Antimicrobial Activity of Aurisin A Against Streptococcus suis and Its Protective Effect on Epithelial Cells

TL;DR

A new compound from a mushroom shows strong antibacterial effects against a dangerous pig and human pathogen, protecting cells from damage.

Contribution

Aurisin A, a natural compound, is shown to inhibit Streptococcus suis growth and protect epithelial cells from its harmful effects.

Findings

Aurisin A inhibits biofilm formation and degrades preformed biofilms of Streptococcus suis at low concentrations.

Aurisin A protects lung epithelial cells by reducing adhesion, cell death, and cytotoxicity caused by S. suis.

Aurisin A reduces the hemolytic effect of S. suis on sheep blood, indicating protective activity against the pathogen.

Abstract

Streptococcus suis is one of the most important zoonotic pathogens threatening the lives of pigs and humans. Increasingly severe antimicrobial resistance in S. suis is becoming a global issue. Therefore, there is an urgent need to discover novel antibacterial alternatives for the treatment of S. suis infections. The current study investigated aurisin A, an aristolane dimer sesquiterpene isolated from the luminescent mushroom Neonothopanus nambi Speg. (Marasmiaceae), against S. suis. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of aurisin A against S. suis strains were in the range of 1.94–62.5 μg/mL. Scanning electron microscopy showed that aurisin A induced alterations in the cellular structure of S. suis, including a significantly wrinkled surface, intracellular content leakage, and cell lysis. The crystal violet staining assay illustrated that aurisin A significantly inhibited biofilm formation of S. suis strains at sub-MICs and exhibited strong degrading activity against the preformed biofilms. Aurisin A significantly inhibited the adhesion, cell death, and cytotoxic activities of S. suis in lung epithelial cells in a concentration-dependent manner. Additionally, aurisin A significantly reduced the hemolytic effect of S. suis on defibrillated sheep blood, indicating protective activity of aurisin A against this bacteria. Taken together, these findings highlight aurisin A as a promising therapeutic candidate for the treatment of S. suis infections, with key roles in inhibiting biofilm formation and hemolytic activity, as well as providing protective effects to epithelial cells, including anti-adhesion, anti-cytotoxicity, and anti-cell death activities.

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1. Introduction

Streptococcus suis is a zoonotic pathogen of both veterinary and human concern [1,2,3,4]. Severe infections can progress rapidly to fatal septic shock, as evidenced by the human epidemics in China in 1998 and 2005 [1]. Currently, 29 serotypes of S. suis have been identified based on antigenic variations in the structure of the capsular polysaccharide (CPS) that encases the bacterium [5]. Of these, serotype 2 is the most prevalent in S. suis infections, although human infections caused by serotypes 4, 5, 9, 14, 16, 21, 24, and 31 have also been reported [2,3,4].

Antimicrobial resistance represents a critical global health challenge; in the absence of effective preventive interventions, such as vaccines, there is a considerable risk of S. suis strains developing enhanced antimicrobial resistance and horizontally transferring resistance genes to other bacteria [6]. The small RNA rss04 in S. suis contributes to the development of meningitis by regulating capsular polysaccharide (CPS) synthesis and promoting biofilm formation [7]. Furthermore, biofilm formation in S. suis plays a critical role in the development of meningitis following intracranial subarachnoid infection [8]. In addition, S. suis can secrete extracellular polymeric substances, including polysaccharides, proteins, nucleic acids, lipids, and bacterial vesicles [9]. These secretions facilitate biofilm formation, thereby enhancing bacterial resistance and immune evasion capabilities in adverse environments involving stress, including antibiotic exposure [9,10]. Biofilms have demonstrated antibiotic resistance that is 10–1000 times higher than that observed in planktonic cells [11]. This may be due to a decreased antibiotic permeability into the biofilm [10]. Consequently, there is an urgent need to identify effective non-antibiotic agents, particularly those derived from traditional medicine, for the prevention and treatment of S. suis infection, thereby providing a novel strategy for controlling this pathogen in clinical practice [12].

Phytomedicines, or traditional medicines, offer promising therapeutic potential, including antimicrobial activity and the ability to prevent cancer progression, due to their low toxicity and minimal side effects [13,14,15]. Numerous plant-based substances have demonstrated antibacterial activity. Interestingly, basidiomycetes have long been widely explored as an important source of mycopharmaceuticals with potential therapeutic properties, including antimicrobial activity [15,16]. Several secondary metabolites synthesized by basidiomycetes are terpenoids, such as diterpenoids, sesquiterpenoids and triterpenoids [17].

Aurisin A is an aristolane dimer sesquiterpene isolated from the luminescent mushroom Neonothopanus nambi Speg. (Marasmiaceae), which is normally found on logs or dead wood in broad-leaved forests in northeast Thailand [18]. This compound exhibits various biological and pharmacological activities, including antimycobacterial activity against Mycobacterium tuberculosis and antimalarial activity against Plasmodium falciparum [16], anticancer potential against lung cancer cells (NCI-H187 and A549) [16,19], as well as against breast cancer cells (BC1), epidermoid carcinoma cells (KB), and cholangiocarcinoma cells (KKU-100, KKU-139, KKU-156, KKU-213, and KKU-214) [16]. Furthermore, aurisin A can potently inhibit the growth of vancomycin-intermediate Staphylococcus aureus (VISA) ATCC 700699 and exhibits rapid, time-dependent, bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA), resulting in 100% mortality within 1 h [15].

Thus, aurisin A is a promising candidate for the development of therapeutic agents effective against multidrug-resistant bacteria. The current study evaluated the effect of aurisin A on the inhibition of growth and induction of cell death in different S. suis serotypes. Additionally, the ability of aurisin A to inhibit biofilm formation and the hemolytic activity of S. suis were evaluated, as well as its protective effects against S. suis-mediated adhesion, cytotoxicity, and cell death in lung epithelial cells.

2. Results

2.1. Antimicrobial Activity of Aurisin A Against S. suis Strains

Based on the results, the MIC and MBC values of aurisin A against all nine S. suis serotypes indicated both inhibitory and bactericidal activities against the tested strains. The MIC and MBC values ranged from 1.94 to 62.5 μg/mL. Notably, the most potent activity was observed against S. suis serotype 2 strain 34537, for which the MIC and MBC values were both 1.94 μg/mL (Table 1). However, penicillin G exhibited stronger inhibitory activity against S. suis than aurisin A, with MIC and MBC values ranging from 0.03 to 0.12 µg/mL and from 0.06 to 0.50 µg/mL (Table 1).

2.2. Effect of Aurisin A on Cell Integrity of S. suis

The effect of aurisin A on cell integrity was examined using scanning electron microscopy (SEM), which revealed marked morphological differences in S. suis serotype 2 cells between the control cells and those treated with aurisin A at 1 × MIC and 2 × MIC (Figure 1). Control cells exhibited smooth surfaces and intact cell walls, whereas cells treated with aurisin A at 1 × MIC showed surface roughening, cellular shrinkage, and partial structural disruption (Figure 1), similar to the results observed with penicillin G treatment. Notably, treatment with aurisin A at 2 × MIC resulted in pronounced cell shrinkage, severe structural disruption, and cell lysis compared with the control group (Figure 1).

2.3. Effect of Aurisin A on Biofilm Formation of S. suis

The effects of sub-MICs of penicillin G and aurisin A on the biofilm formation of S. suis strains are shown in Figure 2A–I. The biofilm formation of S. suis was significantly inhibited in a dose-dependent manner following treatment with both agents. Based on the experimental results, aurisin A exhibited the strongest inhibitory activity against S. suis serotype 14 (Figure 2D), followed by serotype 16 (Figure 2E) and serotype 9 (Figure 2C), with biofilm inhibition rates of 83.23 ± 3.85% (p < 0.001), 56.76 ± 3.20% (p < 0.001), and 53.15 ± 5.69% (p < 0.001), respectively, after treatment with 1/2 × MIC compared with the untreated control (Table S1). However, the biofilm-inhibition capacity of aurisin A was significantly lower than that of penicillin G in all tested strains.

2.4. Effect of Aurisin A on Hemolytic Activity of S. suis

The inhibitory effect of aurisin A at different concentrations (1/4 × MIC, 1/2 × MIC, 1 × MIC, 2 × MIC, and 4 × MIC) on the hemolytic activity of the culture supernatant of S. suis was measured. Based on these results, the hemolytic activity of all S. suis strains was markedly reduced by aurisin A in a dose-dependent manner (Figure 3A–I); the hemolytic activity of S. suis serotype 16 showed the strongest effect, which was significantly reduced from 73.75 ± 6.67% (0 × MIC) to 1.23 ± 0.51% (4 × MIC) (p < 0.001). Furthermore, the hemolytic activity in the culture supernatant of all S. suis strains was impaired by aurisin A at sub-MICs in a dose-dependent manner, indicating a direct interaction between aurisin A and hemolytic toxins.

2.5. Effect of Aurisin A on Adherence of S. suis to Epithelial Cells

The effect of aurisin A (1/4 × MIC, 1/2 × MIC, and 1 × MIC) on the adhesion of S. suis to A549 cells was investigated. As shown in Figure 4A–I, aurisin A significantly inhibited the adhesion of all the tested S. suis strains to A549 cells in a dose-dependent manner compared with the control group. The strongest effect was observed with S. suis serotype 24, with its adhesion rates decreasing from 33.89 ± 3.47% (0 × MIC) to 0.28 ± 0.10% (1 × MIC) (p < 0.001; Table S2 and Figure 4F). Additionally, S. suis serotype 2 strain P1/7 exhibited a marked reduction in adhesion activity, from 90.56 ± 5.36% (0 × MIC) to 17.78 ± 5.09% (1 × MIC) (p < 0.001), as shown in Figure 4I and Table S2. Overall, the adhesion ability of S. suis was significantly reduced in a dose-dependent manner following treatment with both agents.

2.6. Effect of Aurisin A on Cytotoxicity of S. suis on A549 Cells

The inhibitory effect of aurisin A at concentrations of 1 × MIC and 2 × MIC on the cytotoxicity of S. suis toward epithelial cells was investigated. Based on these results, treatment with aurisin A significantly reduced the cytotoxic activity of S. suis toward A549 cells in a dose-dependent manner (Table S3 and Figure 5A–I). Specifically, aurisin A markedly reduced cytotoxicity in A549 cells infected with S. suis serotype 4, with the cytotoxicity decreasing from 69.70 ± 4.90% (0 × MIC) to 31.89 ± 5.76% (2 × MIC) (p < 0.001; Figure 5A).

The effects of aurisin A at concentrations of 0 × MIC and 2 × MIC on cell death in S. suis-infected A549 cells were evaluated using Calcein AM/PI staining. Under an inverted fluorescence microscope, live cells exhibit green fluorescence, whereas dead cells emit red fluorescence. As shown in Figure 6, treatment with aurisin A at 2 × MIC markedly decreased the proportion of dead cells compared with untreated S. suis-infected A549 cells and showed a protective effect comparable to that of the positive control. These results demonstrate that aurisin A exerts a protective effect against S. suis-induced cell death in A549 cells.

3. Discussion

Currently, antibiotics remain the most effective means of controlling S. suis infections. However, the emergence of drug-resistant S. suis strains, including those resistant to fluoroquinolones (FQs), has been attributed to the widespread misuse and overuse of various antibiotics, such as tetracyclines, sulfonamides, macrolides, and β-lactams, as well as FQs [20,21]. The resistance and tolerance of S. suis are influenced by multiple factors, including mutations in antimicrobial resistance genes, alterations in metabolic activity, horizontal gene transfer, the presence of outer membrane vesicles, efflux pump mechanisms, regulation of the quorum-sensing system, and biofilm formation [22,23,24]. In addition, S. suis is capable of secreting extracellular polymeric substances, including polysaccharides, proteins, nucleic acids, lipids, and bacterial vesicles, which facilitate biofilm formation and enhance bacterial resistance and immune evasion under adverse environmental conditions, such as stress and antibiotic exposure [9].

Aurisin A has been shown to exhibit antimalarial activity against Plasmodium falciparum and antimycobacterial activity against Mycobacterium tuberculosis, with an inhibitory concentration (IC_50_) of 0.80 µM and a minimum inhibitory concentration (MIC) of 92.55 µM, respectively [16]. Additionally, this compound showed no cytotoxic effect on normal white blood cells at concentrations ranging from 0 to 394 µM [25]. Aurisin A has been reported to exhibit potent anti-MRSA activity, with MIC and MBC values ranging from 3.91 to 7.81 µg/mL, and can effectively inhibit S. aureus [15]. In comparison, our study found MIC and MBC values against S. suis ranging from 1.94 to 62.5 µg/mL. N. nambi FRIM550 has been reported to produce secondary metabolites with potent antibacterial activity against MRSA. These metabolites exhibit strong inhibitory effects against both reference MRSA and clinical fusidic acid-resistant MRSA (FRMRSA) strains, with TRAI showing MIC values of 3.91 and 7.81 μg/mL against MRSA and FRMRSA, respectively [26]. The present study demonstrated that penicillin G exhibited stronger inhibitory effects against S. suis, as indicated by the MIC and MBC values, compared with aurisin A. However, a previous study reported that aurisin A exhibited potent antimicrobial activity and rapid bactericidal effects against antimicrobial-resistant bacteria, achieving 100% bacterial mortality within 1 h, which is more effective than conventional antibiotics [15]. Additionally, plant extracts offer advantages over single antibiotics because they contain a variety of active compounds that act synergistically to enhance antimicrobial effects and employ multitarget mechanisms to effectively inhibit bacterial growth and reproduction, thereby reducing the occurrence of resistance [27,28].

The structural integrity of the bacterial cell wall is a fundamental prerequisite for sustaining normal physiological activities [12]. The cell membrane functions as a natural protective barrier, preventing the penetration of harmful agents, such as antibiotics, toxins, and degradative enzymes while simultaneously regulating the selective uptake of essential nutrients into the cell [12]. Numerous natural compounds have been reported to target bacterial cell wall integrity. For example, a previous study [29] used transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to demonstrate severe structural damage to the morphology of SC19 cells following TF1 treatment. This damage was characterized by disruption of the cell wall and membrane, resulting in the formation of empty shells due to cytoplasmic leakage. In addition, most cells appeared markedly shrunken and irregular in shape, with indistinct cell walls and collapsed membranes. Furthermore, nisin has been reported to alter the cell morphology and ultrastructure of S. suis, leading to a substantially wrinkled surface, intracellular content leakage, and cell lysis [30]. More recently, TEM and SEM analyses have shown that SC19 cells treated with phillyrin exhibits a crumpled morphology, accompanied by damage to the cell wall and membrane, cytoplasmic loss, and cavitation within the intracellular cavity [12]. In the present study, the SEM analysis found that S. suis cells treated with aurisin A similarly exhibited surface roughening, cellular shrinkage, and structural disruption.

Biofilm formation is one of the major factors contributing to bacterial resistance, and the excessive use of antimicrobials promotes the proliferation of drug-resistant strains [31]. Consequently, there is an urgent need to explore alternative strategies for combating infections and mitigating the emergence of resistant bacteria. Plants represent a vast reservoir of natural products, some of which have demonstrated anti-biofilm properties [32,33]. Therefore, reducing the adhesion of S. suis to surfaces may represent an effective strategy for mitigating biofilm formation. Based on the results of the present study, aurisin A significantly reduced biofilm formation in nine S. suis strains. In addition, a previous study demonstrated that syringopicroside can effectively inhibit S. suis biofilm formation [34]. Similarly, nisin has been shown to suppress S. suis biofilm formation in a concentration-dependent manner and to exhibit potent biofilm-degrading activity [30]. In addition, monoterpenoid glycoside extracts from peony seed meal displayed inhibitory effects on the biofilm-forming capacity of S. suis at sub-MICs, with paeoniflorin in particular substantially reducing biofilm formation and markedly suppressing the production of the S. suis AI-2 signaling molecule [35]. More recently, andrographolide (AP) has been shown to exert synergistic effects with antibiotics, whereby the combination of 125 µg/mL AP and 5 µg/mL tobramycin significantly reduced pre-formed S. suis biofilms, while the combination of 62.5 µg/mL AP and 160 µg/mL streptomycin significantly inhibited S. suis biofilm formation [36].

The hemolytic activity of S suis is mediated by suilysin, a cholesterol-dependent cytolysin that lyses red blood cells and contributes to tissue damage, immune evasion, and severe infections such as meningitis and septicemia [37,38,39,40]. Inhibition of suilysin represents a promising anti-virulence strategy, and recent studies have shown that natural compounds can reduce hemolysis and protect host cells. Nisin exhibits minimal hemolytic activity, with hemolysis rates of 0.35% and 1.46% at concentrations of 64 μg/mL and 128 μg/mL, respectively [30]. Similarly, baicalein substantially inhibits the hemolytic activity of suilysin (SLY) by binding directly to the toxin and disrupting its secondary structure [41]. In addition, TF1 was shown to markedly decrease the hemolytic activity of SC19 and to suppress SLY protein expression in the culture supernatant [29]. More recently, phillyrin was reported to significantly reduce hemolytic activity secretion in a dose-dependent manner [12]. In the present study, the reduction in hemolytic activity in the culture supernatants of the nine S. suis strains was attributed to the direct inhibitory effect of aurisin A on toxin activity [29].

In the current study, the cell infection experiments yielded results consistent with the aforementioned findings. Aurisin A significantly reduced adhesion, cytotoxicity, and cell death in lung epithelial cells. The reduced adhesion observed following aurisin A treatment may be due to modulation of the expression of virulence genes or proteins that play important roles in bacterial adhesion. Similarly, water extracts of Rhizoma Coptidis have been reported to markedly inhibit S. suis adhesion by significantly reducing the expressions of the gapdh, sly, and mrp genes, as well as adhesion-related proteins such as antigen-like protein, CPS16V, and methyltransferase H [42]. In addition, TF1 treatment was shown to substantially decrease the adhesion capacity of SC19 [29]. Furthermore, phillyrin was shown to not only markedly inhibit SC19 adhesion but also attenuate cell damage and enhance the expression of tight junction proteins while exhibiting no cytotoxic effects on NPTr cells infected with SC19 [12]. In addition, quercetin treatment was reported to substantially alleviate S. suis-induced cytotoxicity by reducing suilysin (SLY) activity and decreasing the release of the pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) [43]. Consistent with that report, the present study revealed that aurisin A significantly reduced the cytotoxic activity of S. suis in A549 cells in a dose-dependent manner, thereby mitigating cell damage. The reduced cytotoxic effect observed following aurisin A treatment may be due to attenuation of inflammatory responses and SLY-mediated hemolytic activity, which could protect host cells from S. suis infection by decreasing cytotoxicity in infected cells [43]. Although the host-protective mechanisms underlying aurisin A remain unclear, these findings suggest that this compound warrants further investigation.

4. Materials and Methods

4.1. Bacterial Strains, Cell Culture, and Compounds

The S. suis strains used in this study are listed in Table 1. S. suis was cultured in brain heart infusion broth (BHI) (BHI; HiMedia Laboratories, Mumbai, India) or plated on sheep blood agar (Biomedia, Bangkok, Thailand) at 37 °C under 5% CO_2_. The human lung epithelial cell line (A549) was purchased from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and cultured in RPMI 1640 (Gibco; Thermo Fisher Scientific, Inc.; Grand Island, NY, USA) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.; Paisley, UK) 100 units/mL penicillin, and 100 µg/mL streptomycin (Sigma; Grand Island, NY, USA) at 37 °C under 5% CO_2_. Aurisin A was extracted from the culture medium of the luminescent mushroom Neonothopanus nambi PW1 according to a previously described protocol [16,18,25].

4.2. Minimum Inhibitory (MIC) and Minimum Bactericidal (MBC) Concentrations of Aurisin A

The MICs of aurisin A against the S. suis strains were determined using a broth microdilution assay following the M100 35th edition of the Clinical and Laboratory Standards Institute Guidelines [44]. Aurisin A was prepared at various concentrations (0.48–250 μg/mL) and added to 96-well plates containing bacterial suspensions. Penicillin G (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) at concentrations ranging from (0.003–4 μg/mL) was used as the positive control. After overnight incubation at 37 °C, the MIC was defined as the lowest concentration at which no visible bacterial growth was observed. An aliquot of 20 µL from each well that did not show any visible growth was drop-plated onto chocolate agar plates and incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration at which no apparent growth was observed on the agar plates [15].

4.3. Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) was used to assess the effect of aurisin A on the structural integrity of S. suis cells. The assay was performed following a previously described method with minor modifications [45]. Aurisin A and penicillin G were tested at concentrations equivalent to 1 × MIC and 2 × MIC. Bacterial cultures were inoculated into 6-well plates without treatment, overlaid with sterile glass slides, and incubated overnight at 37 °C. The samples were then washed three times with phosphate-buffered saline (PBS) and dehydrated through a graded ethanol series of 25%, 50%, 75%, 90%, and 100% ethanol (v/v in distilled water), with each step lasting 20 min. The specimens were air-dried and stored in a desiccator until further processing. Subsequently, the samples were mounted onto aluminum stubs using double-sided carbon adhesive tape, sputter-coated with a thin layer of gold, and examined using an SEM instrument (JSM-IT510 SEM instrument; JEOL Ltd., Tokyo, Japan).

4.4. Crystal Violet Staining Assay

The effect of aurisin A on S. suis biofilm formation was assayed as previously described in [34]. S. suis was cultured in 96-well plates in the presence of aurisin A and penicillin G at sub-MICs (1/2 × MIC, 1/4 × MIC, 1/8 × MIC, and 1/16 × MIC) for 72 h. After incubation, the culture medium was removed, and the wells were washed three times with 1 × phosphate-buffered saline (PBS) to eliminate planktonic cells. The remaining biofilms were fixed with 200 µL of 99% methanol per well and stained with a 1% crystal violet solution. Excess crystal violet was removed, and the wells were rinsed three times with 1× PBS. Subsequently, 200 µL of 33% glacial acetic acid was added to solubilize the bound dye, and the optical density (OD) was measured at 595 nm using a Multiskan SkyHigh full-wavelength microplate reader (A51119500C; Thermo Fisher Scientific; Waltham, MA, USA) and the percentage inhibition was quantified using the equation: Biofilm inhibition rate (%) = (OD595 blank control − OD595 experimental group)/OD595 blank control [35].

4.5. Hemolysis Assay

Hemolysis assay was performed to evaluate the ability of aurisin A to inhibit S. suis–induced hemolysis following the previously described protocol in [29] with minor modifications. Briefly, S. suis was treated with aurisin A at various concentrations (0 × MIC, 1/4 × MIC, 1/2 × MIC, 1 × MIC, 2 × MIC, and 4 × MIC) at 37 °C for 4 h and then centrifuged at 4200 rpm for 10 min at 4 °C. A 100 μL aliquot of the supernatant was collected and incubated with 100 μL of 2% defibrillated sheep blood (Clinag Co., Ltd.; Bangkok, Thailand) suspended in 0.85% sodium chloride at 37 °C for 2 h. An equal volume of 2.5% Triton X-100 (Kemaus, Cherrybrook, NSW, Australia) was used as the positive and served as the reference for 100% hemolysis. Then, each sample was centrifuged at 1000 rpm for 10 min at 4 °C, and the absorbance value (OD540 nm) of the supernatant was determined using the Multiskan SkyHigh full-wavelength microplate (Thermo Fisher Scientific, Vantaa, Finland) reader mentioned above.

4.6. Adherence Assay

Human lung epithelial A549 cells were seeded into 24-well culture plates at a density of 1 × 10^5^ cells per well and incubated overnight at 37 °C under 5% CO_2_. The cells were then pretreated with aurisin A and penicillin G at concentrations of 1/4 × MIC, 1/2 × MIC, and 1 × MIC for 3 h. Subsequently, S. suis was added to the 24-well plate at a multiplicity of infection (MOI) of 10 and co-incubated for an additional 2 h. After incubation, the cells were washed three times with 1× phosphate-buffered saline (PBS) to remove unattached bacteria. The host cells with attached bacteria were lysed using 0.025% Triton X-100 (KEMAUS; Cherrybrook, NSW, Australia) on ice for 15 min. Finally, the number of bacteria adhering to the cells was determined by counting colonies in three independent experiments [12,29].

4.7. Cytotoxicity Assay

The protective effect of aurisin A on the S. suis-infected A549 cells was evaluated using CCK-8 staining (Dojindo Molecular Technologies, Inc.; Rockville, MD, USA), with slight modifications to previously described methods [12,46]. Human lung epithelial A549 cells (1 × 10^4^ cells/well) were seeded into 96-well plates and incubated at 37 °C under 5% CO_2_. After incubation, the cells were gently washed three times with sterile 1 × PBS. The cells were then infected with S. suis at an MOI of 100 and incubated for 2 h. Subsequently, aurisin A and penicillin G were added to the wells at concentrations of 1 × MIC and 2 × MIC, respectively, and the plates were incubated for 6 h. The wells were then washed with 1 × PBS, after which, 10 µL of a CCK-8 solution was added to each well and incubated for 2 h. Finally, the absorbance of the supernatant at 450 nm was measured using a Multiskan SkyHigh full-wavelength microplate reader (A51119500C; Thermo Fisher Scientific; Waltham, MA, USA).

4.8. Cell Death Assay

The protective effect of aurisin A on S. suis-infected human epithelial A549 cells was evaluated using Calcein AM/PI staining (MedChemExpress, Monmouth Junction, NJ, USA) using a previously described method with slight modifications [12]. Briefly, human epithelial A549 cells were seeded into 6-well plates at a density of 1 × 10^5^ cells per well and cultured at 37 °C in a humidified atmosphere containing 5% CO_2_. After incubation, the cells were gently washed three times with sterile 1 × PBS. The cultures were then infected with S. suis at a multiplicity of infection (MOI) of 100 and incubated for 2 h. Subsequently, after another PBS wash, aurisin A and penicillin G were added at concentrations corresponding to 0 × MIC and 2 × MIC, and the cells were further incubated for 6 h. Cells cultured in fresh RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS but without aurisin A served as the negative control. The cells were washed with 1× PBS, and a Calcein AM/PI staining solution was added to each well. Following 30 min of incubation in the dark, the stained cells were examined under an inverted fluorescence microscope (TS2-FL; Nikon Corporation, Yokohama, Japan) to visualize live and dead cells.

4.9. Statistical Analysis

All experiments were repeated three times and statistical analyses were performed using the SPSS software (version 26.0; IBM Corp.; Armonk, NY, USA). The results are expressed as the mean ± standard deviation (SD). Statistical significance was analyzed based on one-way ANOVA, with p values of p < 0.05 (), p < 0.01 (), and p < 0.001 () indicating different levels of statistical significance.

5. Conclusions

This study identified aurisin A, a sesquiterpene dimer from Neonothopanus nambi, as a novel antibacterial agent against S. suis, and is the first to demonstrate its in vitro anti-biofilm, anti-hemolytic, and host-protective effects. Infection experiments using a cell model revealed that aurisin A reduced the adhesion ability and virulence of S. suis, as well as its cytotoxicity and ability to induce cell death. Collectively, these findings suggest that aurisin A has strong potential as a promising candidate for the prevention and treatment of S. suis infections.