5% CO2. EOs dilutions (from 16% to 0.0005%
performed at the concentration of 106 CFU/mL. An V/V) were prepared in culture medium with the
inoculum of 100 μL of microbial culture was added to addition of Tween 80 (0.5%) and assessed for 10
100 µL of each concentration of the different samples minutes. The cytotoxicity assay (In vitro toxicology
in 96-well plates and incubated at 37°C for 24 hours. assay kit MTT based, Sigma-Aldrich, Milan, Italy) was
Cultures that showed no visible turbidity were performed according to the manufacturer's instructions.
subcultured on the surface of a Plate Count Agar for Wells were washed twice with PBS and 100 L of
colony counting. MBC was considered as the lowest culture medium without serum plus 1/10 MTT solution
concentration inhibiting 99% of bacterial growth. Each (3-4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
experiment was performed in duplicate and repeated bromide)/PBS was added. After 4 hours of incubation,
three times. M-8910 MTT solubilisation solution - 10% Triton X-
Three replicates of each EOs were analyzed using a 100 plus 0,1N HCl in anhydrous isopropanol was
Hewlett-Packard Model 5890A Gas Chromatograph added. The quantity of formazan (presumably directly
equipped with a flame-ionization detector and fitted proportional to the number of viable cells) was

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Donadu et al. – Lavender essential oils against P. aeruginosa J Infect Dev Ctries 2018; 12(1):009-014.

Table 1. Antimicrobial activity of lavender EOs (% v/v) against 16 multidrug resistant P.aeruginosa.
Strains number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
lavanda SUMIAN Exentiae
16% 16% 16% 8% 16% 8% 16% 16% 16% 16% 16% 8% 8% 16% 8% 8%
s.r.l.
lavanda GROSSO Exentiae
16% 16% 16% 16% 16% 8% 8% 8% 8% 8% 8% 8% 8% 8% 8% 8%
s.r.l.

measured by recording changes in absorbance at 570 cells were induced with mouse - interferon (- INF) (50
nm using a plate reading spectrophotometer. The ng/mL) and Lipopolysaccaride (LPS) (2μg/mL) for 20
percentage of viability was calculated according to the hours. At the end of the incubation NOS activity was
following formula: (OD [570 nm] sample assessed/(OD detected by using the commercial kit Nitric Oxide
[570 nm] negative control) = R; R × 100 = % cells Synthase Detection System Fluorimetric (Sigma–
viability. If the percentage was ≥ 50%, the oil was Aldrich, Milan, Italy)) according to the manufacturer's
considered to have no cytotoxicity. instructions.
A murine macrophage cell line (J774.1A) was Subsequently, cells were seeded on coverslips for
cultured in RPMI-1640 medium (Sigma-Aldrich, observation under a scanning electron microscope
Milan, Italy) supplemented with 10% fetal bovine (SEM). Each sample was tested for three concentrations
serum and 100 U/mL penicillin/streptomycin and of lavender EO: non cytotoxic, superior and inferior.
incubated at 37° C in 5% CO2 air atmosphere. The cells, to be observed by SEM, were fixed in 2%
5×105/mL cells were further seeded in 96-well plates glutaraldehyde solution in 0.1 M cacodylate buffer (1
and incubated at 37°C, 5% CO2 for 24 hours. Some hour). After three washes, 5 minutes each in the same
wells were pretreated with L. sumian and L. grosso EOs buffer, the samples were dehydrated through graded
(dilutions from 4% to 0,0075 % V/V) for 2 hours. The alcohol solutions, air-dried with hexamethyldisilazane

Table 2 Antimicrobial susceptibility of P. aeruginosa strains.

Strains number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Amikacin S S S S S S ND S S S S S R R I I
Amox/ Kclav R R R R R R R R R R R R R R R R
Amp/ Sulbactam R R R R R R R R R R R R R R R R
Ampicillin R R R R R R R R R R R R R R R R
Aztreonam I I I I I I ND I I R I R I R I I
Cefepime S S S S S S ND S S S S S R R R S
Cefpodoxime R R R R R R R R R R R R R R R R
Ceftazidime S S S S S S ND S S S S S R R R S
Ceftriaxone R R R R R R R R R R R R R R R R
Cefuroxime R R R R R R R R R R R R R R R R
Ciprofloxacin S S I S S S ND S R S R R R R R S
Chloramphenicol R R R R R R R R R R R R R R R R
Colistin S S S S ND S ND S S S S S S S S S
Doripenem S S S S S S R S S I S I I R I S
Ertapenem R R R R R R R R R R R R R R R R
Fosfomycin R R R R R R R R R R R R R R R R
Gentamycin S S S S S S ND S S S R S R R R R
Imipenem S S S S S S R S S R S R S R I S
Levofloxacin S S S I S S ND S R I R R R R R S
Meropenem S S S S S S ND S S R S R S R I S
Norfloxacin R R R R R R R R R R R R R R R R
Ofloxacin R R R R R R R R R R R R R R R R
Pip/Tazo S S S S R S ND S S S S S S R S S
Piperacillin S S S S R S ND S S S S S S R R S
Tetracyclin R R R R R R R R R R R R R R R R
Tobramycin S S S S S S ND S S S R S R R R S
Trime/ Sulfa R R R R R R R R R R R R R R R R
Trimethoprim R R R R R R R R R R R R R R R R
R: Resistant; I: Intermediate resistance; S: Sensible; ND: Not Determined.

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Donadu et al. – Lavender essential oils against P. aeruginosa J Infect Dev Ctries 2018; 12(1):009-014.

for 10 minutes, examined and photographed in low Figure 1. Scanning Electron Microscopy micrographs of P.
vacuum using SEM FEI Quanta 200. aeruginosa (sample number one) post treatment with L. grosso
Data are expressed as means ± standard deviations. EO at 16% concentration (V/V): (A) P. aeruginosa control
sample number 1; (B) P. aeruginosa sample number 1, post-
Significant differences between treatments were treatment with EO of L. grosso 16% (v/v)
analyzed by one-way analysis of variance and Kruskal–
Wallis test at p < 0.0001 (Prism 7 software).
Approval from the Ethics Committee/Institutional
Review Board of the Department of Surgery,
Microsurgery, and Medical Sciences, University of
Sassari, Sassari, Italy, was obtained and the study was
conducted in complete agreement with the principles of
the Declaration of Helsinki. Each participant received
detailed information and provided written informed
consent before inclusion. carbapenems. The results of the MicroScan
MIC/Combo Panel are presented in Table 2.
Results EO derived from L. sumian showed cytotoxicity
The EO derived from L. sumian has lower exceeding 50% at concentrations higher than 0.015%
antimicrobial activity when compared to L. grosso. L. (IC50 34 mg/mL). Conversely, for EO derived from L.
Sumian was ineffective on eleven P. aeruginosa strains grosso a cytotoxic effect was found for concentrations
at the concentration of 16%, while L. grosso was exceeding 0.0005% (IC50 4,3 mg/mL). The EOs of L.
effective on the same number of P. aeruginosa at the sumian and L. grosso significantly reduced NOS
concentration of 8% Table 1. This might be a promising activity in macrophage cells only at concentrations
basis for a potential therapeutic approach involving exceeding 8,5mg/mL and it was concentration
natural products alternative to and/or synergic with dependent. No significant differences were found
conventional medicine in the treatment of some chronic between L. sumian and L. grosso. Major compounds of
and acute pathologies. hybrid lavender EO are presented in Table 3. The
All strains showed multiple antibiotic resistance to greatest inhibitory activity was observed at 34 mg/mL
14 antibiotics tested, including amoxicillin/clavulanic and 8,5 mg/mL concentrations.
acid, ampicillin/sulbactam, ampicillin, cefpodoxime, Figure 1 shows the effect of exposure of P.
ceftriaxone, cefuroxime, chloramphenicol, ertapenem, aeruginosa strain number 1 to L. grosso EO at a
fosfomycin, norfloxacin, ofloxacin, tetracycline, concentration of 137 mg/mL. Control showed a cluster
trimethoprim and trimethoprim/sulfamethoxazole. On of well-preserved cells, characterized by intact cell
the other hand, all of them were susceptible to walls (Figure 1 A). Changes in bacterial cells
aminoglycosides, third-generation fluoroquinolones, morphology were observed with the presence of few
third-generation cephalosporins and some rounded bacterial cells compared to the control (Figure

Table 3. Major compounds of hybrid lavender EOs are listed in order of MS detector.
Compound Lavanda Sumian Lavanda Grosso
Eucaliptol 9,89a 0,66a
1,6 Octadien – 3 - ol 32,03 27,27
2 - Bornanone 9,81 9,34
endo- Borneol 3,68 6,77
Terpinen – 4-ol 4,16 5,19
alpha - Terpineol 0,49 1,08
(R) – Lavandulyl acetate 2,85 4,10
Caryophyllene 1,67 2,61
1,3,6 - Octatriene 25,79 34,58
1,6 - Cyclodecadiene 0,47 0,83
Naphthalene 0,35 0,64
alpha - Bisabolol 0,28 0,42
D - limonene 1,35 Tr
(E) Beta - Famesene Tr 1,86
a
RA%, Relative area percentage; Tr: traces.

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Donadu et al. – Lavender essential oils against P. aeruginosa J Infect Dev Ctries 2018; 12(1):009-014.

1 B). Results suggested that L. sumian and L. grosso cases using contact lens care (disinfection) solutions.
Eos might reduce the oxidative stress induced by LPS [14].
and γINF and this reduction might be related to phenolic Molecules composing EOs target cell proteins
compounds. embedded in cytoplasmic membrane. ATP synthases
are known to mediate the active transport of ions and
Discussion molecules thanks to their position within the
These result should be confirmed in vivo, in cytoplasmic membrane and association with lipid
particular for L. grosso, which appeared non cytotoxic molecules. Two mechanisms of cytotoxic activity have
at the lowest concentration tested (0.0005%). The been hypothesized: 1) lipophilic hydrocarbons
chemical structure of single EOs components contained in EOs may accumulate in the lipid layer and
determines the cytotoxic way of action, as confirmed by distort the lipid-protein interaction; 2) other types of
the phenolic hydroxyl group. On the other hand, the interactions excluding lipid component may occur in
position relative to the hydroxyl group does not seem to the proximity of protein hydrophobic portions, resulting
influence significantly the degree of antimicrobial in their destabilization [15]. Among non-phenolic EOs
activity. The different degree of cytotoxicity may be compounds, alkyl substituents (alkenyl groups rather
due to a distinct chemical composition of EOs. Given than alkyls) have been reported to influence cytotoxic
the elevated number of various chemical groups found activity [16]. The present study is in agreement with the
in EOs, it is plausible that their cytotoxic activity may above mentioned considerations regarding the activity
not be attributed to a single mechanism but rather to and efficacy of cytotoxic mechanisms through which
several pathways. Hydrophobicity is an important EOs act on human cells. Moreover, very low EOs
chemical feature of EOs, which let them penetrate concentrations are necessary to achieve a non-cytotoxic
through the cell membrane by increasing permeability effect [17]. Many compounds of plant origin have
– a phenomenon resulting in the spillage of ions and potent antioxidant activities. As expected, the Lavender
molecules leading to cell death. In general, cytotoxicity essential oil presented antioxidant activity and this
of the EOs is caused by a high concentration of phenolic ability was concentration-dependent. Pretreatment of
compounds, such as eucalyptol, linalool or α-terpineol. LPS and γINF induced cells with Lavender EOs
Treating bacterial infections by antibiotics is the significantly reduced NOS activity, primarily due to the
gold standard but their indiscriminate use has led to an presence of phenolic compounds as main components,
alarming antibiotic resistance among microorganisms. which are responsible for the antioxidant properties
A new approach could be a synergistic therapy with [18-20]. Actually, at higher concentrations NOS
traditional antibiotics and Eos with antimicrobial activity was reduced to control levels: this effect could
properties. Antibacterial and cytotoxic activities of Eos be interpreted by different mechanisms such as the
can be attributed to their different constituents. EOs radical scavenging activity or an inhibition of oxidative
contain complex mixtures including monoterpenes and stress generation. However, further studies are
sesquiterpenes: some of them have been shown to have warranted to confirm EOs antioxidant activity.
antimicrobial activity against bacteria and fungi [8,9-
11]. It is believed that EOs antimicrobial activity is not Conclusion
related to one specific mechanism, because they have The investigation on plant extracts, starting with the
different compositions and, therefore, may damage evaluation of the biological activities of
microbial cells in different ways. However, not all these phytocomplexes with further identification of the main
mechanisms work separately. They act through a active components, may provide advantages for the
mechanism most probably shared with other phenols, assessment of new substances to be used either
which usually involves the cell membrane. singularly or in combination with conventional drugs
P. aeruginosa has been repeatedly identified as the already used in clinical practice. In conclusion, there is
most frequently isolated pathogen in all culture-positive an agreement on the fact that a progressive reduction of
cases of contact lens–related microbial infection of the effective antimicrobial drugs and the associated
cornea. [12-13] increase of multidrug resistance, together with a scarce
Current therapeutic interventions involve anti- involvement of the pharmaceutical industry in the
Pseudomonal antibiotics, e.g. aminoglycosides or distribution of orphan drugs, should encourage the
fluoroquinolones, and prevention relies on reducing P. development of new treatments.
aeruginosa contamination of contact lenses and lens

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Donadu et al. – Lavender essential oils against P. aeruginosa J Infect Dev Ctries 2018; 12(1):009-014.

Acknowledgements 11. Cannas S, Usai D, Pinna A, Benvenuti S, Tardugno R, Donadu

This work was supported by grants funded by the Province of M, Zanetti S, Kaliamurthy J, Molicotti P (2015) Essential oils
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guidelines from CLSI to EUCAST: influence on cumulative Matthew Donadu, PharmD
hospital antibiograms. PLoS One 1: 8. Department of Biomedical Sciences,
9. Mohanty S, Maurya V, Gaind R, Deb M (2013) Phenotypic University of Sassari, Viale San Pietro 43b,
characterization and colistin susceptibilities of carbapenem- 07100 Sassari, Italy
resistant of Pseudomonas aeruginosa and Acinetobacter spp. J Phone: +39 3458593956
Infect Dev Ctries 15;7: 880-887. doi: 10.3855/jidc.2924. Email: [email protected]
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medicinal plants [Book in Italian]. Milan: New Techniques 952 Conflict of interests: No conflict of interests is declared.
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