In the present study, ESBL-producing E. coli (KBN10P03217) was used which was isolated from the blood sample of the 70-years old male patient and exhibited resistance to several antibiotics, including ceftazidime, amphotericin B, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotaxime, ciprofloxacin, and trimethoprim/sulfamethoxazole. Bacterial sample was received after the ethical clearance from the ethical committee of Kyungpook National University Hospital, Daegu, South Korea. E. coli was grown in Luria-Bertani (LB) medium purchased from Acumedia (Lansing, MI, United States). Carvacrol, with a purity of ≥98%, was purchased from Sigma-Aldrich (St. Louis, MO, United States) and dissolved in 5% dimethyl sulfoxide (DMSO) (Sigma Aldrich). Phosphate-buffered saline (PBS) with 5% DMSO was used as a negative control for all experiments.

The minimum inhibitory concentration (MIC) of carvacrol was evaluated using the broth micro-dilution technique in LB broth with an initial inoculum of 10⁷ cells/ml in a non-treated polystyrene microtiter plate CC7672-7596; (CytoOne) in accordance with the Clinical and Laboratory Standards Institute (CLSI). The MIC was interpreted as the lowest concentration of carvacrol that completely inhibited visible growth of the bacteria after 24 h of incubation at 37°C. Each concentration was tested in triplicate in at least three independent experiments.

The viable cell count was evaluated according to the method adopted by Chauhan and Kang (2014). Briefly, mid-log phase E. coli cells (10⁷ cells/ml) were inoculated into LB broth containing carvacrol (MIC) and incubated at 37°C. At different incubation times such as 0, 30, 60, 90, and 120 min, the culture was plated onto LB agar medium with appropriate dilutions, followed by incubation at 37°C for 24 h. Colonies were counted after the incubation period as indicated above. Simultaneously, the controls without carvacrol were also set up under the same experimental conditions.

Live and dead cells were evaluated by AO/EB (Acridine orange and ethidium bromide) fluorescent staining (Hameed et al., 2016). Overnight grown E. coli cells (10⁷ cells/ml) were treated with or without carvacrol (MIC) for 15 min at 37°C. After incubation, cells were harvested by centrifugation, washed with PBS and stained with AO/EB (1:1) (100 μg/ml) for 30 min. After 30 min incubation, cells were washed with PBS and visualized under fluorescent microscope. Similarly, to assess the effect of carvacrol on reactive oxygen species (ROS) and membrane potential, H₂DCFDA [5(6) -Carboxy-2′,7′-dichlorofluorescein diacetate] and rhodamine 123 fluorescent staining was carried out, respectively (Warnes et al., 2012). In brief, E. coli cells (10⁷ cells/ml) were treated with or without carvacrol for 15 min followed by centrifugation to harvest the cells. After washing with PBS, cells were stained in dark for 30 min with H₂DCFDA (10 mM) or rhodamine 123 (1 μg/ml). Cells were thoroughly washed with PBS and visualized under EPI fluorescent Eclipse TS100 Inverted Routine Microscope with PAXCam software (Nikon TS 100, Japan). All experiments were repeated three times independently and at least three different fields were observed for each culture.

Alteration of membrane permeability was detected by crystal violet assay (Devi et al., 2010). Suspensions of E. coli (10⁷ cells/ml) were prepared in LB broth. Cells were harvested at 10,000 rpm for 5 min. The cells were washed twice and suspended in 50 mM PBS (pH 7.4). Carvacrol at MIC was added to the cell suspension and incubated at 37°C for 30 min. Control samples were prepared similarly without treatment. The cells were harvested at 10,000 rpm for 5 min and suspended in PBS containing 10 μg/ml of crystal violet. The cell suspension was then incubated for 10 min at 37°C and centrifuged at 10,000 rpm for 5 min, after which the OD₅₉₀ of the supernatant was measured using a UV-VIS spectrophotometer (UV-2120 Optizen, Mecasys, South Korea). The optical density (OD) value of crystal violet solution, which was originally used in the assay, was considered as 100% excluded. The percentage of crystal violet uptake for all samples was calculated using the following formula:

Measurement of release of cellular materials (DNA) from E. coli cells was carried out by inoculating the mid-log phase culture (10⁷ cells/ml) into NaCl (0.85%) in the presence and absence of carvacrol (MIC) and incubated at 37°C. After incubation for 0, 30, 60, and 120 min, 1 ml of broth was transferred to an Eppendorf tube, which was centrifuged at 10,000 rpm. The absorbance of the supernatant was then measured at 260 nm using an UV-VIS spectrophotometer. Results were expressed in the form of OD recorded at each time interval. Simultaneously, the amount of released protein in the presence and absence of carvacrol was determined by using Bradford reagent (Devi et al., 2010). In brief, 5 ml overnight grown cells (10⁷ cells/ml) were harvested at 8,000 rpm, washed thoroughly three times using normal saline (0.85% NaCl) and finally suspended in 1 ml of saline in the presence of carvacrol. A control without carvacrol was also set up under the same experimental conditions. At different time points (60 and 120 min), protein release was quantified.

Furthermore, to confirm the damaging effect of carvacrol on E. coli cell membrane, the supernatant of carvacrol-treated bacterial suspensions was subjected to SDS-PAGE (Devi et al., 2010). Initially, bacterial cells were grown to OD 2.0 and then harvested at 10,000 rpm for 5 min at room temperature; the bacterial pellet was washed twice with PBS (pH 7.4) and re-suspended in PBS. Carvacrol at MIC value was added to the cell suspensions, and the samples were incubated at 37°C for 60 and 120 min. After treatment, the suspensions were centrifuged at 10,000 rpm for 10 min. Control samples were prepared similarly without carvacrol treatment. The supernatant (50 μl) was placed in a centrifuge tube, after which 25 μl of sample buffer (1 M Tris-HCl (pH-6.8), 50% glycerol, 10% SDS, 10% β-mercaptoethanol, 1% bromophenol blue) was added. The samples were then heated at 100°C for 5 min and loaded onto 12% SDS-PAGE. Silver staining was performed to analyze the proteins released due to membrane disruption.

The potential effect of carvacrol on the cell morphology of E. coli was determined with the help of SEM (Khan and Kang, 2016). In brief, bacterial cells were incubated with or without carvacrol for 1 h, washed three times using PBS (pH 7.4), and then centrifuged at 4,000 rpm. The obtained pellet was suspended in PBS, and a thin smear was prepared on a glass slide, which was further fixed in glutaraldehyde (2.5 g/100 ml; Electron Microscopy Science, United States) for 2 h. After fixation, a dehydration step was performed using an increasing order of ethanol ranging from 50 to 100%, after which the cells were dried with liquid CO₂. The dried cells were coated with gold in a sputter coater, and samples were observed under a scanning electron microscope (SEM) (Hitachi-S4300, Japan).

To assess the effect of carvacrol on bacterial motility, a colony grown on LB agar was stabbed into semi-solid medium (1% Bacto tryptone; Difco Laboratories, Detroit, MI, United States), 0.5% (w/v) NaCl, and 0.35% agar (Difco Laboratories, Detroit, MI, United States) with or without carvacrol at sub-inhibitory concentrations (100 and 150 μg/ml). Bacterial swarming was assessed after incubating the stabbed E. coli at 37°C for 24 h. Motility was further confirmed by the non-quantitative hanging-drop technique (Inamuco et al., 2012). In brief, a droplet of bacterial culture was suspended from a glass coverslip over a microscope slide with a central concavity. The motility of bacterial cells was observed at a magnification of 100x under a light microscope.

Human colon HCT-116 cell line was purchased from the Korean Cell Line Bank (KCLB) and cultured in RPMI-1640 medium at 37°C in 5% CO₂ incubator.

Escherichia coli was grown to mid-log phase in the presence of 100 or 150 μg/ml of carvacrol. Bacterial culture was centrifuged and suspended in RPMI-1640 medium to a density of 1 × 10⁸ cells/ml. HCT-116 colon cells were grown to confluence in 12-well tissue-culture plates, washed three times with plain medium, and incubated with 1 ml of the bacterial solution for 1 h at 37°C. HCT-116 monolayers in wells were washed once with 1 ml of warm plain medium and then incubated for 2 h with 1 ml of 100 mg/ml of gentamycin in warm RPMI-1640 medium to kill extracellular bacteria. Cells were washed three times with plain RPMI-1640 medium and finally lysed with 1% Triton X-100. The number of intracellular bacteria was determined by colony-plating as described above (Khan and Kang, 2016).

The cytotoxic effect of carvacrol was first determined by MTT assay (Khan and Kang, 2017) Briefly, 5 × 10⁴ HCT-116 cells/well were incubated in the presence of carvacrol at the concentrations of 50, 100, and 150 μg/ml for 2 h at 37°C in a CO₂ incubator. After incubation, cells were treated with MTT solution (5 mg/ml) to produce dark blue colored formazan crystals, which were further dissolved in 50 μl of DMSO. Finally, absorbance was measured at 540 nm in a microplate reader (Bio-Tek Instrument, Co., Everett, WA, United States).

All experiments were carried out in triplicate. The results were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical significance was calculated using Student’s “t” tests. ^∗Represents P- values < 0.05, ^∗∗represents P- values < 0.01, and ^∗∗∗represents P-values < 0.001.

In this assay, carvacrol exhibited a severe detrimental effect on the growth of E. coli at MIC (450 μg/ml). Especially, viable cell counts of E. coli decreased significantly in the time-dependent manner after exposure of carvacrol. As depicted in Figure 1, log CFU/ml drastically decreased to less than log 2 CFU/ml after 60 min, followed by complete inhibition at 120 min. This proves that carvacrol has a severe toxic effect on the viable count of E. coli, whereas cell number of control sets prepared without carvacrol increased slightly.

Furthermore, live and dead bacterial cells were evaluated by AO/EB staining. In AO/EB staining, AO stains live cells, however; EB stains to cells which have lost membrane integrity. AO stained cells give green fluorescence representing live cells while EB gives red fluorescence denoting dead cells. We found a high red color fluorescence in carvacrol treated cells, indicating its toxicity to E. coli (Figure 2).

A crystal violet uptake assay was used to determine the membrane permeability. Uptake of crystal violet by E. coli was 4.8% in the absence of carvacrol which increased by 26 and 42% at 200 and 450 μg/ml (MIC) of carvacrol treatment, respectively (Figure 3A). Furthermore, we investigated the membrane polarization with rhodamine 123. Carvacrol treatment to E. coli for 15 min significantly reduced the fluorescence intensity of rhodamine 123 (Figure 3B) signifying its adverse effect on electrical potential (ΔΨ) of bacterial membrane. Moreover, the role of carvacrol on the induction of oxidative stress by the involvement of ROS was observed by H₂DCFDA staining (Figure 3B). The fluorescence dye H₂DCFDA was used to detect the accumulated ROS in the bacterial cell where it is deacetylated by esterase and gives green color that visualized under fluorescence microscope. After treatment with carvacrol for 15 min, ROS level found to be elevated in the E. coli representing its impact on the induction of oxidative stress.

In this assay, carvacrol showed potential to disrupt the cell membrane integrity of E. coli, leading to release of cellular materials. As depicted in Figure 4A, carvacrol led to the release of intracellular materials based on absorbance at 260 nm, whereas control experimental sets showed no release of cellular contents. Simultaneously, protein release was detected by using the Bradford method. Protein release serially increased at 60–120 min from 400 to 480 μg/ml, in the carvacrol treated cells (Figure 4B). To confirm membrane disintegration of E. coli by carvacrol, released membrane proteins were also detected by SDS-PAGE. Intensities of separated bands increased from 60 to 120 min and showed a distinct banding pattern as compared to control (untreated) (Figure 4C).

Scanning electron microscopy was carried out to visualize the effect of carvacrol on cell morphology of E. coli. Carvacrol caused cell membrane lysis, as evident by membrane damage and altered cell morphology unlike to control (untreated) where intact cells were observed (Figure 5).

To determine the suitable concentration of carvacrol for motility and invasion experiments, we plotted a growth curve with different concentrations of carvacrol (0–250 μg/ml). As a results, no significant growth inhibition of E. coli was observed at 100 and 150 μg/ml (Figure 6A). Moreover, carvacrol did not exert the cytotoxic effect on HCT-116 colon cells as determined by MTT assay (Figure 6B). Furthermore, carvacrol showed a significant effect on the bacterial motility at the sub-lethal concentrations of 100 and 150 μg/ml. At 150 μg/ml concentration of carvacrol, E. coli showed minimum motility with 39% relative zone diameter as compared to 100% in the control (untreated) (Figure 6C). Light microscopy results of the hanging drop assay revealed complete loss of motility at concentration 150 μg/ml of carvacrol (data not shown). Furthermore, E. coli cells pretreated with carvacrol showed decreased invasion to HCT-116 colon cells as compared to control (untreated) (Figure 6D). E. coli cells pretreated with 150 μg/ml carvacrol represent 31 ± 3.2% invasions as compared to the 100% invasion of the control.

Two concentrations of carvacrol 100 and 150 μg/ml were considered as sub-lethal as bacterial cells were found to grow freely in these concentrations. The finding suggests that carvacrol has strong inhibitory effects against bacterial invasion and motility.