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Desouky et al.
Research Article
WORLD JOURNAL OF PHARMACEUTICAL
World Journal of Pharmaceutical and Medical Research
ISSN
2455-3301
AND MEDICAL RESEARCH
WJPMR
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EFFECT OF SALVIA EGYPTICAE AND FOENICULUM VULGARA EXTRACTS ON
QUORUM SENSING AND BIOFILM FORMATION OF METHICILLIN
RESISTANT/SENSITIVE STAPHYLOCOCCUS AUREUS ISOLATES
Said E. Desouky1*, Mamdouh S. El-gamal1, Mohammed Abo Elghait1*, Saad E. Hassan1, Tamer I. M. Ragab2,
Mahmoud Emam3
1
Department of Botany and Microbiology, Faculty of Science, Al-azhar University, 11884 Nasr, Cairo, Egypt.
2
Natural and Microbial Products Chemistry, National Research Centre, Dokki, Cairo, Egypt.
3
Phytochemistry and Plant Systematic Department, National Research Centre, Dokki, Cairo, Egypt.
*Corresponding Author: Dr. Said E. Desouky
Department of Botany and Microbiology, Faculty of Science, Al-azhar University, 11884 Nasr, Cairo, Egypt.
Article Received on 14/12/2016
Article Revised on 02/01/2017
Article Accepted on 23/01/2017
ABSTRACT
Staphylococcus aureus employ cyclic peptide-mediated quorum sensing (QS) systems, termed agr to regulate the
expression of a series of virulence genes. To identify quorum sensing inhibitors (QSIs) that target agr systems, two
important medicinal plants were investigated. In this study, the use of an S. aureus agr reporter strain that carries
luciferase protein genes under the agr P3 promoter facilitated the development of screening for QSIs. As a result of
screening of Salvia egypticae and Foeniculum vulgara extracts, the two extracts showed reduction of hemolysin
production regulated with agr system and biofilm formation and QS activity against the agr system. Without
growth inhibitory activity the extracts were analyzed by HPLC.
KEYWORDS: Staphylococcus aureus, Salvia egypticae and Foeniculum vulgara extracts.
INTRODUCTION
Staphylococcus aureus is an opportunistic nosocomial
pathogen threatening human health worldwide in order to
the high rat of mortality and morbidity which exceeded
the prevalence in hospitals to community of healthy
persons (Marchese. et al., 2000). This pathogen causing
variety diseases ranging from skin infections to life
threatening endocarditis and toxic shock (TS) the
emergence of methicillin resistant S. aureus (MRSA),
has complicated treatment due to a limited choice of
effective antibiotics (Martinez et al., 2003). Most
infections caused by this pathogen were carried out
through releasing diverse toxins and proteins in the host
cell such as α hemolysin. Regulation of toxins and other
virulence factors undergoes by that so called quorum
sensing (QS) system. This system usually coordinate the
regulation of specific genes which leading to a
phenotypic shift in the organism behavior from nonvirulent to virulent by initiating production of proteins
and toxins (Bjarnsholt and Givskov 2008 & Dunman et
al., 2001). Accessory gene regulator (agr) is a wellstudied quorum sensing of S. aureus. The agr locus
consists of two divergent transcription units driven by
promoters agrP2 and agrP3 (Said et al., 2013). The
former leads to the synthesis of RNAII, which encodes
AgrBDCA, the structural components of the QS system,
while agrP3 drives the synthesis of RNAIII which
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encodes ¥ hemolysin and regulate more than 150
virulence gene. Agr inhibition is recently considered to
be a target for the development of anti-pathogenic agents
that can suppress virulence without influencing cell
growth which may an alternative therapeutic way to
antibiotic. Nowadays medicinal herbs are good
alternative to chemical drugs, one of the major reason for
this is low side effect compared to chemical drugs (Kooti
et al., 2014 & Beyrami et al 2014) From 422 000
flowering plants around the world, more than 5000 ones
are used for medicinal purposes. Among these herbs can
point to fennel (Foeniculum vulgara) (Abe. R, et al.,
2013). This plant has anti-inflammatory, antispasmodic,
antiseptic, carminative, diuretic and analgesic effect and
is effective in gastrointestinal disorder treatment
(Birdane et al., 2007& Delaram et al., 2011) the aim of
the present study was to evaluate the therapeutic
properties of this medicinal plant. Leaves of Salvia
aegyptiaca is used for treating various unrelated
conditions that include nervous disorders, dizziness,
trembling, diarrhea, piles, and famous to possesses
antitumor activity (Jiang et al., 2014c). Salvianolic acid
B (Sal B) increases the level of antioxidant substances
and decrease free radical production (Park et al., 2014).
The present study targeting investigation of two
medicinal plants Salvia aegyptiaca and Foeniculum
vulgara against agr QS system and their phenotypic
effect such as hemolytic activity and biofilm formation
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Desouky et al.
World Journal of Pharmaceutical and Medical Research
in order to searching for alternative therapeutic agents to
antibiotic for solving the antibiotic resistance problem of
MRSA.
strain S. aureus 8325-4 (pSB2035), 7 µg/ml of
chloramphenicol for plasmid selection was added to the
medium.
MATERIALS AND METHODS
2- Antibiotic sensitivity assay for clinical isolates
The standardized disc-diffusion method was performed
on Mueller-Hinton agar using antibiotics oxacillin 1 μg
and vancomycin 30 μg (Oxoid) for testing antibiotic
susceptibility of the isolated clinical samples for
detecting MRSA and MSSA strains according to
(Mamishi et al, 2011& CLSI 2009). In brief, the Muller
Hinton agar (MHA) was poured into sterile petri- plates
and was allowed to solidify. A suspension equivalent to
0.5 McFarland (1.5 x 108 CFU/mL) was prepared from
each isolate. A swab was dipped and streaked on the
surface of a (MHA) plates. Standard antibiotic discs were
introduced on the upper layer of the seeded agar plate.
The plates were incubated at 37°C for 18-24h. The
experiment was carried out three times and the mean
values are presented. The antimicrobial activity was
evaluated by measuring the diameter of zone of
inhibition in mm (Table 1) (Vaghasiya and Chanda,
2010).
1- Bacterial strains and inoculum Preparation
Bacterial strains used in this study were S. aureus
(ATCC 29213), reporter strain S. aureus 8325-4
(pSB2035) and Clinical S. aureus samples collected from
patients of Arab contractor's medical center- CairoEgypt, and identified by traditional biochemical methods
(Manual of Determinative Bacteriology 2001& Miller,
2005). The coded strains as well as the clinical isolates
were maintained in LB broth (Oxoid) with 20% glycerol
and kept at a temperature -20°C. For preparing the
inoculum of the bacterial strains, the Sub-culturing was
done on nutrient agar (Oxoid), and then Two to three
colonies from overnight culture of each organism were
transferred aseptically by sterilized loop into 5 ml sterile
saline in a test tube and mixed thoroughly, using vortex
mixer (TOMMY) for uniform distribution and the
turbidity was compared with 0.5 McFarland standard
(1.5 x 108 CFU/mL). For preparation of the reporter
Table 1: Antibiotic susceptibility patterns classification.
Antibiotics disc
Oxacillin (1 μg)
Vancomycin (30 μg)
Resistant
≤10
≤10
Range of zone of inhibition (mm)
Intermediate
Sensitive
11-12
≥13
11-14
≥15
3- Plant extraction
Leaves of Salvia egypticae and seeds of Foeniculum
vulgara were collected and identified from the Ministry
of Agriculture medicinal plants sale outlets in Giza,
Egypt, then the identification confirmed according to
(Agra, M.F et al, 2007 a,b) by department of botany
faculty of science Alazhar University in Cairo, Egypt.
For extraction, plant parts were dried at room
temperature in shadow, then powdered by using a
mechanical grinder (KM30) and then powder was stored
in a sterile bottle at room temperature. The powder (500
g) was mixed with 3,000 ml of methanol (70%) for 7
days at room temperature. The supernatant of the mixture
was filtered and then collected through filter paper. The
filtrate was concentrated using rotary evaporator
(Heidolh. Germany). The extracted powder was kept at 20°C until used. Successive extraction of plant extracts
were performed using solvents (non-polar to polar) e.g.
hexane, petroleum ether, diethyl ether, chloroform, ethyl
acetate, ethanol, methanol, and water for 16 h in soxhlet
apparatus (Sharma et al., 2013 and Faizi et al., 2008).
4-Determination
of
Minimum
Inhibitory
Concentration (MIC)
Micro broth dilution method, using 96 well microtitre
plates, was performed to evaluate MIC of the plant
extracts (Andrews, 2001& Sampaio et al., 2009). An
inoculum suspension was prepared in Mueller–Hinton
broth. The inocula were adjusted to each bacterial strain
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to 0.5 McFarland standard (1.5 x 108 CFU/mL). A final
volume of 200 μl was achieved in each well (180 μl
bacterial suspension and 20 μl plant extract with different
concentrations) then the plates incubated at 37°C for 18 24 h after the incubation period, the optical density of the
growth of each well were recorded at 620 nm using
(ELISA) reader (BioTecan, ELx808). Two control wells
were maintained for each test batch, these included test
control (well containing extract and the growth medium
without inoculum) and organism control (the well
containing the growth medium and the inoculum). The
lowest concentration (highest dilution) of the extract that
produced no visible bacterial growth (no turbidity) when
compared with the control wells were regarded as MIC.
5- Quantitative hemolytic assay
Hemolysis analysis was modified from the previous
method (Larzabal et al., 2010). The hemolysis efficacy
of human red blood cells was measured with whole
cultures of S. aureus strains grown in the presence of
different concentrations of plant extracts. In brief,
overnight culture of S. aureus cells were diluted to 1:100
in TSB and then cultured with or without all plant
extracts at different concentration under MIC values at
37°C for 16 h with shaking at 250 rpm. The cell cultures
(100 μl including cells and culture supernatant) were
added into diluted human red blood cells that had
previously been separated by centrifugation at 900xg for
5 min, washed with PBS buffer three times and diluted at
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Desouky et al.
World Journal of Pharmaceutical and Medical Research
3 % of red blood cells in PBS buffer. For hemolytic
activity, the mixture was incubated at 37 °C for 3 h with
250 rpm shaking. The supernatant was collected by
centrifugation at 16,600xg for 10 min, and the optical
density was measured at 543 nm. The hemolytic
activities of the isolated organisms were determined by
comparing the optical density of each organism with the
most hemolytic active S. aureus strain ATCC29213 (Qiu
J et al 2010 & Rowe and Welch, 1994). Anti-hemolytic
activity of the tested extracts were determined by
comparing the hemolytic activity of test organisms which
cultured in the presence of extracts with the positive
control, and then calculating the ratio (Lee et al., 2012).
6-Quantitative assay for biofilm formation
Quantitative determination was carried out by the Micro
plate method (MP) proposed by (Afreenish et al., 2011,
Pfaller et al., 1988 & Christensen., et al 1998) using
tissue culture plates of 96 flat bottomed wells. Each well
was filled with 0.2 ml of 105CFU/ml of a bacterial
suspension in TSB. After 24h incubation in aerobic
condition at 37◦C, and reading the optical growth of each
well at 620 nm using (ELISA) reader (BioTecan,
ELx808), the contents were aspirated and plates were
washed twice with phosphate buffered saline (PBS, pH:
7.2).The wells were stained with 0.1% crystal violet for 2
min. The plates were read in an (ELISA) reader
(BioTecan, ELx808) at 492 nm. Sterile TSB was used as
a negative control. All the experiments were repeated at
least twice, and the values of optical density (OD) were
then averaged. Evaluating the strains slime producing
ability by comparing with OD of negative control
(Stepanovic et al., 2007& Pavithra and Doble, 2008).
7- Purification of Plant extracts.
One milliliter of butanol extract from Salvia egypticae
leaves (X1) and Foeniculum vulgara seeds (X2) were
dried, then collected and redissolved in 0.5 ml of
methanol. One hundred microliters of this solution was
mixed with 900 µl of milliQ water and was then applied
to a Sep-Pak C18 cartridge column (100 mg; Waters).
After washing with 2 ml of milliQ, the column was
eluted with 1 ml of 20%, 40%, 60%, 80 %, and 100%
methanol. The QSI activity of each eluate was monitored
by measuring hemolytic activity as described in part 2.
One hundred microliters of these active fractions were
subjected to a high performance liquid chromatography
(Agilent HP1100) with reverse-phase column (Agilent
Zorbax Eclipse XDB-C18 2.1 by 50 mm) and a
photodiode array detector (GMS-T100LC AccuTOF)
The column was eluted at a flow rate of 0.2 ml/min at
30°C with a linear gradient of acetonitrile (20% to 100%
in 30 min after 5 min of 20%). Fractions were collected
every 1 min and the QSI activity of each fraction was
examined as mentioned above.
RESULTS AND DISCUSSION
1-Detection of Methicillin susceptibility of the clinical
isolates
This study found that from the total S. aureus clinical
isolates there are 80% of isolates were exhibited
resistance to oxacillin and another groups of antibiotic
which confined them to be related to methicillin
resistance staphylococcus aureus (MRSA) group, while
20% of the isolated strains exhibited sensitivity to
oxacillin antibiotics which confined them to Methicillin
sensitive staphylococcus aureus (MSSA) group, both
MRSA and MSSA were sensitive to vancomycin, so in
this study neither Vancomycin resistant (VRSA) nor
Vancomycin intermediate were present. Data were
illustrated in (Table 3).
2- Hemolytic activity of the clinical isolates
All clinical isolates in the present study showed
hemolytic activity with β hemolysis type and by
quantitative analysis to it, samples S3 and S41 had
demonstrated hemolytic activity more than the high
hemolysin producing organism ATCC29213 with 11.1%
and 5.3% respectively (Table2), (figure 1).
3- Anti-hemolytic activity of plant extracts
MIC of each plant extracts in the present study were
tested against the two most hemolytic active organisms
S3 and S41 and determined depended on roles of the
clinical and Laboratory Standards institute (CLSI)
guidelines (CLSI, 2015) in order to detect the lethal dose
of each extract. And then sub lethal concentrations under
MIC values of whole extracts of Salvia egypticae leaves
(X1) and Foeniculum vulgara seeds (X2) showed
reducing effect to hemolytic activity of S. aureus MSSA
(S3) strain up to 64.8 % at the dose 1/16 MIC of X1and
up to 81.1% at concentration 1/2MIC of X2 without any
effect on the bacterial growth. While the previous
extracts (X1 and X2) showed reducing effect to hemolytic
activity of S. aureus MRSA (S41) strain up to 49.7% at
the dose 1/4 MIC of X1and up to 56.2% at the dose 1/2
MIC, respectively (Table 3, 4) (fig 2, 3). When we
reducing the dose lower than the effected concentrations,
the organism's hemolytic activity returned to the normal
values.
Table 2: MIC values of extracts of Salvia egypticae (X1) and Foeniculum vulgara (X2).
MIC mg/ml
S. ext.
S1
S34
S35
S40
S41
S42
1.8 0.9 1.8 1.8 1.8 0.9 1.8 0.9
1.8 1.8 0.9
X1
6.2 12.5 6.2 6.2 6.2 12.5 6.2 6.2 12.5 6.2 6.2
X2
S.: sample code. ext.: extract code. MIC: minimum inhibitory concentration
3.7
6.2
1.8
6.2
1.8
6.2
0.9
6.2
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S2
S3
S12
S14
S14
S17
S18
S25
S26
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Desouky et al.
World Journal of Pharmaceutical and Medical Research
Table 3: hemolytic activity of S. aureus clinical isolates.
Serial
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Sample
code
S1
S2
S3
S12
S13
S14
S17
S18
S25
S26
S34
S35
S40
S41
S42
29213
-ve control
source
wound
wound
wound
sputum
wound
tip of urine casther
pus
throat swab
sputum
wound
wound
sputum
wound
abscess
Sputum
ATCC
media
Methicillin
susceptibility
MRSA
MRSA
MSSA
MRSA
MRSA
MSSA
MRSA
MRSA
MRSA
MRSA
MRSA
MSSA
MRSA
MRSA
MRSA
MSSA
-
Zone mm
8
0
18
7
5
20
6
7
0
4
8
18
6
0
5
13
-
Hemolysis
O.D
0.591 ± 0.02
0.583 ± 0.08
0.668 ± 0. 1
0.442 ± 0.02
0.531 ± 0.08
0.432 ± 0.03
0.354 ± 0.2
0.345 ± 0.01
0.55 ± 0.01
0.61 ± 0.02
0.55 ± 0.03
0.45 ± 0.05
0.53 ± 0.01
0.631 ± 0.03
0.536 ± 0.009
0.60 ± 0.05
0.00
Fig 1: growth and hemolytic activity of S. aureus clinical isolates.
Table 4: Effect of Salvia egypticae leaves (X1) extract on hemolytic activity of MSSA strainS3
X1
dose
Growth
O.D 600S3
Hemolysis
O.D 546nm
Hemolysis
%
Control +ve
1.345 ± 0.04
0.6395 ± 0.04
100
Control –ve
0.0013 ± 0.01
0.0015 ± 0.01
0
1/2MIC
1.185 ± 0.03
0.404 ± 0.19
63.2
1/4MIC
1.32 ± 0.01
0.248 ± 0.04
38.8
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World Journal of Pharmaceutical and Medical Research
1/8MIC
1.32 ± 0.04
0.304 ±0.006
47.5
1/16MIC
1.365 ± 0.04
0.2255 ± 0.06
35.2
1/32MIC
1.335 ± 0.04
0.5355 ± 0.01
83.7
Fig 2: Effect of X1 on growth and hemolytic activity of MSSA strain (S3) on the left and MRSA strain (S41) on the
right.
Table 5: Effect of Foeniculum vulgara (X2) extract on hemolytic activity of MSSA strain S3
X1
dose
Growth
O.D 600 S3
Hemolysis
O.D 546nm
Hemolysis
%
Control +ve
1.385 ± 0.02
0.63 5 ± 0.02
100
Control –ve
0.0015 ± 0.01
0.0011 ± 0.01
0
1/2MIC
1.34 ± 0.02
0.1205 ± 0.03
18.88
1/4MIC
1.34 ± 0.01
0.147 ± 0.005
23.1
1/8MIC
1.34 ± 0.014
0.411 ± 0.07
64.4
1/16MIC
1.265 ± 0.06
0.386 ± 0.04
60
1/32MIC
1.365 ± 0.02
0.622 ± 0.057
97.4
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World Journal of Pharmaceutical and Medical Research
Fig 3: Effect of X2 on growth and hemolytic activity of MSSA strain (S3) on the left and MRSA strain (S41) on the
right.
4- Effect of sub inhibitory concentrations of Salvia
egypticae leaves (X1) and Foeniculum vulgara seeds
(X2) extracts on biofilm formation of S. aureus clinical
isolates.
The whole methanol extracts of Salvia egypticae leaves
(X1) and Foeniculum vulgara seeds (X2) were showed in
this study antimicrobial activity against the most virulent
strains of isolated clinical samples with MIC 1.8 and 6.2
mg/ml respectively. The values of sub inhibitory
concentrations (1/2, 1/4 and 1/8 MIC) of this extracts
have been investigated against biofilm formation of
MSSA strain (S3) and MRSA strain (S41). 1/2 and 1/4
MIC of Salvia egypticae leaves extract (X1) was
exhibited reduction effect up to 74.1% and 42.3%
respectively, when compared with positive control,
against biofilm formation activity of MSSA (S3) strain
and up to 54.4% and 40.7% respectively against MRSA
(S41)strain without affecting planktonic cell growth
(Table 6 and fig 4), while 1/2 MIC of Foeniculum
vulgara seeds extract (X2) exhibit reduction activity up
to 58.1%, when compared with positive control, against
biofilm formation activity of MSSA (S3) and up to
35.4% against MRSA (S41) without affecting planktonic
cell growth (Table 7) (fig 5)
Table (6): Anti-biofilm effect of Salvia egypticae leaves (X1) extract against S. aureus.
Strain code
Subinhibitory
concentrations
Control +
1/2 MIC
1/4 MIC
1/8 MIC
OD: optical density, h: hours
MSSA (S3)
Biofilm formation
OD
Reduction
492nm
%
1.27
0
0.328
74.1
0.732
42.3
1.0
21.2
Growth
OD
600nm
1.04
0.99
1.1
1.08
MRSA (S41)
Biofilm formation
Growth
OD
Reduction
OD
492nm
%
600nm
1.35
0
1.2
0.67
54.4
1.14
0.8
40.7
1.19
1.2
11.1
1.22
Table (7): Anti-biofilm effect of Foeniculum vulgara seeds (X2) extract against S. aureus.
Strain code
Subinhibitory
concentrations
Control +
1/2 MIC
1/4 MIC
1/8 MIC
OD: optical density, h: hours
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MSSA (S3)
Biofilm formation
Growth
OD
OD
%
492nm
600nm
1.27
0
1.05
0.531
58.1
0.95
1.11
12.5
0.99
1.2
5.5
1.1
MRSA (S41)
Biofilm formation
OD
%
492nm
1.35
100
0.872
35.4
0.942
64.0
1.197
81.4
Growth
OD
600nm
1.26
1.2
1.3
1.21
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World Journal of Pharmaceutical and Medical Research
Figure (5): Effect of sub inhibitory concentrations of Salvia egypticae leaves extract on biofilm formation against
MSSA and MRSA strain S3 and S4.
Figure (6): Effect of sub inhibitory concentrations of Foeniculum vulgara seeds extract on biofilm formation
against MSSA and MRSA strain S3 and S41.
5- Activity of plant extracts against Enterococcus
faecalis fsr system:
Agr-like QS system in E. faecalis which regulate
production of Gilatinase and serine protease was
addressed as virulent factors. FV7061-6 represented
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pleiotropic effect between QSI and growth inhibition
while SE7042-4 did not affect growth of E. faecalis and
successfully blocked fsr system in dose response manner
(Figure x).
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6- Identification of active natural semi-purified
compounds using HPLC
Promising plant extracts from S. egypticae and F.
vulgara were further purified with guidance of blocking
of hemolysis in S. aureus in every step as follow, first by
using butanol/water separation, followed by sep-pak
column method with 0, 30, 70, 100 % of methanol/water
and finally active fractions were subjected to HPLC.
Eluted fractions from HPLC were collected and
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World Journal of Pharmaceutical and Medical Research
subjected to hemolysis bioassay. Signed group of peaks
which named as FV7061-6 from F. vulgara (Figure x)
showed significant QSI activity compared with other
fractions and SE7042-4 from S. egypticae extract (Figure
x) eluted in fraction xx did not affect bacterial growth
and attenuate regulation of agr gene. However, final
stage of active extract still need further purification to
address chemical identification.
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Desouky et al.
ACKNOWLEDGMENTS
Said Desouky is greatly acknowledged The Science and
Technology Development Fund (STDF) for providing a
short-term fellowship. This work was supported by
Grant-in-Aid for Scientific Research to Said Desouky
(No.12365) from Science and Technology Development
Fund (STDF), Egypt.
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