Attenuation of Adhesion, Biofilm Formation and Quorum Sensing of Campylobacter jejuni by Euodia ruticarpa Katja Bezek, 1,2 Marija Kurinčič, 1 Elvira Knauder, 3 Anja Klančnik, 1 Peter Raspor, 2 Franz Bucar 3 and Sonja Smole Možina 1 * 1 Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana 1000, Slovenia 2 Faculty of Health Sciences, University of Primorska, Polje 42 SI, Izola 6310, Slovenia 3 Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Graz 8010, Austria Thermophilic campylobacters are a major cause of bacterial food-borne diarrhoeal disease. Adherence and bio- film formation are key elements of Campylobacter jejuni persistence in unfavourable environmental conditions. The phytochemical analysis of Euodia ruticarpa fruit ethanol solution extract (EREE) indicated that the major compounds were evodiamine (1), rutaecarpine (2) and evocarpine (9). E. ruticarpa fruit ethanol solution extract, compounds 1 and 2 as well as a mixture of quinolinone alkaloids with 41.7% of 9 were tested for antibacterial, antibiofilm and antiquorum sensing activities against C. jejuni. Minimal inhibitory concentrations varied from 64 to 1024 μg/mL. A mutant strain that lacks the functional gene coding for the CmeB efflux pump protein was the most susceptible. Interestingly, in addition to the wild-type (NCTC 11168) and cmeB mutant, also a mu- tant that lacks autoinducer-2 production (luxS) was able to adhere (1 h) and to produce a biofilm (24, 48 and 72 h). The subinhibitory concentrations of all preparations at least partly inhibited C. jejuni adhesion and biofilm formation with the most visible effect of the quinolinone alkaloid fraction. Using a Vibrio harveyi luminescence assay, the inhibition of autoinducer-2 production was observed in the wild-type and cmeB mutant after 48 h with the most visible effect of EREE and its fraction Q. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: Euodia ruticarpa; Campylobacter jejuni; biofilm; quorum sensing. INTRODUCTION The food-borne pathogen Campylobacter jejuni is one of the leading causes of bacterial gastrointestinal infections in the world (Reuter et al., 2010) and is the most commonly reported food-borne pathogen in the European Union (EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control), 2015). This rather sensitive microorganism can however at- tach to and form a biofilm on a variety of surfaces (Joshua, 2006; Teh et al., 2014). Moreover, attached bacterial cells enclosed in such an extracellular polymeric matrix are more protected from the stressful environmental condi- tions than their planktonic counterparts (Joshua, 2006). The presence of efflux systems is one of the key resistance mechanisms in Gram-negative bacteria, and its contribu- tion to biofilm and quorum sensing (QS) has also been highlighted before (Soto, 2013). The CmeABC efflux pump contributes to intrinsic and acquired resistance to various antimicrobials in C. jejuni (Lin et al., 2002). For some natural phenolic compounds, it has already been sug- gested to be the substrates of CmeABC efflux pump in C. jejuni while inactivation of the transport protein (cmeB) resulted in higher susceptibility (Klančnik et al., 2012). Therefore, a compound that could circumvent this obstacle and inhibit the attachment and biofilm formation would be beneficial in the post-antibiotic era. While biofilm formation is a cooperative activity that includes communication between bacterial cells, the use of QS inhibitors would be of significant interest in influencing bacterial pathogenesis (Li and Tian, 2012). The enzyme LuxS homologous protein, which is involved in the synthesis of autoinducer-2 (AI-2), was found in C. jejuni (Elvers and Park, 2002). Moreover, the presence of AI-2 plays an important role in the development of biofilm by C. jejuni (Reeser et al., 2007). Because the frequency and severity of food-borne outbreaks, there is an increasing interest in introducing an effective antimicrobial compound that would be not only food admissible but also a step towards eliminating microbial resistance to antibiotics (Friedman et al., 2002). One of the promising ways to reduce health hazards and economic losses due to the food-borne microorgan- isms is the use of natural and organic antimicrobials, such as natural plant compounds (Sirsat et al., 2009). In partic- ular, ethanol solution extracts of different plants have been proposed to control food pathogens (Serio et al., 2014) and multidrug resistant bacteria (Valle et al., 2016). Euodia ruticarpa (A. Juss.) Benth. (Tetradium ruticarpum (A.Juss.) T.G.Hartley; Rutaceae) has long been appreciated for its bioactive components, serving as a rich source of quinolinone and indoloquinazole alkaloids, with evodiamine (1), rutaecarpine (2) and cis-evocarpine (9) being the most important ones (Huang et al., 2012). Moreover these compounds exhib- ited promising antimicrobial effects (Pan et al., 2014; Wang et al., 2013). The aim of the study was to test the biofilm formation of C. jejuni NCTC 11168 wild-type, cmeB and luxS mutants on stainless steel (SS) surface * Correspondence to: Sonja Smole Možina, Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, Ljubljana SI-1111, Slovenia. E-mail: sonja.smole@bf.uni-lj.si PHYTOTHERAPY RESEARCH Phytother. Res. (2016) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5658 Copyright © 2016 John Wiley & Sons, Ltd. Received 24 February 2016 Revised 01 May 2016 Accepted 05 May 2016