The L. monocytogenes 10,403 s strain was used in this study and was grown in brain heart infusion (BHI, Haibo, Qingdao, China) broth media supplemented with 1% (w/v) peptone, 1.25% (w/v) dehydrated calf brain infusion, 0.5% (w/v) dehydrated beef heart infusion, 0.5% (w/v) NaCl, 0.2% (w/v) glucose, and 0.25% (w/v) Na₂PO₄ under sterile conditions. The medium was filter-sterilized with a 0.2 μm filter (Beyotime Biotechnology, Shanghai, China). Before exposure to glabridin or macrophages, the bacterial cultures were incubated in sterile 50 mL closed conical tubes (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou, China) at 37°C under static conditions or with shaking at 180 rpm.

Glabridin (molecular weight of 324.40 g/moL) from Glycyrrhiza glabra L. root (licorice) was obtained from Luoyang Lansealy Technology Co., Ltd. (Luoyang, China). The antibacterial activity of glabridin against L. monocytogenes 10,403 s was determined using broth microdilution methods (MIC and MBC), as previously described (Fang et al., 2018). Briefly, 2-fold serial dilutions of glabridin ranging from 1.95 to 500 μg/mL were prepared and then added to wells containing bacteria (1 × 10⁶ CFUs/ml) in a 96-well plate. The plates were incubated overnight at 37°C. Finally, the MIC was defined as the lowest sample concentration that prevented microbial growth according to the OD_{600 nm}. A 1:10 dilution of each concentration in fresh broth was spread onto BHI (Haibo, Qingdao, China) agar plates to determine the MBC. After an incubation at 37°C for 24 h, the MBC was recorded as the minimum concentration at which no apparent growth on agar subculture occurred. Triplicates of this experiment were conducted.

Growth curves were generated to determine the effect of subinhibitory concentrations of glabridin on L. monocytogenes growth, as previously described (Jiang et al., 2019). Briefly, cultures of L. monocytogenes at the exponential phase in the BHI medium were collected by centrifugation and washed once with fresh BHI medium. Then, the cell’s initial concentration was adjusted to a concentration of 1 × 10⁶ CFUs/ml, and the cells were incubated in BHI media supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) or the MIC of glabridin with shaking at 37°C. The optical density at 600 nm was measured each hour. Moreover, viable bacteria were counted by serial dilution, plating onto BHI agar, and incubation at 37°C for 24 h.

Moreover, the colony morphology of L. monocytogenes was evaluated on BHI agar plates. Cultures of L. monocytogenes at the exponential phase in BHI medium were collected by centrifugation and washed once with PBS (10 mM, pH 7.4). Ten microliters (~10⁴ CFUs) of the culture solution were dripped on BHI agar supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) or the MIC of glabridin. Following an overnight incubation at 37°C, the colony morphology was observed and photographed. The colony growth diameter was measured using a micrometer.

Cultures of L. monocytogenes at the exponential phase in BHI medium supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC), the MIC, or MBC of glabridin were collected by centrifugation and washed once with PBS (10 mM, pH 7.4) to observe cellular morphology. Then, the cell suspensions were uniformly spread onto sterile glass coverslips. Bacteria were stained with Gram stain and examined under a light microscope.

Moreover, scanning electron microscopy (SEM) was performed via an earlier procedure (Jalil Sarghaleh et al., 2023). Cultures of L. monocytogenes at the exponential phase in BHI medium supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC), the MIC, or MBC of glabridin were collected by centrifugation and washed once with PBS (10 mM, pH 7.4). Then, the cell suspensions were uniformly spread onto sterile glass coverslips. A 2.5% (v/v) glutaraldehyde solution (Servicebio, Wuhan, China) was used for stabilization and incubated with the samples at 4°C for 2 h. After washing with distilled water, ethanol gradients (25, 50, 75, 95, and 100%) were used for the final dehydration. After air drying at room temperature, critical point drying was performed with liquid CO₂. The samples were covered with a layer of gold and then observed under a JEOL scanning electron microscope (JSM-IT200 InTouchScope™, JEOL, Tokyo, Japan).

The motility phenotype was assayed on soft BHI agar as described previously (Roy et al., 2022), with minor modifications. Briefly, 0.25% (w/v) and 0.5% (w/v) agar were mixed with BHI for the swimming and swarming studies, respectively. Cultures (~10⁴ CFUs) at the exponential phase were point inoculated onto BHI agar plates supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin. The culture medium was incubated at 30°C for 24 h, and the migration zone (mm) was determined to report the bacterial motility in the agar by measuring the diameter using a micrometer. Each group was independently tested three times.

TEM experiments were performed to investigate the flagella presence as described previously (Wang et al., 2024). Bacteria from 0.25% BHI agar plates supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin were fixed with a 3% paraformaldehyde solution on an electron microscopy grid. Then, the grids were washed three times. The bacteria were stained with phosphotungstic acid (2%) for 10 s at room temperature, the excess liquid was gently removed using filter paper, and the grids were air-dried. The dried grids were observed under a Hitachi transmission electron microscope (HT7700, Hitachi High-Tech Corporation, Tokyo, Japan).

The effect of subinhibitory concentrations of glabridin on the formation of biofilms was determined using a crystal violet assay according to the procedure described by Djordjevic et al. (2002), with slight modifications. Assays were performed in sterile 96-well plates. Briefly, overnight cultures were added to the wells with 100 μL of fresh medium supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC), the MIC or MBC of glabridin. The plates were placed in an incubator at 37°C for 48 h. Subsequently, the supernatant was discarded, and then the wells were gently washed three times with aseptic PBS. The plates were blotted dry on a sterilized paper towel after the last wash and allowed to dry naturally for 1 h. Then, the biofilms were stained with 2% (m/v) crystal violet for 45 min. The wells were washed with distilled water to remove the unbound dye. After drying at 37°C for 10 min, the cell-bound dye was dissolved in 200 μL of 95% (w/w) ethanol. The quantitative evaluation of biofilm formation was performed on a microplate reader (Infinite M Nano, TECAN, Switzerland) by measuring the absorbance of the solution at 570 nm.

Moreover, the biofilm formation of L. monocytogenes was investigated using confocal laser scanning microscopy (CLSM) (Adamus-Bialek et al., 2015). Initially, overnight cultures were seeded onto 20-mm glass cover slips in 12-well plates in the absence or presence of glabridin. After an incubation at 37°C for 48 h to allow biofilm formation, the culture medium was discarded, and then the wells were gently washed three times with aseptic PBS. Subsequently, the biofilms were stained with SYTO Green (KeyGEN BioTECH, Nanjing, China) and PI (Beyotime Biotechnology, Shanghai, China), a green and red fluorescent nucleic acid stain, for up to 20 min at room temperature in the dark. The samples were visualized under a confocal laser scanning microscope (FV3000, Olympus, Japan) at an excitation wavelength of 500 nm and emission wavelength of 535 nm, according to the manufacturer’s instructions.

SEM was used to visualize the morphologies of the L. monocytogenes biofilms, as previously described (Wang et al., 2022), with slight modifications. The fresh cultures were placed on 20-mm glass cover slips in the wells of 12-well plates. After the incubation, the attached cells were washed three times with PBS to remove the planktonic cells. The samples were fixed with a 2.5% (v/v) glutaraldehyde solution (Servicebio, Wuhan, China) at 4°C for 10 h. After washes with distilled water, the samples were subjected to dehydration with an increasing series of ethanol solutions (25, 50, 75, 95, and 100%). After air drying at room temperature, critical point drying was performed with liquid CO₂. Subsequently, the samples were covered with a layer of gold using a sputter coater and examined using a JEOL scanning electron microscope (JSM-IT200 InTouchScope^™, JEOL, Tokyo, Japan).

The evaluation of the biofilm-eradicating ability of glabridin was performed according to the previously described methods with further modifications (Shen et al., 2021). Briefly, overnight cultures were added to 96-well plates and incubated for 48 h at 37°C to form a biofilm. After the incubation, the culture medium was removed and replaced with sterile BHI medium by washing three times with PBS. Subsequently, subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC), the MIC, and MBC of glabridin were added to the wells. The plates were incubated at 37°C for 1 h. After the incubation, the methods mentioned above (crystal violet assay, CLSM, and, SEM) were used to evaluate biofilm eradication.

Hemolytic activity was assayed using the blood-plate method as previously described (Kawacka et al., 2022). Briefly, 5.0% of sterile sheep blood was mixed with BHI to prepare a blood agar plate. L. monocytogenes cultures with logarithmic growth (~10⁴ CFUs) at the exponential phase were point-inoculated onto BHI agar plates supplemented with subinhibitory concentrations (1/8, 1/4, and 1/2 of MIC) of glabridin. The culture medium was incubated at 37°C for 24 h. The plates were then visually examined for clear zones or no zones around colonies. The experiments were independently repeated three times.

Moreover, the hemolytic activity was determined by performing a microdilution method according to a previous study (Yue et al., 2023). Briefly, L. monocytogenes cultures with logarithmic growth (~10⁶ CFUs) were treated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin at 37°C and 120 rpm for 12 h. Bacterial cultures were centrifuged at 12,000 rpm for 10 min. Then, the supernatant was filtered through a 0.22 μm pore filter (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou, China). After filtration, 100 μL of the cell-free supernatant was mixed with 100 μL of a 5.0% (v/v) red blood cell suspension in a 96-well plate and incubated for 1 h at 37°C. Subsequently, the tested samples were gathered by centrifugation at 1000 rpm for 5 min. Finally, absorbance measurements were performed at 540 nm with a microplate reader. BHI and 1% Triton X-100 were served as negative and positive controls, respectively. The percentage of hemolysis (%) = (OD₅₄₀ nm of test well – OD₅₄₀ nm of negative control)/(OD₅₄₀ nm of positive control – OD₅₄₀ nm of negative control) × 100%.

L. monocytogenes cultures with logarithmic growth were initially treated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin at 37°C and 120 rpm for 12 h. The samples were centrifuged at 12,000 rpm for 5 min, and the supernatant was discarded. Total RNA was extracted using a TRNpure Total RNA Kit (Nobelab Biotech, Beijing, China) based on the manufacturer’s manual. The RNA concentration, purity, and integrity were assessed using a NanoDrop 2000c (Thermo, Massachusetts, United States). Then, the cDNA strand was synthesized using an EasyScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (TransGen, Beijing, China), according to the manufacturer’s protocol. Reactions (20 μL) containing 100 ng of RNA, the mix, and primers were prepared. The thermal cycling conditions were 42°C for 15 min and 85°C for 5 s using a T100 Thermal Cycler (T100™; Bio-Rad Laboratories, California, United States). cDNA was quantified using a NanoDrop 2000c. Beacon Designer (version 8.0) software was used to design specific primers for the hly (GenBank: DQ054589.1; forward: 5′-TGCAAGTCCTAAGACGCCA-3′, reverse: 5′-CACTGCATCTCCGTGGTATACTAA-3′) gene (Tamburro et al., 2015). The 16S rRNA (GenBank: CP002002.1; forward: 5′-TTAGCTAGTTGGTAGGGT-3′, reverse: 5′-AATCCGGACAACGCTTGC-3′) gene is considered a housekeeping gene for L. monocytogenes (Fraser et al., 2003). The amplification profile was as follows: predenaturation at 94°C for 30 s and 40 cycles of 5 s at 94°C and 30 s at 60°C. Reactions (20 μL) containing PerfectStart^® Green qPCR SuperMix (TransGen, Beijing, China), cDNA, and specific primers were performed in a CFX96 real-time system (Bio-Rad Laboratories, California, United States). All samples were prepared in triplicate. The mRNA expression level of the hly gene was normalized to the expression level of 16S rRNA. The expression levels in the untreated group were normalized to 1 for comparison with those in the treated group. The relative expression levels of the hly gene were analyzed using the 2^−ΔΔCT relative expression quantification method: Δ^ΔCt = (mean Ct value of hly in the sample – mean Ct value of 16S rRNA in the sample) – (mean Ct value of hly in the control – mean Ct value of 16S rRNA in the control).

RAW264.7 cells were purchased from Procell Life Science and Technology Co., Ltd. (Wuhan, China). The RAW264.7 cells and Caco-2 cells were stored in the Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control. The cells were grown in DMEM (HyClone, United States) supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS; Clark Bioscience, Dalian, United States) and maintained at 37°C with 5% CO₂ in a humidified incubator. Then, 100 U/mL penicillin and 100 μg/mL streptomycin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) were added when needed. The cells were passaged in 25 cm² flasks (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou, China). After the cells were counted using a hemocytometer, they were seeded into 24-well plates (Guangzhou Jet Bio-Filtration Co., Ltd., Guangzhou, China) for subsequent procedures.

The cytotoxicity of glabridin was evaluated using a lactate dehydrogenase (LDH) assay kit as previously described (Hou et al., 2023). Briefly, RAW264.7 cells and Caco-2 cells were seeded at a density of 2 × 10⁵ cells per well in a 24-well cell culture plate, respectively. The cells were incubated in DMEM supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC), the MIC, or MBC of glabridin for 6 h. The cells treated with 0.2% Triton X-100 or DMEM only were used as the positive and negative controls, respectively. The cell culture supernatants were centrifuged and collected. The levels of LDH release were measured using an LDH Cytotoxicity Assay Kit (Beyotime Biotechnology, Shanghai, China) in accordance with the manufacturer’s instructions. The absorbance at 490 nm was read using a microplate reader. The results are shown as (OD₄₉₀ nm of sample – OD₄₉₀ nm of negative control)/(OD₄₉₀ nm of positive control – OD₄₉₀ nm of negative control) × 100%.

The intracellular growth assay was performed as previously described (Meng et al., 2015; Zhou et al., 2017). RAW264.7 cells were seeded at a density of 2 × 10⁵ cells per well in 24-well plates with antibiotic-free medium. The cells were incubated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin, infected with bacteria at an MOI of 10:1, and then incubated at 37°C with 5% CO₂ for 1 h. The cells were rinsed three times with PBS and incubated with 100 μg/mL gentamicin for 1 h to kill the extracellular bacteria. Then, the cells were washed three times with PBS to remove the killed extracellular bacteria. The infected RAW264.7 cells were then incubated with fresh DMEM supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin and 10% FBS for 3h. The cells were subsequently lysed with 0.1% Triton X-100 in PBS for 5 min. The number of viable intracellular bacteria was determined by plating serial dilutions of the resulting lysates on BHI agar plates. The experiments were repeated in three independent assays.

Macrophage extracellular traps (METs) were visualized as previously described (Cogen et al., 2010; Brinkmann et al., 2012). RAW264.7 cells (2 × 10⁵ cells per well) were plated onto laser confocal Petri dishes in DMEM without antibiotics or fetal calf serum. The cells were incubated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin, infected with L. monocytogenes at an MOI of 10:1, and then incubated at 37°C with 5% CO₂ for 2 h. The cells were stained with Hoechst 33342 (Beyotime Biotechnology, Shanghai, China) and observed via confocal laser scanning microscopy (FV3000, Olympus, Japan) at 350 nm excitation and 460 nm emission. Images of random fields were acquired from each group (Zhang X. W. et al., 2023). The MET formation in the PMA-treated macrophages was set as 100%. The data are presented as percentages of the PMA-treated group.

Intracellular ROS generation was detected according to a previously described procedure (Liu et al., 2014). RAW264.7 cells (2 × 10⁵ cells per well) were seeded onto laser confocal Petri dishes in DMEM without antibiotics or fetal calf serum. The cells were incubated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin, infected with L. monocytogenes at an MOI of 10:1, and then incubated at 37°C with 5% CO₂ for 2 h. DCFH-DA (5 μM, Beyotime, Shanghai, China) was added, and the cells were continuously incubated in the dark. The samples were observed under a confocal laser scanning microscope at 488 nm excitation and 525 nm emission.

The MET-mediated extracellular killing assay was performed as described previously (Liu et al., 2014). Briefly, 2 × 10⁵ RAW264.7 cells per well were seeded into a 24-well plate. The cells were incubated with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin, infected with L. monocytogenes at an MOI of 10:1, and then incubated at 37°C with 5% CO₂ for 2 h. Cytochalasin D (10 μg/mL; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) or 100 U/mL protease-free DNase I (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) was added to inhibit phagocytosis or MET formation at 20 min before infection with L. monocytogenes. Pretreatment with cytochalasin D and DNase I was used as the 100% survival control group. The experiments were repeated five times. Bacteria were serially diluted and evenly plated onto BHI agar to determine the number of CFUs.

All experiments were performed in three independent replicates. The data are presented as means ± SEMs. Statistical significance was determined using two-tailed Student’s t-test. p values indicated in the figures (*p < 0.05 and **p < 0.01) were considered significant.

As determined using the microdilution method, the MIC value for L. monocytogenes was 31.25 μg/mL of glabridin. The results indicated that the MBC of glabridin against L. monocytogenes was 125 μg/mL. The optical densities of the bacterial suspension were measured at 600 nm to determine the effect of subinhibitory concentrations of glabridin on the growth of L. monocytogenes. Following an overnight incubation in BHI medium, the growth profiles of glabridin-treated and -untreated L. monocytogenes were similar (Supplementary Figure S1A). As shown in Figure 1, the growth of L. monocytogenes was not significantly affected by glabridin concentrations of 3.91, 7.81, or 15.63 μg/mL, which are equal to 1/8, 1/4, or 1/2 of the MIC of glabridin, respectively (Figure 1A). Moreover, the colony morphologies of the glabridin-treated and -untreated L. monocytogenes strains were compared. The detailed results are presented in Figure 1B. The colony morphology of the glabridin-treated L. monocytogenes after incubation with subinhibitory concentrations was similar to that of the glabridin-untreated L. monocytogenes in BHI agar plates (Figure 1B). As the glabridin concentration increased, no significant differences in colony size were observed (Supplementary Figure S1B). The colonies of all groups appeared as milky white, opaque colonies with smooth surfaces. Therefore, our results suggested that subinhibitory concentrations of glabridin did not affect the growth of L. monocytogenes.

Gram staining and SEM were performed to determine the effect of subinhibitory concentrations of glabridin on the morphology of L. monocytogenes. The light microscopic examination of bacterial staining indicated that L. monocytogenes treated with 1/8, 1/4, or 1/2 of the MIC for glabridin had the same cellular morphology as the glabridin-untreated L. monocytogenes (Supplementary Figure S1C). The microscopic appearance of the glabridin-treated L. monocytogenes was further observed via SEM. SEM images of L. monocytogenes before and after treatment with subinhibitory concentrations of glabridin (1/8, 1/4, or 1/2 of the MIC) are shown in Figure 1C. The structure of L. monocytogenes is naturally rod-shaped. Treatment of the bacteria with glabridin (1/8, 1/4, or 1/2 of the MIC) did not cause changes in the surface morphology (Figure 1C). Under a light microscope, the results revealed that the number of bacteria in the group treated with the MIC of glabridin was significantly lower than the number of bacteria in the groups treated with subinhibitory concentrations of glabridin (Supplementary Figure S1C). However, SEM confirms the shrunken, crumpled appearance of the membranes of some bacteria in the group treated with the MIC of glabridin (Figure 1C). Moreover, the bacteria were disrupted after the application of the MBC of glabridin (Figure 1C). Therefore, in the following experiments in this study, subinhibitory concentrations of glabridin, 1/8, 1/4, or 1/2 of the MIC, were used since they did not affect the growth or morphology of L. monocytogenes.

Flagellum-mediated motility is involved in the bacterial pathogenicity, biofilm formation, drug resistance, and chronic infections. Thus, we examined whether subinhibitory concentrations of glabridin affect the motility of L. monocytogenes in vitro. A semisoft agar assay with 0.3% (w/v) and 0.5% (w/v) agar was used to monitor the swimming and swarming of L. monocytogenes, respectively. In the swimming test, we observed a significant decrease in the mobility of L. monocytogenes exposed to glabridin (1/4 or 1/2 of the MIC) through soft agar (Figure 2A). In addition, the quantitative experimental results showed that the swimming expansion rate of L. monocytogenes colonies in the presence of 1/8 of the MIC of glabridin did not differ from that of glabridin-untreated L. monocytogenes colonies (Figure 2B). As depicted in Figure 2, for the swarming test, the motility of L. monocytogenes decreased with increasing concentrations of glabridin (1/8, 1/4, or 1/2 of the MIC) in 0.5% BHI agar, and the effect was concentration-dependent (Figure 2A). The zone size of L. monocytogenes exposed to glabridin at a concentration of 1/2 of the MIC was significantly lower than that at a concentration of 1/8 of the MIC (Figure 2B). Therefore, subinhibitory concentrations of glabridin inhibited bacterial swimming and swarming motility in L. monocytogenes.

TEM was used to further determine whether subinhibitory concentrations of glabridin adversely affected flagella synthesis. Interestingly, the results showed that L. monocytogenes exposed to different concentrations of glabridin were able to produce flagellar filaments, as shown by negative staining of the glabridin-untreated L. monocytogenes (Figure 2C). No significant difference was found. Therefore, the results clearly showed that subinhibitory concentrations of glabridin did not interfere with flagellar production in L. monocytogenes.

Bacteria were grown on polystyrene in BHI plates supplemented with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin to study whether subinhibitory concentrations of glabridin influence biofilm formation of L. monocytogene in vitro. Biofilm formation was quantified after 48 h by performing crystal violet staining. As shown in Figure 3A, L. monocytogenes produced a biofilm in the absence of glabridin. Although a declining trend in biomass was observed after the bacteria were exposed to subinhibitory concentrations of glabridin, a statistically significant difference was not observed after culture in the absence or presence of glabridin (Supplementary Figure S2A). The corresponding fluorescence microscopy images are shown in Figure 3A. Consistent with the above results, L. monocytogenes showed a similar phenomenon in terms of the biofilm biomass with or without glabridin treatment (Figure 3A). Since a green color is used to stain cells, the cells treated with or without glabridin were clearly green and observed in the form of dots. Moreover, very small amounts of biofilm production were observed after treatment with glabridin at the MIC but not at the MBC (Figure 3A). SEM imaging revealed biofilm morphologies and supported the CLSM results. Based on the SEM images, many microcolonies that held together were observed in the absence of glabridin, whereas no change occurred after treatment with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) of glabridin (Figure 3B). However, compared with those in the control group, a few cells were attached to the glass coupons after treatment with glabridin at the MIC, and the bacteria exhibited a normal morphology. Interestingly, the bacteria were disrupted after the application of glabridin at the MBC (Figure 3B), and the bacterial morphology was obviously wrinkled.

According to the crystal violet staining results, none of the subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) or the MIC of glabridin could eradicate the complete biofilms of L. monocytogenes (Figure 4A; Supplementary Figure S2B). What is more, at the MBC, glabridin did not eradicate the formed biofilms. Furthermore, CLSM images indicated that subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) or the MIC of glabridin had no obvious effect on the biofilms (Figure 4B). Then, SEM images of the bacterial biofilms were recorded. The thick and dense layer of bacteria in the control group was smooth and intact (Figure 4B), similar to that observed after treatment with subinhibitory concentrations (1/8, 1/4, or 1/2 of the MIC) or the MIC of glabridin (Figure 4B). When the bacteria were exposed to glabridin at the MBC, the bacteria exhibited obvious wrinkling (Figure 4B). These results suggested that subinhibitory concentrations of glabridin did not alter L. monocytogenes biofilms.

A bacterial plate assay was used to determine hemolytic activity. As shown in Figure 5A, L. monocytogenes 10403S without glabridin treatment exhibited hemolytic activity on blood agar plates. No difference in the hemolytic ability was observed between untreated L. monocytogenes and L. monocytogenes treated with glabridin at 1/8 of the MIC. However, none of the L. monocytogenes strains cultured in the presence of glabridin at 1/4 or 1/2 of the MIC showed hemolytic activity (Figure 5A). The microdilution method also indicated that the addition of glabridin at 1/4 or 1/2 of the MIC resulted in a dramatic decrease in hemolytic activity (Figure 5B). Moreover, the expression of the hly gene was significantly higher in L. monocytogenes treated with glabridin at 1/4 or 1/2 of the MIC than in untreated L. monocytogenes. The expression of the hly gene in L. monocytogenes treated with glabridin at 1/4 or 1/2 of the MIC decreased significantly by more than 5-fold compared with that in L. monocytogenes treated with glabridin at 1/8 of the MIC (p < 0.001), as shown in Figure 5C. Therefore, these results clearly showed that subinhibitory concentrations of glabridin decreased the hemolytic activity of L. monocytogenes.

The cytotoxic effects of subinhibitory concentrations of glabridin on epithelial cells and macrophages were investigated via the LDH method following 6 h of treatment. As shown in Figure 6, different concentrations of glabridin (1/8, 1/4, or 1/2 of the MIC) had no toxic effect on Caco-2 and RAW264.7 cells (Figure 6). The results showed that subinhibitory concentrations of glabridin did not induce the release of LDH from Caco-2 and RAW264.7 cells. Concentrations of 1/8, 1/4, or 1/2 of the MIC were used for additional cell studies.

Gentamicin survival assays were performed to evaluate the influence of glabridin at subinhibitory concentrations on the intracellular growth of L. monocytogenes in RAW264.7 cells, as previously described. The results of the intracellular growth assay are illustrated in Supplementary Figure S3. After exposure to subinhibitory concentrations of glabridin at 1/4 or 1/2 of the MIC, the number of intracellular bacteria in RAW264.7 cells was much lower than that in the glabridin-untreated L. monocytogenes (Supplementary Figure S3). Glabridin inhibited the intracellular growth of L. monocytogenes in a concentration-dependent manner. However, treatment with glabridin at 1/8 of the MIC did not affect the intracellular growth of L. monocytogenes in RAW264.7 cells. This result suggested that subinhibitory concentrations of glabridin could improve L. monocytogenes clearance in RAW264.7 cells.

We incubated RAW264.7 cells with glabridin (at 1/8, 1/4, or 1/2 of the MIC) for 2 h to examine the effect of glabridin on MET formation in macrophages. The fluorescence imaging analysis revealed that compared with the control treatment, treatment with PMA, an inducer of METs, could stimulate macrophages to release METs (Figure 7). The untreated macrophages did not release extracellular DNA. However, treatment with glabridin (1/8, 1/4, or 1/2 of the MIC) did not induce MET formation in macrophages (Figure 7). However, the presence of glabridin at the MBC significantly induced the release of extracellular DNA from macrophages (Supplementary Figure S4). In addition, we also observed intracellular ROS generation in macrophages exposure to glabridin (1/8, 1/4, or 1/2 of the MIC) (Figure 7). No significant difference in ROS production was detected among the glabridin treatments.

Next, we asked ourselves whether subinhibitory concentrations of glabridin would have any effect on the ability of L. monocytogenes-stimulated macrophages to release METs. For this experiment, we incubated RAW264.7 cells with L. monocytogenes treated with glabridin for 2 h. Figure 8 shows that treatment with L. monocytogenes alone induced MET release from macrophages (Figure 8A). A similar phenomenon was also observed in which L. monocytogenes induced the release of extracellular reticulation structures after exposure to glabridin; however, the extent of MET formation was not significantly different from that of L. monocytogenes alone (Figure 8B). Based on these results, we showed that subinhibitory concentrations of glabridin did not affect the L. monocytogenes-induced MET release from macrophages.

The ET-mediated extracellular bactericidal effect is a novel defense mechanism of innate immune cells. L. monocytogenes was co-incubated with RAW264.7 cells in the presence of glabridin to determine whether subinhibitory concentrations of glabridin contribute to MET-mediated killing of extracellular bacteria by macrophages. We found that after a co-incubation with RAW264.7 cells and L. monocytogenes at 37°C for 2 h, L. monocytogenes was killed by macrophages compared with the control group (Figure 9). Efficient bacterial killing by macrophages was observed after the addition of glabridin. Cytochalasin D (10 μg/mL) was used to block phagocytosis to assess the effect of glabridin on extracellular L. monocytogenes clearance by METs. The results showed that the survival of L. monocytogenes treated with 1/4 of the MIC glabridin was similar to that of L. monocytogenes not treated with glabridin (Figure 9). This result showed that glabridin had no effect on the ability of macrophages to kill L. monocytogenes via METs.