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Int. J. Adv. Eng. Pure Sci. 2021, 33(2): 187-194
DOI: 10.7240/jeps.741412
Corresponding Author: Nüzhet Cenk SESAL, Tel: +90 216 330 11 98 /1581, e-posta: csesal@marmara.edu.tr
Submitted: 22.05.2020, Revised: 21.01.2021, Accepted: 26.01.2021
RESEARCH ARTICLE / ARAŞTIRMA MAKALESİ
Inhibition of Pseudomonas aeruginosa Biofilm Formation and Quorum
Sensing System by Extracts of Prunus avium Stalk
Prunus avium Sapından Elde Edilen Özütlerle Pseudomonas aeruginosa’ya ait Quorum
Sensing Sisteminin ve Biyofilm Oluşumunun İnhibisyonu
Ayla YILDIZ1 , Arhun Ali BALKAN1 , Didem BERBER2,3
Barış GÖKALSIN1 , N. Cenk SESAL2
1Marmara University, Institute of Pure and Applied Sciences, Biology Department, İstanbul, Turkey.
2Marmara University, Faculty of Arts and Sciences, Biology Department, İstanbul, Turkey.
3Maltepe University, Fine and Arts Faculty, Gastronomy and Culinary Department, Marmara Egitim
Koyu, İstanbul/Turkey.
Abstract
Recently, misuse or overuse of antibiotics has led to antibiotic resistance problem, a global healthcare problem. Most virulence
factors and biofilm formation in Pseudomonas aeruginosa are controlled by quorum sensing (QS). The inhibition of QS system
by inhibitor molecules has been suggested as a novel alternative antivirulence approach in which no need to kill the bacteria.
In the present study, QS and biofilm inhibitory potentials of the methanol and acetone extracts of Prunus avium stalk against
P. aeruginosa were evaluated. The extracts were tested at the concentrations of 240, 120, and 60 μg/ml. lasB-gfp, rhlA-gfp,
pqsA-gfp biosensor strains and P. aeruginosa PAO1 were used to monitor QS and biofilm inhibition, respectively. Fluorescence
and absorbance measurements were performed on Cytation 3 multimode microplate reader. QS inhibition ratios for las, rhl,
and pqs systems and biofilm inhibition ratios of the acetone extracts were recorded as 70.43%, 47.25%, 76.31%, and 47.76%
(±6,60) and of the methanol extracts as 74.96%, 40.10%, 71.89%, and 38.54% (±3,56) at a certain concentration of 240 μg/ml,
respectively. As a result, anti-QS and anti-biofilm properties of acetone extracts were better than that of methanol extracts.
Further investigations are needed to discover inhibitor compounds of P. avium and also their effects on human cells and then
these compounds may be used in new drug discoveries.
Keywords: Pseudomonas aeruginosa, quorum sensing, biofilm, quorum quenching, Prunus avium stalk, sweet
cherry.
Öz
Son zamanlarda, antibiyotiklerin yanlış veya aşırı kullanımı, küresel bir sağlık sorunu olan antibiyotik direnci sorununa yol
açmıştır. Pseudomonas aeruginosa'da çoğu virülans faktörü ve biyofilm oluşumu, quorum sensing (QS) ile kontrol edilir. QS
sisteminin inhibitör moleküller tarafından inhibisyonu, bakterileri öldürmeye gerek olmayan yeni bir alternatif antivirulens
yaklaşımı olarak önerilmiştir. Bu çalışmada, Prunus avium sapından elde edilen metanol ve aseton özütlerinin P. aeruginosa'ya
karşı QSI (QS inhibitörleri) ve anti-biyofilm potansiyellerini değerlendirdik. Ekstraktlar 240, 120 ve 60 μg/ml'lik
konsantrasyonlarda test edilmiştir. QS ve biyofilm inhibisyonunu izlemek için lasB-gfp, rhlA-gfp, ve pqsA biyosensör suşları
ve P. aeruginosa PAO1 kullanıldı. Floresans ve absorbans ölçümleri Cytation 3 çok modlu mikroplaka okuyucu üzerinde
gerçekleştirildi. 240 μg/ml konsantrasyonunda aseton özütlerinin las, rhl ve pqs sistemleri üzerine QS ve biyofilm inhibisyon
oranları sırasıyla % 70.43, % 47.25, % 76.31 ve % 47.76 (± 6,60) ve metanol özütlerinin sırasıyla % 74.96, % 40.10, % 71.89
ve % 38.54 (± 3,56) olarak kaydedilmiştir. Sonuç olarak, aseton özütlerinin anti-QS ve anti-biyofilm özellikleri metanol
özütlerinden daha başarılı olmuştur. P. avium’un inhibitör bileşikleri ve bu bileşiklerin insan hücreleri üzerindeki etkilerini
keşfetmek için daha fazla araştırmaya ihtiyaç vardır ve daha sonra bu bileşikler yeni ilaç keşiflerinde kullanılabilir.
Anahtar Kelimeler: Pseudomonas aeruginosa, quorum sensing, biyofilm, quorum quenching, Prunus avium stalk,
sweet cherry.
I. INTRODUCTION
Bacterial pathogenicity is defined as the potential of an organism to cause any disease. As known, the production
of virulence factors by microorganisms has great importance in the clinical course of a disease. These factors may
cause damages on the host immune system due to failure in the balance between bacterial pathogenicity and host
resistance (1). Unfortunately, antibiotics are not sufficiently efficient in the treatment of bacterial infections.
Therefore, an antivirulence approach has been proposed to treat infections. By this approach, expressions or
Int. J. Adv. Eng. Pure Sci. 2021, 33(2): 187-194 P. avium stalk on QS
188
activities of virulence properties can be prevented and
bacteria cannot colonize the host. Furthermore, it has
been assumed that there is probably less evolutionary
pressure to develop resistant clones than conventional
antibiotics because this strategy does not directly kill
bacteria but prevent bacterial infections and damages to
their host. These anti-virulent drugs can potentially be
used in combination with synergistically established or
new antimicrobials to prolong the life of these drugs (2,
3).
The World Health Organization (WHO) declared the
priorities of antibiotics for pathogens as critical, high,
and medium highlighting the need for new antibiotics.
According to this list, Pseudomonas aeruginosa is
categorized as critical for the discovery of new
antibiotics (4). P. aeruginosa can cause nosocomial
infections such as cystic fibrosis, especially in
immunocompromised patients. In 2004, the U.S. Cystic
Fibrosis Foundation Patient Registry reported that P.
aeruginosa was identified in 57.3% of all respiratory
cultures (5). The data from newborn screening
programs showed that the total number of cystic
fibrosis (CF) patients in sixteen European countries,
CF adults and CF children in 2025 would rise by 50%,
75%, 20% respectively (6). The clinical importance of
this opportunistic pathogen attributed to its resistance
to multiple antimicrobial agents, its quorum sensing
mediated virulence factors (exoproteases, siderophores,
exotoxins and, rhamnolipids, etc.), and ability to form
biofilm formation resulting in the community- or
hospital-acquired infections (7-9).
Bacteria communicate via quorum sensing system (QS)
which allows controlling their social behaviors. A high
density of bacterial population in the surrounding
environment trigger QS system for intra-species, inter-
species, or inter-kingdom interactions (10, 11).
Autoinducers (AIs), small molecules that can easily
diffuse across inner and outer membranes, are secreted
into bacterial local milieu. Gram-negative bacteria
utilize homoserine lactones (HSLs). It is well
documented that P. aeruginosa has las, rhl, pqs and iqs
systems for interspecies communication (12).
Approximately a tenth of the total P. aeruginosa genes
are coordinated by QS. These genes are responsible for
many virulence factors, antibiotic resistance, regulation
of metabolic pathways under stress and biofilm
structure of P. aeruginosa (13). The critical importance
of biofilm structures in chronic infections has been
emphasized in the literature. Biofilms are sessile
community complexes in which bacterial cells attach
onto various surfaces in an exopolysaccharide matrix.
Biofilm forms are more resistant to antibiotics
compared to planktonic forms (14).
Most researchers have focused on an alternative
antivirulence approach to combat bacterial antibiotic
resistance by disrupting the QS system, called quorum
quenching (QQ) (15). In this way, several compounds
and enzymes with quorum sensing inhibitory (QSI)
potential have been identified to quench the QS
mechanism. The criteria for QSI molecules are notified
as high specificity, efficiency, stability, having low-
molecular-weight (16).
Since ancient times, different parts of plants have
traditionally been used in the treatment of various
disorders. Nowadays, they are globally valuable
resources of new drugs not only in developing
countries but also in modern countries (17). For this
reason, compounds with QSI properties are
investigated especially in plants and anti-QS potentials
of plant species collected from different localities are
investigated as direct extracts or based on the
substances they contain (18).
Prunus avium L., (sweet cherry) is a member of
Rosaceae family and is distributed around the world
with a temperate climate but especially in Europe,
North Africa, South Australia, New Zealand, USA,
Canada, Argentina and Chile (19, 20). P. avium has
several beneficial effects on various illnesses such as
cancer, cardiovascular disease, diabetes, Alzheimer’s
disease, neurodegenerative diseases and, other
inflammatory diseases as well as being consumed as
food (21, 22). Different parts of P. avium such as its
fruit, stem, and bark are used for medicinal and
therapeutic purposes (20). Furthermore, its
antibacterial, antioxidant, and anti-inflammatory
activities have been demonstrated (23- 29).
To our knowledge, there is no study investigating the
QSI and anti-biofilm potentials of P. avium stalk
against P. aeruginosa. In the view of an urgent need for
new alternative approaches that can solve the global
health problem due to the current antibiotic resistance,
the potential impact of P. avium (sweet cherry) stalk
was investigated to inhibit the QS system and biofilm
formation of P. aeruginosa. For this purpose, QSI
potentials of acetone ad methanol extracts of P. avium
stalk samples were tested on the biosensor strains of P.
aeruginosa, lasB-gfp, rhlA-gfp and pqsA-gfp, and anti-
biofilm activities were tested on the PAO1 wild type
strain.
II. MATERIAL AND METHODS
2.1. Sample Collection and Extraction of P. avium
Stalk Samples by Acetone and Methanol
Solvents
Following the washing and drying of P. avium stalk
samples, ten grams of each sample were weighed and
pulverized. Acetone and methanol solvents were added
into sterile bottles including the samples and stored in
a dark place for 3 days. They were evaporated in a
rotary evaporator at 40 °C and 100 rpm. The acetone
and methanol extracts of P. avium stalk samples were
weighed again to obtain the weight of crude extracts.
To evaluate the anti-QS and anti-biofilm properties, a
P. avium stalk on QS Int. J. Adv. Eng. Pure Sci. 2021, 33(1): 187-194
189
stock concentration of these extracts was prepared as
16 mg/ml and then dissolved in 100% DMSO. Finally,
these extracts were diluted with a physiological saline
solution.
2.2. Monitor Strains
lasB-gfp, rhlA-gfp, and pqsA-gfp were used as QS
monitor strains of P. aeruginosa (30-32). These
monitor strains included lasR, rhlR, pqsR regulated
promoters and a gene for an unstable green fluorescent
protein (gfp). In the present study, M9 minimal media
supplemented with 2.5 mg/l thiamine, 0.5% (wt/vol)
glucose, and 0.5% (wt/vol) casamino acids were used
for the growth of the test bacteria.
2.3. QSI Screening
QSI potentials of the acetone and methanol extracts of
P. avium stalk samples were examined in 96-well black
microplates (Nunc, Thermo Scientific) (33). 100 µl of
the prepared growth medium given above was added to
each well. The test extracts were then diluted three-fold
to obtain final concentrations of tested extracts as 240,
120 and 60 µg/ml in 96-well black microplates. The
total volume in each well was then adjusted to 200 µl
by adding overnight cultures of the lasB-gfp, rhlA-gfp
and pqsA-gfp monitor strains with an OD 450 nm of
0.1. The positive and negative control groups were also
tested. The experiments were performed in three
replicates. The bacterial growth and gfp expressions
were measured every 15 minutes using Cytation 3
multimode microplate reader (Biotek) for 16 h.
The measurements of fluorescence were recorded at
485 nm excitation and 535 nm emission wavelengths.
2.4. Biofilm Experiments
P. aeruginosa PAO1 strain was incubated overnight in
a prepared M9 growth medium at 37°C. In 96-well
microplates, the acetone and methanol extracts of P.
avium stalk samples were tested at the concentrations
of 240, 120 and 60 µg/ml, respectively. The
experiments included positive and negative controls.
Three replicates were made for the tests. The biofilm
forms were stained with 0.1% crystal violet and
measured at OD 590 nm in the microplate reader
(Cytation 3-BioTek).
III. RESULTS
Three concentrations (240, 120 and 60 µg/ml) of
acetone and methanol extracts of P. avium stalk
samples were tested on lasB-gfp, rhlA-gfp and pqsA-gfp
monitor strains of P. aeruginosa. Since azithromycin
was reported to reduce the transcription of lasI by 80%
and of rhlI by 50% in the literature (34), azithromycin
was also tested as a positive control to inhibit tested QS
systems (las, rhl and pqs). We determined that
azithromycin was significantly able to inhibit gfp
production of tested monitor strains (The data was not
shown). In our experiments, maximum QS inhibition
ratios on las, rhl and pqs systems were detected at a
certain concentration of 240 µg/ml for acetone and
methanol extracts of P. avium stalk samples. The
acetone extracts of P. avium stalk at a concentration of
240 µg/ml inhibited las, rhl and pqs systems of P.
aeruginosa in ratios of 70.43%, 47.25%, and 76.31%
respectively. The related dose-response curves of lasB-
gfp, rhlA-gfp and pqsA-gfp monitor strains of P.
aeruginosa treated with the acetone extracts of P.
avium stalk at certain concentrations of 240, 120 and 60
µg/ml were given in Figure 1A-C.
On the other hand, QS inhibitory potentials for las, rhl
and pqs systems of methanol extracts of P. avium stalk
were recorded as 74.96%, 40.10%, and 71.89%,
respectively Figure 2.
Anti-biofilm properties of acetone and methanol
extracts of P. avium stalk samples were tested against
PAO1 strain. The inhibition percentages for biofilm
formation belonging to the extracts of P. avium stalk
samples at a dose of 240 µg/ml were found to be
slightly different and recorded as 47.76% (±6.60) and
38.54% (±3.56), respectively. Biofilm inhibition ratios
of the acetone and methanol extracts of P. avium stalk
at the concentrations of 240, 120, 60 µg/ml against
PAO1 strain were given in Figures 3-4. These results
indicate that acetone extracts have a more pronounced
effect in comparison to methanol extracts.
Int. J. Adv. Eng. Pure Sci. 2021, 33(2): 187-194 P. avium stalk on QS
190
Figure 1. Dose-response curves of biomonitor strains of P. aeruginosa treated with the acetone extracts of
P.avium stalk at certain concentrations of 240, 120 and 60 µg/ml. Data are shown as relative fluorescence unit
over OD 450 nm.
Figure 2. Dose-response curves of biomonitor strains of P. aeruginosa treated with the methanol extracts of
P.avium stalk at the certain concentrations of 240, 120 and 60 µg/ml. Data are shown as relative fluorescence
unit over OD 450 nm.
P. avium stalk on QS Int. J. Adv. Eng. Pure Sci. 2021, 33(1): 187-194
191
Figure 3. The percentage of anti-biofilm properties of
the acetone extracts of P.avium stalk at concentrations
of 240, 120, 60 µg/ml against PAO1 strain.
Figure 4. The percentage of anti-biofilm properties of
the methanol extracts of P.avium stalk at the
concentrations of 240, 120, 60 µg/ml against PAO1
strain.
IV. DISCUSSION
As known, antibiotic misuse or overuse led to the
problem of antibiotic resistance as a global healthcare
problem. P. aeruginosa controls its’ virulence factors
and biofilm formation by QS mechanism.
Alternatively, QS system and biofilm formation can be
inhibited by inhibitor molecules as an antivirulence
approach without killing bacteria. To the best of our
knowledge, anti-QS and anti–biofilm effects of P.
avium stalk samples against P. aeruginosa have not
been studied yet. In this study, we demonstrated the
potential inhibitory properties of acetone and methanol
extracts of P. avium stalk samples on QS mechanisms
and biofilm formation.
It has been well documented that the different parts
(fruit, seed, stem bark and roots) of P. avium (sweet
cherry) has several bioactive compounds (35-39).
Several chemical compounds such as flavonoids,
phenolics, polyphenols, alkaloids, tannins were
reported in P. avium (37, 40-42). There are many
studies in the literature focusing on the antibacterial
activities of extracts or fractions of different parts of P.
avium obtained by different solvents or of its’ fruit
juice. Rovčanin et al. (2015), reported that ethanol
extracts of P. avium petiole had an antibacterial effect
against multiple antibiotic-resistant Escherichia coli
and they detected also high concentrations of phenols
and flavonoids in the ethanol extracts (26).
Accordingly, the inhibitory activities of leaf and stem
bark ethanol extracts of P. avium were found to be more
effective against Gram-negative compared to the
Gram-positive bacteria. In this study, researchers
detected that the stem bark extracts were more potent
when compared to the leaf extracts (39).
On the other hand, it was demonstrated that ethyl
acetate and butanol fractions of P. avium had high
antibacterial activity against Listeria monocytogenes,
Staphylococcus aureus, and Salmonella typhimurium
but the extracts and fractions had no inhibitory effect
against P. aeruginosa (23). Propionibacterium acnes,
acne-inducing bacteria, was also inhibited by the juice
of P. avium. The juice and/or methanol extracts of P.
avium inhibited the growth of Streptococcus pyogenes
and P. acnes but not Staphylococcus epidermidis (24).
The reason for differences in the sensitivity of Gram-
positive and Gram-negative bacteria might be
dependent on variables in the pattern including the cell
wall such as peptidoglycan structure or high levels of
lipopolysaccharides (39). Taken together, P. avium has
noticeable antibacterial potentials against Gram-
positive, or Gram-negative bacteria depending on the
species. Nevertheless, studies about the antibacterial
properties of P. avium against P. aeruginosa are scarce.
To our knowledge, there is no study investigating the
QSI and anti-biofilm potentials of P. avium stalk
against P. aeruginosa. As mentioned before, quorum
quenching, by other words quorum sensing inhibition,
is a novel approach to overcome bacterial antibiotic
resistance. In this meaning, plant-based QSIs with
fewer side effects, considerable bioavailability, low
costs and no toxicity may serve alternatively promising
treatment strategies individually or along with
conventional antibiotics. The stalk parts of P. avium are
generally discarded. The evaluation of stalk parts of
this plant that will be disposed may provide highly
possible add value to the country's economy since P.
avium stalk may ensure beneficial effects in terms of
health without any cost. Therefore, there are many
positive aspects of utilization of P. avium stalk in
healthcare because they are plant-based material and
have cost-effectiveness as well as their potential
therapeutic effects. Besides the evaluation of the
material to be discarded can be achieved.
Considering the extract concentrations that we applied
in our biofilm experiments, we can suggest that all our
three concentrations (240, 120, and 60 µg/ml) are
considerably low according to other studies testing the
anti-biofilm properties of extracts from various plants.
For example, Ravichandiran et al. (2013) reported that
Melia dubia bark extracts reduced biofilm formation by
Int. J. Adv. Eng. Pure Sci. 2021, 33(2): 187-194 P. avium stalk on QS
192
84% and QS system by 75% in E.coli at a concentration
of 30 mg/ml (43). In another study, it was shown that
Capparis spinosa extract inhibited biofilm formation of
some pathogen Gram-negative bacteria at a
concentration of 2 mg/ml (44). Sandasi et al.,
demonstrated that Rosmarinus officinalis, Mentha
piperita, and Melaleuca alternifolia exhibited anti-
biofilm activity against Listeria monocytogenes at a 1
mg/ml (45). Trentin et al. (2011) tested the antibiofilm
properties of several medicinal plants from the
Caatinga in Brazil at the concentration of 0.4 mg/mL
and 4.0 mg/mL against S. epidermidis (46).
In other respects, QS inhibition is usually assessed by
the detection of AHL-related inhibitory activity based
on the violacein pigment production in
Chromobacterium violaceum strain CV026, which is
unable to synthesize its AHL in the literature.
Fluorescence-based biosensor strains have more
advantages to evaluate QS response of bacterial cells
due to their ability for screening directly QS-related
gene expressions. Because we tested our samples on
biomonitor strains, we could easily observe the
inhibition rates by gene expression levels. According to
our results, both extracts of P. avium stalk inhibited the
las and pqs system but they provided less inhibition on
rhl system of P. aeruginosa. Anti-biofilm properties of
P. avium stalk extracts were tested against PAO1 strain.
The inhibition percentages for biofilm formation
belonging to the acetone and methanol extracts of P.
avium stalk samples at a concentration of 240 µg/ml
were found to be slightly different and recorded as
47.76% (±6,60) and 38.54% (±3,56), respectively.
Accordingly, to the QS experiments, our acetone
extracts were found to be more effective on biofilm
inhibition in contrast to methanol extracts.
From this point, taken into consideration of
ethnobotanical importance and bioactive potential of P.
avium stalk, antibiotic resistance problems may be
solved by interrupting the QS system as an alternative
strategy. Further detailed studies about the bioactive
compounds of this plant should be performed. These
compounds may be used in the manufacture of new
drugs or alternatively in combination with antibiotics in
drug discovery.
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