Materials and methods

1. Isolation, identification and biochemical detection of the virulence factors of the

bacterial strains
The microorganisms used in this study were 8 Pseudomonas aeruginosa and 6 Acinetobacter
baumannii clinical strains, isolated from different pathological products and collected from
The Cardiovascular Disease Institute C.C.Iliescu in Bucharest during september 2014 till
march 2015 (table 1). A standard bacteria strain of Pseudomonas aeruginosa ATCC 27853
was also used as control.
Table 1 Isolation sources for the analyzed bacterial strains
No. Bacterial strain
Secretion type
1
Pseudomonas aeruginosa 49
anal discharge
tracheal
2
Pseudomonas aeruginosa 147
secretion
tracheal
3
Pseudomonas aeruginosa 156
secretion
tracheal
4
Pseudomonas aeruginosa 157
secretion
urinary tract
5
Pseudomonas aeruginosa 160
infection
tracheal
6
Pseudomonas aeruginosa 164
secretion
tracheal
7
Pseudomonas aeruginosa 165
secretion
urinary tract
8
Pseudomonas aeruginosa 234
infection
9
Acinetobacter baumannii 64
catheter
tracheal
10 Acinetobacter baumannii 110
secretion
11 Acinetobacter baumannii 230
urine culture
12 Acinetobacter baumannii 286
nasal exudate
13 Acinetobacter baumannii 288
nasal exudate
pharyngeal
14 Acinetobacter baumannii 388
exudate
Identification of bacterial strains was performed using the automatedVITEK 2
Compact system.
Soluble virulence factors expression of the tested strains were assessed by biochemical
enzymatic methods on specific culture media used for detecting each soluble virulence factor
hemolysins, lecitinaze, DN-aze and indirect virulence factors - gelatinase, cazeinaze. Each
medium was inoculated with 10 L of the bacterial suspension, 1.5 x 10 8 CFU/ mL. The
media were incubated for 48h at 37 C.
2. Profiling the antibiotic resistance of the tested strains
Evaluation of the antibiotic susceptibility of Pseudomonas aeruginosa and
Acinetobacter baumannii strains was performed by disk diffusion method recommended by
CLSI (Clinical and Laboratory Standards Institute, Ed.2015). Briefly, bacterial suspensions
adjusted to 1.5 X 108 CFU/mL according to 0.5 McFarland nephelometrycal standard were
inoculated by evenly seeding the plates. Certain -lactamase characteristic phenotype were
highlighted by specific distribution of the antibiotics discs on the plate i.e. imipenem disc
was placed in the center, near to another -lactam antibiotic (piperacillin + tazobactam,
ceftazidime) for the detection of a inducible cephalosporinase producing, chromosomally
encoded by Pseudomonas aeruginosa strains. The antibiotics used for profiling the resistance
1

phenotype of the tested strains are summarized in table 2. The results, i.e. the inhibition zones
diameters have been reading after 24h of the plates incubation at 37 oC.
Table 2 The antibiotics used for profiling the resistance phenotype of Pseudomonas
aeruginosa and Acinetobacter baumannii strains
Concentrati
Antibiotic
Abbreviation
The class of antibiotics
on (g/mL)
Imipenem
IPM
Carbapenem
10
Piperacillin +
Extended spectrum -lactam antibiotic
TZP
110
Tazobactam
+ -lactamase inhibitor
Ceftazidime
CAZ
IIIrd generation cephalosporin
30
Piperacillin
PRL
Extended spectrum -lactam antibiotic
100
Tobramycin
TOB
Aminoglycoside
10
Polymyxin B
PB
Polipeptide
300
Ciprofloxacin
CIP
Fluoroquinolone
5
3. Genotypic characterization of bacterial strains by detection of antibiotic resistance
genes and virulence genes
The screening for antibiotic resistance and virulence genes was made by simplex or
multiplex Polymerase Chain Reaction assay.
Genomic bacterial DNA was extracted using the alkaline extration method. One to five
colonies of bacterial cultures were suspended in 1.5 ml tubes containing 20 L solution of
NaOH (sodium hydroxide) and SDS (sodium dodecyl sulphate). For the permabilization of
cell membrane the tubes were heated on thermoblock at 95 oC for 5 minutes. 180 L TE
buffer (Tris + EDTA)1X was added in the tubes and the tubes were centrifuged at 13000 rpm
for 3 minutes. The DNA in the supernatant was kept and stored at -4oC before analysis. All
PCR assays were performed on Corbett Research thermocycler. The PCR products were
verified using 1.5% agarose gel electrophoresis, stained with ethidium bromide 3.5 g/mL, at
90 V for 45 minutes and were identified on their size by comparing with specific DNAladders (molecular-weight size markers).
Several multiplex or simplex PCR assays were performed to detect in Pseudomonas
aeruginosa strains the metallo--lactamase MBL genes - blaVIM, blaIMP, the extended spectrum
-lactamase ESBL genes blaTEM, blaCTX-M, the virulence genes for hemolytic and
nonhemolytic phospholipase C plcH and plcN, the virulence exotoxins genes exoU and exoT,
the alginat gene algD and the outer membrane porin protein gene oprD.
Several multiplex PCR assays were performed to detect in Acinetobacter baumannii
strains the metallo--lactamase genes - blaVIM, blaIMP, the extended spectrum -lactamase
genes blaTEM, blaCTX-M, the oxacillinase genes blaOXA-23 and blaOXA-24 and the the virulence
genes for hemolytic and nonhemolytic phospholipase C plcH and plcN.
The primers sequences and the amplicon sizes are presented in table 2 and the
amplification conditions are listed in table 3.

Table 2 The primerssequences and the product sizes (R = G/A, W = A/T)

Gene
blaVIM
blaIMP
blaCTX-M
blaTEM
blaOXA-23
blaOXA-24
plcH
plcN
exoU
exoT
algD
oprD

Primers
VIMF
VIMR
IMPF
IMPR
CTX-MF
CTX-MR
TEMF
TEMR
OXA23F
OXA23R
OXA24F
OXA24R
plcHF
plcHR
plcNF
plcNR
exoUF
exoUR
exoTF
exoTR
algDF
algDR
oprDF
oprDR

Primer sequence
5-GATGGTGTTTGGTCGCATA-3
5-CGAATGCGCAGCACCAG-3
5-GGAATAGAGTGGCTTAA(C/T)TCTC-3
5-GGTTTAA(C/T)AAAACAACCACC-3
5-CGCTGTTGTTAGGAAGTGTG-3
5-GGCTGGGTGAAGTAAGTGAC-3
5-ATAAAATTCTTGAAGACGAAA-3
5-GACAGTTACCAATGCTTAATCA-3
5-CCCCGAGTCAGATTGTTCAAGG-3
5-TACGTCGCGCAAGTTCCTGA-3
5-GCAGAAAGAAGTAAARCGGGT-3
5-CCAACCWGTCAACCAACCTA-3
5-GAAGCCATGGGCTACTTCAA-3
5-AGAGTGACGAGGAGCGGTAG-3
5-GTTATCGCAACCAGCCCTAC-3
5-AGGTCGAACACCTGGAACAC-3
5-CCGTTGTGGTGCCGTTGAAG-3
5-CCAGATGTTCACCGACTCG-3
5-AATCGCCGTCCAACTGCATGCG-3
5-TGTTCGCCGAGGTACTGCTC-3
5-ATGCGAATCAGCATCTTTGGT-3
5-CTACCAGCAGATGCCCTCGGC-3
5-CGCCGACAAGAAGAACTAGC-3
5-GTCGATTACAGGATCGACAG-3

Amplicon size (bp)

390
232
754
1080
501
270
307
466
134
152
1310
1412

Table 3 Amplification conditions used in PCR assays

Gene
blaVIM
blaIMP
blaCTX-M
blaTEM
blaOXA-23
blaOXA-24
plcH
plcN
exoU
exoT
algD
oprD

PCR type

Multiplex
Multiplex
Multiplex
Multiplex
Multiplex
Simplex
Simplex

Initial
denaturation
94oC,
10 min
94oC,
10 min
94oC,
10 min
94oC,
3 min
94oC,
2 min
94oC,
3 min
94oC,
5 min

Cycles

Amplification conditions
Denaturation
Annealing
94oC,
30 s
94oC,
30 s
94oC,
30 s
94oC,
30 s
94oC,
30 s
94oC,
30 s
94oC,
30 s

30
30
36
30
30
30
30

52oC,
40 s
52oC,
40 s
52oC,
40 s
55oC,
1 min
59oC,
30 s
51oC,
1 min
60oC,
30 s

Extension
72oC,
50 s
72oC,
50 s
72oC,
50 s
72oC,
1,5 min
68oC,
1 min
72oC,
1,5 min
72oC,
30 s

Final
extension
72oC,
5 min
72oC,
5 min
72oC,
5 min
72oC,
5 min
68oC,
7 min
72oC,
5 min
72oC,
10 min

4. Extraction of essential oil

Four 200g samples of dry plant material were hydrodistillated in a Neo-Clevengertype apparatus for 4 hours according to European Pharmacopoeia 7th ed. The extraction yield
was considered as the medium yield of the four experiments, expressed as the volume (EO)
per weight (plant material) (v/w). The EOs portions were mixed, dried with anhydrous
magnesium sulfate and stored in brown vessels at 4oC prior to the physico-chemical analysis
and antimicrobial properties assessment.
5. Physico-chemical characterization of the volatile oils from Rosmarinus officinalis
herba
For the GC-MS analysis a 1:50 dilution of the EO sample in hexane was made.
Chemical components identification was conducted on GC-MS system, 7890A coupled with
3

5975C MSD (Agilent Techonologies, Santa Clara, CA). A DB-5 MS capillary column (60 m x
0.25 mm x 0.25 m) was used. The acquisiton was conducted with oven temperature different
ramps and temperature ranging between 90o and 280oC. Split mode injection was selected and
the split ratio was 1:300. The injection volume was set up at 1 l and the flow rate at 1.3
mL/min. MS acquisition parameters were scan mode; relative EMV mode; scanning range (50
400) a.m.u.; MS source 230oC; MS quad 150oC. The compoundsidentification was made
by using the NIST mass spectral library.
6. Qualitative screening for the antimicrobial activity of essential oil and eucalyptol
An adapted diffusimetric method was used for the antimicrobial activity qualitative
screening. According to the standardised method, the plates have been seeded as described
before. The Rosmarinus offcinalis EO and eucalyptol diluted in dimethyl sulfoxide (DMSO)
1:1 were used for spoting the seeded plates. Spotsvolumes were 10 l each. The plates were
incubated for 24 h at 37oC and the results were quantified according to the growth inhibition
diameters.
7. Quantitative screening for the antimicrobial activity of essential oil and eucalyptol
and determination of MIC (Minimum Inhibitory Concentration)
The quantitative determination of antimicrobial activity was performed by serial
binary microdilution method in Mller-Hinton liquid medium in 96-well plates. Dimethyl
sulfoxide (DMSO) was used to facilitate mixing the essential oil and eucalyptol with the broth
in a ratio 1:1. 9 serial binary dilutions were performed in a ratio 2.5:1 broth with EO/
eucalyptol + DMSO. 25 l of adjusted bacterial suspension to 1.5 X 108 CFU/mL according
to 0.5 McFarland nephelometrycal standard were used for inoculating the plates in final
volume per well of 250 l. Positive controls made in duplicate were bacterial suspensions
growing in broth medium. The plates were incubated for 24 h at at 37oC.
Minimum inhibitory concentration MIC was defined as the lowest concentration of
essential oil and eucalyptol that inhibited bacterial growth macroscopically observed, and
assessed by spectrophometric measurement of the OD620 nm.
8. Modulation screening of antibioticsactivity by the essential oil and eucalyptol
(Evaluation of synergy-antagonism between essential oil/eucalyptol and antibiotics)
This evaluation was performed by serial binary microdilution method in MllerHinton liquid medium in 96-well plates. 9 serial binary dilutions were performed in a ratio
2.5:1 broth with EO/ eucalyptol + DMSO. The tested antibiotics were Ciprofloxacin at
concentration of 3 g/mL and Colistin at concentration of 6 g/mL, antibioticssolutions
being prepared in liquid broth Mller-Hinton. The antibiotics concentration was chosen
according breakpoint for sensitive strains recommended by CLSI 2015: CMI Ciprofloxacin 1
g/mL and CMIColistin 2 g/mL. 25 l of adjusted bacterial suspension to 1.5 X 108
CFU/mL according to 0.5 McFarland nephelometrycal standard were used for inoculating the
plates in a final volume per well of 250 l. The controls made in duplicate were bacterial
suspensions growing in untreated broth medium and bacterial suspension treated with
antibiotic. The plates were incubated for 24 h at at 37oC.
Evaluation of sinergy-antagonism between essential oil/eucalyptol and antibiotics was
done by calculating Fractional Inhibitory Concentration Index FICI, using the formula:
FICI = FICEO/eucalyptol + FICantibiotic , where
FICEO/eucalyptol = MICEO/eucalyptol + antibiotic/MICEO/eucalyptol
FICantibiotic = MICantibiotic+ EO/eucalyptol/MICantibiotic
9. Flow citometrys assessment of bacterial efflux pump activity for the natural
compounds and antimicrobial mixtures
Flow citometry was carried out in order to evaluate the influence of natural
compounds and antimicrobial mixtures on bacterial efflux pump activity. For the cell staining

two intercalant fluorochromes with affinity for DNA were used: propidium iodide (PI
10g/mL) and ethidium bromide (EB 5g/mL). PI was used for the cellullar viabilitys
determination, as living cells are impermeable to this dye due to their intact membranes. EB
was used for the detection and quantification of bacterial efflux activity due to its low
fluorescence outside the cells and its high fluorescence through intracellular accumulation. So
the fluorescence intensity is a measure of EBs cellular content and a ratio between EBs
influx due to bacterial cell walls permeabilization and EBs extracellular extrusion due to
efflux activity (Martins & colab., 2013).
The study was conducted in two different experiments: (1) for the EO/eucalyptol and
(2) for the combinations EO/eucayptol: antibiotics, the testing concentrations being MIC and
MIC/8. Staining procedures were applied to the harvested cells grown in the presence of the
tested compounds. The cells were centrifuged 13000 rpm for 3 minutes, washed 2 times,
resuspended in PBS, stained with 10L PI or EB and incubated for 10 minutes at 4oC in dark.
Heat-treated cells for 30 minues at 100C were used as positive controls and viable cells were used as
negative controls, the bacterial inoculum being adjusted to 1.5 X 108 CFU/mL according to 0.5

McFarland nephelometrycal standard. The samples were analyzed with a FACS Calibur
instrument equipped with a 488 nm Argon laser, using 670 nm long pass filter for the samples
stained with PI and (58542) nm band pass filter for the samples stained with EB. The
measured parameters were forward scatter FSC, side scatter SSC and fluorescence and the
back gating procedure was used, excluding all non fluorescent particles.Typical
photomultiplier tube voltage were SSC 550 V (log scale), 670 nm long pass filter (log scale)
and (58542) nm band pass filter 550V (log scale). 10000 events were collected in all runs.
Results and discussions

3. Genotypic characterization of bacterial strains by detection of antibiotic resistance

genes and virulence genes
Molecular screening of carbapeneme resistance was performed by PCR assay and aimed the
detection of carbapenemase encoding genes in Pseudomonas aeruginosa and Acinetobacter
baumannii strains. The results of PCR assays for -lactamase encoding genes and for outer membrane
porin protein encoding gene are listed in table 4 and the agarose gel electrophoresiss image is

presented in figure 1.
Table 4 The prevalence of -lactamase and porin encoding genes in analyzed bacterial
strains
Bacterial
strain
Ps 49
Ps 147
Ps 156
Ps 157
Ps 160
Ps 164
Ps 165
Ps 234

Isolation source
anal discharge
tracheal secretion
tracheal secretion
tracheal secretion
urinary tract
infection
tracheal secretion
tracheal secretion
urinary tract
infection

Metallo-lactamase genes

Extended
spectrum lactamase genes

blaVIM
-

blaIMP
-

blaCTX-M
-

blaTEM
+
+
+

Oxacillinase genes
blaOXA-23

blaOXA-24

Porin
gene
oprdD
-

Ac 64
Ac 110
Ac 230
Ac 286
Ac 288

catheter
tracheal secretion
urinary tract
infection
nasal exudate
nasal exudate

+
+

+
+

+
+

Figure 1 Amplicons electrophoresis for blaCTX-M, blaTEM, blaOXA-23, blaOXA-24 genes:

L Gene Ruler 100bp (Fermentas), 20 negative control; for blaCTX-M and blaTEM:
2-13 Ps.aeruginosa and Ac.baumannii strains (2 - Ac288, 3 Ac230, 4 Ps49,
5 Ps156, 6 Ac64, 7 Ps164, 8 Ps234, 9 Ps147, 10 Ps160, 11 Ps157,
12 Ac110, 13 Ps165); for blaOXA-23 and blaOXA-24: 15-19 Ac. baumanii strains
(15 Ac288, 16 Ac286, 17 Ac64, 18 Ac110, 19 Ac230)
Genotypic screening of carbapeneme resistance prooved the presence of blaTEM genes
in 60% Acinetobacter baumannii strains and in 37.5% Pseudomonas aeruginosa strains and
the presence of blaOXA-23 in 80% Acinetobacter baumannii strains.
The first class A -lactamase encoding gene was discovered in 1965 in Greece on a
plasmid from Escherichia coli and was named TEM-1 after the pacients name (Temoneira).
Then it was discovered TEM-2 with the same hydrolytic profile as TEM-1, but with a
different isoelectric point, and the encoding gene had a more active promoter. One of the first
extended spectrum -lactamase ESBL enzyme with a higher activity on cephotaxime was
TEM-3 and the encoding gene was isolated in 1987 in France on a plasmid from Escherichia
coli. Nowadays over 100 variants of blaTEM codifying ESBL enzymes have been described
((Paterson & Bonomo, 2005). The ESBL enzymes are able to hydrolyze a large spectrum of
cephalosporins, including oxyimino-cephalosporins as ceftriaxone, ceftazidime or cefotaxime,
and monobactams, but they are not able to efficiently hydrolyze carbapeneme and
cephamycin, being sensible to -lactamase inhibitors.
The class D enzymes were initially named oxacillinase due to their efficient
hydrolyzing capacity on oxacillin. OXA-type -lactamases can induce resistance phenotypes
to penicillins, extended spectrum cephalosporins and carbapenems.
The first OXA-type -lactamase with carbapenemase activity OXA-23 was isolated in
1985 in Scotland in an Acinetobacter baumannii strain and was initially named ARI-1
(Acinetobacter resistant to imipenem). OXA-23 genes originated in Acinetobacter
radioresistens, which is a non-pathogenic species (Ehlers & Kock, 2012). OXA-23 is the most
widespread carbapebemase in Acinetobacter baumannii and has been identified worldwide.
The blaOXA-23 gene was identified as part of different transposons structures: Tn2006, Tn2007
and Tn2008 (Poirel & colab., 2011)
The class D oxacillinases are classified in 4 subgroups:
- the OXA-23 group, including OXA-27 and OXA-49, plasmidial encoded;

- the OXA-24 group, including OXA-25, OXA-26 and OXA-40, chromosomally or

plasmidial encoded;
- the OXA-51/69 group, chromosomally encoded;
- the OXA-58 group, chromosomally or plasmidial encoded (Ehlers & Kock, 2012).
OXA-51-type enzymes are able to hydrolyze penicillins and carbapenems as
imipenem and meropenem, but do so only weakly. The contribution to -lactame resistance by
OXA-51-type enzymes requires the presence of an insertion sequence ISAba1 upstream of the
blaOXA-51 gene, which acts as a strong transcriptional promoter, inducing the enzymes
overexpression. The most frequent resistance phenotypes to carbapenems are encoded by the
genes from blaOXA-23, blaOXA-40/24 and blaOXA-58 groups (Howard & colab., 2012).
OXA-type carbapenemase producing bacteria are less susceptible to imipenem and the
presence of an OXA-23-type gene can explain the imipenem resistance.
As the carbapenems resistance in Gram-negative non-fermentative bacteria is often
associated with mutations in porins, which induce decreased outer membrane permeability,
the presence of oprD gene was tested in Pseudomonas aeruginosa strains. The results of PCR
assay prooved the absence of this gene in all analyzed Pseudomonas aeruginosa strains.
Porin protein OprD in Pseudomonas aeruginosa strains contains a 16-strand
transmembrane -barrel structure. The channel formed by this porin is narrower than other
porins channels, which causes a lower outer membrane permeability. The OprD porin permits
the passive uptake across the outer membrane of basic amino acids and dipeptides with a
basic residue and forms pores that are permeable for carbapenems. The external loops 2 and 3
were identified as entrances for basic amino acids and binding sites for imipenem. Any
substitution or deletion within loop 2 or 3 induce changes in conformation and can cause
imipenem resistance (Li colab., 2012).
The loss of OprD porin in Pseudomonas aeruginosa strains is often associated with
imipenem resistance, as imipenem requires the porins presence in order to pass through the
outer membrane.
Molecular characterization of virulence factors in Pseudomonas aeruginosa and
Acinetobacter baumannii strains performed by PCR assays aimed the detection of specific
virulence factors encoding genes, virulence factors which are undetectable by phenotypic
methods. The results of PCR assays for phospholipase C, exotoxins and alginat encoding
genes are summarized in table 5 and the agarose gel electrophoresis images are presented in
figures 2 and 3.
The PCR results for the detection of virulence genes in analyzed Pseudomonas aeruginosa
strains showed the presence of 4 virulence factors codifying genes: plcH, plcN, exoU and exoT.

Table 5- The prevalence of virulence genes in analyzed bacterial strains

Bacterial
strain
Ps 49
Ps 147
Ps 156
Ps 157
Ps 160
Ps 164
Ps 165
Ps 234

Isolation source
anal discharge
tracheal secretion
tracheal secretion
tracheal secretion
urinary tract
infection
tracheal secretion
tracheal secretion
urinary tract

Phospholipase C
encoding genes

Exotoxins encoding
genes

Alginat
encoding
gene

plcH
+
+
+

plcN
+
+
+
+

exoU
+
+
+
+

exoT
-

algD
-

+
+
+

+
+
+

+
+
+

+
-

Ac 64
Ac 110
Ac 230
Ac 286
Ac 288

infection
catheter
tracheal secretion
urinary tract
infection
nasal exudate
nasal exudate

Figure 2 Amplicons electrophoresis for plcN and plcH genes: L- Gene Ruler 100 bp
(Fermentas), 16 negative control; 2-9 Ps. aeruginosa strains (2 - Ps234,
3 Ps164, 4 Ps147, 5 Ps165, 6 Ps49, 7 Ps156, 8 Ps157, 9 Ps160),
11-15 Ac.baumanii strains ( 11 Ac230, 12 Ac64, 13 Ac110, 14 Ac288,
15 Ac286)
Pseudomonas aeruginosa produces 2 homologous phospholipase C enzymes a
hemolytic phospholipase PlcH and a nonhemolytic phospholipase PlcN, encoded by 2
nontandem genes plcH and plcN. These phospholipases have an important role in
pathogenesis of Pseudomonas infections, mainly in pulmonary infections and cystic fibrosis,
through their synergic hydrolytic action on phospholipids found in eucaryotic cell membranes
and lung surfactant. Production of PlcH is post-transcriptionally regulated by PlcR1 and
PlcR2, proteins encoded by adjacent genes in the same operon (Engel, 2003). Experimental
studies showed that purified PlcH is cytotoxic and induces alterations of signaling processes
in different eucaryotic cells (Barker & colab., 2004).
The PCR results prooved the presence of plcN gene in all analyzed Pseudomonas
aeruginosa strains and the presence of plcH gene in 75% strains.

Figure 3 Amplicons electrophoresis for exoU and exoT genes: L Gene Ruler 100 bp
(Fermentas), 10 negative control; 2-9 Ps. aeruginosa strains (2 Ps49, 3 Ps147,
4 Ps156, 5 Ps157, 6 Ps160, 7 Ps164, 8 Ps165, 9 Ps234)

Pseudomonas aeruginosa produces four effector proteins through the type III secretion
system: ExoS, ExoT, ExoU and ExoY. These bacterial exotoxins are directly translocated into
the cytoplasm of the host cell through the pore formed in the cell membrane and are
associated with acute invasive infections, high cytotoxicity and increased mortality, being
major determinants of virulence.
The four effector proteins are expressed variably in different Pseudomonas
aeruginosa strains. No strain expresess all four effector proteins. Experimental studies stowed
that nearly all strains express one of the exotoxins ExoS or ExoU, but very rarely both
(Gellatly & Hancock, 2013).
ExoU is a 100 more potent toxin than exotoxin S with phospholipase A2 activity and
is capable of causing disorganization and lysis of cell membranes and rapid necrosis. ExoT is
a bifunctional toxin, which inhibits bacterial internalization by macrophages and endothelial
cells and in association with ExoS induces the actin cytoskeleton reorganization (Schulert &
colab., 2003).
The PCR results prooved the presence of exoU gene in all tested strains and the
presence of exoT gene in 25% strains.
Typical invasive Pseudomonas aeruginosa strains are associated with exoS gene,
while non-invasive strains, with a cytolytic phenotype, are associated with exoU gene
(Kaufman & colab., 2000).
The PCR results showed the absence of hemolytic and nonhemolytic phospholipase C
encoding genes in all tested Acinetobacter baumannii strains.
7. Quantitative screening for the antimicrobial activity of essential oil and eucalyptol
and determination of MIC (Minimum Inhibitory Concentration)
The MIC values for the Rosmarinus officinalis essential oil were in the range of (13,07
52,26) mg/ml, while the MIC values for 1,8-cineole were in the range of (1,86 41,86) mg/ml. On
quantitative level 1,8-cineole (eucalyptol) possessed the best antimicrobial activity.
Both rosemary essential oil and 1,8-cineole exhibited antimicrobial activity against tested
Gram-negative non-fermentative bacterial strains Pseudomonas aeruginosa and Acinetobacter
baumannii as seen in figure 4.

Figure 4 Antimicrobial activity of Rosmarinus officinalis essential oil and 1,8-cineole

(eucalyptol) against Pseudomonas aeruginosa and Acinetobacter baumannii strains

Conclusions
Both the microbial resistances emergence and spreading and the absence of
antimicrobial agents with efficient activity against multi-drug resistant strains started to raise
doubts about the anti-infectious therapeutic strategy, mainly in immunocompromised pacients.
In this context the present study enabled the establishment of some prerequisites for
pharmacognosy research of Rosmarinus officinalis essential oil and analytical standard 1,8cineole (eucalyptol).
Rosmarinus officinalis essential oil possessed an antibacterial activity closed to
eucalyptols, one of its monoterpenoid components, against Gram-negative non-fermentative
Pseudomonas aeruginosa and Acinetobacter baumannii strains.
Both the essential oil and eucalyptol exhibited a significant synergistic activity in
combination with Ciprofloxacin (2nd generation quinolone) against the analyzed
Pseudomonas aeruginosa and Acinetobacter baumannii strains, opportunistic pathogens that
are on the top of the nosocomial agents list.
The antipathogenic activity of the essential oil was less significant, the strongest effect
observed being the reduction of lecithinases and DN-ases expression.
In recent decades vegetal extracts, essential oils and their fractions are under
investigation and represent viabile solutions for new anti-infectious strategies, a systematic
analysis of specific combinations between antibiotics and natural compunds being required, in
order to decrease the antibiotics selective pressure and to achieve efficient therapies for the
MDR strainsinfections.
This study might extent to molecular level in order to assess the vegetal extracts
influence on the expression of some antibiotic resistance or virulence factors codifying genes.

10