Essential Oils and Eugenols Inhibit Biofilm Formation and the Virulence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) has caused foodborne outbreaks worldwide and the bacterium forms antimicrobial-tolerant biofilms. We investigated the abilities of various plant essential oils and their components to inhibit biofilm formation by EHEC. Bay, clove, pimento berry oils and their major common constituent eugenol at 0.005% (v/v) were found to markedly inhibit EHEC biofilm formation without affecting planktonic cell growth. In addition, three other eugenol derivatives isoeugenol, 2-methoxy-4-propylphenol, and 4-ethylguaiacol had antibiofilm activity, indicating that the C-1 hydroxyl unit, the C-2 methoxy unit, and C-4 alkyl or alkane chain on the benzene ring of eugenol play important roles in antibiofilm activity. Interestingly, these essential oils and eugenol did not inhibit biofilm formation by three laboratory E. coli K-12 strains that reduced curli fimbriae production. Transcriptional analysis showed that eugenol down-regulated 17 of 28 genes analysed, including curli genes (csgABDFG), type I fimbriae genes (fimCDH) and ler-controlled toxin genes (espD, escJ, escR, and tir), which are required for biofilm formation and the attachment and effacement phenotype. In addition, biocompatible poly(lactic-co-glycolic acid) coatings containing clove oil or eugenol exhibited efficient biofilm inhibition on solid surfaces. In a Caenorhabditis elegans nematode model, clove oil and eugenol attenuated the virulence of EHEC.

and of adhesive factors. For example, carvacrol and eugenol 14 , grapefruit limonoids 15 , β-sitosterol glucoside from clementine peel 16 , ginkgolic acids from Ginkgo biloba 17 , and cinnamaldehyde and eugenol from cinnamon bark oil 18 , which are found in essential oils, have been reported to inhibit EHEC biofilm formation. However, few studies have been undertaken to compare the antibiofilm characteristics of large numbers of essential oils.
In this study, 83 essential oils were initially screened for their ability to inhibit EHEC biofilm formation. Three oils, namely, bay, clove, and pimento berry oils demonstrated strong anti-biofilm activity against EHEC at sub-inhibitory concentrations, but not against laboratory E. coli strains. Chemical structure-activity assays revealed that eugenol and three other eugenol derivatives had anti-biofilm activity. In order to understand their action mechanisms, transcriptional analysis, motility analysis, and electron microscopy were utilized. In addition, a biocompatible poly(lactic-co-glycolic acid) surface coatings containing biofilm inhibitors were prepared and their antibiofilm effects were examined. Finally, an in vivo Caenorhabditis elegans model was used to study the effects of eugenol and of clove oil to confirm their antivirulence effects on EHEC.

Results
Anti-biofilm effects of essential oils against EHEC. To identify new anti-biofilm agents, 83 essential oils were initially screened in 96-well plates at a concentration of 0.005% (v/v) to minimize antimicrobial effects. Several essential oils were found to inhibit EHEC biofilm formation, but with widely different efficiencies. Detailed information on EHEC growth and biofilm formation in the presence of the 83 essential oils is provided in Supplementary Table S1. Notably, four essential oils, namely bay, cinnamon bark, clove, and pimento berry oil inhibited EHEC biofilm formation by more than 75%. No growth reduction of EHEC cells above 30% at OD 620 was observed at 0.005% (v/v) as compared with untreated controls. Kim et al. found that cinnamon bark oil 18 had antibiofilm activity against EHEC, but this is the first time that bay, clove, and pimento berry oils have been reported to have antibiofilm activity. In the present study, more detailed study showed bay, clove, and pimento berry oil all dose-dependently inhibited EHEC biofilm formation in 96-well polystyrene plates (Fig. 1a-c). Since bacteria form biofilms on the bottoms and sides of these plates, confocal laser microscopy and EHEC expressing green fluorescent protein were used to observe biofilm formation on glass, and our microscopic observations confirmed that all three essential oils dramatically inhibited biofilm formation on the bottom of glass (Fig. 1d). Biofilm inhibition was further confirmed by COMSTAT analysis. More specifically, bay, clove, and pimento berry oils reduced all three measured parameters (biomass, mean thickness, and substratum coverage) of EHEC (Table 1), and biomass (volume/area) and mean thickness were reduced by > 80% by all three oils at 0.005% (v/v).

Identification of the active anti-biofilm components in essential oils.
To identify the active anti-biofilm components in the above three essential oils, GC-MS analysis was performed and as a result 33 different compounds were identified (Table 2). Eugenol was the predominant component and accounted for more than 62% of all three oils. In addition, myrcene, chavicol, methyleugenol, and β-caryophyllene were found to be present at > 7.8%. This result concurs with previous reports, in which bay oil 19 , clove oil 20 , and pimento berry oil 21 were found to contain about 60% eugenol. As was expected, eugenol was also found to dose-dependently inhibit EHEC biofilm formation (Fig. 2b), whereas other compounds, such as, myrcene and β-caryophyllene, did not show anti-biofilm activity at concentration under 0.005% (data not shown). These results suggest that eugenol is largely responsible for the anti-biofilm activity of these oils. Figure 1. Effects of bay, clove, and pimento berry oils on EHEC biofilm formation. Biofilm formation (OD 570 ) by EHEC was quantified in the presence of each of the three essential oils selected for further study after culture for 24 h in 96-well plates (a-c). Biofilm formation by EHEC/pCM18 tagged with green fluorescent protein in the presence of essential oils (0.005%) was confirmed by confocal laser microscopy (d). Scale bar = 50 μ m.
Scientific RepoRts | 6:36377 | DOI: 10.1038/srep36377 The antimicrobial activities of essential oils and eugenol were investigated by measuring minimum inhibitory concentrations (MICs) and EHEC planktonic cell growth. The MICs of the three oils and eugenol against EHEC were > 0.1%, which is consistent with previously reported values 12,22 . Notably, MICs against EHEC were 20-times higher than the concentrations (~0.005%) required for antibiofilm activity. Furthermore, the three oils and eugenol at concentrations ≤ 0.005% did not retard planktonic cell growth, whereas at 0.01%, they reduced final cell  Table 2. GC-MS analysis results for bay, clove, and pimento berry oils. Components present in essential oils at greater than 7% are indicated by bold font. a SI: Library search purity value. b Compounds are listed in order of elution from a DB-5 capillary column. c Percentages were calculated based on normalized FID peak areas.
density by about 20% (Supplementary Fig. S1). These findings indicate reduced biofilm formation by the three oils or eugenol was due to antibiofilm activity and not to antimicrobial activity.

Anti-biofilm activities of eugenol derivatives on EHEC.
To identify the structural motif present in eugenol responsible for antibiofilm activity against EHEC, we investigated the antibiofilm activities of eleven eugenol-related compounds (Fig. 2a). We found eugenol, 4-ethylguaiacol, isoeugenol, and 2-methoxy-4-propylphenol at 0.005% markedly inhibited EHEC biofilm formation by ≥ 50% versus untreated controls. Specifically, eugenol at 0.005% decreased EHEC biofilm formation by 87%, whereas the other seven compounds (guaiacol, 4-ethylcatechol, thymol, vanillin, carvacrol, methyl eugenol, and 2-methoxyhydroquinone) at concentrations of up to 0.005% did not significantly inhibit EHEC biofilm formation. Although, it has been previously reported that vanillin inhibits biofilm formation by Aeromonas hydrophila 23 , it did not inhibit EHEC biofilm formation in the present study. Interestingly, antibiofilm activity against EHEC was found to be closely related to the presence and size of a C-4 alkane chain, and the presence of a hydroxyl group and a methoxy group at the C-1 and C-2 positions of the benzene ring, because eugenol, 4-ethylguaiacol, isoeugenol, and 2-methoxy-4-propylphenol all possess a C-4 alkane or alkyl chain and C-1 hydroxyl and C-2 methoxy groups (Fig. 2a). However, methyl eugenol, which has an alkyl chain and a methoxy unit at C-1 and C-2 lacked inhibitory activity, indicating the eugenol backbone and the C-1 hydroxyl and C-2 methoxy groups are required for antibiofilm activity. Because eugenol reduced biofilm formation most, we focused on it and eugenol-rich clove oil for further study. Microscopic observations confirmed that eugenol and 4-ethylguaiacol at 0.005% (v/v) markedly inhibited EHEC biofilm formation (Fig. 2c). In addition, COMSTAT analysis confirmed that eugenol and 4-ethylguaiacol reduced biofilm biomass, mean thickness, and substratum coverage ( Table 1).

Effects of the three essential oils and eugenol on biofilm formation by other E. coli strains.
It is important that we develop therapeutic compounds that inhibit pathogenic biofilm formation but leave beneficial commensal biofilms unharmed 24 . Thus, the effects of the three essential oils and eugenol were investigated on three laboratory E. coli strains: BW25113, MG1655, and TG1. Unlike that observed for EHEC, neither eugenol nor the three oils had any biofilm inhibitory effects on these three E. coli K-12 strains (Fig. 3a-c). The three laboratory E. coli strains developed poor biofilms in comparison to the EHEC strain as judged by crystal violet staining. The biofilm formation of the untreated laboratory strains was similar to that of the EHEC strain treated with 0.005% eugenol. It is intriguing how eugenol specifically inhibits biofilm formation by EHEC. Because curli fimbriae are critical for E. coli biofilm development 25 , we investigated fimbriae productions by EHEC and the three laboratory strains. Interestingly, EHEC produced more fimbriae than the three laboratory E. coli strains on Congo red plates (Fig. 3d), which suggests eugenol inhibits the development of fimbriated E. coli like EHEC.
Effect of the three essential oils and eugenol on EHEC motility. The impacts of three essential oils and eugenol on the swarming and swimming motilities of EHEC were also investigated because motility plays an important role in E. coli biofilm formation 9,26 . While the three oils clearly reduced swarming motility, eugenol did not ( Supplementary Fig. S2a), and neither the three oils nor eugenol changed EHEC swimming motility ( Supplementary Fig. S2b). Additionally, the presence of elongated swarming and swimming was measured as the diameter (in millimeters) of the zone of expansion ( Supplementary Fig. S2c,d). The result suggests that the anti-biofilm effect of eugenol on EHEC is not closely related to motility.

Transcriptional changes in EHEC cells induced by clove oil or eugenol.
To investigate the genetic bases of EHEC biofilm inhibition by clove oil and eugenol, qRT-PCR was performed to examine the differential expressions of biofilm-and virulence-related genes in treated and non-treated EHEC cells. Most noticeably, both clove oil and eugenol most markedly inhibited the expression of curli genes (csgABDFG) by 8-fold to 155-fold, while they less appreciably changed the expression of type I fimbrial and other fimbrial genes (Supplementary Table S2), whereas they did not affect the expression of rrsG (a housekeeping gene) (Fig. 4). Also, clove oil down-regulated several motility genes (swarming genes fimA and fimH and swimming genes flhD, fliA, and motB), and LEE transcriptional regulator ler gene (Fig. 4a), which concur with its inhibition of motility ( Supplementary Fig. S2). On the other hand, eugenol altered the expression of motility genes less but decreased the expressions of ler and ler-controlled toxin genes (espD, escJ, escR, and tir) (Fig. 4b). Neither clove oil nor eugenol appreciably affected the expressions of quorum sensing genes (luxS, luxR and tnaA) or Shiga-like toxin genes (stx1 and stx2). Previously, it has been reported that curli fimbriae production is crucial for biofilm formation by EHEC strains, particularly ATCC 43895 strain or called EDL933 strain used in this study [4][5][6] . This qRT-PCR result indicates that clove oil and eugenol strongly reduce the transcription of curli genes, as well as reducing the transcription of several other EHEC genes, including type I fimbriae genes.
Eugenol reduced fimbriae formation by EHEC. Because fimbriae are important for EHEC biofilm formation 4,6,27 , and the gene expression of the csg operon producing curli fimbriae production was found to be markedly down-regulated by clove oil and eugenol (Fig. 4), we investigated curli fimbriae production by SEM and Congo red that specifically binds curli fimbriae. In-line with our gene expression data, both clove oil and eugenol at 0.005% clearly reduced fimbriae production (Fig. 5a) and specifically curli production (Fig. 5b), which indicates down-regulation of curli genes and of curli fimbriae inhibition underlie the antibiofilm activities of clove oil and of eugenol. Biofilm inhibition using a biocompatible polymer coating. To prevent biofilm formation on a solid surface, biofilm inhibitors were incorporated into a biocompatible poly(D,L-lactide-coglycolide) (PLGA; a polymer with many medical, pharmaceutical, and industrial uses). As was expected, PLGA coatings containing clove oil or eugenol (0.005%) markedly inhibited EHEC biofilm formation (Fig. 6). COMSTAT analysis showed that both clove oil and eugenol reduced biofilm biomass, mean thickness, and substratum coverage by ≥ 90% (Table 3). These results complement observed biofilm reductions observed on polystyrene plates (Figs 1a-c and 2b) and glass (Figs 1d and 2c). Also, the result demonstrates that biocompatible polymer coatings could be potentially used to prevent pathogenic biofilm formation on biomedical and food processing surfaces.
Clove oil and eugenol reduced EHEC virulence in the nematode model. Since EHEC colonizes and replicates in the digestive tract of C. elegans and has the ability to kill the nematode 28 , the effects of clove oil and eugenol on EHEC virulence were investigated by assessing C. elegans survival. In the agreement with previous results 28 , EHEC significantly reduced the lifespan of C. elegans as compared with E. coli OP50, which is a common food source for the nematode. Moreover, it was found that clove oil or eugenol at 0.005% prolonged C. elegans survival in the presence of EHEC. In fact, the effects of clove oil and eugenol were similar (Fig. 7). This result is in-line with the observed down-regulation of biofilm formation and of the LEE transcriptional regulator ler by eugenol (Fig. 4). In addition, to examine the toxicity of eugenol, C. elegans survival was investigated using E. coli OP50. E. coli OP50 fed eugenol at a concentration of 0.005% was found to have no effect on C. elegans survival (Fig. 7). Discussion EHEC infection is problematic worldwide because of the lack of an effective therapy and the formation of antibiotic-resistant biofilms by EHEC serotypes. The present study demonstrates that three essential oils (bay, clove, and pimento berry oils) exhibit high antibiofilm activity against EHEC without affecting its planktonic cell growth. Hence, unlike antibiotics that aim to inhibit cell growth, biofilm inhibitors that do not inhibit bacterial growth could reduce the risk of drug resistance. In the present study, identification of the active compound and a chemical structure-activity relationship investigation revealed the antibiofilm activities of eugenol and its derivatives. Furthermore, transcriptional and phenotypic assays provided clues regarding the mechanism of biofilm inhibition and virulence attenuation by eugenol and eugenol-rich oils.
Our investigation of fimbriae production, motility, and laboratory E. coli biofilm formation, and qRT-PCR analysis showed that the antibiofilm activities of eugenol and the three oils were due to the suppression of fimbriae production (Figs 3 and 5) and other genes (Fig. 4) in EHEC. It is well known that fimbriae or pili play important role in the biofilm formation of laboratory and pathogenic E. coli such as EHEC 9,27,29 . EHEC strains contain a series of fimbriae such as curli fimbriae (Csg), type I fimbriae (Fim), E. coli common pilus (Ecp), F9 fimbriae (Z2200), and other fimbrial proteins 29 . It has been also reported that several phytochemicals, such as, 3-indolylacetonitrile 30 , phloretin 31 , resveratrol (and its dimer viniferin) 32,33 , cinnamaldehyde 18 , coumarin 34 , and    ginkgolic acids 17 inhibit EHEC biofilm formation primarily by inhibiting curli fimbriae production. Hence, it appears that fimbriae-reducing ability is not rare in the plant kingdom and that the fimbriae inhibition could be viewed a practical target for suppressing EHEC biofilm formation.
To date, a dozen essential oils have been shown to possess antibacterial and antibiofilm activities against wide range of pathogenic bacteria 13,35,36 . Previously, eugenol was found to inhibit biofilm formation by Staphylococcus aureus 37 , Candida albicans 38 , Listeria monocytogenes 14 , and EHEC 14,18 , and to inhibit quorum sensing of Pseudomonas aeruginosa 39 . The present study shows for the first time that eugenol-rich oils (bay, clove, and pimento berry oils) at 0.005% (about 50 μ g/ml) have antibiofilm activity against EHEC (Fig. 1), and provides detail of chemical structure-activity relationships and action mechanism involved. Eugenol is used in perfumes, flavorings, and as a local antiseptic and anaesthetic 40 , and is abundant found in clove oil, nutmeg, cinnamon, basil, and bay leaves. This study suggests another application for inexpensive eugenol-rich oils to control EHEC infection.
Our chemical structure-activity relationship study revealed that the C-1 hydroxyl, the C-2 methoxy, and a C-4 alkyl or alkane chain on the aromatic ring of eugenol are required for antibiofilm against EHEC (Fig. 2). Many eugenol derivatives can be synthesized 41,42 , and several phytochemicals, such as, zingerones, gingerols, shogaols, and paradols bearing the eugenol motif are found in plant extracts. Thus, further investigations of eugenol derivatives are probably worthwhile to identify more potent antibiofilm and anti-virulence compounds.
EHEC produces potent Shiga-like toxins and encodes the LEE operon genes, which are both required for forming attaching and effacing lesions to host epithelial cells 2 . Furthermore, it has been shown the majority of LEE genes are positively regulated by the ler gene (LEE-encoded regulator) 43 . In the present study, eugenol was found to down-regulate the expression of ler and ler-controlled toxin genes (espD, escJ, escR, and tir) and curli-producing genes (csgABDFG) (Fig. 4). The pathogenicity of EHEC was attenuated by eugenol and clove oil (Fig. 7), but they did not harm laboratory E. coli biofilms (Fig. 3). Therefore, it appears eugenol could be used as a lead compound for the development of biofilm inhibitors and toxin producing inhibitors for fimbriae-rich and LEE-encoding E. coli strains, such as, enteropathogenic Escherichia coli 44 , uropathogenic Escherichia coli 45 , and enteroaggregative Escherichia coli 46 .
In the present study, eugenol, eugenol derivatives, and essential oils were found to reduce biofilm formation by and the virulence of EHEC. Furthermore, PLGA coatings containing eugenol or eugenol-rich clove oil effectively prevented EHEC biofilm formation on solid surfaces (Fig. 6). These results suggest that eugenol and its derivatives have potential uses in antivirulence strategies against persistent EHEC infection. In addition, it would be interesting to explore the uses of inexpensive eugenol-rich oils in the food, cosmetics, fishery, agricultural, and environmental industries and in the medical field.

Materials and Methods
Bacterial strain, essential oils, and growth conditions. All experiments were conducted at 37 °C in Luria-Bertani (LB) medium, which was used to culture E. coli O157:H7 (ATCC 43895, EDL933 strain) and three laboratory E. coli K-12 strains (MG1655, BW25113, and TG1). To culture E. coli O157:H7/pCM18 tagged with green fluorescent protein, LB broth containing 300 μ g/ml of erythromycin was used to maintain the pCM18-GFP plasmid. Bacterial cells were initially streaked from − 80 °C glycerol stock on LB plates, grown on plates, and then were cultured from a fresh single colony in LB broth. For phenotypic assays, overnight cultures (stationary phase cells) were inoculated again in LB broth at an initial turbidity of 0.05 at 600 nm. The 83 essential oils (Supplementary Table S1) were obtained from Berjé (Bloomfield, NJ, USA) or Jin Aromatics (Anyang, Gyeonggi Province, Korea). Other chemicals (guaiacol, 4-ethylcatechol, thymol, 2-methoxyhydroquinone, vanillin, carvacrol, methyl eugenol, 2-methoxy-4-propylphenol, isoeugenol, 4-ethylguaiacol, and eugenol) were purchased from Sigma-Aldrich (St. Louis, USA). For cell growth measurements, turbidity was measured at 600 nm using a spectrophotometer (Optizen 2120UV, Mecasys, Korea). Crystal-violet static biofilm formation assay. A static biofilm formation assay was performed in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously described 30 . Briefly, overnight cultures were inoculated in LB broth (total volume 300 μ l) at an initial turbidity of 0.05 at 600 nm and cultured with or without bay, clove, or pimento berry oils or components for 24 h without shaking at 37 °C. To quantify biofilm formation, cell cultures were washed three times with H 2 O to remove all non-adherent cells. Biofilms were stained with crystal violet for 20 min, rinsed three times with H 2 O, extracted with 95% ethanol, and absorbances were measured at 570 nm. Results are the averages of at least twelve replicate wells.

Gas chromatograph/mass spectroscopy (GC-MS) analysis.
Detailed chemical compositions of the three active essential oils were obtained by GC/MS using an Agilent 6890N GC DB-5 MS fused silica capillary column (30 m × 0.25 m i.d., film thickness 0.25 μ m) and a Jeol JMS 700 mass spectrometer. Capillary column details and temperature conditions for the analysis were as previously described 47 . GC-MS was performed using an electron ionization system at 70 eV. Helium was used as the carrier gas at a flow rate of 1 ml/min. The temperatures of the GC injector and MS transfer line were 280 °C and 250 °C, respectively. An initial oven temperature of 50 °C was maintained for 2 min and then increased to 250 °C at a rate of 10 °C/min and this was followed with a holding period at 250 °C for 10 min. Diluted samples (1.0 ml, 1/100 (v/v) in methanol) were injected manually in split-less mode. The relative percentages of oil components were calculated by normalizing peak areas and are expressed as percentages. Components were identified using GC retention times and by matching mass spectra with entries in the Wiley and NIST libraries.
Confocal laser scanning microscopy and COMSTAT analysis. Bacterial cells (E. coli O157:H7/pCM18 tagged with green fluorescent protein) were cultured in glass-bottomed dishes (SPL Life Sciences, Korea) without shaking with or without essential oils or eugenol. Planktonic cells were removed by washing with PBS three times, and biofilms were visualized by excitation using an Ar laser 488 nm (emission wavelengths 500 to 550 nm) under a confocal laser microscope (Nikon eclipse Ti, Tokyo) using a 20 × objective 48 . Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were chosen for microscopic analysis.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software 49 was used to determine biomasses (μ m 3 per μ m 2 ), mean thicknesses (μ m), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.

Fimbriae assay using a Congo red and scanning electron microscopy (SEM).
To measure curli fimbriae production, LB agar medium containing 20 μ g/ml Congo red (Sigma), 10 μ g/ml Coomassie brilliant blue (Sigma), and 15 g L -1 agar was used, as previously described 50 . Curli fimbriae production was visualized after 24 h of incubation at 37 °C on Congo red plates. In addition, a Congo red binding assay was performed, as previously described 51 . Briefly, overnight culture of E. coli O157:H7 EDL933 was reinoculated (100:1) with 0.01% eugenol or clove oil in LB medium with 20 μ g/ml Congo red and 10 μ g/ml Coomassie brilliant blue in 14-ml round bottom tubes and incubated at 37 °C for 24 h with 250 rpm shaking. Incubated cells were collected by centrifugation at 16,600 × g for 15 min. Optical density of the supernatants was measured at 490 nm. Also, SEM was used to observe fimbriae production, as previously described 30 . Briefly, EHEC cells were incubated for 2 h at 37 °C with agitation at 250 rpm, and then re-incubated for 3 h more with or without clove oil or eugenol (0.005%) at 37 °C with shaking. After prompt fixation with glutaraldehyde and formaldehyde, cells were collected by filtering through a 0.45 μ m nylon filter under vacuum. The filter was then cut into 0.5 × 0.5 mm squares and post-fixed in sodium phosphate buffer, osmium, ethanol, and isoamyl acetate, and critical-point dried. Specimens were examined using an SEM (S-4100; Hitachi, Japan) at 15 kV and magnifications ranging from 2,000X to 10,000X.
Swimming and swarming motility. Swimming motility was assayed using 0.3% agar plates containing 1% tryptone and 0.25% NaCl, and swarming motility was assayed using LB broth supplemented with 0.8% glucose and 0.5% agar, as previously described 32 . Essential oils, eugenol or DMSO (the control) were added to motility agar. EHEC was grown to an OD 600 of 1.0 and then ~0.2 μ l aliquots of cultures were placed in motility plates using a sterilized pipette tip. Sizes of swimming halos were measured 16 h later. Each experiment was performed using at least three independent cultures. RNA isolation and quantitative real-time RT-PCR. For transcriptional analysis, EHEC was inoculated into 25 ml of LB broth in 250 ml shake flasks at a starting OD 600 of 0.05, and then cultured at 37 °C for 3 h with agitation (250 rpm) in the presence or absence of clove oil or eugenol (0.005%) for another 2 hours. RNase inhibitor (RNAlater, Ambion, TX, USA) was added to prevent RNA degradation. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).
qRT-PCR was used to investigate the transcription levels of curli genes (csgA, csgB, csgD, csgF and csgG), type I fimbriae genes (fimA, fimC, fimD, and fimH), other fimbriae genes (ecpA, ecpR, and Z2200), cellulose gene (bcsA), motility genes (flhD, fliA, motB, and qseB), AI-2 quorum sensing genes (luxS and luxR), indole-synthesis gene (tnaA), shiga-like toxin genes (stx1 and stx2), and LEE-encoded regulator genes (ler, espD, escJ, escR, and tir) in EHEC treated with or without clove oil or eugenol (0.005%). Gene specific primers were used and rrsG was used as a housekeeping control (Supplementary Table S3). The qRT-PCR method used was an adaptation of a previously described method 30 . qRT-PCR was performed using a SYBR Green master mix (Applied Biosystems, Foster City, USA) and an ABI StepOne Real-Time PCR System (Applied Biosystems) on two independent cultures. Surface coating with biofilm inhibitors. To fabricate a coating containing biofilm inhibitor, we used biodegradable poly(D,L-lactide-coglycolide) (PLGA) as previously described 52 . Briefly, essential oils or eugenol Scientific RepoRts | 6:36377 | DOI: 10.1038/srep36377 (final concentration, 0.005% v/v) were mixed into 2% PLGA dissolved in chloroform and 25 μ l of these mixtures were applied to slide-glass bottomed dishes to produce a 0.7~0.8 cm diameter coatings, which were then air-dried for 1 h and sterilized by UV exposure for 4 h. To induce biofilm formation on coated glass surfaces, cells (E. coli O157:H7/pCM18 tagged with green fluorescent protein; 4 × 10 7 CFU/ml) were re-inoculated into LB. Samples were then incubated at 37 °C for 24 h. Planktonic cells were removed by washing with PBS twice, and biofilm cells in PBS buffer were visualized by confocal laser microscopy.
The Caenorhabditis elegans nematode model. The C. elegans killing assay was performed as described previously 53 . In brief, E. coli O157:H7 was cultured with or without clove oil or eugenol (0.005%) at 37 °C for 18 h, and then 10 μ l of E. coli O157:H7 was spread onto NGM plates. E. coli OP50 was used as a control strain. L4/young adult fer-15;fem-1 54 worms (n = 60) were infected by placing them on the lawns. Nematodes were then incubated at 20 °C and scored as alive or dead on a daily basis by gently touching them with a platinum wire. Three independent experiments were conducted. Statistical analysis. Sample sizes of all experiments are indicated in 'Material and Methods' . Results are expressed as means ± standard deviations. The Student's t-test was used to determine the significances of differences between samples and non-treated controls. Statistical significance was accepted for p values < 0.05, and significant changes are indicated by asterisks in figures.