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. 2020 Jul 24;99(10):5127–5136. doi: 10.1016/j.psj.2020.06.067

Antibacterial and antibiofilm activities of thyme oil against foodborne multiple antibiotics-resistant Enterococcus faecalis

Fang Liu ∗,†, Panpan Jin ∗, Hansheng Gong ‡, Zhilan Sun ∗, Lihui Du §,1, Daoying Wang ∗,1
PMCID: PMC7598324  PMID: 32988551

Abstract

The inhibitory and bactericidal activities of thyme oil against the foodborne multiple antibiotics-resistant Enterococcus faecalis biofilm were evaluated in this study. Gas chromatography-mass spectrometry revealed that more than 70% of the composition of thyme oil is thymol. Crystal violet staining assay showed that 128 and 256 μg/mL thyme oil significantly inhibited the biofilm formation of E. faecalis. The cell adherence of E. faecalis, as shown by its swimming and swarming motilities, was reduced by thyme oil. The exopolysaccharide (EPS) quantification assay showed that thyme oil inhibited the EPS synthesis in E. faecalis biofilms. The 3D-view observations through confocal laser scanning and scanning electron microscopy suggested that cell adherence and biofilm thickness were decreased in thyme oil–treated biofilms. Quantitative real-time analyses showed that the transcription of ebp and epa gene clusters, which were related to cell mobility and EPS production, was inhibited by thyme oil. Thus, thyme oil effectively inhibited the biofilm formation of E. faecalis by affecting cell adherence and EPS synthesis. Furthermore, 2,048 and 4,096 μg/mL thyme oil can effectively inactivate E. faecalis population in the mature E. faecalis biofilms by 5.75 and 7.20 log CFU/mL, respectively, after 30 min of treatment. Thus, thyme oil at different concentrations can be used as an effective antibiofilm or germicidal agent to control E. faecalis biofilms.

Key words: plant essential oil, biofilm, Enterococcus faecalis, exopolysaccharide, cell adherence

Introduction

Enterococci are gram-positive bacteria which are natural components of the intestinal flora of humans and many animals (Foulquié Moreno et al., 2006; Rehaiem et al., 2016; Chajęcka-Wierzchowska et al., 2017). They can easily survive for long periods and can contaminate the animal carcass and processed products (Foulquié Moreno et al., 2006; Rehaiem et al., 2016; Chajęcka-Wierzchowska et al., 2017). Moreover, two Enterococcus species (Enterococcus faecalis and Enterococcus faecium) are important opportunistic pathogens and caused a wide variety of infections including endocarditis, urinary tract infections, prostatitis, intra-abdominal infection, cellulitis, and wound infection as well as concurrent bacteremia, which mainly occurred among hospitalized patients (Gao et al., 2018; Fiore et al., 2019). Enterococci have intrinsic resistance to antibiotics and can form biofilms on abiotic surfaces (Holmberg and Rasmussen, 2016; Qayyum et al., 2018). A biofilm is a community of matrix-enclosed microorganisms that adhere to a surface (Costerton et al., 1995). The bacterial cells in biofilms are protected by the extracellular matrix of proteins, polysaccharides, and nucleic acids. As a result, these bacterial cells are more resistant to antibiotics or antibacterial agents than planktonic cells (Whitehead and Verran, 2015; Liu et al., 2017). Enterococci biofilms that form in food-processing environments are difficult to eliminate, which make enterococci as one of the common opportunistic pathogens and spoilage bacteria in meat products (Giaouris et al., 2014; Pesavento et al., 2014; Rizzotti et al., 2016; Liu et al., 2020). Therefore, novel, safe, and effective antibiofilm agents that can inhibit the formation of enterococci biofilm during food processing and storage should be developed.

Plant essential oils are a class of natural antimicrobial compounds with good antimicrobial activities against bacteria, viruses, and fungi (Wattanasatcha et al., 2012; Marchese et al., 2016; Zhang et al., 2016; Cui et al., 2019). Secondary metabolites with a phenolic structure are the main antimicrobial compounds in these plant essential oils (Wattanasatcha et al., 2012; Marchese et al., 2016). Thymol (2-isopropyl-5-methylphenol) is a monoterpene phenol found in the essential oils extracted from Thymus, Moschus, Dracocephalum, and Origanum (Wattanasatcha et al., 2012; Marchese et al., 2016). Thyme oil possesses strong inhibitory activity against many foodborne pathogens or spoilage bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, E. faecalis, Escherichia coli, Pseudomonas aeruginosa, Vibrio alginolyticus, and Salmonella typhimurium (Wattanasatcha et al., 2012; Marchese et al., 2016; Miladi et al., 2016). The application of thymol will expand if it exhibits good antibiofilm activity against foodborne pathogens at concentrations lower than the minimum inhibitory concentration. In this work, the changes in the biofilm formation of E. faecalis in the presence of thyme oil were studied using the crystal violet biofilm and 3D morphological analyses. The possible molecular mechanism of its inhibitory action was further studied by analyzing the changes of cell motility, exopolysaccharide (EPS) production, and the expression levels of genes related to biofilm formation. Finally, the effects of thyme oil at concentrations higher than the minimal inhibitory concentration (MIC) to inactivate E. faecalis cells in mature biofilms were also evaluated.

Material and Methods

Bacterial Strain and Culture Condition

E. faecalis R612Z1 was isolated from the product of Chinese water-boiled salted duck (Liu et al., 2020) and naturally resistant to multiple antibiotics, including vancomycin, erythromycin, streptomycin sulfate, cephalexin, tetracycline, ampicillin, and kanamycin. This bacterium was stored in a refrigerator at −80°C and inoculated in a brain heart infusion (BHI) broth (Beijing Land Bridge Tech. Co., Beijing, China) at 37°C for approximately 6 h to obtain the logarithmic phase cells for subsequent studies.

Chemical Composition of Thyme Oil

Thyme oil was extracted from thyme by Hubei Xin-runde Chemical Industry Co., Ltd. (Wuhan, China). The composition analysis of thyme oil was determined using gas chromatography-mass spectrometry (GC-MS) (Thermo Scientific TSQ 8000 EVO). The sample was separated using the Thermo Scientific TraceGOLD TG-5MS GC (30 m × 0.25 mm × 0.25 μm). GC-MS was performed using an electron ionization system at 70 eV, and the injector and detector temperatures were maintained at 280°C. The temperature setting of the chromatographic column was 40°C for 1 min and 280°C for 2 min (heating rate = 6°C min−1). The diluted samples (1.0 mL, 1/100 (v/v) in methanol) were injected manually in splitless mode. The individual components in thyme oil were identified on the basis of their retention times and through the comparison of mass spectra with entries in IST105 and Wiley7.0 libraries. Each component percentage was calculated on the basis of the GC peak area via the normalization method. For all experiments, thyme oil was dissolved in absolute ethanol to obtain a stock solution of 20,480 μg/mL and twice diluted with BHI broth or sterile distilled water to obtain a series of thyme oil dilutions.

Determination of MIC

Each well in the 96-well microliter plate was added with 125 μL 100-diluted logarithmic phase E. faecalis culture of approximately 7 log CFU/ml and added with thyme oil solution diluted with 125 μL of BHI broth, obtaining final concentrations of 16, 32, 64, 128, 256, 512, and 1,024 μg/mL. The bacteria were further cultured at 37°C, and cell growth was monitored at 600 nm by using an Infinite M200 Microplate Reader (Tecan, Mannedorf, Switzerland). The lowest concentration of thyme oil that inhibited the visible growth of E. faecalis was designated as the MIC value.

Determination of Antibiofilm Activity

The antibiofilm activity of thyme oil against E. faecalis was evaluated in accordance with previously described methods (Sandasi et al., 2010; Bazargani and Rohloff, 2016; Liu et al., 2019). The 100-diluted logarithmic phase E. faecalis cultures of approximately 7 log CFU/mL were cultured in BHI broth in a 96-well microplate for 72 h at 37°C and supplied with thyme oil at final subminimal concentrations of 0, 64, 128, and 256 μg/mL. The BHI broth with or without thyme oil was changed every 24 h. At predetermined sampling times (12, 24, 48, and 72 h), the biofilms in the plate were dried and stained with 0.1% crystal violet for 30 min. After removing the excess dyes, the biofilms were destained using 150 μL of 95% ethanol, and the OD570 nm values were measured by using a multimode plate reader (Infinite M200 PRO).

Changes in Cell Motility

The changes in the cell motility of E. faecalis after adding thyme oil were analyzed in accordance with previously described procedures (Cong et al., 2011; Hidalgo et al., 2011). Two agars for swimming (10 g/L tryptone, 5 g/L NaCl, 2.5 g/L glucose, and 0.3% agar) and swarming (25 g/L LB, 0.5 g/L glucose, and 0.5% agar) analyses were used. The 3-μL 100-diluted logarithmic phase E. faecalis culture was spotted on the agar plate surfaces containing varying concentrations of thyme oil (0, 64, 128, and 256 μg/mL). The diameters of bacterial growth after incubation at 30°C for 8 or 20 h were measured for swimming or swarming motilities.

Changes in EPS

The contents of EPS in E. faecalis biofilms were detected in accordance with previous studies (Harimawan and Ting, 2016; Liu et al., 2020). The logarithmic phase E. faecalis cultures were 100-fold diluted in BHI broth with or without thyme oil (0, 64, 128, and 256 μg/mL). A 1-mL cell suspension was added into 24-well plates and incubated for 3 D at 37°C. The BHI broth with or without thyme oil was changed every 24 h. The content of each well was harvested after the addition of 1 mL of phosphate-buffered saline (PBS) after washing and drying of the biofilm samples. The contents of ten wells represented ten replicates for one treated concentration and were mixed and centrifuged for 30 min at 5,000 g and 4°C. The concentrated precipitates were resuspended in 10-mL aqueous solution (0.85% NaCl, 0.22% formaldehyde) for 30 min at 80°C, and the EPS dissolved in the formaldehyde solution was extracted by centrifugation for 30 min at 15,000 g and 4°C. The concentrations of extracted EPS solutions were quantified using the phenol-sulfuric acid (PSA) method according to the previous articles (Dubois et al., 1956; Jiang et al., 2013). Briefly, the 5% phenol solution was mixed with 98% concentrated sulfuric acid in volume ratio of 1:5. One milliliter of standard and EPS solutions was transferred into microcentrifuge tubes, and 5 mL of the prepared PSA reagent was added. The test tubes were placed in a water bath at 100°C for 15 min, and the absorbance were measured at 490 nm using a multimode plate reader (Infinite M200 PRO).

Confocal Laser Scanning Microscopy

The biofilms grown in the presence of thyme oil were observed through confocal laser scanning microscopy (CLSM) (Liu et al., 2017; 2018). The logarithmic phase E. faecalis cultures were 100-fold diluted in BHI broth with or without thyme oil (0, 64, 128, and 256 μg/mL) and cultivated in 8-well chamber slides (Nunc Lab-Tek; Fisher Scientific) at a volume of 400 μL/well at 37°C for 3 D. The BHI broth with or without thyme oil was changed every 24 h. The biofilms in the wells were washed with 0.01-M PBS and dyed using the LIVE/DEAD BacLight viability kit (Molecular Probes; Life Technologies, Eugene, OR) at indicated sampling times (12, 24, 48, and 72 h). The samples were visualized using the Leica Ultra View VOX CLSM (Leica Microsystems, Ltd., Wetzlar, Germany) and analyzed using the Volocity software (Improvision; PerkinElmer, Cambridgeshire, UK). The excitation and emission wave lengths of SYTO 9 and PI were 485 and 498 nm and 535 and 637 nm, respectively.

Scanning Electron Microscopy

The bacterial cell microstructures of E. faecalis biofilms were visualized through scanning electron microscopy (SEM). The biofilms were cultured similar to that for CLSM and fixed with 2.5% glutaraldehyde at 4°C for 30 min at the indicated sampling times (12, 24, 48, and 72 h). The slide was cut in different parts corresponding to each sample, and the fixed samples were dehydrated using a graded ethanol series of 25, 50, 70, 90, and 100% for 10 min. The final biofilm samples were examined through SEM (EVO-LS10; Zeiss, Oberkochen, Germany) at an accelerating voltage of 20 kV and magnification of 5,000×.

Isolation of RNA and Quantitative Real-Time Reverse Transcription PCR

The changes in biofilm-related genes in transcriptional levels were analyzed using quantitative real-time reverse transcription PCR (qRT-PCR). The control and thyme oil–treated E. faecalis biofilms were cultured in 24-well plates at 37°C for 24 h. The total RNA from the bacterial cells in biofilms was isolated using the TIANamp RNAprep pure Cell/Bacteria kit (Tiangen, China). The isolated RNAs were reverse-transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Takara, China). The transcription levels of the selected biofilm-related genes in Ebp pili and Epa polysaccharide clusters were analyzed using the SYBR Premix Ex Taq Ⅱ (Takara, China) in the Roche LightCycler 480 Real-Time PCR System (Indianapolis, IN). The primers of the selected genes are listed in Table 1. Data were analyzed using the 2−ΔΔCT method (Liu et al., 2020). All samples were analyzed in triplicate.

Table 1.

Primer sequences for real-time PCR.

Gene Primers Sequence (5′-3′)
Housekeeping gdhA Forward 5′-GGAATTGATGTGGCGTTAG-3′
Reverse 5′-GTGTTGCACATGGTAACG-3′
Endocarditis and biofilm-associated Ebp pilus subunits ebpA Forward: 5′-CTACGACACTCTTGCTGGAA-3′
Reverse: 5′-AATTTCGCTCCTTTAAGTGG-3′
ebpB Forward: 5′-AACAGAAATTCCTTTTACGG-3′
Reverse: 5′-ATAGCCAAATGACTACCAGC-3′
ebpC Forward: 5′-TTGAAGTGGTGACAGGTGGG-3′
Reverse: 5′-GCTGCTTTCGTTGTTTCATC-3′
Epa polysaccharide synthesis epaA Forward: 5′-TTGCATCACCGCTTGTTATC-3′
Reverse: 5′-TCGCCAACTAGACCGATTAG-3′
epaB Forward: 5′-CGGATACAGAAACAACGGAT-3′
Reverse: 5′-TAGAGAATCCGATAGCCTGC-3′
epaE Forward: 5′-CGAAGTCAAATTACGCAGTG-3′
Reverse: 5′-AGGATTCGTGTGTGCCTGTA-3′
epaG Forward: 5′-CCGATGGTGAAGAAGACAAT-3′
Reverse: 5′-TGATTGTTTCTGCCAAGCCT-3′
epaH Forward: 5′-TCCAAGAAATCCTGGACGAC-3′
Reverse: 5′-CAAATGCCATAAATTCGGCT-3′
epaM Forward: 5′-GAGATACGATGGACGTGACT-3′
Reverse: 5′-TGCTTTACCATTTCTAAGGG-3′
epaQ Forward: 5′-TGGTTTTGTCGGAGTAGCTG-3′
Reverse: 5′-CCACAAAAGGTTCCATACCG-3′
epaR Forward: 5′-AATTTAG`AACGATGTCGGCA-3′
Reverse: 5′-CCGACAATGGACATATCTCC-3′
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Bactericidal Activities of Thyme Oil Against Bacterial Cells in Mature E. faecalis Biofilms

The bactericidal effect of thyme oil at concentrations higher than MIC against the E. faecalis bacterial cells in mature biofilms was evaluated. The E. faecalis biofilms cultivated in 24-well plates for 72 h were used and treated with different concentrations of thyme oil (512, 1,024, 2,048, and 4,096 μg/mL) for 5, 15, 30, and 60 min (Liu et al., 2017). After treatment, sterile water (negative control) or thyme oil solution was removed from the wells, and the biofilms were washed using 0.01-M PBS. The bacterial cells in each well were suspended in 9 mL of 0.01-M PBS buffer and added with 9 mL of 0.01-M PBS. The diluted 100-μL cell suspension was cultured on BHI plates in duplicate and incubated at 37°C for bacterial enumeration.

Statistical Analysis

All experiments were performed in triplicate, and results were presented as mean ± standard deviation. A statistical analysis except for qRT-PCR data was carried out by ANOVA by using the SPSS software (version 19.0; IBM-SPSS Inc., Armonk, NY). P < 0.05 was considered significant.

Results and discussion

Identification of the Active Components of Thyme Oil

The active compositions of the thyme oil used in this study were analyzed via GC-MS, and the result is presented in Table 2. Nine components representing 98.74% of the total detected constituents were identified, which included thymol (70.76%), 1,1,1,3,5,5,7,7,7-Nonavmethyl-3-(trimethylsiloxy) tetrasiloxane (22.42%), heptasiloxane, hexadecamethyl- (1.71%), decane, 3-ethyl-3-methyl- (1.48%), and 1-Decanol, 2-hexyl- (1.06%). The result suggested thymol was the predominant component of thyme oil used in this study.

Table 2.

Chemical compositions of thyme oil used in this study.

Composition Formula Proportion (%)
Decane, 4-methyl- C11H24 0.12%
Hexane, 3,3-dimethyl- C8H18 0.20%
Decane, 2-methyl- C11H24 0.17%
Thymol C10H14O 70.76%
Decane, 3-ethyl-3-methyl- C13H28 1.48%
Nonadecane, 2-methyl- C20H42 0.82%
1,1,1,3,5,5,7,7,7-Nonamethyl-3-(trimethylsiloxy) tetrasiloxane C12H36O4Si5 22.42%
Heptasiloxane, hexadecamethyl- C16H48O6Si7 1.71%
1-Decanol, 2-hexyl- C16H34O 1.06%
Total 98.74
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Antibiofilm Activity of Thyme Oil Against E. faecalis

The results of E. faecalis R612-Z1 growth curves under different concentrations of thyme oil are shown in Figure 1A. The MIC value of thyme oil was 512 μg/mL, which was the concentration in which the bacterial cells did not grow. The antibiofilm activity of 64, 128, and 256 μg/mL thyme oil against E. faecalis R612-Z1 was detected through crystal-violet biofilm assays. As shown in Figure 1B, the biofilm formation of E. faecalis after 72 h of growth was decreased significantly by 128 and 256 μg/mL thyme oil (P < 0.05) (Figure 1B).

Figure 1.

Figure 1

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Effects of different concentrations of thyme oil on planktonic cell growth (A) and biofilm formation (B) of Enterococcus faecalis R612-Z1. The different capital letters in the figures indicate different treatments with significant differences (P < 0.05).

Foodborne pathogens that form biofilms in food processing environments are difficult to eliminate. Many studies have focused on the antibiofilm activities of plant oils against foodborne pathogens and spoilage bacteria. Kim et al. (2016) reported that bay, clove, pimento berry oils, and their major common constituent eugenol are effective in inhibiting the E. coli O157:H7 biofilm formation without affecting planktonic cell growth. Lee et al. (2014) reported that the extracts of Ginkgo biloba inhibit E. coli O157:H7 and S. aureus biofilm formation on the surfaces of polystyrene and glass. Szczepanski et al. (2014) studied the antibiofilm activities of thyme, oregano, and cinnamon essential oil at sublethal concentrations on the biofilm formation of Sphingomonas, Acinetobacter, and Stenotrophomonas and found that thyme oil is more effective in inhibiting biofilm development than the other two oils. Nithyanand et al. (2015) reported that Pogostemon oil can inhibit the growth of streptococcal biofilms. These antibiofilm plant oils are plant secondary metabolites that contain phenolics. The present study showed that thyme oil can also inhibit the biofilm formation of E. faecalis effectively.

Effect of Thyme Oil on Cell Motility of E. faecalis

Bacterial adhesion plays a main role in the initial stage of biofilm formation, and bacterial cell motility can affect this adhesion (Borges et al., 2012; Monte et al., 2014). Some nonflagellated cocci, including S. aureus and E. faecalis, are historically regarded as nonmotile bacteria. In the recent years, some articles have reported that S. aureus, a nonflagellated bacterium, can spread across agar surfaces and is actively motile under certain conditions (Borges et al., 2012; Pollitt et al., 2015). The biofilm-associated Ebp pili of E. faecalis play an important role in bacterial adherence to different surfaces (La Rosa et al., 2016; Afonina et al., 2018). Besides Ebp pili, type IV Pili are also important in cell motility, adherence, and biofilm formation of E. faecalis (Kohler et al., 2018; Schmitt et al., 2018). The effects of thyme oil on E. faecalis motility were analyzed, and the diameters of bacterial halos corresponding to swimming and swarming motilities of bacterial cells treated with 64, 128, and 256 μg/mL thyme oil significantly decreased compared with those of the control (P < 0.05) (Figure 2). Thus, the cell motility of E. faecalis can be inhibited by thyme oil. The cell motility of E. faecalis may be due to the effect of bacterial surface pili, and the decrease of cell motility means the decrease of bacterial adherence. So, the antibiofilm activity of thyme oil may be partly attributed to the inhibition of bacterial adherence. Bai et al. (2019) reported that the cell motility of S. aureus is also inhibited by shikimic acid.

Figure 2.

Figure 2

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Effects of different concentrations of thyme oil on cell swimming (A) and swarming (B) motilities of Enterococcus faecalis R612-Z1. The different capital letters in the figures indicate the significant differences in different treatments (P < 0.05).

Other plant oils inhibit biofilm formation by interfering with bacterial motility. Merghni et al. (2018) reported that the Eucalyptus globulus essential oil and its main component 1,8-cineole can inhibit the swarming behavior of methicillin-resistant S. aureus strains. Lee et al. (2018) studied the antibiofilm activity of two different plant extracts and found their different inhibition activities against cell motility. Harmaline can reduce swarming motility, whereas norharmane has a remarkable inhibitory effect on swimming motility. As cell motility plays an important role in quorum sensing-mediated biofilm formation, these plant-oriented antibiofilm agents may interfere with bacterial quorum sensing at different levels to inhibit the cell swimming or swarming ability of bacteria.

Inhibition of Biofilm EPS Production by Thyme Oil

Bacteria can continue to grow and secrete the extrapolymeric biofilm matrix, which includes EPS, proteins, fatty acids, and nucleic acids, when they adhere to the solid surface (Flemming and Wingender, 2010). The extrapolymeric matrix accounts for 80% of biofilms and forms the mature biofilm architecture (Jung et al., 2013; Roy et al., 2018). EPS is the major component of the extrapolymeric matrix (Flemming and Wingender, 2010). Thus, the inhibition or reduction of EPS production is important in controlling biofilm formation.

The EPS content in the control or thyme oil–treated E. faecalis biofilms was detected via a quantification assay. As shown in Figure 3A, the EPS contents in E. faecalis biofilm were significantly inhibited by addition of 128 and 256 μg/mL thyme oil (P < 0.05) and can be potentially inhibited by addition of 64 μg/mL thyme oil. Thus, treatment with 128 and 256 μg/mL thyme oil can inhibit EPS production of E. faecalis cells in biofilms.

Figure 3.

Figure 3

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Change in the polysaccharide contents (A) and microstructure (B) of Enterococcus faecalis biofilms. The polysaccharide contents were detected by phenol-sulfuric acid method. The 3D images of confocal laser scanning microscopy corresponding to the microstructure and height of E. faecalis biofilms were analyzed using the E. faecalis R612-Z1 biofilms grown at 37°C for 12 (C12, T12), 24 (C24, T24), 48 (C48, T48), and 72 h (C72, T72). C, controls; T, 128 μg/mL thyme oil–treated groups. The different capital letters in the figures indicate that the different treatments had significant differences (P < 0.05). The different small letters in the figures indicate significant differences in the treatments (P < 0.05).

The 3D architecture changes corresponding to the contents of extrapolymeric matrix of E. faecalis biofilms in the presence of thyme oils were further visualized through CLSM. As shown in Figure 3B, the bacterial cells in control E. faecalis biofilms were closely adherent and formed well-organized structures at 12, 24, 48, and 72 h of incubation (Figures 3B–C12, C24, C48, and C72), whereas those treated with 128 μg/mL thyme oil had porous structures with uniform holes and decreased thickness (Figures 3B–T12, T24, T48, and T72). These biofilm images showed that the extrapolymeric matrix production of bacterial cells in biofilms was inhibited in the presence of thyme oil. This finding was in agreement with the results obtained via the EPS quantification assay. Chen et al. (2018) reported that a small molecule ST056083 can inhibit the secretion of EPS and biofilm formation of E. faecalis. Liu et al. (2019) reported that anionic chitosan can also inhibit EPS synthesis during the formation of P. aeruginosa biofilms.

Scanning Electron Microscopy

The aggregation states of bacterial cells in the biofilms at different cultivation stages during 24, 48, and 72 h of culture were observed through SEM. In Figure 4, the bacterial cells in the control biofilms aggregated densely and thickly, which were typical characteristics of biofilm formation. The dense aggregates of cells were reduced upon treatment with 128 and 256 μg/mL thyme oil. Thus, thyme oil can reduce bacterial cell-cell adhesion by inhibiting EPS production. This antibiofilm effect of thyme oil is in accordance with many other antibiofilm agents. Bai et al. (2019) reported that shikimic acid can make bacterial cells in S. aureus biofilms loose and discrete, suggesting the role of shikimic acid in preventing the attachment of bacterial cells.

Figure 4.

Figure 4

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SEM images of Enterococcus faecalis R612-Z1 biofilms grown at 37°C for 24 (A24, B24, C24), 48 (A48, B48, C48), and 72 h (A72, B72, C72). A, control; B, 128 μg/mL thyme oil–treated group; C, 256 μg/mL thyme oil–treated group.

Transcriptional Changes of Biofilm-Related Genes Induced by Thyme Oil

Bacterial pili and polysaccharide synthesis are important in different biofilm formation stages, including aggregation, adherence, and maturation (Montealegre et al., 2015; Afonina et al., 2018). The endocarditis and biofilm-associated pili encoded by ebp operon play an important role in the aggregation and adherence of E. faecalis (Nallapareddy et al., 2006; Sillanpää et al., 2013). The polysaccharide biosynthesis of E. faecalis depends on the epa operon (Rigottier-Gois et al., 2015; Dale et al., 2017). Thus, the differential expressions of ebp and epa operons were studied via qRT-PCR to investigate the genetic bases of E. faecalis R612-Z1 biofilm inhibition by thyme oils. RNAs were extracted from the bacterial cells in control and 128 μg/mL thyme oil–treated biofilms grown for 24 h. As shown in Figure 5, the expressions of the Ebp pili (ebpABC) and the Epa polysaccharide (epaABEGHMQR) genes were markedly inhibited 2- to 9-fold in the presence of 128 μg/mL thyme oil. Thus, thyme oil can inhibit biofilm formation by affecting the transcription of pili and polysaccharide genes.

Figure 5.

Figure 5

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Changes in the transcription levels of genes (ebpA, ebpB, ebpC, epaA, epaB, and epaE) in the two clusters of ebp and epa in Enterococcus faecalis R612-Z1 biofilms grown at 37°C for 24 h after treatment with 128 μg/mL thyme oil.

Inactivation of Bacterial Cells in Mature Biofilms

The effectiveness of thyme oil at concentrations higher than MIC to inactivate E. faecalis cells in mature biofilms was further evaluated. The mature biofilms were cultivated on 24-well plates at 37°C for 3 D. The bacterial cells in the biofilms were treated with 512, 1,024, 2,048, and 4,096 μg/mL thyme oil for 5, 15, 30, and 60 min. Results are shown in Table 3. The initial E. faecalis counts in biofilms were approximately 9.3 log CFU/mL. The bacterial counts in the biofilms did not have significant differences when treated with selected thyme oil solutions for 5 min (P > 0.05). When the treatment time of 512 and 1,024 μg/mL thyme oil was prolonged to 15 min, the bacterial counts did not show any significant difference. However, the bacterial count was significantly decreased after treatment with 2,048 and 4,096 μg/mL thyme oil compared with that of the control (P < 0.05). The effectiveness of treatments by using 1,024, 2,048, and 4,096 μg/mL thyme oil was significantly increased when the treatment time was prolonged from 15 min to 30 min (P < 0.05) but did not increase with prolonged treatment time from 30 min to 60 min.

Table 3.

Bacterial counts in the biofilms of E. faecalis R612-Z1 grown on 24-well flat-bottom polystyrene plates at 37°C for 3 D after treated with thyme oils at different concentrations for 5, 15, 30, and 60 min.

Treatment time The bacterial counts in biofilms (log CFU/mL)
Control 512 μg/mL 1,024 μg/mL 2,048 μg/mL 4,096 μg/mL
5 min 9.39 ± 0.19A,a 9.29 ± 0.15 9.55 ± 0.20A,a 9.32 ± 0.03A,a 9.29 ± 0.07A,a
15 min 9.28 ± 0.28A,a 9.06 ± 0.17A,a 9.42 ± 0.15A,a 5.91 ± 0.82B,b 5.38 ± 0.21B,b
30 min 9.33 ± 0.16A,a 9.12 ± 0.24A,a 7.78 ± 0.59B,b 3.57 ± 0.38C,c 2.09 ± 0.13C,d
60 min 9.15 ± 0.09A,a 9.25 ± 0.11A,a 7.54 ± 0.35B,b 3.63 ± 0.21C,c 2.54 ± 0.15C,d
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The different capital letters in the figures indicate that the different treatments had significant differences (P < 0.05). The different small letters in the figures indicate significant differences in the treatments (P < 0.05).

Based on the results, the bacterial cells in the biofilms were more difficult to inactivate than the planktonic bacterial cells. The difficulty of inactivating bacterial cells in biofilms may be due to the protection provided by the extrapolymeric matrix (Liu et al., 2017), reflecting the importance of increasing the germicidal efficacy of sanitizers to inhibit the secretion of this matrix. Foodborne pathogens are difficult to eliminate once they form biofilms on the surface of food equipment. Thus, effective methods must be designed to inhibit biofilm formation.

Conclusions

In this study, different concentrations of thyme oils were found to have effective antibiofilm or germicidal effects against E. faecalis R612-Z1 biofilms. Biofilm formation was significantly reduced after treatment with subinhibitory concentrations (128 and 256 μg/mL) of thyme oil. The antibiofilm effect of thyme oil was associated with cell motility reduction and EPS production. Thyme oil at concentrations of 2,048 and 4,096 μg/mL effectively inactivated the bacterial cells in 3-day-old mature biofilm of E. faecalis grown on 24-well plates after treatment for 30 min. This study revealed that thyme oil can be used as an effective green antibacterial agent in food processing.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (31871866), the Innovation of Agricultural Science and Technology of Jiangsu Province (CX(18)1006), and China Agriculture Research System (CARS-41).

Conflict of interest statement: The authors declare that they have no conflicts of interest regarding the contents of this article.

Contributor Information

Lihui Du, Email: ddabc_2000@163.com.

Daoying Wang, Email: daoyingwang@yahoo.com.

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