In Vitro Evaluation of Antimicrobial and Antibiofilm Potentials of Essential Oil of Tanacetum argenteum ( Lam . ) Willd . subsp . canum ( K . Koch ) Grierson var . canum

This study was performed to determine the antimicrobial and antibiofilm activities of essential oil of Tanacetum argenteum (Lam.) Willd. subsp. canum (K.Koch) Grierson var. canum. The plant was collected from Amasya. The antimicrobial activity was determined by disc diffusion and microdilution broth methods. Essential oil was active against indicator organisms. The maximal inhibition zone diameter were as follows: E. cloacae ATCC 28355 (14mm), P. fluorescens ATCC 55241 (16mm), P. aeruginosa ATCC 27853 (19mm), S. sonnei RSKK 8177 (18mm), E. coli ATCC 25922 (21mm), E. coli O157:H7 (13mm), Y. enterocolitica RSKK 1501 (11mm), C. jejuni ATCC 33291 (12mm), K. pneumoniae ATCC 27736 (13mm), S. enteritidis RSKK 171 (12mm), S. aureus ATCC 33862 (26mm) and S. aureus ATCC 25923 (29mm), M. luteus NRLL-B-4375 (20mm), E. faecalis ATCC 19433 (19mm), B. cereus NRRL-B-3711 (30mm), B. cereus RSKK (32mm), C. albicans ATCC 10231 (19mm) and C. tropicalis (17mm). The minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) were ranged from 62.5-900 μg/ml and 125-1000 μg/ml, respectively. Plant essential oil was the most active against B. cereus NRRL-B 3711 and B. subtilis RSKK 867. Also, the essential oil has shown a strong anti-candidal activity against two Candida species. For determining the antibiofilm effect, various dilutions of oil were studied on M. luteus, E. cloacae, P. fluorescens, Y. enterocolitica and S. enteritidis by using microplate biofilm assay. According to results, antibiofilm effect was found as weak. Maximum antibiofilm effect was determined on E. cloacae ATCC 28355 with 32.92% biofilm inhibition rate. ARTICLE HISTORY Received: 17 July 2017 Revised: 30 October 2017 Accepted: 11 November 2017


Introduction
The incidence of infectious agents such as human, animal and plant pathogens has increased dramatically over the past few decades.The most common of these pathogens are developed multi-drug resistance and there is a reduction in the number of effective drugs.
Because of this, the new and effective natural compounds against pathogens are searched by scientists.Aromatic plants and their essential oils are commonly used for the treatment or prevention of disease in traditional medicine as antimicrobial agents.Moreover, their pharmacological effects such as antimicrobial, antioxidant and anticholinesterase are scientifically proven [1,2].The chemical analysis of Tanacetum species has been well documented [3][4][5][6][7][8][9][10][11].Also, the essential oils and sesquiterpenes isolated from some Tanacetum species have various biological properties, such as antifeedant, insecticidal, antimicrobial, larvicidal, cytotoxic, anticoagulant and antifibrinolytic, etc. [12][13][14][15][16][17][18][19].Plant oils in traditional medicine are used to treatment of infectious diseases.Particularly, searching of new drugs against to resistant microorganisms and/or biofilm forming microorganisms has received much attention.Because, treatment of chronic infections related with biofilm are very difficult as biofilms are exceptionally resistant to antibiotics and host immune response.Therefore, natural products have also been screened for their antimicrobial or antibiofilm activity.
T. argenteum (Lam.)Willd.subsp.Canum (K.Koch) Grierson var.canum used in our study has distributed in south Anatolian regions of Turkey [20].Its local name is Bodur Pireotu.In the literature, there are many works about the composition essential oil and sesquiterpenoids of T. argenteum subsp.canum [4,21].But, we have not found any published report on the antimicrobial and antibiofilm effects of essential oil of this subspecies.Therefore, present study will be the first scientific research to provide data that the antimicrobial and antibiofilm activity of this essential oil.Also we expect that results obtained from this study will become a good reference for detailed screening of other biological and pharmacological properties.

Material and Methods
Plant materials: T. argenteum (Lam.)Willd.subsp.canum (K.Koch) Grierson var.canum plant was collected during the flowering period and natural populations in A5 Amasya (between Direkli village and Yassıçal town, rocky areas and steppes, at 1300 m, 20.06.2010, Cansaran 5402) which is a city in the Black Sea Region of Turkey.The identification of plant was confirmed by Assoc.Prof. Dr. Arzu Cansaran according to the description given by Davis [20] and Cansaran et al. [22].Extraction of the essential oil: Air-dried aerial parts of plants were subjected to hydrodistillation for 3-5 h using a Clevenger-type apparatus to obtain essential oil in a yield of 1.905% (v/w) for T. argenteum subsp.canum var.canum based on the dry weight of the samples.The essential oil was stored in a sealed dark vial at 4 °C.

Determination of inhibitory effect by the disc diffusion method:
The antimicrobial activity of essential oil was assigned by disc diffusion method (23).100 µl suspensions of microorganisms were spread on the solid medium plates.Empty sterilized disks of 6 mm (Schleicher and Schuell, No. 2668, Germany) were each impregnated with 50 µl of essential oils.The discs injected with essential oils were placed on the inoculated agar.Petri dishes were sealed with sterile laboratory parafilm to avoid eventual evaporation of the essential oils.The dishes were left for 2 h at 4 o C to allow the diffusion of oil, and then were incubated at 37 o C for 24 h (at 30 o C for M. luteus NRRL-B-4375, at 42 o C for C. jejuni ATCC 33291 and at 28 o C for yeasts).The diameters of the inhibition zones formed on the medium were evaluated in millimetres.All the tests were performed in duplicate.The inhibition zones were compared with those of Meropenem and Nystatin as antibiotic and antifungal.

Analysis of the minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC):
MIC value of essential oils was determined by the broth microdilution method in 96-well microplates [24].95 µl of TSB, 100 µl of the essential oil and 5 µl of the culture were added into each wells.Our controls (with no cells and no essential oils) were also included on each microplate.The MIC was defined as the lowest concentration of the essential oils where no growth was visually observed.The growth of the microorganism was indicated by turbidity.After determination of the MIC, the microplates were mixed and 25 µl from each well (MIC and higher concentrations) was plated on solid medium.After 24 hours for bacteria and 48 h for yeasts, the lowest concentration of essential oil which no growth of colonies (98%) was considered as MLC.Standard powder of Meropenem and Nystatin were dissolved in distilled water and DMSO, respectively.
Antibiofilm activity: Crystal violet method was applied using 96-well polystren plate for antibiofilm activity [25].The culture suspensions was adjusted at 0.5 McFarland standard tube and 100 µl was dispensed into each well of 96-well plates in the presence of 100 µl essential oil (1/2 MIC, ¼ MIC and 1/8 MIC) or 100 µl TSB (control).The plates were incubated for 48 h at 37 °C and 30 °C.Following incubation, crystal violet staining assay was performed.Measure the optical density (OD) of each of these samples at a wavelength of 550 nm.Each experiment was performed in duplicate.And the biofilm inhibition percentage was calculated by using the following formula: [(OD growth control -OD sample) / OD growth control] x100

Results and Discussion
Undoubtedly, infectious disease agents cause major problems in the worldwide and antimicrobial resistance is a critical public health issue.Therefore, the scientific community has been focused on various fields to combat pathogens.Biological and pharmacological properties of aromatic plants and their seconder metabolites are scientifically screened to control the colonization and growing of pathogens in environment.It is the known fact that essential oils obtained from aromatic plants are very rich in complex compounds and the biological and pharmacological properties of some of them are confirmed scientifically.Moreover, it was reported their potential applications as natural and safe food preservatives or an antibacterial agent for both pharmaceutical and pesticide industries [26,27].According previous reports, camphor, borneol and 1,8-cineole (eucalyptol) were common compounds of the essential oils of Tanacetum species and antimicrobial potentials of these compounds were well determined [28][29][30][31].The antimicrobial and cytotoxic activities of caryophyllene oxide and thujone, the main constituents of the oil of T. argenteum subsp.canum var.canum [21], were also verified by some researchers [32,33].
In the present research, the antimicrobial activity of essential of T. argenteum subsp.canum var.canum collected from Amasya (between Direkli village and Yassıçal town) was examined against some pathogens on the basis of disc-diffusion and microdilution assay.The activity results, quantitatively estimated by the presence or absence of inhibition zones and also MIC and MLC values, were given in Table 1 and 2. According to the initial antimicrobial screening test, it was shown that the activity of essential oil was good against all the used organisms.But, E. coli 0157:H7, Y. enterocolitica RSKK 1501, Campylobacter jejuni ATCC 33291, Klebsiella pneumoniae ATCC 27736 and Salmonella enteritidis RSKK 171 were more resistant than other bacteria.The inhibition zones of disc, MIC and MLC values for the microorganisms were in the range of 11-32 mm, 62.5-900 µg/ml and 125-1000 µg/ml, respectively.The essential oil was more active against gram-positive bacteria than gram negative bacteria.As known, spore forming bacteria are critical issue in food industry.For example, Bacillus cereus is an important bacterium for the safety of prepared foods.Because, spores of B. cereus are found widely in environment such as soil, water, dust, etc. and they produce toxins in foods [34].In our study, Bacillus cereus NRRL-B-3711 (30 mm) and Bacillus cereus RSKK 867 (32 mm) were the most sensitive bacteria among all tested organisms.The MIC and MLC values of essential oil against these bacteria were 62.5 µg/ml and 125 µg/ml, respectively.On the other hand, it was noted that the bacteria such as Escherichia coli and Pseudomonas aeruginosa, known to be multi-resistant to antibiotics and concern to public health, were moderate sensitive to the essential oil.For example, Escherichia coli O157:H7 is an enterohemorrhagic foodborne pathogen and causes human disease [35].Campylobacter jejuni is the most important source of gastrointestinal illness.Moreover, its infection happens more frequently than do infections caused by Salmonella and Shigella species, or Escherichia coli O157:H7 [36].It is well known that such infections are fatal in children and elderly.Therefore, the use of natural antimicrobial agents to combat pathogens is important issue.Because, there are no toxic effects of the plant extracts.MIC values of the essential oil against E. coli 0157:H7 and C. jejuni ATCC 33291 were 850 µg/ml and 875 µg/ml, respectively.MIC and MLC values of essential oil against P. aeruginosa were also determined as 150 µg/ml and 300 µg/ml.Also, S. aureus was another pathogen species for public health in our study and it was found that the essential oil has strong antibacterial activity against Staphylococcus aureus ATCC 33862 (26 mm) and S. aureus ATCC 25923 (29 mm).Essential oil was active against Candida tropicalis and C. albicans.It was clear from Table 1 and 2 that essential oil of T. argenteum subsp.canum was not only active against bacteria but it was also effective against yeast species tested in present study.It is considered from this result that this oil is a broad-spectrum.Particularly, searching of new drugs against to resistant microorganisms and/or biofilm forming microorganisms has received much attention.Because, treatment of chronic infections related with biofilm are very difficult as biofilms are exceptionally resistant to antibiotics and host immune response [37].Therefore, natural products have also been screened for their antibiofilm activity.In this study, T. argenteum subsp.canum oil was also studied for its antibiofilm activity.The antibiofilm effect of the oil was tested at the ratio of 1/2, 1/4 and 1/8 MICs.It was found that, the oil exhibited moderate antibiofilm activity on E. cloacae ATCC 28355 (32.92, 29.30 and 18.40% for ½ MIC, ¼ MIC and 1/8 MIC, respectively) and less antibiofilm activity on S. enteritidis RSKK 171 (maximum 10.26%), P. fluorescens ATCC 55241 (maximum 15.62%) and Y. enterocolitica RSKK 1501 (maximum 12.54%).Moreover, any antibiofilm activity could not be determined on other bacteria and candida species (Table 3).

Conclusion
In conclusion, essential oil of T. argenteum subsp.canum var.canum showed antimicrobial and antibiofilm activity various pathogens.In the literature, there are many works about the biologic and pharmacologic activity and content of essential oils Tanacetum species.But, there was no published report about the antimicrobial or antibiofilm effect of T. argenteum subsp.canum var.canum.Since, to the best of our knowledge, no or limited data is available about antimicrobial and antibiofilm activity of this essential oil, our aim was to investigate its effect on some pathogen bacteria and yeasts.The results of the present study have confirmed our hypothesis that this essential oil has the antimicrobial and antibiofilm effect on viability of tested pathogens.Last of all, present study will be the first scientific research to provide data that the antimicrobial and antibiofilm activity of this essential oil possesses on some pathogen organisms.

Table 1 .
Growth inhibition zones of pathogens in millimetres (±standard deviation) of Tanacetum argenteum subsp.canum var.canum NT: Not tested; ATCC: American Type Culture Collection; NRRL: a culture collection of the Agricultural Research Service (ARS); RSKK: Refik Saydam National Type Culture Collection

Table 2 .
Values of MIC and MLC of Tanacetum argenteum subsp.canum var.canum essential oil