SCREENING FOR ANTIQUORUM SENSING AND ANTI-BIOFILM ACTIVITY IN Viscum album L . EXTRACTS AND ITS BIOCHEMICAL COMPOSITION

Many opportunistic pathogenic bacteria use the Quorum Sensing (QS) system to coordinate their virulence expressions. Thus, QS can likely be a new approach to control bacterial infections. The aim of this study was to evaluate the antimicrobial, anti-biofilm, and anti-quorum sensing activities of ethanol, chloroform, and dichloromethane: methanol extracts of leaf, stem, and fruits of the European mistletoe Viscum album L. on 2 Gram-positive and 7 Gram-negative pathogenic bacteria. The extracts at concentrations ranging from 50 to 250mg/ml were tested depending on the extracts of the plant parts and the test bacteria. The extract with 50mg/ml concentration, in which no antimicrobial activity was observed, was used for anti-quorum sensing and antibiofilm studies. The dichloromethane: methanol extracts were found to show the highest biological activities. QS activities of the plant extracts were also determined using the recently established Chromobacterium violaceum CV026 reporter strain and the signaling molecule N-(ß-ketocaproyl)-L-homoserine lactone (3-oxo-C6-HSL) agar well diffusion assay. Biofilm was quantified using the microtiter plate test and the crystal violet assay. Anti-microbial, anti-biofilm, and anti-quorum sensing activity of leaf and stem extracts showed higher efficiency than fruit extracts. It was concluded that the extracts of V. album had the potential to treat microbial infections by biofilm inhibition or inhibition of QS.


Introduction
Viscum album L. subsp.album (mistletoe), belonging to the family Santalaceae, is an evergreen plant growing semi-parasitically on its host.It is a Eurasian and North African species and its distribution in Turkey covers mainly the north, west, and south-west of Anatolia.Its leaves are opposite and parallel-veined, and its fruits are viscid berries.The epithet name of Viscum album in Latin was assigned to the plant considering the white color of the fruits (Ergun & Deliorman 1995).Seed distribution of V. album is mediated by birds who achieve this task by eating the fruits and leaving their stools on trees.The seeds germinating on the host trees send out their rootlet to penetrate into the bark and absorb nutrients and water from the trees which are used by the growing plant.The high amount of loss in volume and diameter of the host trunk occurs due to this parasitic feature of V. album (Eroğlu & Usta 1993).
Viscum album has long been known as one of the most magical, mysterious, and sacred plants in nature.Therefore, the use of V. album as a herbal medicine probably dates back to prehistoric times.The Druids of Britain used to harvest V. album from their sacred oaks to use them in rituals and medicine.Dioscorides  and Hippocrates (460-377 BC) used it to treat diseases of the spleen and complaints of menstruation.Plinius  was also reported to cure epilepsy, infertility, and ulcers using this plant.Celcus the Platonist remarked that mistletoe was used in the treatment of swellings or tumors (around 150 AC).Tabernaemontaunus stated that its leaves healed hepatitis, leprosy, and mumps.At the end of the 19 th century, mistletoe-containing ointment was reported to be effective in the treatment of eczema, burn diseases, and some wounds (Bussing 2003).V. album has also drawn attention as a possible anti-cancer agent since the 1920s (Habeck 2003).Although it is a parasitic plant, its medical importance has been gradually increasing due to the diverse phytochemicals that the plants contain such as lectins, viscotoxins, alcholoids, amines, amino acids, flavonoids, glycosides, lignans, carbocyclic acids, phenylpropanes, polypeptides, polysaccharides, sugar alcohols, polyphenolic, and terpenoid compounds all which are known to have rich biological activities (Deliorman et al. 2001, Arda et al. 2003, Sengul et al. 2009, Nazaruk & Orlikowski 2016).However, the use of the European mistletoe V. album is common in medicine since the American mistletoe Phoradendron flavescens (Pursh Nutt.) is toxic (Ogunmefun et al. 2013).The compounds obtained from V. album are prevalently used for gastro-intestinal, diabetes, blood pressure problems, treatment of cancer, the hepatitis C virus (HCV), HIV, and human parainfluenza virus type 2 (HPIV-2) (Stoss & Gorter 1998, Tusenius et al. 2001, Karagöz et al. 2003), but the field of the use of the plant may vary based on the chemical properties of host tree which in turn affect compounds present in V. album.
Viscum album has been successfully used for the treatment of infection diseases in consequence of the effects of plant on microorganisms (Hussain et al. 2011).A bacterial infection begins with the organization of bacteria acting in unison (Rumbaugh et al. 2009, Antunes et al. 2010).This system is known as "Quorum-sensing" (QS) system (Waters & Bassler 2005).Microorganisms cannot activate their resistance mechanisms if this step is blocked in the treatment of infection (Adonizio et al. 2006, Musthafa et al. 2010).Biofilms are thought to be associated with microbial infection and their formations are considered to be regulated by QS (Brackman & Coenye 2015).Therefore, maturation and eradication of biofilms have great importance in fighting infection (Chung & Toh 2014).
In the present study, we evaluated the anti-biofilm activities of ethanol, chloroform, and dichloromethane:

Collection of V. album samples and extract preparation
Viscum album samples collected at an altitude of 1475m around Ihlara-Kulaköy in Aksaray province in October and November 2015 were used.The Flora of Turkey and The East Aegean Islands (Davis 1965) and The Checklist of the Flora of Turkey -Vascular Plants (Güner 2012) were used for identification of the plant specimens.Fresh plant samples were separated as leaves, stems, and fruits in sterile conditions in the laboratory and were left to dry at room temperature.Dried plant parts were grinded by a pulverizer, and 100gr/500ml of each dry plant part was extracted with ethanol, chloroform, and dichloromethane:methanol (w/v) (at 60°C for 6h) in soxhlet apparatus.The extracts were then concentrated in a rotary evaporator after the residues had been centrifuged (at 3.000g) and washed with physiological saline solution for 5 minutes.The supernatants of the extracts were kept at 4°C and used for future investigations.The residues were used for anti-quorum sensing and anti-biofilm experiments.

Bacterial Strains and Culture Conditions
The microorganisms used in the study were produced from the microbial culture collection kept in microbiology laboratory of Scientific

Antibacterial Activity Assays
The antibacterial influence of Viscum album leaf, stem, and fruit extracts on the nine pathogenic bacteria were investigated by the agar well diffusion method.Each purified extract was dissolved in dimethyl sulfoxide and stored at 4°C.The antibacterial activity of the substances was shown by a clear zone of inhibition around the application point.All bacterial strains were grown in Brain Heart Infusion Broth (Merck) for 24h at 37°C.The concentration of bacterial suspensions was adjusted to 10 8 cells/ml in Brain Heart Infusion Broth and 100µl of each culture of bacteria was spread on agar plate surfaces.Wells 5mm in diameter were opened on the agar with a cork borer to load 20µl of each sample incubated at 25°C for 3h.Gentamycin (10UI) was used as positive control, while Dimethyl sulfoxide (DMSO) was used as the negative control.The plates were incubated at 37°C for 24h before they were examined for inhibition zones of growth.All tests were performed in triplicate.

Antiquorum Sensing Activity Assay
Plant extracts at a concentration of 50mg/ml were used depending on the results of the antibacterial effects.The bacterial culture of C. violaceum CV026 biosensor strain, which was grown at 30°C for 15 hours, was adjusted to a McFarland standard 0.5 (10 6 CFU/ml).The ideal wavelength of the absorbance was established, the absorbance of the standards was measured, and the cell counting in UV-Vis spectrophotometrically was performed.Chromobacterium violaceum CV026 and the extract of 50µl N-(ß-ketocaproyl)-L-homoserine lactone (3-oxo-C6-HSL) on 10ml soft Luria Bertani (LB) agar medium was added for agar diffusion test.LB agar medium was prepared with 0.9% agar, 100µl C. violaceum CV026, and 50µl N-(ß-ketocaproyl)-Lhomoserine lactone (3-oxo-C6-HSL) extract and after solidification, 4mm diameter wells were scooped out from the LB agar medium.The wells were filled with the plant extracts (50mg/ml) in different solvents.The agar plates were incubated at 30°C for 48 hours.Agar diffusion tests were also conducted in triplicate (Adonizio et al. 2006, Bezek et al. 2016, Oliveira et al. 2016).
Violacein Pigment Isolation 100μl of 20ml of fresh C. violaceum CV026 strain grown LB medium in 15 hours was inoculated according to the procedure given in Table 1 for the extraction of violacein pigment from the liquid culture.The bacterial culture was incubated at 30°C for 24 hours and then vortexed.200µl of the culture from the tube was transferred to a 1.5ml Eppendorf microcentrifuge tube.
200μl of 10% sodium dodecyl sulfate (SDS) solution was added in the culture for cell fractionation, and the culture was kept at room temperature for 5 minutes after vortexing for 5 seconds.900μl of water-saturated butanol (50ml n-butanol + 10ml distilled water) was added into the tube for violacein pigment isolation, and the tube was vortexed for 5 seconds.It was then centrifuged at 10,000rpm for 5 minutes.The upper phase was transferred to a new tube.It was read spectrophotometrically at 595nm, and the amount of viola was determined (Khan 2009).

Anti-biofilm Activity Assays
Biofilm formation was confirmed with the Crystal Violet Method.The test bacteria which were incubated in Tryptic Soy Broth at 37°C for 24 hours were diluted according to Mc Farland 0.5.100µl of the bacterial cultures was inoculated in 5ml Tryptic Soy Broth (TSB) in a shaking incubator at 120rpm for 24 hours.Following the incubation, polystyrene microplates with 24 wells were filled with 900µl TSB medium + 50µl plant extract (50mg/ml) + 50 µl test bacteria, and left at 37°C for 48 hours.The mediums were removed from microplates, and washed by 1xPBS buffer three times.The microplates were dried at 65°C in an incubator, and then were left to be dyed with 1% Crystal Violet for 2 minutes.The microplates were washed three times with distilled water at the end of the staining process and dried at room temperature.The Crystal Violet solutions in microplates solved by 30% acetic acid solution were read in the spectrophotometer at 595 nm against the control group.Thus, the ability of biofilm formation of the test bacteria was determined (Stepanović et

Statistical analyses
Arithmetic means and standard deviations of the obtained data were calculated in Microsoft Excel 2016.

Results
In recent years, despite the development of technology and medicine, the fight against the infection factors remains inadequate.This leads the scientists to the use of natural resources.The use of plant extracts in healthcare industry has recently gained popularity, and it is a prevalent application in not only in Turkey but also in many developing countries worldwide.Scientists have been interested in the availability of new compounds in plants which have not yet been discovered for the inhibition of anti-quorum sensing recently.The effects of compounds in plant extracts on microorganisms have been reported to vary according to the plant species, amount of plant parts in the extract, and their maturation.
The utilization of V. album in traditional medicine implies that it can also be used in modern medicine, especially in cancer and cardiovascular diseases (Gray & Flatt 1999).According to epidemiological studies, essential oil and phenolic compounds have inhibitive effects against the progress of bacterial infections (Ghuman et al. 2016, Martinelli et al. 2017).GC-MS chromatogram analysis of leaf extracts of V. album showed different peaks, which indicates the presence of 8 main phytochemical constituents (Fig. 1), whilst the stem of V. album was found to include 10 main components (Fig. 2).These constituents were characterized and identified by comparing their mass spectra with the NIST library (Tables 2,3).
The GC-MS chromatogram analysis of fruit extracts showed that the content of fruit extracts appeared to be rich in some compounds such as 5-hidroximetilfurfural, glucose, Inositol, Palmitic acid, Stearic acid, p-coumaric acid, and phenolic components (Fig. 3).The identification and quantification of phenolic compounds in fruits were analyzed by the GC-MS analysis (Table 4).The overall results of the GC-MS analysis showed that both stem and leaf extracts contained polyphenolic compounds that are biologically more important compared to compounds in fruits.Although the fruit extracts of V. album appeared to be rich in antioxidant content, they should be used with caution because they have cytotoxic activity.The pharmacologically active chemical content of the V. album extracts varies depending on the host plant and the harvesting time (Büssing & Schietzel 1999).In conclusion, this study revealed the anti-quorum sensing, antibiofilm, and antimicrobial activities of the main chemical components of the V. album.
Viscum album is known to possess healing properties and was successfully used in treatment applications of various infectious diseases in humans.The antibacterial activity of V. album stem and leaf extracts in our study was determined on different pathogenic bacteria.The results of the antimicrobial screening of fruit, stem and leaf extracts are shown in Tables 5 and 6.The extracts prepared at concentrations of 150mg/ml, 200mg/ml, and 250mg/ml in methanol: dichloromethane, ethanol, and chloroform were shown to stop the bacterial growth.
The dichloromethane: methanol extract prepared at a concentration of 50mg/ml inhibited only on Proteus vulgaris.In general, it was found that the effectiveness of chloroform and ethanol extracts prepared at 150mg/ml concentration on bacteria was lower, but the efficiency of the same concentration of dichloromethane: methanol extract on bacterial growth was higher.The dichloromethane: methanol extract at concentrations of 150, 200, and 250mg/ml showed the highest inhibition on E. faecalis ATCC 29212.All extracts prepared at concentrations 150, 200, and 250 mg/ml were found to be effective on P. aeruginosa PA14.We found that the dichloromethane: methanol extract at concentrations from 100 to 250mg/ml prevented spread of Klebsiella pneumoniae ATCC 700603 with the zones ranging from 11-15mm.The efficacy of dichloromethane:methanol extracts at concentrations of 100, 150, 200 and 250mg/ml was higher on Klebsiella pneumoniae ATCC 700603 strain than E. coli O157: H7.Different concentrations of V. album extracts were also found to stop the growth of L. monocytogenes ATCC 7644 (8-13mm), B. cereus RSKK 863 (7-13mm), S. epidermitis wt (9-13mm), and P. aeruginosa ATCC 27853 (10-13mm) (Table 6).
According to the antimicrobial activity analysis results, the highest antimicrobial effect was observed in dichloromethane: methanol extracts for all plant parts tested.Therefore, anti-biofilm and anti-quorum sensing assays were performed using dichloromethane: methanol extracts.The lowest concentration of fruit extract for biological activity was determined as 150 mg/ml.Therefore, anti-quorum sensing and anti-biofilm tests were performed at a concentration of 100mg/ml in accordance with the concentration of leaf and stem extracts.When we analyzed the antimicrobial activity of dichloromethane: methanol extracts of fruit, we found that the maximum effect was on E. faecalis ATCC 29212 with a zone diameter of 15 mm.Klebsiella pneumoniae ATCC 700603 and E. coli O157: H7 are among the most inhibited species by the fruit extracts.
The fruit extract (150mg/ml) was found to be less effective against P. aeruginosa PA14 (10 mm inhibition zone).The fruit extracts of prepared with ethanol and chloroform resulted in an inhibition of 5-9mm.It was also proved that DMSO solution selected as the control had no effect on any of the strains.When our results are evaluated in general, it is clear that the extracts whose antibacterial activities were tested have inhibitory effects on the bacterial strains included in the study.The antimicrobial activities of different concentrations of N-hexane extracts of V. album L. subsp.abietis (Wiesb.)was investigated on Candida albicans (Robin) Berkhout, Bacillus subtilis (Ehrenberg) Chon, Staphylococcus aureus Rosenbach, Escherichia coli Escherich, Pseudomonas aeruginosa (Schröter) Mihgula, Enterobacter cloacae (Jordan) Hormaeche & Edwards, and Proteus vulgaris and the 6 th and 7 th hexane fractions were found to be effective (Ertürk et al. 2004).
The antimicrobial activities of dichloromethane, chloroform, and water extracts of Viscum capense L. were tested against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans (Amabeoku et al. 1998).The results showed that the chloroform extract inhibited the growth of S. aureus, whereas it was not effective on other microorganisms.
As a result of the present study, V. album extracts were found to be effective on 3-oxo-C6-HSL which is a bacterial communication molecule used by many Gram-negative pathogenic bacteria.C. violaceum CV026 is a mini Tn-5 mutant of C. violaceum ATCC 31532 (Mc Clean et al. 1997).The mutant produces violacein pigment when 3oxo-C6-HSL is in the medium.It was seen that violacein pigment production was reduced using both agar diffusion test (zone formation) and the extraction of violacein pigment from broth medium when the Viscum extract was released into the medium.We came to the conclusion that this molecule was affected as a result of these tests.The dichloromethane: methanol extracts of stem, leaf and fruit of V. album showed promising anti-quorum sensing activity, and a clear white opaque zone of inhibition was observed in the biosensor plate containing the reference strain C. violaceum CV026 (Fig. 4).The production of violacein by C. violaceum CV026 was inhibited by dichloromethane: methanol extracts of leaf, stem, and fruits (Fig. 5).The presence of plant extracts had no influence on the growth of C. violaceum CV026.The violacein production and cell counts were similar in the control group with solvents (Group B) and the group without solvent (Group A).
Viscum album was reported to be highly effective on both bacteria and fungi (Dulger & Gonuz 2004).The components of V. album were proved to be effective as anti-diabetic and anti-hyperlipidemic in diabetic rats, which supports the use in traditional medicine (Adaramoye et al. 2012).The positive effect on lipid profile in diabetic rats eliminates secondary complications of diabetes.Önay-Uçar et al. (2012) stated that chloroform extracts of V. album can inhibit oxidative DNA damage, and that biological activity of V. album depends on its host tree.
The biological activity (anti-tumor and anti-bacterial) of the European mistletoe V. album collected from 13 different host trees was evaluated by (Turker et al. 2012).The water extract of the plants collected from Prunus divaricata Ledeb.showed the best antitumor activity (87.3% inhibition).The anti-quorum sensing activity of E. angustifolia extracts were determined in another study (Erdonmez et al. 2016).In our study, V. album was also collected from Elaeagnus angustifolia L. as the host plant.When the results of effects of V. album extracts are compared with the effects of E. angustifolia extracts, it is seen that the extracts of V. album are more efficient than the extracts of E. angustifolia on QS process.Even so, V. album may contain the chemical components of its host and these components may increase the biological activity of V. album.In future, the prevention of bacterial communication by the help of plant extracts instead of anti-microbial activity will increase the chance of success in infectious diseases.Anti-quorum sensing activity of V. album may play an important role in antibacterial activity, and therefore, it ensures an extra strategy in the struggle against bacterial infections.However, molecular researches are needed to explain the mechanism of antibacterial activity completely.
Many studies showed that the biofilm formation by pathogens leads to an increase in their virulence (Vuong et al. 2004, Antunes et al. 2010).Therefore, if the biofilm formation is inhibited, the bacterial infection can be prevented.Nowadays, biological materials are mostly preferred for use in the inhibition methods of biofilm formation and also in protection against bacterial infections (Rutherford & Bassler 2012).Although many antimicrobial studies of V. album are available in literature, no studies related to antibiofilm and anti-quorum sensing activity of this species has been carried out (Chandrashekhara et 6).The formation of bacterial biofilm decreased after the addition of plant extracts.A decrease ranging from 20% to 80% depending on the extract used was recorded in the spectrophotometric measurements.The effects of biofilm inhibition of leaf and stem extracts were higher than effects of fruit extracts.
Bazargani & Rohloff (2016) investigated the vitro anti-biofilm activities of essential oils and plant extracts of Coriandrum sativum L. through Crystal Violet (CV) and XTT tests and found that the plant extracts and their essential oils could reduce the bacterial biofilm at a rate of 50% and that the activity of essential oils of coriander was particularly high.The ethanol extract of stem bark of Terminalia fagifolia Mart.showed a distinct biofilm inhibition, and it could block biofilm formation of some strains above 80% (de Araujo et al. 2015).Stem, leaf and fruits of V. album contain very large amounts of phytochemicals such as flavonoids, alkaloids, steroids, glycosides, and phenols showing that V. album is suitable for medical use.
In conclusion, this study demonstrated that V. album extracts are remarkable anti-biofilm agents against biofilm formed by pathogens and inhibit signal molecules, which form the basis of bacterial communication mechanism.

Fig. 4 .
Fig. 4. Inhibition of violacein production by (a) fruit, (b) leaf, and (c) stem extracts in dichloromethane: methanol using Chromobacterium violaceum CV026 biomonitor strain and the agar well diffusion method.The inhibition was detected by a colorless, opaque halo around the discs.(d) DMSO was used as control.In accordance with the data acquired from well diffusion studies, the pigment isolation of violacein is carried out to understand the inhibition of the signal molecule (Fig.5).
Klebsiella pneumoniae ATCC 700603, Proteus vulgaris Hauser, Pseudomonas aeruginosa PA14, Enterococcus faecalis ATCC 29212, Escherichia coli O157: H7, and Bacillus cereus RSKK 863 and the interaction of these plant extracts with bacterial QS.Viscum album was selected as the study material because of the wide use of natural and easily available herbal drugs obtained from it.
*Group A and B are the control groups.Group C is the experimental group.

Table 2 .
GC-MS Analysis of V. album leaf extracts.

Table 3 .
GC-MS Analysis of V. album stem extracts.

Table 4 .
GC-MS Analysis of V. album fruits extracts.

Table 5 .
Growth inhibition activity of V. album fruit exracts against the pathogenic bacteria tested.