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Antimicrobial and Virulence-Modulating Effects of Clove Essential Oil on the Foodborne Pathogen Campylobacter jejuni Judit K. Kovács,a Péter Felso˝,a Lilla Makszin,b Zoltán Pápai,c Györgyi Horváth,d Hajnalka Ábrahám,e Tamás Palkovics,a Andrea Böszörményi,f Levente Emo˝dy,a,g György Schneidera Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungarya; Institute of Bioanalysis, University of Pécs, Pécs, Hungaryb; Department of Biochemistry and Medical Chemistry, University of Pécs, Pécs, Hungaryc; Department of Pharmacognosy, University of Pécs, Pécs, Hungaryd; Central Electron Microscope Laboratory, University of Pécs, Pécs, Hungarye; Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungaryf; Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungaryg

ABSTRACT

Our study investigated the antimicrobial action of clove (Syzygium aromaticum) essential oil (EO) on the zoonotic pathogen Campylobacter jejuni. After confirming the clove essential oil’s general antibacterial effect, we analyzed the reference strain Campylobacter jejuni NCTC 11168. Phenotypic, proteomic, and transcriptomic methods were used to reveal changes in cell morphology and functions when exposed to sublethal concentrations of clove EO. The normally curved cells showed markedly straightened and shrunken morphology on the scanning electron micrographs as a result of stress. Although, oxidative stress, as a generally accepted response to essential oils, was also present, the dominance of a general stress response was demonstrated by reverse transcription-PCR (RT-PCR). The results of RT-PCR and two-dimensional (2D) PAGE revealed that clove oil perturbs the expression of virulence-associated genes taking part in the synthesis of flagella, PEB1, PEB4, lipopolysaccharide (LPS), and serine protease. Loss of motility was also detected by a phenotypic test. Bioautographic analysis revealed that besides its major component, eugenol, at least four other spots of clove EO possessed bactericidal activity against C. jejuni. Our findings show that clove EO has a marked antibacterial and potential virulence-modulating effect on C. jejuni. IMPORTANCE

This study demonstrates that the components of clove essential oil influence not only the expression of general stress genes but also the expression of virulence-associated genes. Based on this finding, alternative strategies can be worked on to control this important foodborne pathogen.

C

ampylobacter jejuni has emerged as one of the most important bacterial causes of foodborne diseases in the world. It is responsible for severe gastrointestinal symptoms characterized by diarrhea, fever, abdominal cramps, nausea, and in certain cases by postinfectious sequelae, like Guillain-Barré syndrome and MillerFisher syndrome (1). The most important sources of infection are undercooked contaminated poultry meat and raw milk (2, 3). After ingestion, chemotaxis-driven motility, adhesion, invasion, and intracellular survival are the major stages during the pathogenic process (4). The helical shape of C. jejuni is postulated to be an important factor in colonization, inducing host interactions, and thus having an advantageous impact on its pathogenic attributes. The spiral morphology and motility, mediated by one or two polar flagella, steer the bacterial cell toward the surface of the epithelial cells and help it penetrate the mucinous layer (5). For the proper function of the flagellar apparatus, a sophisticated chemosensory network is required. Impaired function of one of its subunits could lead to disturbed motility and reduced colonization potential, as has been demonstrated in earlier studies based on nonflagellated mutants (6). Other authors have revealed a more complex role of flagella in C. jejuni pathogenesis mediated by its channel-forming feature also contributing to adhesion and invasion (7, 8). Adhesion onto the surface of epithelial cells is crucial for successful colonization. The role of the fibronectin-binding outer membrane protein CadF (9) and the periplasmic binding proteins PEB1 (10) and PEB4 (11) have been studied in detail, and their important role in the colonization process has been clearly dem-

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onstrated. Several other bacterial factors, like the capsular polysaccharide (CPS) (12) and sialylation of the lipooligosaccharide (LOS) outer core (13) are, however, still currently under scrutiny with respect to determining their exact roles in the pathogenic process of C. jejuni. These phase-variable surface structures not only greatly promote successful colonization but also contribute to the survival under various circumstances (14) and stress conditions (15). Compared to other bacteria, C. jejuni is more sensitive to different stresses, and for this reason, it is prone to transforming itself into a less-vulnerable and viable but nonculturable (VBNC) form that is characterized by coccoid cell morphology (16). Its enhanced sensitivity to environmental stresses was partially understood when the first genome sequences of C. jejuni became

Received 20 April 2016 Accepted 4 August 2016 Accepted manuscript posted online 12 August 2016 Citation Kovács JK, Felso˝ P, Makszin L, Pápai Z, Horváth G, Ábrahám H, Palkovics T, Böszörményi A, Emo˝dy L, Schneider G. 2016. Antimicrobial and virulencemodulating effects of clove essential oil on the foodborne pathogen Campylobacter jejuni. Appl Environ Microbiol 82:6158 –6166. doi:10.1128/AEM.01221-16. Editor: C. A. Elkins, FDA Center for Food Safety and Applied Nutrition Address correspondence to György Schneider, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AEM.01221-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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available (17). Astonishingly, this bacterium lacks the global stationary-phase regulator, the sigma factor RpoS, which induces expression of numerous proteins involved in different forms of stress response (18). Furthermore, C. jejuni also lacks the oxidative stress response regulatory elements SoxRS and OxyR (17) but expresses Dps, SodB, AhpC, and KatA in order to dispose of reactive oxygen species (ROS), and it also expresses GroES, GroEL, and DnaK in order to manage general and chemical stresses (19). There are undoubtedly permanently presenting food safety problems caused by C. jejuni, which are further aggravated by an increasing spread of antibiotic resistance among the isolates. For this reason, the search for alternative antimicrobials, both for prevention and therapy, has a pivotal role. With their remarkable antibacterial features, essential oils may represent a possible solution for these problems. Different mechanisms are known and have been hypothesized to play a role in their antimicrobial effects (20). The hydrophobic feature of certain components can cause partition of the lipids in the bacterial cell membrane, resulting in increased membrane permeability, lipid depolymerization, and therefore, disturbance in coordinated ion flow leading to decreased membrane potential and a halt in ATP synthesis. Other EO components are able to interfere with cell wall proteins usually involved in the transport of essential molecules into the cell. The exact mode of antibacterial action of most EOs, however, has not been entirely elucidated until now, although some studies have emphasized the possible importance of pore formation and subsequent oxidative stress in this process (20). The essential oil of clove is widely known for its medicinal properties as an antiseptic and analgesic in medical care. The crude essential oil of clove is classified and generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (21), and it is widely used in the food industry as a flavoring additive. Clove oil has been shown to have a wide range of antimicrobial activities against both Gram-positive and Gram-negative bacteria, including major human pathogens, like Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli (22). Clove has also been tested successfully in food systems against Listeria monocytogenes (23). Recently, clove EO has been proven to have effective antiCampylobacter activity (24–26), but until now, no further studies have been carried out with the aim to clarify the affected pathways and consequently to reveal its possible routes of antimicrobial action in C. jejuni. Eugenol, the main component, is thought to be responsible for the strong biological and antimicrobial properties of clove EO (27, 28); however, no antibacterial activities have been ascribed to the other components. The purpose of this study was to explore the potential role of clove EO in controlling C. jejuni and to identify those processes that accompany and elicit the antimicrobial effect. Sublethal concentrations of clove EO were used, as it has recently been revealed to be the most suitable concentration to elucidate those molecular changes that are in the background and may reveal the antibacterial modes of action of EOs. Cellular changes were analyzed using phenotypic, proteomic, and genomic approaches, and besides the activation of stress response genes, clove EO was found to have the potential to selectively suppress the expression of certain virulence-associated factors as well. MATERIALS AND METHODS Bacterial strains, essential oil, and culture conditions. The antibacterial effect of clove EO was screened on four reference strains (NCTC 11168,

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81168, 81-176, RM1221) and 50 human Campylobacter jejuni isolates representing independent clones (29). For detailed analysis aimed at revealing the molecular background of clove EO on C. jejuni, the extensively studied reference strain NCTC 11168 was used throughout the study. Bacteria were grown on charcoal cefoperazone deoxycholate agar (CCDA) at 42°C under microaerophilic conditions for 24 h, unless stated otherwise. For each antimicrobial experiment, Luria-Bertani broth (LB) (bacteriological 1% peptone [Oxoid, Basingstoke, United Kingdom], 0.5% yeast extract [Scharlab, Debrecen, Hungary], 0.5% sodium chloride [Reanal, Budapest, Hungary]) was used. Prior to each experiment, bacterial cell counts were synchronized by setting the optical density at 600 nm (OD600) to 1.0 (4 ⫻ 108 CFU/ml) in LB broth. C. jejuni NCTC 11168 was exposed to 330 ␮g/ml clove EO treatment for 2 h at 42°C under microaerophilic conditions, unless stated otherwise. Clove EO was obtained from Aromax Co. (Budapest, Hungary). The quality of the essential oil was consistent with the standards described in the European Pharmacopoeia (30). The compound composition of the clove EO was determined by gas chromatography-mass spectrometry (GC-MS) analyses (see Table S1 and Fig. S1 in the supplemental material). Determination of the MIC and MBC of clove EO. For identifying the MIC and minimal bactericidal concentration (MBC) of clove oil on C. jejuni, the broth dilution method was used. The suspensions (OD600, 1.0) were diluted 1:10 (OD600, 0.1), and 5-ml aliquots were placed into the wells of six-well tissue culture plates in three parallel experiments. All plates were sealed with a plastic cover to prevent evaporation of the essential oil from the wells. Each broth dilution was performed in tubes three times in triplicate. The tested clove EO concentrations ranged from 25 ␮g/ml to 6,400 ␮g/ml. No EO was added to the control wells/tubes. After 24 h of incubation, under microaerophilic conditions at 42°C, samples were taken, and the CFU were determined by dropping 10 ␮l · s⫺1 out on CCDA plates from each step of the serial dilution. The MIC was defined as the lowest concentration of clove EO completely inhibiting visible bacterial growth, while the MBC was defined as the lowest concentration that killed at least 99.9% of the presenting bacteria. Time-kill analysis. Based on the formerly obtained MIC and MBC data, a sublethal concentration (330 ␮g/ml) of clove EO was applied against strain NCTC 11168 at different incubation times ranging from 0 to 24 h. The bacterial living cell counts of control and treated samples were subsequently counted and compared in the case of (i) clove EO and three of its major compounds, (ii) eugenol, (iii) beta-caryophyllene, and (iv) alpha-humulene. Statistical analysis involved bacterial counts recovered with or without treatment after 2, 4, 6, 12, and 24 h, which were compared with a nonparametric Mann-Whitney test (two-tailed). The difference was considered statistically significant at a P value of ⬍0.05. Statistical analysis was performed using GraphPad Prism 5.0. Protein assays. (i) Protein chip analysis. Protein profiles of an untreated NCTC 11168 strain and clove EO-treated NCTC 11168 strain were analyzed in three parallel experiments by the high-sensitivity protein 250 LabChip (Agilent, Santa Clara, CA, USA). Bacterial protein preparations were made according to the manufacturer’s instructions and diluted 1:10 with standard labeling buffer (300 mM Tris-HCl [pH ⬎ 8.5]; Agilent). For fluorescent labeling, 0.5 ␮l of fluorescent dye-dimethyl sulfoxide (DMSO) (Agilent) solution was added to 4.5 ␮l of diluted sample and subsequently incubated for 10 min at room temperature. Four microliters of the 1:2 diluted sample preparation was combined with 2 ␮l of denaturing solution and then incubated at 100°C for 5 min. Centrifuged (8,000 ⫻ g for 15 min) supernatants were used for the electrophoretic analyses. For electrophoresis, the original protein analysis protocol was used. Six microliters of labeled sample was then loaded into the gel matrix. Injection was carried out at 1,000 V for 80 s, and separation was continued at 1,000 V for 60 s at 30°C. Raw data were plotted by the 2100 Expert software. Peak areas for the components were obtained by manual integration (31). Each experiment was done three times, each time in triplicate. (ii) Two-dimensional SDS-PAGE. Untreated and clove EO-treated NCTC 11168 cultures were disrupted by sonication in 50 mM Tris-HCl

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and 1 mM EDTA (Scharlab, Debrecen, Hungary) (pH 7.4). The protein concentrations of C. jejuni lysates were measured with the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Then, 100 ␮g of the total protein content of the lysates was supplemented with 2D sample buffer (8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], 50 mM dithiothreitol [DTT], 0.2% BioLyte 3/10 ampholytes, and 0.002% of bromophenol blue [all Bio-Rad]) to a total volume of 125 ␮l, and then the 7-cm immobilized pH gradient (IPG) strips (pH 3 to 10) were incubated for rehydration overnight (Bio-Rad). Isoelectric focusing (IEF) was performed by (i) 250 V, for 2 h, in linear mode; (ii) 500 V, for 2 h, in linear mode; (iii) 4,000 V, at 10,000-V · h rapid phases, followed by two equilibration steps in an equilibration buffer (6 M urea, 2% sodium dodecyl sulfate [SDS], 20% glycerol, 0.002% of bromophenol blue) with (a) 2% DTT for 10 min, and (b) 2.5% iodoacetic acid (IAA) for 10 min. Second-dimension (12% SDS-PAGE, 8 by 6 cm) electrophoresis was performed at 80 V for 20 min and at 120 V. Gels were stained with Coomassie blue R-250 (Bio-Rad) and evaluated by a Pharos FX scanner (Bio-Rad). The ProSieve QuadColor protein marker, 4.6 to 300 kDa (Lonza, Alpharetta, GA, USA), was used as a standard protein ladder. (iii) In-gel digestion. Protein bands were excised from the gel, cut into small pieces, and digested using a modified version of the protocol developed by Shevchenko et al. (32). Coomassie blue and SDS were removed with 100 mM ammonium bicarbonate (Bio-Rad, Hercules, CA, USA), and then the gel slabs were dehydrated with acetonitrile. Disulfide bridges were reduced using 10 mM DTT (Bio-Rad), and then the free -SH groups were alkylated with 55 mM iodoacetamide (Bio-Rad) solution. The modified proteins were in-gel digested with side chain-protected trypsin (Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate overnight at 37°C. The digested peptides were extracted from the gel using 5% formic acid (Sigma-Aldrich, St. Louis, MO, USA) solution in a 2:1 acetonitrile-water mixture (Sigma-Aldrich). The extracted digests were evaporated to dryness, and prior to the mass spectrometric measurements, they were dissolved in 10 ␮l of 0.1% trifluoroacetic acid (TFA) in water. (iv) LC-MS analysis. Excised spots from the gels were prepared according to Shevchenko et al. (32) for analysis with liquid chromatography (LC) using Waters nanoACQUITY ultraperformance high-performance liquid chromatography (HPLC) equipment in combination with a nanoelectrospray ionization (ESI) mass spectrometry (MS) instrument (maXis 4G ultrahigh-resolution– quadrupole time of flight [UHR-QTOF] MS; Bruker, Billerica, MA, USA). After digestion, aliquots (5 ␮l) of the samples were injected and separated on a 1.7-␮m BEH130 C18 analytical column (75 ␮m by 100 mm) using gradient elution at a flow rate of 350 nl/min. Eluent A was an aqueous formic acid solution (0.1%), and eluent B was acetonitrile-formic acid (99.9%/0.1% [vol/vol]). The scanning range was set to m/z 100 to 3,000. Nitrogen was used as nebulizer gas (60 kPa), the drying-gas flow rate was set at 4 liters/min at 180°C, and the capillary voltage was set to 3.8 kV. Each intensive peptide was fragmented, and the completed data were processed with the Data Analysis 3.4 software. Protein identification was carried out by searching for taxonomically restricted bacteria in the databases of the NCBI and Swiss-Prot using Mascot version 2.4.1. Search parameters were set in a way to allow one missed cleavage site, accepting 80 ppm mass error at the MS1 mode and 0.3 Da at the MS2 mode. We searched for variable modifications, including methionine oxidation and fixed carbamidomethylation on cysteine. Nucleic acid assays. (i) RNA isolation and cDNA synthesis. Ten milliliters of synchronized clove EO-treated and -untreated cells of strain NCTC 11168 were centrifuged (8,000 ⫻ g for 15 min) and suspended in RNAzol (Molecular Research Center, Cincinnati, OH, USA). DNA remnants were removed by DNase (Roche Ltd., Basel, Switzerland) treatment for 20 min at 30°C, and then the reaction was stopped using 2 ␮l of 0.2 M EDTA (10 min at 75°C). RNA samples were purified using the RNeasy minikit (Qiagen, Hamburg, Germany) and served as a template for cDNA synthesis applying SuperScript reverse transcriptase III (Invitrogen, Carlsbad, CA, USA). Here, 10 pg to 0.5 ␮g of extracted total RNA, 50 ␮M random primers (Applied Biosystems, Foster City, CA, USA), and 10 mM

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dinucleoside triphosphates (dNTPs; Thermo Fisher Scientific, Waltham, MA, USA) were applied. The reaction mixture was incubated for 5 min at 65°C, followed by 1 min on ice. RNA amounts were quantified using the ND-1000 NanoDrop spectrophotometer (Thermo Fisher Scientific). (ii) RT-PCR analysis. Primers for reverse transcription-PCR (RTPCR) were designed with the Vector NTI software. SYBR green (Bio-Rad, Hercules, CA, USA) master mix was used for the PCRs performed in triplicate using the Rotor-Gene 3000 (Qiagen, Hamburg, Germany) apparatus. The conditions were 15 s at 96°C, 15 s at 50°C, and then 25 s at 72°C for 45 cycles. Melting-curve analysis was performed immediately after each amplification. Each specific amplicon was verified by the presence of both a single melting temperature peak and a single band of expected size on a 2% agarose gel after electrophoresis. In the negative control, no RNA template was present. Samples were normalized using phosphoglucosamine mutase (pgm) as an internal standard. Relative nfold changes in the transcription of the examined genes between the treated and untreated samples were calculated by the 2⫺⌬⌬CT method (33). Group-wise comparison and statistical analysis of the relative expression results were performed with the Relative Expression Software Tool (REST) 2009 (34). (iii) SEM. The methods of Hazrin-Chong and Manefield (35) and Xie et al. (36) were combined and modified in order to prepare strain NCTC 11168 cells for scanning electron microscopy (SEM) analyses. At first, 100 ␮l of 2.5% (vol/wt) glutaraldehyde in phosphate-buffered saline (PBS) (pH 7.4) was added as a primary fixative solution to 100 ␮l of clove EO-treated and -untreated bacteria (OD600, 1.0), which then remained for 2 h at room temperature. After centrifugation (12,000 ⫻ g for 15 min), the supernatant was discarded, and a gradual dehydration of the bacteria was carried out by subsequent ethanol treatments (50%, 80%, and 96%), with three changes, for 10 min at each concentration. Samples were dried with 50% and 100% hexamethyldisilazane (HMDS) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min each. Bacterial cells were then mounted on aluminum stubs coated with a layer of gold using the JFC 1100 fine-coat ion sputter (Jeol, Welwyn Garden City, United Kingdom) and examined using a JSM 6300 scanning microscope (Jeol) at 16 kV and magnifications of 10,000⫻ and 20,000⫻. (iv) Motility assay. A motility assay was performed in three parallel experiments as described previously (37). The bacterial suspension was set at an OD600 of 1.0 in LB broth, and 10 ␮l of this suspension was spotted onto the middle of 0.3% agar plates lacking or containing clove EO (330 ␮g/ml and 50 ␮g/ml, respectively). After 24 h of incubation at 42°C under microaerophilic conditions, the diameter of the growth zone was measured. Determination of clove EO composition. (i) Determination of oil composition by GC-MS. Essential oil compounds were identified by the Agilent 6890N/5973N GC-MSD (Agilent, Santa Clara, CA, USA) system equipped with an Agilent HP-5MS capillary column (30 m by 250 ␮m by 0.25 ␮m). The GC oven temperature was programmed to increase from 60°C (3 min isothermal) to 200°C at 8°C/min (2 min isothermal), from 200 to 230°C at 10°C/min (5 min isothermal), and finally from 230 to 250°C at 10°C/min (1 min isothermal). The following conditions were used for the measurements: high-purity helium as carrier gas at 1.0 ml/ min (37 cm/s) in constant flow mode; ionization potential, 70 eV; and scan range, 41 to 500 A/s. Data were evaluated using the MSD ChemStation D.02.00.275 software (Agilent). GC analysis was also performed using a Fisons GC 8000 gas chromatograph (Carlo Erba, Italy), equipped with a flame ionization detector (FID). The oven temperature was increased at a rate of 8°C/min from 60°C to 230°C, with a final 5 min at 230°C. Identification of peaks was made by retention time and standard addition; percentage evaluation was carried out by area normalization. Three parallel measurements were carried out; relative standard deviation (RSD) percentages were below 4.5% (38). (ii) Active-component determination by direct bioautography. Active-component visualization and bioautography of clove EO were performed in parallel on two preconditioned (100°C for 30 min) 5 by 10-cm

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FIG 1 Demonstration of the effects of clove EO and its three major compounds on the growth of C. jejuni strain NCTC 11168. Bacterial counts recovered with 330 ␮g/ml clove (A), 222.4 ␮g/ml eugenol (making up 67.4% of clove EO composition) (B), 44.2 ␮g/ml beta-caryophyllene (making up 13.4% of clove EO composition) (C), and 18.5 ␮g/ml alpha-humulene (making up 5.6% of clove EO composition) (D) after 2, 4, 6, 12, and 24 h. In each case, they are depicted against the presence of the nontreated control. Graph shows individual datum points with means. A difference was considered statistically significant at a P value of ⬍0.05.

60G F254 thin-layer chromatography (TLC) plates (Merck, Darmstadt, Germany), as was described recently (39). Briefly, 100-␮l aliquots of essential oil were dissolved in 500 ␮l of absolute ethanol, and then 0.3- to 0.5-␮l aliquots were separated on the plates. Eugenol (Sigma-Aldrich, St. Louis, MO, USA) was used as a control (1 ␮l). After sample application, the TLC plates were developed with the mobile phase of toluene-ethyl acetate (95:5). Ascendant development was determined using a saturated twin-trough chamber (Camag, Muttenz, Switzerland). Ethanolic vanillinsulfuric acid reagent (40) was applied to visualize the separated compounds. The developed layers were dipped into this reagent and heated for 5 min at 90°C in order to remove the solvent completely. Identification of the separated compounds was performed using the ratio of fronts (Rf) and color of the standards. For bioautography, the other plate was incubated for 1 h at 42°C under microaerophilic conditions in 50 ml of an LBbacterium suspension (3 ⫻ 108 CFU/ml). The plates were then placed into an aqueous solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.05 g/90 ml) for 10 s and were further incubated for 2 h. White spots against the bluish background indicated the lack of dehydrogenase activity due to the antibacterial activity of the separated compound.

RESULTS

MIC and minimal bactericidal concentration of clove EO against C. jejuni. MICs and minimal bactericidal concentrations (MBC) of the clove EO preparation we used were determined to be 200 ␮g/ml and 800 ␮g/ml, respectively, in a 24-h experiment. Effect of incubation time on antimicrobial activity of clove EO. The growth of C. jejuni in LB was comparable to that observed in standard medium, e.g., brain heart infusion broth (data not

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shown). A concentration of 330 ␮g/ml proved to have a moderate killing effect not only on the four reference C. jejuni strains but also on all 50 clinical isolates (data not shown). In the case of strain NCTC 11168, clove EO reduced the living cell counts to approximately one-third in 2 h, to one-fourth in 4 h, and to around one-tenth in 6 h compared to the control samples. Regrowth of the bacteria after a 24-h treatment was not observed. After 2 h, 4 h, 6 h, and 24 h, the reduction in living bacterial cells was significant (Fig. 1). Clove EO-induced changes in proteome. Marked differences were identified in the total protein profile of the clove EO-treated strain NCTC 11168 by the protein chip assay, compared to the untreated bacteria. Eleven discrete peaks could be identified on the electropherogram of the untreated bacteria (see Fig. S2 and Table S2 in the supplemental material), but in order to reveal the proteins affected most in the presence of clove EO after a 2-h treatment, a 2D polyacrylamide gel electrophoresis and subsequent LC-MS analysis were carried out. Four spots that were well identifiable on the control gel presented drastically decreased expression in the profile of the clove EO-treated counterpart (Fig. 2A). Three spots corresponded to proteins known to be involved in the synthesis of three virulence-associated factors: (i) PEB1, an important factor in host colonization (10, 41); (ii) PEB4 (11), a temperature-dependent colonization factor; and (iii) HtrA, a serine protease, which has a role in adherence and invasion (42). Furthermore, a protein was identified without having any specific function. Additionally, two spots were

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FIG 2 Two-dimensional polyacrylamide gel of the total protein profile of C. jejuni strain NCTC 11168. (A and B) Untreated (A) and 330 ␮g/ml clove EO-treated (B) bacterial cells. Proteins were isolated, and samples were run on 2D PAGE using a standard protocol by applying pH gradient gels (pH 3.0 to 10.0). Proteins designated by numbers display increased expression and are identified in Table 2.

found manifesting elevated expression levels compared to the control (Fig. 2B). They were identified as a chaperonin (43) and the elongation factor Tu (44) (Table 1). Peptide mass fingerprints of the digested proteins and detailed information on the identified proteins are presented in Fig. S3 in the supplemental material. Gene expression profile of C. jejuni in response to clove EO. Altogether, 45 genes (see Table S3 in the supplemental material) were targeted by reverse transcription-PCR, with the aim to reveal their incidental alterations in expression as a result of clove EO treatment. These genes are known to be involved in (i) stress responses, (ii) pathogenic processes, and (iii) basic metabolism (housekeeping genes). Regarding the stress genes, it was revealed that four of them were evidently upregulated (Table 1). The katA gene (45), encoding catalase, and groEL (43), encoding a molecular chaperone, proved to be upregulated 51- and 20-fold, respectively. Furthermore, groES, encoding a cochaperonin, and dnaK (46), encoding

TABLE 1 Significant transcription intensity changes of open reading frames of C. jejuni NCTC 11168 in the presence of clove EOa

Gene ID/name

Direction of change in expression level

Fold change

Cj1385/katA Cj1220/groES Cj1131c/galE Cj0335/flhB Cj0536/oorA

Up Up Down Down Down

51.967 3.937 0.158 0.131 0.104

Cj0415 Cj1221/groEL Cj0759/dnaK C8J_0494/sucC Cj0268c Cj0891c/serA Cj1259/porA Cj0670/rpoN Cj0061c/fliA

Down Up Up Down Down Down Down Down Up

0.195 20.350 3.704 0.050 0.206 0.21 0.33 0.32 1.73

Specific functionb Catalase Cochaperonin GroES UDP-GlcNAc/Glc 4-epimerase Flagellar biosynthesis protein FlhB 2-Oxoglutarate-acceptor oxidoreductase subunit OorA GMC oxidoreductase subunit Molecular chaperone GroEL Molecular chaperone DnaK Succinyl-CoA synthetase beta-subunit Transmembrane protein D-3-Phosphoglycerate dehydrogenase Major outer membrane protein RNA polymerase factor sigma-54 Flagellar biosynthesis sigma factor

a Transcripts of the selected 45 genes were quantified by real-time quantitative PCR (RT-qPCR), and data were analyzed using the comparative critical threshold (⌬⌬CT) method. Statistically significant changes (P ⬍ 0.001) in transcription levels were observed for 14 open reading frames. b GMC, glucose-methanol-choline; CoA, coenzyme A.

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another chaperone, were upregulated to similar degrees, with nearly 4-fold upregulation (3.937 and 3.704, respectively). The results of the RT-PCR showed that at least two virulenceassociated genes were downregulated in the presence of clove EO. The UDP-glucose 4-epimerase (galE) (47), involved in the lipooligosaccharide (LOS) biosynthesis, and flhB (8), which codes for a flagellar biosynthesis protein, were downregulated nearly 7-fold. A 3-fold downregulation was observed in the case of porA, a major outer membrane protein gene possessing strong antigenic capacity (48). Expression of the three known global transcriptional regulators of C. jejuni (49) was influenced in different ways. The rpoN (␴54) was downregulated to one-third, in contrast to fliA (␴28), which was 1.73 times upregulated, while expression of rpoD (␴70) remained unaltered (data not shown). The investigated housekeeping genes were transcribed in equal rates in both treated and untreated cells. Effect of clove EO on C. jejuni morphology. The influence of clove EO (330 ␮g/ml) on C. jejuni morphology was examined by electron microscopy. After a 2-h clove EO treatment, the originally curved C. jejuni cells (Fig. 3A and B) presented with a shrunken and straightened morphology (Fig. 3C and D). No significant amounts of coccoid forms were found as described by others in relation to other environmental stresses (36). Effect of clove EO on the motility of C. jejuni. After a 24-h microaerophilic incubation, untreated cells showed a 2-cm-diameter swarming area. Surviving C. jejuni cells treated with the generally applied 330 ␮g/ml of clove EO completely lost their ability to move. If the subinhibitory (50 ␮g/ml) concentration of clove EO was applied, bacterial cells survived but showed a decreased capacity to swarm. Demonstration of the antibacterial activities of clove essential oil components. Applying TLC with ethanolic vanillin-sulfuric acid reagent, eight constituents could be observed. The major component, eugenol, was identified as an orange-brown zone (Rf ⫽ 0.58) (Fig. 4). Direct bioautography revealed that at least 1 characteristic and 3 minor spots on the TLC layer possessed antibacterial activities against C. jejuni. Besides the major and the most frequently observed components of clove EO (see Table S1 in the supplemental material), GC analyses of the other three spots revealed the presence of the components in various levels of excess, e.g., linalool, terpinen-4-ol, and calamenene (data not shown).

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FIG 3 Scanning electron microscopic images of C. jejuni strain NCTC 11168. (A to D) Untreated (A and B) and 330 ␮g/ml clove EO-treated (C and D) cells. C. jejuni showed the typical slightly curved spiral morphology, while clove EO-treated cells became shortened and less intensely curved. The scale bar at the lower right corner of each image represents 2 ␮m.

DISCUSSION

FIG 4 Compound composition of clove EO and their antimicrobial effects were revealed by thin-layer chromatography-based analyses. Separated components were visualized by ethanolic vanillin-sulfuric acid reagent (1 and 2), while antibacterial effects of the compounds against C. jejuni were revealed by bioautography developed with a tetrazolium salt-base reagent (MTT) (3 and 4). In both cases, eugenol was used as a reference (2 and 4). Sample application: lanes 1 and 3 containing 0.5 ␮l of clove EO (100 ␮l/ml); lane 2 containing 1 ␮l of eugenol as standard (1 mg/ml).

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Foodborne diseases, such as campylobacteriosis, are a growing public health concern across the globe (50). In this study, the molecular background of the antibacterial effects of clove EO were first investigated in C. jejuni, and this is the first extensive study on the antibacterial mechanisms of an essential oil. It is a widely accepted view that the antibacterial effect of EOs is the consequence of pore formation and a subsequent oxidative stress (20). Although the 51.9-fold activation of katA could be a clear indication of that (51), the expression of three other important genes (dps, sodB, and ahpC) involved in oxidative stress responses (52) did not increase (Table 1). It suggests that although an oxidative effect is present, the induced dominant stress response is a general phenomenon. Overexpression of groEL (43) and dnaK (45, 51), two important molecular chaperons, characteristic of the general stress response, also supports this hypothesis. It is also in accordance with recent findings (43), arguing against the role of groEL in the oxidative stress response. Additionally, the upregulation of dnaK, one of the three members of the prokaryotic Hsp70 family, has been demonstrated in chemical stresses (45, 46). Campylobacter is prone to respond to stresses by the transformation into a more resistant, viable, and potentially pathogenic but nonculturable (VBNC) state (16). The VBNC state is developed by forming rounded cells from the normal spiral form, due to, as has been described, a low-osmolarity medium (53), oxidative stress (54), low or high temperatures (55, 56), and the presence of zinc oxide (36). We found that as a result of clove EO

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TABLE 2 LC-MS analysis of proteins showing increased expression on 2D SDS-PAGE separation Spot no.a

Protein

Mascot score

Molecular mass (kDa)

No. of peptides

Uni-Prot or NCBI accession no.

1 2 3 4 5 6

Major antigen Peb4A Major cell-binding factor Peb1A Uncharacterized protein Serine protease HtrA 60-kDa chaperonin Elongation factor Tu

497.4 964.1 201.6 555.0 694.6 269.5

30.4 28.2 21.0 51.0 57.9 44.9

11 22 5 15 27 9

CBF2_CAMJJ PEB1A_CAMJE Q5HW47_CAMJR gi|57238208 CH60_CAMJE gi|488955902

a

For spot numbers, see Fig. S2 in the supplemental material. (See also Fig. 2.)

treatment, the bacteria became shrunken and straightened (Fig. 3). A similar morphology has been described in C. jejuni as a result of a 10- to 30-h exposure to atmospheric concentrations of oxygen (57). Some essential oils have been described as having modulating effects on the morphology of other bacteria: cumin EO on Klebsiella (58), lemon EO on E. coli (59), and oregano EO on Pseudomonas aeruginosa and Staphylococcus aureus (60). In line with these findings, our observations raise the possibility of one or some components of EOs having an effect on the structures determining cell shape, e.g., peptidoglycan (61) and certain other proteins, mostly those anchored in the cell membrane. The altered morphology can spoil the pathogenic potential, especially in the case of C. jejuni, in which the helical structure was revealed to be acutely advantageous during the pathogenic process (5). Clove has this kind of potential. Furthermore, we also revealed that eugenol alone was able to evoke this change in morphology, in contrast to alpha-humulene, another compound of clove EO we investigated in this study. Alpha-humulene has neither an influence on morphology nor an influence on viability (data not shown). The effect of beta-caryophyllene was, however, mixed, since it showed a mild effect on cell morphology but did not have any effect on proliferation (Fig. 1). In the case of clove treatment on C. jejuni, we detected the dominance of a general stress response. This type of response seems to be essential for the C. jejuni cell for survival, downregulating its virulence-associated gene pool. Our results are in accordance with the reports on perilla oil, affecting exotoxin production in S. aureus (62), and oregano, affecting Shiga toxin production in enterohemorrhagic Escherichia coli (63). A more detailed analysis was carried out with Cronobacter sakazakii in the presence of two garlic-derived organosulfur compounds, ajoene and diallyl sulfide, both possessing antibacterial features (64). By using whole-transcriptome analysis and Raman spectroscopy, the authors revealed that the chemical stresses evoked by these two compounds differ fundamentally. While ajoene caused the downregulation of virulence-associated motilityrelated genes, diallyl sulfide treatment resulted in an increased expression of cell wall synthesis genes and promoted cellular defense mechanisms. A similar metabolic shift toward the suppression of virulenceassociated factors was observed at low temperatures (65) and under starvation conditions (66). Under these conditions, the bacterium cell strictly focuses on survival by maintaining its basic metabolic functions and by not wasting energy on virulence. The observed low virulence gene expression and unaltered expression levels of all the 11 investigated housekeeping genes (67) (see Table S3 in the supplemental material) during clove treatment suggest that to facilitate survival, the stressed C. jejuni cell tries to stabilize

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its basic metabolic functions. Marked upregulation of elongation factor thermounstable (EF-Tu) (Table 2) also points to the abovementioned findings, since the crucial role of EF-Tu in ensuring translational accuracy is more pronounced under stress conditions when the proper function of housekeeping genes is a matter of life or death (44). Chemotaxis, motility, adhesion, invasion, and intracellular survival are the major stages in the pathogenic process of C. jejuni (4). Detection of the downregulation of flhB by RT-PCR was a clear indication of clove EO having an effect on motility, which was also verified by the motility assay. Knowing that the buildup in the flagellar apparatus is very complex (49), and that its proper function is based on a sophisticated chemosensory network, we tend not to believe that flhB is the only affected gene in this machinery. Our results suggest the involvement of sub-sigma factor targets, since flhB, a class I flagellar gene, proved to be downregulated, although the expression of its sigma factor rpoD (␴70) was constant (49). Since FlhB, through its channel-forming feature, was able to inhibit the secretion of Cia proteins (68), and downregulation of PEB1 (10) and PEB4 (11) was also shown, our results demonstrate that clove has a multitargeted effect on the pathogenic process of Campylobacter jejuni, affecting not only morphology and motility but also adhesion and invasion. It is, however, highly probable that these effects of clove EO are not evoked by one component only. An antibacterial effect had already been ascribed to its major component eugenol (69), which we also confirmed. Besides, we have revealed that beta-caryophyllene and alpha-humulene, the only two representative compounds present at ⬎5% (see Table S1 in the supplemental material), have not shown any antibacterial features (Fig. 4) against C. jejuni NTCC 11168, but the results of the bioautography experiments suggest that at least one additional minor compound of clove EO has to possess antibacterial features. We can conclude that although clove EO treatment elicited a marked general stress response, oxidative stress was also firmly present. Components of clove EO were demonstrated to selectively influence the expression of certain genes involved in stress and virulence. With respect to flagellar function, this observation was also confirmed by a functional assay. A more systematic study with separated clove EO components could identify the affected groups of genes or proteins in order to precisely locate potential target sites on and in the Campylobacter cell. ACKNOWLEDGMENTS We wish to thank Ferenc Kilár, László Seress, and László Márk for the availability of scanning electron microscopy, protein chip, and LC-MS facilities, respectively, and Béla Kocsis for all his practical advice in con-

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nection with bioautography. The authors thank Valéria Szíjártó for her help with the statistical analysis. This work was financially supported by the Hungarian Government (MFCDiagn–TECH_08-A1-2008-0279), the Hungarian Scientific Research Fund (OTKA) grant PD 104660, and by the University of Pécs (PTE ÁOK-KA-2013/23). The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary.

16. 17.

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