Göse, Hacıoğlu Doğru, 2019, Biologica Nyssana 10(2) 10 (2) December 2019: 169-173 DOI: 10.5281/zenodo.3600197 Antibiofilm activity of Verbascum pinnatifidum Vahl. ethanolic extract Original Article Mehmet Göse Department of Plant Physiology, Institute of Biology, Graduate School of Natural and Applied Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey m_gose@hotmail.com Nurcihan Hacıoğlu Doğru Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey nurcihan.n@gmail.com (corresponding author) Received: July 1, 2019 Revised: September 9, 2019 Accepted: September 13, 2019 Abstract: Microbial biofilms pose health risks in clinical environments, food industry and drinking water systems. Microorganisms within biofilms are more resis- tant to antibiotics and chemical agents than planktonic cells in suspension. New alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. Here, we investigated in vitro antibiofilm activity of ethanol extract of Verbas- cum pinnatifidum Vahl. against Escherichia coli NRRL B-3704, Pseudomo- nas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 and Candida albicans ATCC 10231 test microorganisms based on crystal violet binding assay. The highest antibiofilm activity was shown against biofilm formed by B. subtilis ATCC 6633 and S. haemolyticus ATCC 43252 at the lowest MIC values of 2.5 and 10 µg/mL, respectively. The current findings indicated that bacterial biofilm formation can be potentially managed using V. pinnatifidum plant extracts. Key words: antibiofilm activity, Verbascum pinnatifidum Apstract: Antibiofilm aktivnost etanolnog ekstrakta Verbascum pinnatifidum Vahl. Biofilmovi mikroorganizama predstavljaju zdravstveni rizik u kliničkim uslovima, prehrambenoj industriji i sistemima pijaće vode. Unutar biofilmova, mikroorganizmi su rezistentniji na antibiotike i hemijske agense od planktonskih ćelija u suspenziji. Predložene su nove alternative za kontrolisanje infekcija, koje su fokusirane na terapeutske osobine medicinskih biljaka i njihovu antibiofilm aktivnost. Ovde je, korišćenjem kristal violet testa, vršeno istraživanje in vitro antibiofilm aktivnosti etanolnog ekstrakta Verbascum pinnatifidum Vahl. u odnosu na vrste Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 i Candida albicans ATCC 10231. Najveća antibiofilm aktivnost, sa najnižim MIK vrednostima od 2,5 i 10 µg/mL, utvrđena je za biofilmove formirane od strane vrsta B. subtilis ATCC 6633 i S. haemolyticus ATCC 43252, respektivno. Ovi nalazi ukazuju na to da stvaranje bakterijskih biofilmova potencijalno može biti sprečeno korišćenjem ekstrakta V. pinnatifidum. Ključne reči: antibiofilm aktivnost, Verbacum pinnatifidum Introduction Microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (Vasudevan, 2014). They are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses (Grant and Hung, 2013). Biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type IV secretion systems, decreased permeability, and the action of antibiotic-modifying enzymes (Alekshun and Levy, 2007). The increased biofilm resistance © 2019 Göse, Hacıoğlu Doğru. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non- commercially under the same license as the original. 169 13th Symposium on the Flora of Southeastern Serbia and Neighboring Regions to conventional treatments enhances the need to develop new control strategies (Simões et al., 2007; Sanchez et al., 2016). Verbascum plants have been used medicinally since ancient times in folk medicine as a remedy for respiratory problems such as bronchitis, dry coughs, whooping cough, tuberculosis, and asth- ma. The leaves, roots and the flowers have been used also as anodyne, sedative, diuretic, sudorific, expectorant and antidiarrheal agents in tradition- al World and Turkish medicine (Baytop, 1999; Georgiev et al., 2011). Therefore Verbascum plant species have been also previously reviewed for their antiviral, antimicrobial, antimalarial, antioxi- dant, anti-inflammatory, antinociceptive, antitumor, anticancer, cytotoxic, immunomodulatory, anticho- linesterase, antiulcerogenic, antihepatotoxic, anti- hyperlipidemic, anthelminthic, antitussive and an- tigermination activities (Tatli and Akdemir, 2006; Dulger and Hacioglu, 2009; Kahraman et al., 2010; Kahraman et al., 2011; Kozan et al., 2011; Ozcan et al., 2011; Boğa et al., 2016). However, there is no any literature about Verbascum plants antibiofilm activity, except Moghaddam et al. (2015), which evaluated the antibiofilm activity of Verbascum pin- natifidum Vahl. ethanol extract. Material and methods Plant materials Verbascum pinnatifidum was collected from Canakkale, Kumkale near the lantern d.s. sandy area, (40.008101 N, 26.203127 E) in 2018 and identified with the aid of Flora of Turkey (Davis et al., 1988) by Dr. Ersin Karabacak. Voucher specimens were deposited in the Biology Department at Çanakkale Onsekiz Mart University, Çanakkale, Turkey. Preparation of plant extracts The plant parts were air-dried. Dry powdered plant material (10 g) was extracted with 300 mL of 80% ethanol (Merck, Darmstadt, Germany) for 24 h by using Soxhlet equipment (Khan et al., 1988). The extracts were filtered using Whatman filter no.1, and the filtrates were then evaporated under reduced pressure and dried using a rotary evaporator at 55 oC. Dried extracts were stored in labelled sterile screw- capped bottles at + 4 oC. Test Microorganisms Gram negative bacteria - Escherichia coli NRRLB 3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606 and Gram positive bacteria – Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, S. haemolyticus ATCC 43252 and yeast culture - Candida albicans ATCC 10231 were used as test microorganisms. Minimum inhibitory concentration assay To determine the plant extract doses to be used in biofilm inhibition study, Minimum inhibitory concentration (MIC) values of all samples were determined. MIC was investigated as recommended instruction of the Clinical and Laboratory Standards Institute (CLSI, 2006). The lowest concentration of extract inhibiting the visible growth of each test microorganisms was taken as the MIC. The medium, 0.1% (w/v) Streptomycin (ST), Nystatin (NYS100) and 10% DMSO were used as the non- treated, positive and negative controls, respectively (Teanpasian et al., 2017). Biofilm inhibition assay Microplate biofilm method (Merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by V. pinnatifidum plant ethanol extract against test microorganisms. Cultures were incubated in 5 mL Tryptic Soy Broth (TSB) medium containing 5% glucose. Cultures were diluted 1:100 in TSB and loaded into each well in 4 sterile microplates. Different concentrations of plant extract (MIC and sub-MIC concentrations: 50, 25, 12.5% of MIC) were prepared and transferred to each microplate well. After incubation at 37 ± 0.1°C for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. 200 μL of 0.1% crystal violet solution was added to each well (20 minutes). The crystal violet bounded extracts were poured and washed until the crystal violet removed. The microtiter plates were inverted, and the remaining liquid was drained and dried in room heat. Finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%), and the absorbance was measured at 550 nm by using an automated ELISA reader. All experiments were repeated thrice in triplicate. The measurement of the antibiofilm effect of the extract was made by the percentage reduction formulation. % Inhibition = (Acontrol – Asample / Acontrol) x 100 Acontrol: Absorbance of the control (containing 100 µL TBS instead of plant extract) reaction Asample: Absorbance of the test compound Results and discussion Ethanol extract of V. pinnatifidum was tested against eight clinical bacterial and fungal strains (Tab. 1). Ethanol was observed as the best solvent for extracting antimicrobial substances in a previous study (Jonathan and Fasidi, 2003). Results of MIC were quite variable between each test microorganisms ranging from 2.5 to 20.0 µg/ 170 BIOLOGICA NYSSANA ● 10 (2) December 2019: 196-173 Göse, Hacıoğlu Doğru ● Antibiofilm activity of Verbascum pinnatifidum Vahl. ethanolic extract Biofilm is considered to be one of the essential virulence factors that cause persistent chronic and recurrent infections, because of their high resistance to antibiotics and host immune defensive system (Grant and Hung, 2013). Bacteria are more resistant to antibiotics than planktonic cells because they are preserved by exopolysaccharide. This necessitated the screening of new and natural antibiotic sources in the fight against biofilm. New alternatives for controlling infections have been proposed focusing on the therapeutic properties of medicinal plants and their antibiofilm activities. 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 (Erdönmez et al., 2018). Biofilm formation can be controlled by quorum sensing, a bacterial communication system which causes a rapid and coordinated change of expression pattern in the bacterial population in response to population density (Pratiwi et al., 2015). The fact that at MIC and sub-MIC concentrations, V. pinnatifidum extract is capable of disturbing biofilm formation and causes biofilm breakdown, suggests that this disturbance may have been caused by the presence of compounds that inhibit quorum sensing. Other plant compounds could attenuate biofilm development by inhibiting bacterial peptidoglycan synthesis (Ogunlana et al., 1987), disrupting the permeability barrier of microbial membrane structures, causing the cell to leak out (Cox et al., 2000), modify bacterial membrane structure hydrophobicity (Türi et al., 1997; Das, 2014), or disturbing the extracellular polymeric matrix in the biofilm to release biofilm from the surface of the solid substratum (Traba and Liang, 2011; Pratiwi et al., 2015). Further studies need to be performed mL (Tab. 1). It was observed that almost all tested microorganisms were sensitive towards the ethanol extract of V. pinnatifidum. There is no data about V. pinnatifidum antimicrobial activity in the literature. Our findings about MIC ranges confirmed the observations of some other researchers, which stated that some Verbascum species have antimicrobial activity against Gram positive and negative bacteria and yeast and mold cultures (Dulger and Hacioglu, 2008;2009; Ozcan et al., 2010; Morteza-Semnani et al., 2012; Noori et al., 2012; Anil et al., 2016; Dulger and Dulger, 2018). However, we found that V. pinnatifidum extract was also effective against E. coli NRRLB 3704 and C. albicans ATCC 10231, contrary to some studies with the other Verbascum species in the literature (Dulger and Hacioglu, 2008; Morteza-Semnani et al., 2012; Amin et al., 2015). The results of potential inhibition of test microorganism’s biofilm formation by ethanol extract of V. pinnatifidum were shown in Tab. 1. The results indicated that V. pinnatifidum extract in 2.5 to 10.0 µg/mL concentrations could inhibit the biofilm formation of all the tested microorganisms except E. coli NRRLB 3704 and C. albicans ATCC 10231. Throughputs also showed that antibiofilm function of extract was in a dose-dependent manner. The highest antibiofilm activity was noticed against biofilm formed by B. subtilis ATCC 6633 and S. haemolyticus ATCC 43252 at the lowest MIC values of 2.5 µg/mL and 10 µg/mL, respectively. Verbascum pinnatifidum has not been investigated in terms of antibiofilm activity. In a study conducted by Moghaddam et al. (2015), ethanol extract of V. thapsus had inhibitory effect on biofilm formation of Streptococcus mutans, S. sanguinis, and S. salivarius. The current findings indicated that biofilm forming of bacteria could be potentially managed using V. pinnatifidum plant extracts. 171 BIOLOGICA NYSSANA ● 10 (2) December 2019: 169-173 Göse, Hacıoğlu Doğru ● Antibiofilm activity of Verbascum pinnatifidum Vahl. ethanolic extract Table 1. The percentage of inhibition of biofilm structure of test microorganisms MIC (µg/mL) % Inhibition of biofilms formation Test Microorganisms Plantextract Control ST/ NY100 MIC MIC/2 MIC/4 MIC/8 E.coli NRRLB 3704 20.0 4.0 7.27±0.33 3.34±0.08 - - P. aeruginosa ATCC 27853 10.0 1.0 74.65±6.23 65.33±5.67 43.56±4.22 34.76±1.76 P. vulgaris ATCC 13315 10.0 4.0 70.50±0.54 60.03±1.55 45.54±1.00 40.01±0.23 A. baumanii ATCC 19606 10.0 2.0 57.42±0.12 43.12±0.10 - - B. subtilis ATCC 6633 2.5 4.0 90.02±0.01 70.01±0.10 55.78±1.23 43.26±1.43 S. aureus ATCC 6538P 10.0 4.0 70.54±0.60 - - - S. haemolyticus ATCC 43252 10.0 5.0 76.12±3.11 58.41±3.12 44.45±4.11 23.56±1.56 C.albicans ATCC 10231 2.5 2.5 10.45±1.14 5.45±0.99 - - -: No inhibition of biofilm formation was observed due to the lack of antibiofilm effect. to confirm the actual mode of action of anti-biofilm activity from these extracts. Conclusion Our investigation was the first report on the antibiofilm property of the V. pinnatifidum ethanol extract. In this research, we found that forming of bacterial biofilm could be potentially being managed using V. pinnatifidum plant extract, especially against Gram (+) bacteria. Detailed phytochemical investigations are required to determine the types of substances responsible for the biological activities of V. pinnatifidum plant species. We hope that our results will provide useful data for discovering new compounds with better activity than agents currently available. Acknowledgments. This investigation is a part of Master thesis of Mehmet GÖSE. This study was financially supported by the Çanakkale Onsekiz Mart University Scientific Research Projects Coordination Unit, Turkey (FYL-2018-2693). This paper was presented at the Symposium on The Flora of Southeastern Serbia and Neighboring Regions, June, 20-23 2019, Serbia. Authors would like to thank Assoc. 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