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

Abstract

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.

Figure 1. Effects of bay, clove, and pimento berry oils on EHEC biofilm formation.

Biofilm formation (OD₅₇₀) 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 μm.

Figure 2. Inhibition of biofilm formation by eugenol-like compounds.

Chemical structures are shown (a). Biofilm formation by EHEC was quantified in the presence of selected chemicals after incubation for 24 h in 96-well polystyrene plates without shaking (b). At least two independent experiments were conducted (total 12 wells). Error bars indicate standard deviations. Biofilms formed in the presence of 4-ethylguaiacol or eugenol (0.005%) were observed under a confocal laser microscope (c). Scale bar = 50 μm. *P < 0.05 versus non-treated controls.

Figure 3

Effects of bay, clove, pimento berry oils and eugenol on biofilm formation by three laboratory E. coli strains and fimbriae production. Biofilm formation (OD₅₇₀) by E. coli BW25113 (a), MG1655 (b), and TG1 (c) was quantified in the presence of essential oils or eugenol after culture for 24 h in 96-well plates without shaking. Fimbriae production was assessed on Congo red plates after 24 h (d). At least three independent experiments were conducted.

Figure 4. Transcriptional profiles of EHEC cells treated with or without clove oil or eugenol.

EHEC was cultivated in LB broth with or without clove oil (a) or eugenol (b) at 0.005% (v/v) for 2 h with agitation (250 rpm) at 37 °C. Transcriptional profiles were measured by qRT-PCR. Relative gene expressions represent transcriptional levels after treatment with clove oil or eugenol versus untreated controls (value 1.0). The experiment was performed in duplicate.

Figure 5. Effects of clove oil and eugenol on curli fimbriae production by EHEC.

Fimbriae productions by EHEC cells grown with or without clove oil or eugenol (0.005%) were observed by SEM (a). For SEM analysis, cells were cultured in LB broth with or without clove oil or eugenol at 37 °C for 2 h. The scale bar represents 3 μm. Congo red binding assay for the detection of curli fimbriae production (b). EHEC cells were grown in LB broth in the presence of Congo red and Commassie brilliant blue at 37 °C for 24 h. Culture supernatants were measured at 490 nm and cell pellets were photographed. Dark red color indicates curli production stained by Congo red and Commassie brilliant blue.

Figure 6. Inhibition of biofilm formation by PLGA coatings containing clove oil or eugenol.

Biofilms formed by EHEC/pCM18-GFP were investigated by confocal laser microscopy. Clove oil or eugenol (0.005%, v/v) was incorporated into PLGA and biofilms were grown at 37 °C for 24 h without shaking. Scale bars represent 50 μm. At least three independent experiments were conducted.

Figure 7. Effects of clove oil or eugenol on the survival of nematodes infected with EHEC.

Solid killing assays of C. elegans strain fer-15;fem-1 infected with EHEC or E. coli OP50 (control) in the presence of clove oil or eugenol (0.005%, v/v) at 20 °C. The experiment was performed in triplicate (n = 60).